Macrophage activation and anti-tumor activity of biologic and synthetic agents. Systemic administration of the synthetic immunopotentiator pyran, was as effective as the use of the biologic immunopotentiator BCG in activating macrophages and in inhibiting the Lewis lung carcinoma and MCA 2182 sarcoma. Several other synthetic polyanions also activated macrophages and exhibited some anti-tumor activity, but none were as effective as pyran. Cell-wall fractions such as the Ribi vaccine and MER were considerably less effective than BCG. The anti-tumor activity of pyran against the virtually non-immunogenic Lewis lung carcinoma involved non-specifically activated macrophages, and both anti-tumor activity and macrophage activating ability persisted over a 100-fold range of drug from 0.5 mg/kg to 50 mg/kg. The ability of activated macrophages to destroy tumor cells was abrogated by treatment with trypan blue, an inhibitor of macrophage lysosomal enzymes. In addition, preincubation of tumor cells with activated peritoneal cells at effector-cell:target-cell ratios of 20:1 and 5:1 markedly decreased tumor incidence and mortality. Glycogen-stimulated or unstimulated peritoneal cells were completely inactive in inhibiting tumor growth in vivo or exhibiting cytotoxicity in vitro, demonstrating the requirement for activated macrophages selective for tumor-cell destruction.


Dietary aflatoxins and human liver cancer. A study in Swaziland. A study in Swaziland to assess the possible relationship of aflatoxin contamination and the incidence of primary liver cancer is reported. Aflatoxin ingestion levels have been determined in "food from the plate" samples collected over a 1-year period. A significant correlation between the calculated ingested daily dose and the adult male incidence of primary liver cancer in different parts of Swaziland has been established. Samples of foodstuffs other than the plate samples also reflected the correlation of aflatoxin contamination and liver cancer. This study extends and amplifies the findings of an earlier study in the Murang'a district of Kenya and supports the hypothesis that aflatoxin ingestion is a factor in the genesis of primary liver cancer in Africa.


Recent trends in breast-cancer incidence and mortality in relation to changes in possible risk factors. Breast cancer incidence and mortality in England and Wales and the United States increased between 1950 and 1973, mainly in women aged between 45 and 64 years. These increases appeared to be partly cohort-specific, beginning with cohorts born around 1899, and partly cross-sectional, beginning in the mid-1960s. In both countries, cohort-specific decreases in fertility paralleled the cohort-specific increases in breast cancer rates and may, at least in part, have been responsible for them. Changes in other factors, such as age at menarche and menopause, use of rauwolfia derivatives and oestrogens, consumption of fat and meat, and breast cancer treatment were considered in relation to the cross-sectional increases in breast cancer rates. On the evidence available, it was not certain that any of these could explain the breast cancer increases.


Cellular immunity in mammary cancer patients as measured by the leukocyte migration test (LMT). A follow-up study. In 58 cases of mammary cancer treated by surgery the leukocyte migration test (LMT) has been applied to the study of cellular immunity using homogenate from autochthonous and homologous tumors as antigens. A positive test, i.e. inhibition of migration by antigen, was observed in 52 patients from 1 day up to 40 days after surgery. Six patients were negative. There was extensive immunological cross-reactivity among mammary cancer in that a positive test was also obtained with homogenate from homologous mammary tumors. Generally, antigens from other tumors did not react. The test was also negative when the leukocytes were derived from healthy persons. Sera from mammary cancer patients abrogated the inhibitory effect of tumor antigen. The disappearance of positivity about 40 days after surgery and its reappearance during the metastatic process indicates that the positive LMT is associated with progressive disease.


Rat liver microsome-mediated binding of benzo(a)pyrene metabolites to DNA. Individual metabolites of benzo (a) pyrene were isolated from rat liver microsomal incubation of the parent hydrocarbon, and subsequently bound to DNA in separate incubations with microsomes. Of the metabolites examined, by far the greatest binding resulted with 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (BP-7,8-diol); the binding od the benzo (a) pyrene phenols (BP-OH) was about 50% that of the 7,8-diol. Analysis of the hydrocarbon-deoxyribonucleoside derivatives obtained by enzymic degradation of these DNA samples revealed that the binding of BP-7,8-diol was accounted for mainly by a single product identical to one of the product identical to one of the products obtained from DNA with bound benzo(a)pyrene. Furthermore, the microsome-induced binding of BP-OH to DNA yielded mainly a single product identical in chromatographic behaviour to the major product derived from benzo(a)pyrene when bound to DNA during incubation with microsomes.


[Concentration course of lithium in liquor and aqueous humor after single oral application in man]. Kinetic examinations of lithium concentrations in the aqueous humour and in the liquor in man were carried out. The passage of lithium into both extravascular compartments took place in different manners. Lithium - liquor concentrations seem to be only of a rather limited positive evidence as to the concentrations at the spot of action; the possible causes for this fact are discussed.


Distribution and diaplacental passage of infused 3H-fenoterol hydrobromide (partusisten) in the gravid rabbit. Tritium-labelled fenoterol hydrobromide was infused into an ear-vein of pregnant rabbits over a period of 4 hours. Two doses were used. The tritum concentrations in the blood of the dams and the amniotic fluid were determined from samples obtained during infusion and up to 11 hours after completion of infusion. The tritium concentrations were also determined in selected organ samples from dams and fetuses, taken immediately and 5 and 11 hours after infusion. Within 1.5 hours after commencement of infusion, the tritium activity in the whole blood of the dams reached a plateau on a level corresponding to the dose administered. Immediately after completion of infusion, the level of radioactivity fell rapidly. The tritium concentration in the amniotic fluid began to rise far more slowly after a time lag, so that approximately 2 hours after completion of infusion, the concentrations in maternal whole blood  and amniotic fluid were equal. As the amniotic fluid level fell at a slower rate than the maternal whole blood level, the former somewhat exceeded the latter at this advanced stage of the study. The tritium distribution patterns of the dams indicate rapid elimination via kidneys and intestine. The tritium concentration in all the tissue fell rapidly after completion of infusion. The lung and uterus have a slightly greater affinity. The tritium concentrations in the fetal organs remained below those in the organs of the dams for the whole duration of the study. The highest radioactive concentration in the fetuses was found in the urine. Further observation of the rapid conversion of fenoterol hydrobromide into conjugates leads us to expect only minimal amounts of active substance to be found in the fetuses after infusion.


Analgesic abuse and personality characteristics. The Freiburg Personality Inventory (FPI) was mailed to a group of 880 female industrial workers who were participating in a 5-year prospective study aimed at elucidating eventual renal toxity of phenacetin-containing analgesics. 338 completely filled in test-sheets were returned. The study sample was divided into five sub-groups according to the amount of drug which could be located. 1. Consumers who had a positive test for analgesic-intake in at least one urine sample (paracetamol = N-acetyl-p-aminophenol as principal metabolit of phenacetin),N = 65.2. "High intakers", paracetamol values over 0.499 o.D. (o.D. = optical density in the spectrophotometric determination of paracentamol [total of three tests]), N = 73.3. "Medium intakers", paracetamol values 0.200-0.499 o.D. (total of three tests), N = 25.4. "Low intakers", paracetamol values 0.049-0.199 oD., N = 84.5. "None-consumers" with negative urine tests, N = 199. It was shown that for some personality features, consumers could be distinguished from non-consumers and "high intakers" from "low intakers", but not "low intakers" from non-consumers nor "high intakers" from "medium intakers". The areas contributing mainly to differentiation were general psychosomatic disturbance, irritability tinged with dysphoria, psychosocial and emotional immaturity, and lability. The importance of early registration is consequently demonstrated.


Investigation on the serum and lung tissue level of rifampicin in man. After the oral administration of 600 mg rifampicin, we determined the rifampicin level of the lung tissue of 18 operated patients, as well as the rifampicin level of the pleural callus of one patient, that of the pericardial cyst of another, and the serum level of all the 20 patients. The serum level amounted to an average of 6.5 mcg/ml between 2 and 6hours after administration, and to 3.3 mcg/ml between 8 and 9 hours. The lung tissue level amounted to an average of  2.3 mcg/g. The lung rifampicin level reached 32-44-62 per cent of the serum level 2 to 9 hours after its administration. This concentration was found to meet treatment requirements of Mycobacterium tuberculosis, Gram-positive cocci, and certain Gram-negative bacteria-induced airway infections.


[Efficacy of prolonged therapy with vitamins C and A and radiologic changes in the Ehlers-Danlos syndrome]. Two children of the same sex, almost of equal age, and suffering from the Ehlers-Danlos syndrome were followed up for a period of seven years. One of the children was subjected to a systematic treatment with vitamins C and A, Lipovitan, sulphuric mineral waters, and kinesitherapy. The non-treated child is serving as control person for the treated one. Thus it has been possible to evaluate the efficacy of the therapy administered to the first child and also to state the osseous progressive changes in the non-treated child.


Influence of diabetes mellitus on drug metabolism in man. Drug metabolizing capacity was assessed in 29 normal subjects and in 28 diabetics on various treatment regimes using the rate of antipyrine elimination as reflux. Plasma half-life of antipyrine was markedly shorter (38%), and the plasma clearance of this drug greater (40%) in diabetics receiving insulin than in controls. There was also an indication of a small increase in rate of test drug elimination in diabetics taking chlorpropamide. Patients being controlled solely on diet or those on tolbutamide did not exhibit an altered rate of drug elimination. The rate of metabolism and inactivation of other drugs may be altered in diabetics taking insulin so that optimal doses of some comcomitantly administered drugs may be different in this group of patients.


An improved synthesis of tert-butyloxycarbonyl-L-histidine. In our investigations on the effect of replacement of histidine by homohistidine (1) on the biological activity of some peptide-hormones, relatively large quantities of Boc-homohistidine were required. Homohistidine being difficult to synthesize, it was essential to find an effective way of introducing the Boc-group. In the past, many syntheses of Boc-histidine were reported (2-6), none of which proved to be quite satisfactory. However, by modifying the procedure of Flouret et al. (6) a convenient method resulting in a high yield of Boc-histidine and Boc-homohistidine was found.


Protamines, histones and the genetic code. New evidence for code evaluations. A new approach is presented to give evidence for the theories of Jukes and Crick (1-3) that at a more primitive stage the genetic code consisted of doublets separated by "comma-bases" rather than true triplets and that G and C or A and U are the exclusive bases used by the primordial code. This approach makes use of the conservation of the histone IV sequence over extremely long periods of time by comparing the amino acid composition of the average vertebrate protein with the one of histone IV, a reconstructed ancestral polypeptide and various nuclear proteins, homologous or otherwise related to it. All protamines studied and the majority of histones show deviations from the average vertebrate protein which are statistically highly significant if the amino acids sufficiently coded for by the first two bases are compared. A similar result is obtained for those amino acids which are sufficiently coded for by the first two bases of the codon and have codons composed of G and C only.


Amino acids and peptides. XLII. Synthesis of an octapeptide sequence (A20-A27) of rubredoxin. A protected octapeptide corresponding to a section found in the first half of the protein chain of rubredoxin has been prepared by standard peptide methods. Alternative approaches of this fragment are discussed in some detail. This work completes the series of subunits necessary to synthesize the molecule.


On the conformation of the disulphide group in L-cystine and its derivatives. The terminal groups in cystine are asymmetric and hence the dissulphide group with right and left chirality may not have equal energy. An attempt is made to resolve this problem through energy calculation using semi-empirical methods involving non-bonded and electrostatic interactions and simple Pauling type torsional potential for the disulphide group. The results are discussed in the light of experimental data.


Heat denaturation of human serum albumin. Migration of bound fatty acids. Prolonged heat treatment of solutions of human serum albumin at 60 degrees C resulted in formation of one aggregate fraction and one fraction that was stable against further heat treatment. Fatty acid analyses of these fractions indicate that the heat stable fraction is formed by migration of fatty acids from the aggregating molecules to the remaining monomer thereby stabilizing the latter against heat denaturation. Disulphide and SH groups are involved in the aggregation process.


Synthesis of peptides by fragment condensation on a solid support. II. A scheme for preparation of 4,8-disubstituted vasopressins evaluated on 8-arginine-vasopressin. A convenient scheme for the synthesis of 4,8-disubstituted vasopressins has been designed and its usefulness evaluated in the preparation of one of the parent hormones, 8-arginine-vasopressin. The main feature of the scheme involves preparation of two protected tripeptide fragments, Z-Cys(Bzl)-Tyr(Bzl)-Phe and Boc-Asn(Mbh)-Cys(Bzl)-Pro which are incorporated in a synthesis on a solid support. Both tripeptides were prepared conventionally with the carboxyl groups protected as benzyl esters. The benzyl-ester groups were removed by transesterification with 2-dimethylaminoethanol and subsequent hydrolysis. To avoid racemization in the coupling step with the fragment containing a C-terminal phenylalanine, N-hydroxysuccinimide was added. After removal of the peptide from the resin, deprotection, oxidation and desalting, final purification was effected by ion-exchange chromatography. Apart from the main product, which exhibited full pressor activity, only small amounts of impurities could be isolated.


Isolation of a trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) by affinity adsorption through Kunitz inhibitor-sepharose. A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.


Relative reactivities of sulfhydryl groups with N-acetyl dehydroalanine and N-acetyl dehydroalanine methyl ester. The reaction rates in aqueous solutions of aminothiols, thiols, and other compounds with N-acetyl dehydroalanine and its methyl ester (2-acetamindoacrylic acid and methyl 2-acetamidoacrylate) were studied as a function of the structure of the thiol compound in aqueous solutions. Correction of the observed second-order rate constants to identical thiol anion concentration gave a series of computed rate constants whose logarithms showed a linear dependence on the pK's of the thiol group in similar steric environments. Comparison of the addition rates of penicillamine to N-acetyl dehydroalanine and its methyl ester showed the methyl ester to react approximately 11,400 times faster than the acid. Addition rates for thiol acids and aromatic and heterocyclic thiols were also compared; each showed sluggish reactivity with dehydroalanine, but each reacted readily with methyl dehydroalanine. The kinetic data were applied in developing a method for preparing lanthionine in high yield.


Studies on trypsin inhibitors. Part I. Synthesis of protected peptides related to sequence 1-10 of porcine pancreatic secretory trypsin inhibitor II (Kazal). The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.


Studies on trypsin inhbitors. Part II. Synthesis of the protected tetrapeptide (sequence 11-14) of porcine pancreatic secretory trypsin inhibitor II (Kazal). Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.


Studies on trypsin inhibitors. Part III. Synthesis of the protected decapeptide (sequence 15-24) of porcine pancreatic secretory trypsin inhibitor II (Kazal). The synthesis of the amino-protected decapeptide tert-butyloxycarbonylhydrazide corresponding to positions 15-24 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal inhibitor) is described. The tripeptide free base threonyl-beta-tert-butylaspartylglycine tert-butyloxycarbonylhydrazide (sequence 22-24) was acylated with 1-succinimidyl o-nitrophenylsulfenylvalyl-S-acetamidomethylcysteinylglycinate (sequence 19-21). Removal of the amino protecting group from the resulting hexapeptide followed by acylation of the free base with either benzyloxycarbonylisoleucyl-O-tert-butyltyrosylasparaginylproline or O-nitrophenylsulfenylisoleucyl-O-tert-butyltyrosylasparaginylproline, via the pyrazoline active ester method, yielded the decapeptide tert-butyloxycarbonylhydrazide (sequence 15-24) in the form of Nalpha-benzyloxycarbonyl or Nalpha-O-nitrophenylsulfenyl derivative. The stereochemical homogeneity of the two decapeptides was assessed, after partial deprotection with liquid hydrogen fluoride, or thioacetamide and aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.


Observations on the functional cytochemistry of pars intermedia of the rat hypophysis. Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH2-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no alpha-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.


Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid. The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0-4 degrees C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.


Esterase. XXII. Cellular and subcellular localisation of the Es-9 esterase in mouse kidney. Among the various esterases of the mouse kidney the Es-9 esterase is outstanding in that it is fully repressed in the cortical proximal tubulus in the absence of testosterone but is constitutive in the terminal parts of the proximal tubulus. Thus, this enzyme is regulated in a different way in closely neighboured sections of the tubular system. Comparing the disc electrophoretic patterns of the kidney esterases with the distribution of esterases in ultrathin sections as revealed by electron microscopy it is concluded that the Es-9 esterase is located in the mitochondria.


Effect of clofibrate application on morphology and enzyme content of liver peroxisomes. Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.


Cryosurgical treatment of ocular squamous cell carcinoma of cattle. A total of 718 ocular squamous cell carcinomas of cattle were treated with various cryosurgical units and techniques during a 2.5-year study. In 609 of the lesions, a single freeze was used, with a cure rate (total regression) of 66%. In 109 lesions treated with a double freeze-thaw cycle (rapid freeze to - 25 C, and unaided thaw followed immediately by refreezing to -25 C), the cure rate was 97%. Cryosurgical units designed for delivery of liquid nitrogen provided a more rapid rate of freezing and, thus, a higher cure rate than did units designed for delivery of nitrogen vapor. It was concluded that cryosurgery for treatment of squamous cell carcinoma is a simple and rapid procedure; is economical; provides analgesia itself, due to sensory nerve injury; requires minimal preoperative and no postoperative medication; causes minimal side effects; may be repeated; and is excellent for use on suspect premalignant lesions.


Histochemical studies on the morphology of the Golgi apparatus and on the enzymes of carbohydrate metabolism in various nuclei of the rat mesencephalon. Detailed histochemical studies have been conducted on the distribution of various enzymes such as thiamine pyrophosphatase, alpha-glucan phosphorylase, hexokinase, glucose-6-phosphate dehydrogenase, aldolase, lactate dehydrogenase and succinate dehydrogenase in various components of the nucleus Edinger-Westphali, nucleus n. oculomotorii, nucleus ruber and nucleus niger of healthy adult male Wistar strain rats. The thiamine pyrophosphatase reaction showed the morphological patterns of the Golgi apparatus characteristic for each nucleus. The Golgi apparatus was well developed in the nucleus Edinger-Westphali, composing a network of highly fenestrated plates in the nucleus n. oculomotorii and nucleus ruber, and a simple network in the nucleus niger. These results indicate that the former three nuclei need a rich energy supply and argue against the possibility that the four nuclei have a secretory role. The neurons of the nucleus Edinger-Westphali may derive their energy mainly from glucose of the circulating blood, but glial cells may serve as energy donators to the neurons in the pars compacta of the nucleus niger, and the neurons of the other nuclei may derive energy from both sources. These conclusions are consistent with the morphological patterns of the Golgi apparatus.


Observations on bacteriologic isolation from pairs of quarter-milk samples. Observations are reported on the agreement of bacteriologic isolation between 11,917 pairs of quarter-milk samples collected as either duplicate samples (both samples collected during 1 farm visit) or consecutive samples (2 samples collected at different milkings). Better agreement was found for duplicate samples (95.5%) than for consecutive samples (88.5% for interval of 2 to 7 days and 81.3% for interval of 8 to 16 days). From these observations, it was estimated that for diagnosing an intramammary infection, the examination of a single quarter-milk sample would be expected to have an error of approximately 3%.


Pathogenicity of field isolants of hemagglutinating encephalomyelitis virus for neonatal pigs. The pathogenicity of hemagglutinating encephalomyelitis virus isolated from the brain of each of 5 pigs with clinical signs and microscopic lesions of encephalomyelitis (1 pig from each of 5 naturally infected herbs) was tested under experimental conditions. Each isolant was administered intranasally to 5 neonatal pigs. Clinically affected pigs developed either an acute or chronic form of illness, commencing 4 to 8 days after exposure. Listless, inappetence, and vomiting were clinical signs common to both forms. Additional signs of the acute form were paddling, muscle tremors, and hyperesthesia. Additional signs of the chronic form were loss of condition and either loss of weight or failure to gain weight at a rate similar to nonaffected littermates.


Porcine encephalomyelitis caused by hemagglutinating encephalomyelitis virus. Six epizootics of encephalomyeltis in suckling pigs in Minnesota were attributed to infection with hemagglutinating encephalomyelitis virus. The disease occurred in 74 litters of pigs and was characterized by sudden onset of tremors, inappetence, weakness, atazia, and hyperesthesia, with high morbidity and case fatality rate. Pathologic changes consisted of marked nonsuppurative, nondemyelinating encephalomyelitis characterized by perivascular mononuclear cuffing, gliosis, neuronal death, and satellitosis. Clinical disease was limited principally to suckling pigs during a single farrowing period and did not recur in the herds involved during the ensuing 18 months.


Semiautomated processing of systolic time intervals. Systolic time intervals are commonly used to identify changes in ventricular function. The method described facilitates measurement and calculation of many such intervals. This method utilizes a printed polygraph recording of electrocardiogram, phonocardiogram, and carotid pulse contour; a digitizing device for reading the necessary coordinates from the record; and a minicomputer for calculation of the intervals and for data analysis. Intervals related to approximately 100 pulse beats can be read, calculated, printed in chart and graph form, and subjected to some analyses in about an hour.


Nonrebreathing valve system for small animal respiratory measurements. Flow resistence, frequency response, and backflow characteristics of a nonrebreathing valve system applicable to measuring small animal respiratory volumes are described. Unidirectional valves, molded from liquid resin, were fitted into a modified Y tube for direct use on an endotracheal tube or face mask. Valve size and dead space volume can be scaled to fit several small animal species. In this system, valve resistance was 2.0 and 5.6 mm of water for steady flow rates of 0.5 and 3.0 1/min, respectively.


Validation of an automated determination of pulmonary resistance by electrical subtraction. An analog computer to determine dynamic pulmonary compliance (C) and pulmonary resistance (R) on a breath-by-breath basis was tested in guinea pigs and dogs. C was determined by dividing volume by transpulmonary pressure at instants of zero flow. R was determined by the method of electrical subtraction at predetermined flows. In both species the computer outputs and the results of direct analysis were in close agreement. In guinea pigs, the device reliably followed the rapid three- to fourfold changes in C and R resulting from histamine infusion. In unanesthetized dogs, the dispersion and mean values of C and R were similar by the two methods.


Biplane videoroentgenographic analysis of dynamic regional lung strains in dogs. A method is described for determining the spatial distribution of pulmonary parenchymal strains in the intact canine thorax, using measurements of displacement of metallic (1-mm-diam)) markers percutaneously implanted throughout the parenchyma of the right lung. Dogs are supported head up or head down in a water-immersion respirator with the animal's airway connected to ambient air. Tracking of the parenchymal markers is accomplished by stereo biplane videoroentgenographic recordings, which allow high temporal (60/S) and spatial (+/- 1.5 mm) resolution measurements of the "tagged" lungs during various respiratory maneuvers. After transferring the video information to a stop-action video disc, an operator-interactive computer program is used to input the geometric coordinates of the markers into the computer. The true spatial coordinates are then determined after correction for pincushion and magnification distortions. Spatial and temporal distributions of regional parenchymal strains are obtained by determining the distance between markers on a frame-by-frame basis over the extent of the respiratory cycle. Data indicate nonuniformity in regional lung parenchymal strains.


Force-velocity relations and fiber composition in human knee extensor muscles. Standardized measurements of dynamic strength of the kneee extensor muscles were performed in 25 healthy male subjects (17-37 yr) by means of isokinetic contractions, i.e., knee extensions with constant angular velocities. Overall variation between double determinations of maximal torque throughout the 90 degrees arc of motion (0 degrees = fully extended leg) averaged 10% for the different constant velocities chosen. At any given angle of the knee the torque produced was higher for isometric than for dynamic contractions. Dynamic torque decreased gradually with increased speed of shortening. Peak dynamic torque was reached at knee angles in the range: 55-66 degrees, with a displacement toward smaller knee angles with higher angular velocities. Correlations were demonstrated between peak torque produced at the highest speed of muscle shortening and percent as well as relative area of fast twitch fibers in the contracting muscle. In addition muscles with a high percentage of fast twitch fibers had the highest maximal contraction speeds. These observations on intact human skeletal muscle are consistent with earlier findings in animal skeletal muscle preparations.


Transcapillary exchange of strontium and sucrose in canine tibia. The walls of haversian capillaries have been proposed as a physiologic membrane controlling flux of solute between blood and bone. In this study, capillary permeability to 85Sr and [14C]sucrose was estimated in the dog tibia by using indicator dilution techniques. Sucrose was chosen as a tracer because it is inert and has no known transport system. The mean (+/-SD) observed ratio of permeabilities of 85Sr and sucrose was 2.36 +/- 0.46 (N = 14) which is not substantially different from the ratio of their free diffusion coefficients, 2.55. This ratio was not influenced when the dogs were made hyperparathyroid by injection of parathyroid hormone (2.16 +/- 0.55; N = 11). This suggests that free diffusion is the principal mechanism for moving 85Sr across the bone capillary wall.


Central neural stimulation of respiration in unanesthetized decerebrate cats. A previously reported central neural respiratory control process was restudied in unanesthetized decerebrate cats during spontaneous breathing, and during conditions of constant chemical stimulation where phrenic nerve activity was used to quantitate respiratory output. Respiration was increased by carotid sinus nerve stimulation. The pattern of respiration was examined at the cessation of such stimulation. In spontaneously breathing animals, active hyperventilation (HV) was followed by hyperpnea for up to 30 s and never by apnea. Passive HV was always followed by apnea. In animals with controlled chemical conditions, the transient at the end of stimulation consisted of two components, the first an immediate decrease in respiratory output and the second a slow decrease with a period of over 5 m. It is suggested that a facilitatory feedback process, probably located in the reticular activating system, maintains respiratory output for some time after cessation of a stimulus. This study duplicates the results of previous studies and shows that no area of the brain above the pons is required for the mechanism's operation.


Effects of cholinergic stimulation on human esophageal peristalsis. The effects of cholinergic stimulation on esophageal peristalsis were studied in normal subjects. An intraesophageal transducer assembly was used to measure the dynamics of esophageal peristalsis before and after edrophonium chloride, 80 mug/kg intravenously. Following endrophonium, there was a marked increase in amplitude of esophageal persistalsis associated with a significant reduction in velocity of esophageal peristalsis and a significant increase in width and time of appearance of the contraction wave following a swallow. Graded doses of endrophonium all resulted in significant increases in peristaltic amplitude with the maximal response occurring at doses of 80 and 160 mug/kg. Similar results were obtained with a more direct-acting cholinergic agent, bethanechol, 80 mug/kg subcutaneously. The relevance of these results as an indication of the importance of cholinergic innervation in regulating esophageal motility are discussed.


Frequency and displacement amplitude relations for normal hand tremor. Spectral analysis of hand tremor records obtained from normal subjects during continuous extension of the hand for 15-45 min revealed that the root-mean-square (rms) displacement amplitude of the tremor increased from control levels of about 30 mum to levels on the order of 100-1,000 times control. Associated with this increase in the displacement was a systematic decrease in the hand tremor frequency from control values of 8-9 Hz to values of 4-6 Hz. Spectral analysis of demodulated extensor EMG records indicated a consistent relation between EMG modulation amplitude at the tremor frequency and the tremor displacement amplitude for tremor records with rms displacement above about 100 mum. No consistent relation was found between these two variables for tremor records with displacements below 100 mum. Consideration of both mechanical and neural reflex effects indicated that a viscoelastic-mass mechanism primarily determined the small-amplitude (less than 100 mum) tremors, while the large displacement tremors may have involved both mechanical and neural feed back factors.


Body fluid responses of heat-tolerant and intolerant men to work in a hot wet environment. Acclimatization to heat before proceeding underground is a requirement for each South African mine laborer. Certain individuals among this large population cannot be acclimatized to heat (33.3 degrees C db, 31.7 degrees C wb) and are classified as heat intolerant. In this study certain body fluid responses to heat and work were compared between a group of 19 heat-tolerant (HT) and of 15 heat-intolerant (HI) subjects. To the factors known to affect heat tolerance such as age, weight, and oxygen consumption must now be added differences in body fluid responses. The HI group of subjects failed to hemodilute to the same degree as the HT group though working at the same relative work loads (30% and 50% VO2 max). As the 4-h work period (33.3 degrees C db, 31.7 degrees C wb) continued, the HI group did not maintain hemodilution in spite of the lower absolute work loads, sweat rates, and water deficits suffered by this group. From analysis of blood constituent changes it was suggested that the reason for the differences noted in body fluid dynamics concerned plasma protein equilibrium across capillary walls as well as the protein population of interstitial spaces.


Muscle water and electrolytes following varied levels of dehydration in man. In an effort to assess the effects of dehydration on the content of water and electrolytes (Na+, K+, Cl-, and Mg2+) in plasma and muscle tissue, eight men exercised in the heat (39.5 degrees C, 25%). Blood urine, and muscle biopsy samples were obtained before exercise and after the subjects had reduced their body weight by 2.2, 4.1, and 5.8%. On the average, plasma and muscle water (H2Om) contents were found to decline 2.4 and 1.2% for each percent decrease in body weight. Muscle sodium (Na+m) and chloride (Cl-m) content remained unchanged with dehydration, while muscle magnesium (Mg2+m) declined 12% as a result of the 5.8% dehydration. In terms of intracellular concentrations, K+i increased 7.2 and 10.6% at the 2.2 and 4.1% dehydration levels, respectively. Calculations of the resting membrane potential suggest that the water and electrolyte losses observed in these studies do not significantly alter the excitability of the muscle cell membrane.


Augmentation of sickling process due to turbulent blood flow. The purpose of this study was to determine if the fluid mechanical stresses associated with turbulent blood flow can contribute to the sickling process. Blood from seven patients with sickle cell disease was subjected to intermediate and high levels of turbulent flow in vitro. Turbulence was quantitated by hot film anemometry. Control samples showed 20 +/- 3% sickled cells. Cells subjected to intermediate levels of turbulent flow showed 26 +/- 4% sickling (P less than 0.01); and blood subjected to high intensities of turbulence showed 31 +/- 4% sickling (P less than 0.01). A quantitative count by electronmicroscopy, performed in one patient, showed polymerization of the hemoglobin indicative of sickling in more cells subjected to turbulence than in the control sample. A turbulence-reducing agent, polyethylene oxide, diminished the augmentation of the sickling process as it reduced turbulence at comparable Reynolds numbers. These results support the hypothesis that a deleterious effect upon hemoglobin SS erythrocytes may occur due to the mechanical stresses of turbulent flow. The agitation associated with turbulent flow presumably modifies the stabilizing factors of the intracellular colloidal solution of hemoglobin, thereby contributing to sol-gel transformation. Such hydrodynamic stresses may supplement the previously described factors which contribute to sickle cell crises.


Effect of hyperinflation on inspiratory function of the diaphragm. The inspiratory efficiency of the diaphragm during unilateral and bilateral phrenic stimulation (UEPS and BEPS) with constant stimulus was studied in seven dogs from FRC to 120% TLC. Alveolar pressures (PAl) were recorded during relaxation, BEPS and UEPS at each lung volume in the closed respiratory system. From the PAl-lung volume curves, tidal volume (VT), and pressure developed by the diaphragm (Pmus) were derived. Results are summarized below. a) Hyperinflation impaired the inspiratory efficiency of the diaphragm which behaved as an expiratory muscle beyond the lung volume of 103.7% TLC (Vinef). b) The diaphragm during UEPS became expiratory at the same Vinef as during (BEPS. C) The VT-lung volume relationship was linear during BEPS, allowing simple quantitation of VT loss with hyperinflation and prediction of Vinef. d) With only one phrenic nerve stimulated, the functional loss is less pronounced in VT than in Pmus, as compared to BEPS, indicating that the respiratory system was more compliant during UEPS than BEPS. This compliance difference from UEPS to BEPS diminished with severe hyperinflation.


A quantitative description of normal AV nodal conduction curve in man. The AV nodal conduction curve generated by the atrial extrastimulus technique has been described only qualitatively in man, making clinical comparison of known normal curves with those of suspected AV nodal dysfunction difficult. Also, the effects of physiological and pharmacological interventions have not been quantifiable. In 50 patients with normal AV conduction as defined by normal AH (less than 130 ms), normal AV nodal effective and functional refractory periods (less than 380 and less than 500 ms), and absence of demonstrable dual AV nodal pathways, we found that conduction curves (at sinus rhythm or longest paced cycle length) can be described by an exponential equation of the form delta = Ae-Bx. In this equation, delta is the increase in AV nodal conduction time of an extrastimulus compared to that of a regular beat and x is extrastimulus interval. The natural logarithm of this equation is linear in the semilogarithmic plane, thus permitting the constants A and B to be easily determined by a least-squares regression analysis with a hand calculator.


Interactions of human cord dorsum potential. The slow positive wave (P2 wave) of the evoked spinal electrogram was recorded from the posterior epidural space in wakeful man, and studied by applying several modes of peripheral nerve stimulation. With graded stimulation the P2 wave amplitude rapidly reached the maximum at weaker stimulation than that required for the initially positive spikes (P1) and the preceding negative (N1) wave. The "second" component of the P2 appeared during stronger stimulation or during excitemenpt of the subjects. With prolonged repetitive stimulation the P2 wave increased its duration with several summits on the decaying phase. Two interactions were observed between the P2 waves produced by conditioning and testing stimulations in the same or different nerves: inhibition or occlusion by strong stimulation and faciliation by weak stimulation. Thus, the characteristic of the P2 wave in man was similar in part to that of the positive wave observed in decerebrate animals, and differnt in other ways presumably due to influences from supraspinal structures or species differences.


Metabolic and cardiovascular adjustment to work in air and water at 18, 25, and 33 degrees C. By use of successive increments of discontinuous work with an arm-leg cycle ergometer the VO2, Q, SV, and HR were studied in six male subjects at rest and during exercise in air and in water at 18, 25, and 33 degrees C. The Q values obtained by CO2 rebreathing were reproducible. VO2 was linearly related to work with the plots for air and 33 degrees C water being similar. However, during work in 25 and 18 degrees C water, the VO2 averaged 9.0% (150 ml) and 25.3% (400 ml) higher, respectively, than values observed in 33 degrees C water, with the largest differences observed in leaner subjects. The plot of HR-VO2 was linear and almost identical during work in air and 33 degrees C water, but shifted significantly to the right in cooler water. VO2 averaged 250-700 ml higher in cold water compared to air and 33 degrees C water at a given mean heart rate. The Q vs. VO2 line was similar during work in air and in water with no effect of water or temperature. At similar levels of VO2, SV was significantly larger (P less than 0.05) in 25 and 18 degrees C water than in air or 33 degrees C water. Consequently, the reduction in heart rate during work in cold water was entirely compensated for by a proportionate increase in the SV of the heart. Q was therefore maintained at similar levels of energy expenditure in air and in 18, 25, and 30 degrees C water.


Sustained venoconstriction in man supplemented with CO2 at high altitude. Venoconstriction occurs at high altitude. This study sought to determine whether hypoxia or hypocapnia is the cause of the venoconstriction. Five male subjects were exposed to 4,000-4,400 m (PB 440-465 mmHg) with supplemental 3.77 +/- 0.02% CO2 in a hypobaric chamber for 4 days. Similar alveolar O2 tensions were obtained in four control subjects exposed to 3,500-4,100 m (PB 455-492 mmHg) without CO2. A water-filled plethysmograph was used to determine forearm flow and venous compliance. Systemic blood pressure was measured with the cuff procedure. Catecholamines were measured in 24-h urine collections. Venous compliance fell at high altitude in both groups and was less (P less than 0.01) than control values. Forearm flow and resistance were unaltered at altitude in the group with CO2 supplementation while forearm flow decreased and resistance increased in the hypocapnic group at 72 h of exposure. Urinary catecholamines increased in the group with CO2 and remained unaltered in the hypocapnic group. It is concluded that hypoxia is responsible for decreasing venous compliance, and hypocapnia for increasing resistance and decreasing flow. Group differences observed in urinary catecholamines may be explained by differences in arterial pH.


Collection of normal canine tracheobronchial secretions. A technique is described for the collection of tracheobronchial secretions from the upper trachea of anesthetized dogs. The method is simple, relatively quantitative, and provides secretory material in quantity suitable for biochemical or rheological studies. In 42 experiments comprising 184 individual samples we have found the collection rate of tracheobronchial secretions to be 1.0 +/- 0.1 mg/min per kg (mean +/- SEM).


Effects of temperature and wind on facial temperature, heart rate, and sensation. Skin temperature measurements of the face have shown that the cheek cools faster than the nose and the nose faster than the forehead. The cooling effect of wind is maximum at wind speeds between 4.5 and 6.7 m/s. Cold winds produce significant bradycardia, which is, however, much more pronounced during the expiratory phase of respiration. A significant correlation was noted between cooling of face and the reflex bradycardia observed. Similarly, a very significant correlation was noted between drop in skin temperature and subjective evaluation of cold discomfort. Consequently, the drop in skin temperature, reflex bradycardia, and subjective evaluation are parameters which are directly affected by cold wind and can be used as adequate indicators of the degree of discomfort. When comparing the present results with the windchill index, it was found that in the zone described as "dangerously cold" the index fits well with the physiological measurements. In the zone described as "bitterly cold," the index by comparison with actual  skin temperature measurements and subjective evaluation underestimates the cooling effects of combined temperature and wind by approximately 10 degrees C.


Synchronization of respiratory frequency by somatic afferent stimulation. In cats anesthetized with chloralose-urethan, vagotomized, paralyzed, and artifically ventilated, superficial radial (cutaneous) and hamstring (muscle) nerve afferents were stimulated while phrenic nerve electrical activity was recorded. The results obtained with both types of nerves were similar. Stimulation in mid and late expiration advanced the onset of the next inspiration, shortening its duration. Stimulation in early inspiration advanced, while that in late inspiration delayed, the onset of the next expiration. These effects were often accompanied by changes in phrenic motoneuron firing patterns (earlier recruitment, increased discharge frequency, increased slope of integrated phrenic neurogram). Repetitive somatic afferent stimulation produced sustained increases in respiratory frequency in all cats and in half of them entrainment of respiratory frequency to the frequency of stimulation occurred at ratios such as 4:3, 4:5, 1:2, 1:3, 1:4, and 1:7. The lowest stimulus intensity required for evoking these phase shifts was between 5 and 10T (threshold of most excitable fibers) for muscle afferents and between 1 and 2T for cutaneous afferents. These results demonstrate the existence of a reflex mechanism capable of locking respiratory frequency to that of a periodic somatic afferent input. They also provide an experimental basis for the hypothesis that reflexes are resposible for the observed locking between step or pedal frequency and respiratory rate during exercise in man.


Effects of exercise on excretion rates of urinary free cortisol. Excretion rates of urinary free cortisol were studied in 20 men assigned to four treadmill exercise groups: walking at 3 mph for 10 min or 30 min, or running at 7.5 mph for 10 min or 30 min. Free cortisol in urine was measured before and 30, 60, and 90 min after exercise, and again on a control day. Patterns of free-cortisol excretions after exercise at 7.5 mph for 10 and 30 min were significantly different from the control day (P less than 0.05) with the largest changes occurring in the 30-min group. Exercise and control patterns were not different for the other two conditions (P greater than 0.05). Within the 7.5 mph-30 min group the postexercise cortisol excretion rates were directly related to the relative intensity of exercise (%VO2max) and the respiratory exchange ratio. It is concluded that changes in free cortisol excretion rates depend on the duration as well as the intensity of exercise.


Time-motion and physiological assessments of ice hockey performance. Ice hockey performance was studied during 10 contests to assess time-motion characteristics and associated physiological changes that occur for each position. Depending on the position played, the actual playing time per game for forwards and defensemen ranged between 20.7 and 28.0 min. The number of shifts ranged from 14 to 21 with an average playing time of 85.4 s/shift. Further analysis of each shift indicated that there was an average of 2.3 play stoppages which averaged 27.1 s, producing a continuous playing time of 39.7 s. Telemetered recordings of heart rate during each shift revealed sustained rates of between 170 and 174 beats/min over the three periods of the game. For both the forwards and defensemen, values for blood lactates were highest during the first and second periods (x = 78.0 and 66.1 mg/100 ml), then declined considerably during the third period (x = 44.5 mg/100 ml). Blood glucose showed a similar tendency (x = 139.3, 133.7, 114.1 mg/100 ml), while hematocrit and total protein showed little tendency to change.


Mechanical properties of the dog renal capsule. The renal capsule is an important determinant of whole kidney volume/pressure relationships. To gain further insights into its possible role we examined the mechanical properties of the dog renal capsule using standard materials testing procedures. From each of four locations on the kidney surface, the following mechanical properties of the renal capsule were determined: elastic modulus (force/unit of cross-sectional area theoretically required to double the length of the specimen), tensile stiffness (force/unit width theoretically required to double the length of the specimen), ultimate strength (stress at time of fracture of the specimen), and maximum strain (percent strain at time of the fracture of the specimen). We found that the elastic modulus of the renal capsule from all capsular sites was substantially greater than values previously reported for dog aorta. The stiffness of the capsule covering the anterior-posterior surface of the kidney was found to be about 50% greater than the stiffness of the capsule covering the lateral and polar surfaces of the kidney. The ultimate strength of the anterior-posterior capsule was significantly greater than that of the lateral or polar capsule. This finding may explain the clinical observation that the spontaneous rupture of the renal capsule and parenchyma associated with the acute swelling of transplant rejection is confined almost exclusively to the lateral and polar portions of the renal capsule and cortex. The mean maximum strain at each capsular site was about 35%. This degree of circumferential expansion corresponds to about a doubling of kidney volume. Thus, this observation suggests that the renal capsule is at risk to undergo spontaneous rupture when renal volume increases of this magnitude are observed.


Arterial pressure contour analysis for estimating human vascular properties. The form of an arterial blood pressure curve during the diastolic portion of the cardiac cycle was here employed to identify parameters in a third-order model of the vascular system. Calculated elastic and intertial characteristics of this fitted model then became clinically accessible indices of corresponding real vascular properties. This technique incurred no risk and little discomfort for the patient. Tested in theory, in animal experimentation, and in human observations, our procedure utilized a Gauss-Newton algorithm via digital computer to provide rapid model solutions from different starting values, from multiple measurements sites, and from normal or diseased patients. Model parameters thus determined defined ranges of normal variation and suggested a less compliant arterial bed in hypertensive than in normotensive patients.


Respiratory volume-time relationships during resistive loading in the cat. The first-breath (neural) effects of graded resistive loads added separately during inspiration and expiration was studied in seven anesthetized cats before and after bilateral vagotomy. Additions of airflow resistance during inspiration reduced the volume inspired (VI) and increased inspiratory duration (TI). The duration of the ensuing unloaded expiration (TE) was unchanged. Vagotomy eliminated the TI modulation with inspiratory loads. Tracheal occlusion at the onset of inspiration yielded TI values similar to the fixed values observed following vagotomy. Resistive loads added during expiration produced similar results. Expired volume (VE) decreased and (TE) increased approaching the values obtained after vagotomy. Unlike the inspiratory resistive loads, loading during expiration results in an upward shift in the functional residual capacity (FRC). The FRC shift produces a time lag between the onset of diaphragmatic (EMG) activity and the initiation of airflow of the next (unloaded) inspiration. These studies suggest separate volume-time relationships for the inspiratory and expiratory phases of the breathing cycle. Both relationships are dependent upon vagally mediated volume feedback.


Comparative evaluation of pressure and time factors in estimating left ventricular performance. Left ventricular and ascending aortic pressures were measured in open chest mongrel dogs under pentobarbital anesthesia. The data were digitized, averaged, and subgrouped by mean systolic aortic pressures (MSAP), end-diastolic pressure (EDP), and heart rate (HR). Seven raw and 32 derived variable from the pressure, as a function of time, wave forms were analyzed in each subgroup in the control state and following the infusion of catecholamines. A plot of control variability versus sensitivity to norepinephrine indicates that time to peak ventricular pressure (PVP time) is a more sensitive indicator of changes in the inotropic state than such other commonly used variables as max dP/dt, integrated isometric tension, and (max dP/dt)/developed pressure. PVP time also showed less variability with HR, EDP, and MSAP. Regression lines were also fit to the data using a second-order model. This permitted evaluation of experimentally varying either HR, EDP, or MSAP while maintaining the other two constant. PVP time was again one of the better variables in terms of sensitivity to HR, EDP, or MSAP. Vmax, fractional rate of change of power, preejection period, and systolic time were also analyzed and compared with PVP time using averaged data.


Effects of hypoxia, heat, and humidity on physical performance. The effects of hot, humid environment were compared with the effects of high altitude on the physical performance capacity of Ne-palese residents by measuring oxygen uptakes and heart rates at various work rates. The following groups of men were selected: 66 residents of a hot and humid environment in the Terai at sea level; 24 residents and 16 sojourners at 3,8000 m. The maximal oxygen uptake of the sea-level residents was, on the average, 2.55 1.min-1, at which a maximal heart rate of about 200 beats/min was reached. The sojourners at 3,800 m showed a higher maximal oxygen uptake (2.94 1. min-1) at their maximal heart rate of about 175 beats/min. The residents of 3,800 m achieved a similiar oxygen uptake as the sojourners, but did not show a similar maximal heart rate limitation, suggesting that they were capable of achieving a higher maximal oxygen uptake. This study shows that hot, humid environment at sea level is as much incapacitating as is hypoxia at high altitude.


Local regulation of pulmonary blood flow and ventilation-perfusion ratios in the coatimundi. Small catheters (ca. 3 mm diam at tip) were wedged in subsegmental bronchi in anesthetized coatimundi (Nasua nasua) during spontaneous breathing. Mixed expired gases of a group of lobules were sampled continuously without contamination from neighboring units, and local tidal volume, frequency, carbon dioxide production, and oxygen consumption were measured, as well as mixed venous PO2 and PCO2. Local ventilation-perfusion ratio, alveolar PO2, PCO2, and blood flow were calculated. There was a 22% reduction (range 15-38) in local perfusion (as percent of flow at PAO2 100 mmHg) per 10 mmHg fall in local alveolar oxygen tension over the PAO2 range 150-36 mmHg. Local hypercapnia had little effect on local flow. Local tidal volume (ca. 1% of total tidal volume) was unaffected by changes in alveolar gas tensions. The contribution of vasoconstriction or vasodilatation, as a negative feedback system, to the stability of local PAO2 was greatest close to the physiologic range (65-85 mmHg) falloderate efficiency.


Decompression limits for compressed air determined by ultrasonically detected blood bubbles. The direct decompression limits for a group of divers over a range of pressure-time air exposures was determined using ultrasonic detection of venous gas emboli (VGE). In addition to dry chamber exposures, ranging from 233 ft for 7 min to 25 ft for 720 min, we exposed six divers to open ocean dives at 165 ft for 10 min. Findings demonstrated a strong individual propensity to form VGE, correlating with susceptibility to bends. No bends developed without the prior detection of precordial VGE. The present concept of no problems after any period of time at 30 fsw was not confirmed. Isopleths of equal percentage occurrence of VGE were computed between 10 and 60%. Open ocean exposures increased the percentage of VGE and bends, when compared to dry chamber exposures. Limiting tissue half times computed from the 20% VGE isopleth suggested that saturation exposures are controlled by a greater sensitivity of the short-half-time tissues than previously appreciated, rather than by additionally extended half times.


Physiological adjustments of young men to five-hour desert walks. Seven young men undertook a desert walk of 30 km at a rate of 100 m/min. Six finished; the seventh stopped after 24 km. Each satisfied his thirst with cool tap water each hour. Periodic observations included metabolic rate, blood pressure, heart rate, rectal and skin temperature, body weight, and volume of water drunk. Hand sweat was collected each hour and body sweat residues on the skin were collected at the end of the walk. Subjective reports revealed portents of breakdown: aching muscles, painful joints, hot or blistered feet, hunger, and boredom. Cardiovascular adjustment and temperature regulation maintained tolerable conditions. The volumes of water evaporated by the 5-h walkers were about the same. Wet bulb temperatures were below 25 degrees C; all sweat evaporated and was available for temperature regulation. The volume of water drawn from body reserves was closely correlated with concentration of chloride in body sweat; the volume of water that satisfied thirst maintained osmotic pressure.


An ultrasensitive isometric force transducer for single smooth muscle cell mechanics. The principles of operation, design, and performance of a differential photooptical transducer of very high sensitivity are described. Useful range is 10-2,000 mugf with sufficiently low drift to allow recordings of contractile responses of isolated single smooth muscle cells. Comparison with several previous designs is presented.


Can impedance characterize the heart?

Can impedance characterize the heart? When considering the use of Fourier series in hemodynamics, the question is whether one can relate the frequency components of the flow oscillations to the corresponding ones of pressure using impedance concepts. For the arteries, this method provided the basis for great advances in understanding. However, it is precisely because the arterial tree acts almost linearly while its properties do not change markedly within one beat that frequency analysis achieved such success. For the ventricle, in which the mechanical properties vary widely over the course of one heart cycle, Fourier analysis loses its usefulness. Consequently, we must return to the time domain for formulating a description of the heart as a pump. A time-domain method, the impulse response, is suggested as a possible alternative to impedance.


An inexpensive blood pressure-heart rate computer for laboratory or teaching use. A special-purpose analog computer circuit is described that, given a signal proportional to arterial blood pressure, generates signals equal to systolic, diastolic, pulse, and mean pressure at a calibration of 100 mmHg/V. Heart period and rate are also derived at calibration of 1 s/V and 100 bpm/V, respectively. The outputs are step-function beat-by-beat changes in these six variables suitable either for analog recording, or presentation to a panel meter for digital display.


Probe for production and measurement of acute mitral regurgitant flow in dog. A probe for production and measurement of acute mitral regurgitation in dogs is described. It consists of a tube that is introduced into the mitral valve through the left atrial appendage. Regurgitant flow through the tube is measured by an electromagnetic device. Variation of flow and zero flow are achieved by narrowing or occluding the tube with a rubber cuff. In animals weighing 30-50 kg, the probe does not produce significant mitral stenosis and the mitral leaflets fit closely around the probe during ventricular systole. The instantaneous relationship between mitral regurgitant flow (MRF) and the gradient between left ventricular and left atrial pressure shows a marked delay of MRF at the beginning and end of regurgitation. This delay can be attributed to some extent to electrical phase lag and to the small movement of the probe relative to the mitral valve during the cardiac cycle. Measurement of regurgitant stroke volume is affected by this movement only to a small extent.


Construction of picoliter-nanoliter self-filling volumetric pipettes. A technique is described whereby-volume, self-filling micropipettes can be constructed with minimal time and effort. The delivery volumes of such pipettes may range from several picoliters to more than 100 nl. The delivery of a given pipette of even the smallest volume is precise, generally yielding standard deviations much less than 1% of the mean volume as measured by liquid-scintillation counting of isotope dilutions. The technique offers considerable advantage to previously described techniques for constructing small constant-volume pipettes.


Nonobstructive chronic cannulation of the left circumflex coronary artery of the dog. A cannulation technique for creating a nonobstructive port in a proximal coronary artery is described. The use of this technique in awake animals allows the blood pressure to be measured in a coronary artery distal to an experimentally produced stenosis. This port can also be used to inject drugs directly into the coronary bed.


Application of the hot-wire anemometer to respiratory measurements in small animal. A hot-wire anemometer was evaluated to determine its suitability for measurement of small tidal volumes. Used with a constant background flow of gas, the output of the hot-wire anemometer was linear and independent of respiratory frequency, temperature, and humidity. The change in output with CO2 concentration was negligible within the physiologic range. The use of a background flow eliminates the need for one-way valves, minimizes dead space, and maintains the flow velocity past the hot wire within its range of linear response.


Gas-liquid chromatographic determination of capsaicin. A simple and accurate method has been developed for the quantitative determination of capsaicin in capsicum spices and their oleosresins. Capsaicin is extracted with acetone and separated from interfering substances, using an activated alumina column. The column is first eluted with acetone, and then the capsaicin is eluted with acetone-methanol-water (75+25+2). The capsaicin is quantitated by gas-liquid chromatography, using a Carbowax 20M-Teflon column and piperine as an internal standard. Results can be calculated as per cent capsaicin or Scoville units. Recovery of added capsaicin averaged 101%.


Simple method for simultaneous detection of aflatoxin and zearalenone in corn. A method is described for the simultaneous detection of alfatoxin and zearalenone in corn at 5 and 200 ppb, respectively. No evaporation of solvent is required and the procedure is simple enough to be considered for use at marketing locations. The presence of absence of these myocotoxins can be determined in 10-20 min/sample. The procedure involves an initial blender extraction with methanol, partitioning of fat and pigments into 1-1,2-trichlorotrifluoroethane (Freon-113) from an aqueous ammonium sulfate layer, followed by extraction of aflatoxin from the aqueous layer with chlorobenzene. The chlorobenzene extract can be spotted directly onto a thin layer chromatographic plate which requires only 4 min development. Concentrations of aflatoxin and zearalenone can be estimated by visual comparison of sample spots with standards.


Collaborative study of a screening method for the detection of aflatoxins in mixed feeds, other agricultural products, and foods. A screening method for aflatoxins was collaboratively tested on 11 different agricultural and food products: white and yellow corn, peanuts, peanut butter, pistachio nuts, peanut meal, cottonseed meal, chicken, pig, and turkey starter rations, and dairy cattle feed. The method involves a rapid extraction and cleanup procedure followed by the detection of total aflatoxins (B1 + B2 + G1 + G2) as a fluorescent band on the Florisil layer of a Velasco-type minicolumn. The results of 32 collaborators from 10 different countries are presented. Samples containing 0, 5, 10, 15, 20, and 25 mug aflatoxins/kg were analyzed. Eighty-four per cent of the negative samples and 89% of the samples containing 10-25 mug total aflatoxins/kg were correctly identified. This method has been adopted as official first action for the detection of aflatoxins in corn, peanuts, peanut butter, peanut meal, cottonseed meal, mixed feeds, and pistachio nuts.


Survey for aflatoxins and zearalenone in 1973 crop corn stored on farms and in country elevators. A total of 315 marketable and 57 obviously damaged corn samples were collected at 116 different farms and country elevators located in the United States in countries selected from among those producing more than 1 million bushels of corn in 1972. The samples were analyzed for aflatoxins and zearalenone. The most striking correlations observed were between geographical area and mycotoxin contamination. Aflatoxin contamination was most frequently encountered in the Southeast-Appalachia areas with a 44% incidence of marketable corn with detectable aflatoxins. Zearalenone was most frequently encountered in the Corn Belt with 10% incidence in marketable corn from that region. When mycotoxin contamination was found in an establishment, most of the samples from that establishment were contaminated. There was no correlation between mycotoxin contamination and storage practices nor could the observed contamination of marketable corn be related to the contamination of the obviously damaged grain. These observations plus correlations with the geographic incidence and aflatoxin level distribution of published field contamination data suggest the possibility of a common contamination mode.


Survey of U.S. wheat for ochratoxin and aflatoxin. A total of 291 hard red winter wheat samples, 286 hard red spring wheat samples, and 271 soft red winter wheat samples were analyzed for the presecne of ochratoxin and aflatoxin. Samples in all grades came from those collected during crop years 1970-1973 for grade determinations by the Agricultural Marketing Service, U.S. Department of Agriculture. Sensitivity limits of the analytical method as carried out were 1-3 ppb aflatoxin B1 and 15-30 ppb ochratoxin A. No aflatoxin was detected in any sample. Three samples of hard red winter wheat (Grades U.S. No. 4 and 5 and Sample Grade) contained ochratoxin A (trace, 35, and 25 ppb, respectively). Eight of the hard red spring wheats contained ochratoxin A (15-115 PPB); these were in Grades U.S. No. 4 and 5 and Sample Grade.


Screening method for the detection of aflatoxin, ochratoxin, zearalenone, penicillic acid, and citrinin. A modification of the official method for ochratoxins and a screening method for zearalenone, aflatoxin, and ochratoxin is described and expanded to include citrinin and penicillic acid. The method uses 0.5N phosphoric acidchloroform (1+10) in the initial extraction; the extract is divided and eluted from 2 columns to provide a quantitative thin layer chromatographic (TLC) method for aflatoxin and ochratoxin in corn and dried beans. Aflatoxin and zearalenone are eluted from one column and ochratoxin, penicillic acid, and citrinin from the other. Ochratoxin A recoveries are low (50%) in peanuts. Zearalenone, penicillic acid, and citrinin were qualitatively recovered from corn and beans; zearalenone and penicillic acid were recovered from peanuts but citrinin was not. Several TLC solvents were used to separate interferences.


A spectrophotometric procedure, using carboxypeptidase A, for the quantitative measurement of ochratoxin A. In the method described, ochratoxin A is eleaved into ochratoxin alpha (free isocoumarin chromophore) and phenylaline, using carboxypeptidase. Detection is based on the difference in fluorescence excitation spectra of ochratoxin A (380 NM, maximum) and ochratoxin alpha (340 nm, maximum). The quantitation of ochratoxin A is based on the loss of fluorescence intensity at 380 nm. The method has been used for the quantitative determination of as little as 4 mug ochratoxin A/kg barley and barley meal but it could be extended to other products.


Detection of volatile solvents in gastric contents by gas phase infrared spectrophotometry. A standard procedure for the identification of volatile solvents in the gas phase by infrared spectrophotometry has been modified and applied to the analysis of gastric contents. A 10 ml sample of the vapor from the gastric aspirate is removed with a hypodermic syringe, injected on a spectrophotometer from 2.5 to 15 nm. The sample is then identified by comparison with known reference spectra. Solvent concentrations cen be estimated by comparing the ratios of the major peaks.


Collaborative study of a semiautomated method for the determination of crude protein in animal feeds. A semiautomated method consisting of digestion of animal feeds in a block digestor and determination of ammonia by ammonia-salicylate reaction has been studied collaboratively, along with the official final action Kjeldahl method, sec. 7.016. Each collaborator analyzed 16 feed samples, tryptophan, ammonium dihydrogen phosphate NBS standard, and ammonium sulfate primary standard. Statistical analysis showed that the 2 methods agreed. The semiautomated method has been adopted as official first action.


Determination of saccharin, sodium benzoate, and caffeine in beverages by reverse phase high-pressure liquid chromatography. A reverse phase high-pressure liquid chromatographic method is presented for the simultaneous separation and determination of sacchrain, sodium benzoate, and caffeine in soft drinks, fruit juices, fruit cocktails, fruit punches, coffee, and artificial sweetener concentrates. Decarbonated soft drinks, fruit punches, and artificial sweetener concentrates are injected directly into the chromatograph. Fruit juices and coffee solutions require filtration through a 0.45 mum pore membrane filter prior to injection. Samples are eluted from a mu-Bondapak/C18 column with 5% glacial acetic acid and are quantitated with an ultraviolet detector. The results of saccharin, sodium benzoate, and caffeine determinations in 34 soft drinks (representing 11 manufacturers and 20 flavors); 8 fruit juices, cocktails, and punches; 7 coffees; and 6 artificial sweetener concentrates are presented. Average recoveries of saccharin, sodium benzoate, and caffeine from soft drinks are 99.0, 99.3, and 100.2%, respectively.


Collaborative study of an automated method for the determination of crude protein in animal feeds. An automated macro Kjeldahl instrument determines per cent protein at the rate of 20 samples/hr. The methodology involved is similar to the present official final action Kjeldahl method, sec. 7.016. The 2 methods were compared in a collaborative study. Sixteen animal feeds, 4 meats, tryptophan, ammonium dihydrogen phosphate NBS standard, and ammonium sulfate primary standard were analyzed by the participating laboratories. The data were agreement between the 2 methods. The automated method has been adopted as official first action for the determination of crude protein in feeds, plants, and cereal foods.


Collaborative study of effect of light on cadmium and lead leaching from ceramic glazes. A limited interlaboratory study was carried out to determine the effect of lighting conditions on the release of cadmium from ceramic glazes by 4% acetic acid. Cadmium release increased with increased exposure to light. Further interlaboratory study on plates manufactured under controlled conditions showed that if temperature, intensity of illumination, and time of exposure are specified, reproducible results for leaching of both lead and cadmium can be obtained. A modification of the official AOAC method, 25.031-25.034, to increase sensitivity was collaboratively studied by 8 laboratories. Each received 6 solutions of lead in 4% acetic acid (3 sets of blind duplicates at the 0.1, 0.5, and 0.8 mug/ml levels) plus a reagent blank. Average recoveries were 0.1006, 0.5056, and 0.8194 mug/ml, respectively, with coefficients of variation of 11.4, 3.3, and 2.8%, respectively. The proposed modification has been adopted as official first action, and a parameter for exposure to light during extraction has been included in the method.


High-speed liquid chromatographic determination of o-phenylphenol residues in citrus products. A method is presented for the quantitative analysis of o-phenylphenol residues in citrus oils, encapsulated flavors, and dried meal. The method utilizes high-speed liquid chromatography for the determination after specific sample preparations for each material. These preparations include hexane extraction of acidified basic extracts or steam distillation and extraction. The limit of the analysis is less than 1 ppm with an analysis time of less than 45 min.


Partition chromatographic separation of pesticide residues from fats. A simple, rapid, and efficient partitioning column consisting of acetonitrile on Florisil has been developed for the separation of pesticides from fish, beef, and butter fat. The efficiency of the cleanup column is between 97 and 100%. Nine pesticides having partition coefficients between n-hexane and acetonitrile of less than or equal to 0.05 were satisfactorily separated from fat with good recoveries. When the column was used to clean up temephos in a fish extract, 99.91% of the fat was eluted with 20 ml n-hexane with no loss of the pesticide.


Collaborative study of an extension of the Mills et al. method for the determination of pesticide residues in foods. Fifteen laboratories collaboratively determined parathion, o,p'-DDT, p,p'-DDT, and p,p'-DDE in kale, and lindane, dieldrin, heptachlor epoxide, and p,p'-DDE in eggs, using electron capture gas-liquid chromatography (potassium chloride thermionic detector for parathion). Pesticide levels ranged from 0.033 ppm heptachlor epoxide to 3.32 ppm p,p'-DDT. Mean recoveries (+/- standard deviations) were: parathion 91+/-15%, o,p'-DDT 99+/-8%, P,P'-DDT 97+/-8%, lindane 94+/-4%, dieldrin 106+/-8%, heptachloro epoxide 106+/-5%, p,p'-DDE 94+/-8% in eggs and 101+/-9% in kale. The methods have been adopted as official first action.


Determination of quinomethionate (6-methylquinoline-2,3-diyldithiocarbonate) residues in crops by in situ fluorometry. A simple method is described for the quantitative determination of quinomethionate (6-methylquinoline- 2,3 - diyldithiocarbonate) in crops. The pesticide residue is extracted with acetonitrile and partitioned in petroleum ether. After separation from the co-extractives by thin layer chromatography (TLC), the fluorescence is measured directly on a silica gel TLC plate. An average of 89% recovery is obtained at the 0.05 ppm level in apples, peaches, pears, and tomatoes.


Reference Raman spectra of DDT and five structurally related pesticides and of five pesticides containing the norbornen group. The Raman spectra of DDT and 5 structurally related pesticides, TDE (DDD), Perthane, methoxychlor, dicofol, and tetradifon, and of 5 pesticides containing the norbornene group, heptachlor, chlordane, dieldrin, endrin, and endosulfan, have been recorded. These spectra are presented, along with tables giving values for the frequencies.


Collaborative study of the determination of endosulfan, endosulfan sulfate, tetrasul, and tetradifon residues in fresh fruits and vegetables. A method for the determination of endosulfan I, endosulfan II, endosulfan sulfate, tetrasul, and tetradifon residues in fresh fruits and vegetables was studied collaboratively. The method consists of extraction of the product with acetonitrile, ether and elution from a Florisil column with mixtures of hexane, methylene chloride, and acetonitrile, and determination of the residues by electron capture gas-liquid chromatography. This method was studied by 8 collaborators, using apples and cucumbers as the samples. Each sample was spiked with 2 levels of each pesticide. The average per cent recoveries for apples and cucumbers, respectively were as follows: endosulfan I, 103.6 and 101.5; endosulfan II, 102.9 and 100.0; endosulfan sulfate, 100.9 and 92.9; tetrasul, 98.8 and 102.2; and tetradifon, 106.4 and 101.9. The method has been adopted as official first action.


Substances interfering with the gas-liquid chromatographic determination of T-2 mycotoxin. Two substances interfering with the gas-liquid chromatographic (GLC) detection of T-2 mycotoxin were identified as 1-glyceryl-monooleate and 1-glycerylmonolinoleate. These monoglycerides are natural products formed by species of Fusarium growing on cereal grains and are also additives contained in liquid vegetable and animal fats added to the feed mixture. The monoglycerides can be removed from the analytical sample by resolution by thin layer chromatography prior to separation by GLC. Trimethylsilyl ether derivatives of the monoglycerides and T-2 toxin have almost identical retention times on 3% OV-1 columns, whereas the trifluoroacetyl and pentafluoropropionyl derivatives give baseline separation on the same column. The monoglycerides can be misidentified as the T-2 toxin in analyses involbing GLC.


Gas-liquid chromatographic headspace method for vinyl chloride in vinegars and alcoholic beverages. Vinyl chloride (VC) is determined in vinegars and alcoholic beverages by gas-liquid chromatographic headspace analysis. The lower limit of detection is 10 ppb and confirmation by gas chromatography-mass spectrometry, single ion monitoring at m/e 62, is possible at this level. Comparison of the headspace and the direct injection methods for the determination of VC in vinegars and alcoholic beverages showed that the results obtained by the 2 methods were not significantly different (P greater than 0.05).


The relative nitrogen utilization method for evaluating protein quality. A single-dose rat assay for protein quality called relative nitrogen utilization (RNU) is described. The method includes a factor for the protein utilized for maintenance. The protein quality of the test protein is expressed as a per cent of the value for lactalbumin, the reference protein. For good quality proteins RNU gives results that are similar to those obtained by net protein ratio and relative protein value; for poor quality proteins RNU results fall between values by the other 2 methods. The protein efficiency ratio (PER) gives much lower values for poor quality proteins than any of the other 3 aforementioned methods and is influenced more by both level of protein and lipid in the diet. RNU is a more precise assay then PER as judged by the coefficient of variation.


Collaborative study for the comparison of two methods for the determination of total cholesterol in multicomponent foods. The gas chromatographic method for determining total cholesterol in multicomponent foods, collaboratively studied by the AOAC in 1974 (Method 1), has been evaluated by 9 collaborating laboratories and compared with the Interim Methodology Instructions No. 2 modified method (Method 2). The 5 samples selected for collaboration were deviled ham sandwich spread, vegetable beef stew, frozen chicken pot pie, frozen fish sticks, and mayonnaise. The recovery data were obtained from a sample of wheat germ oil spiked with 0.297% cholesterol as cholesteryl palmitate. Collaborators performed 2 replicate analyses on all samples by both methods. The statistical evaluation of the results showed that Method 1 is superior to Method 2. Average recoveries from the spiked wheat germ oil samples were 91.4% (9 laboratories) and 85.8% (7 laboratories) with coefficients of variation of 12.5 and 14.4%, respectively. Based on the collaborative results and statistical evaluation, Method 1 has been adopted as official first action.


Analysis of bromide ion in wine by ion selective electrode. The use of the bromide ion selective electrode for the determination of bromide ion in wine has been found to be rapid and reliable. The method has been used for still wines and carbonated wines and is applicable to all wines regardless of their country of origin. The method consists of treating a 50 ml aliquot of wine with 2 ml each of 3.75M H3PO4, saturated KNO3, and 1M CuSO4. After 10 min the electrodes are immersed in the samples and a millivolt reading is obtained. One hundred mul 500 ppm bromide ion standard is added and the millivolt reading is taken. Bromide ion concentration in the wine equals (Cdelta x 1)/((antilog deltaE/S)-1) where Cdelta equals 1, deltaE equals the change in potential expressed in millivolts, and S equals the electrode slope.


Detection of sulfonamides in animal feeds. The animal feed sample is extracted with ethanol or acetone and the extract is evaporated to dryness. Another portion of the same sample, spiked at the 3 ppm level with 8 sulfonamides, is similarly extracted and the extract is evaporated to dryness. The residue from each solution is dispersed with 5 ml 0.1N NaOH and, following the addition of 1 ml 1N HCl and mixing, the solution is filtered. The filtrate is mixed with Celite, transferred to a column, and eluted with ammoniacal ether. Aliquots of the concentrated sample and control eluates are spotted on a neutral Adsorbosil-1 thin layer chromatographic (TLC) plate. Following development in chloroform-methanol (95+5) and drying, the plate is sprayed with an alcoholic solution of p-dimethylaminobenzaldehyde until the control chromatogram shows 6 yellow spots which are, from top to bottom; sulfadimethoxine (SO), the combined sulfamerazine (SM)-sulfamethazine (SH)-sulfaquinoxoline (SQ) spot, sulfadiazine (SD), sulfapyridine (SP), sulfathiazole (SZ), and sulfaguanidine (SG). A spot on the sample chromatogram can be identified if the Rf is identical to one of the 5 sulfonamides not overlapping with another compound. If the Rf of the sample spot is approximately the same as that of the combined SM-SH-SQ spot, more definite identification can be obtained by using basic Adsorbosil-1 TLC plates, with chloroform-methanol (92+8) as the developing solvent.


Detection of arsanilic acid in animal feeds. The animal feed sample is extracted with alcohol, the extract is evaporated to a small volume, and an aliquot is spotted on a silica gel H plate. After development of the plate in chloroform-methanol (80+20), the spotting area is scraped and extracted with ethanol, which is evaporated to dryness. The residue is dissolved in 3 ml alcohol, and an aliquot is reacted with p-dimethylaminobenzaldehyde solution; another aliquot of the extract is used as a sample blank. A control is prepared by spiking another portion of the same feed with about 5 ppm arsanilic acid. The method is applicable in the presence of procaine and sulfonamides.


Interlaboratory study of acid orange 12 dye binding for measuring protein in meat. Six samples of beef and pork were analyzed by 4 laboratories, using the dye binding method. Dye binding protein was calculated by using a dye binding capacity of meat of 0.410 mg dye bound/g protein. Correlations of 0.976, 0.987, 0.996, and 0.995 were found between dye binding and Kjeldahl protein values. An analysis of covariance showed that the slopes of regression between dye binding and Kjeldahl protein for 3 laboratories were not significantly different at the 5% level. The adjusted means of the regression lines for the same 3 laboratories were significantly different at the 1% level. The results show that 3 of the laboratories were finding the same relationship between dye binding and Kjeldahl protein but were not in calibration with one another.


Identification of some analogs of the hallucinogen phencyclidine. The drugs 1-[1-(2-thienyl)cyclohexyl]piperidine, 1-[1-2-thienyl)cyclohexyl]pyrrolidine, 1-(-phenylcyclohexyl) morpholine, and 1-(1-phenyleyclohexyl) pyrrolidine are identified by spectroscopic techniques. The ultraviolet and proton magnetic resonance spectra of analogs are similar, but mass and infrared spectra are distinctly different, and reference spectra are provided. Gas-liquid ant thin layer chromatographic systems for the analysis are discussed.


Collaborative study of a column chromatographic method for bendroflumethizide and cyclothiazide. Bendroflumethiazide and cyclothiazide are eluted from a sodium carbonate column with chloroform-acetic acid (98+2) and are measured directly by ultraviolet spectrophotometry. The method was collaboratively studied by 8 analysts. The average per cent recovery and standard deviations for simulated mixes of bendroflumethiazide and cyclothiazide were 99.61+/-0.78 and 99.3+/-1.97. The method has been adopted as official first action for the determination of bendroflumethiazide.


Gas-liquid chromatographic determination of nikethamide in injectable preparations. A gas-liquid chromatographic (GLC) method, using a 4% XE-60 on 80-100 mesh Gas-Chrom Q column, a flame ionization detector, and anthracene as the internal standard, has been developed for the direct determination of nikethamide. Eight collaborators analyzed 4 samples, using methanol as the solvent; the coefficients of variation obtained ranged from 1.19 to 3.20%. In a limited study with acetone as the solvent, the coefficients of variation ranged from 0.59 to 1.96%. The GLC method with acetone as a solvent has been adopted as official first action.


Studies on the synthesis of rat liver fatty acid synthetase. The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.


Biosynthesis of basement membrane collagen by rabbit corneal endothelium in vitro. The synthesis of collagen has been demonstrated in endothelial cells of Descemet's membrane isolated from rabbit cornea. Incorporation of [14C]proline and [14C]lysine into nondialyzable protein was measured in the medium and cell fraction after incubating Descemet's membrane for up to 5 hours. In the [14C]collagen synthesized by the endothelium, 15% of the hydroxy[14C]proline was present as the 3-isomer. About 98% of the hydroxy[14C]lysine in the 14C-labeled-protein found in the medium was glycosylated; 95% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosyl-galactosyl-hydroxy[14C]lysine. Time course experiments with [14C]proline indicated that there was a delay of about 60 min before significant amounts of [14C]collagen were secreted into the medium. The initial polypeptides of [14C]collagen synthesized by the corneal endothelium had an apparent molecular weight of 155,000. The chemical and physical properties of the [14C]collagen synthesized by rabbit corneal endothelium are consistent with those of basement membrane collagen synthesized by other cell types.


Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography. Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3  M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.


Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin. Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.