Low renin essential hypertension in a child. Elevated blood pressure (160/120 mm Hg) was found in an 8 1/2-year old body and remained unchanged during the following 3 years. The only abnormal findings were undetectable plasma renin activity (PRA less 0.16 ng/ml-3 h) even after sodium restriction and upright posture and a slight tendency for hypokalemia (3.7 mEq/1, lowest value 3.3 mEq/1). Plasma aldosterone (PA) and plasma cortisol (PC) were normal. Administration of dexamethasone and spironolactone resulted in only a minimal decrease of blood pressure whereas hydrochlorothiazide was slightly more effective. The circadian rhythm of PRA, PA and PC was studied by otaining frequent blood specimens during one night. The diurnal variation of PA and PC was normal despite undetectable PRA (less than 0.16 ng/ml-3 h). Primary aldosteronism and other conditions associated with mineralocorticoid excess were excluded. The patient seems to represent an example of low renin essential hypertension. This is the second recorded case of this syndrome in childhood.


[Fitness program for infants].

[Fitness program for infants]. The young mother should be instructed not only about feeding and immunizations, but also about her own motherly behaviour with the baby. A simple "fitness programme" to be executed before and after the feeds is described which will help insecure mothers and improve the emotional and physical well being of the baby. The paediatrician should stimulate the mother's ability before he claims the help of expensive paramedical specialists.


Tricuspid atresia, hypoplastic right ventricle, intact ventricular septum and congenital absence of the pulmonary valve. A case of tricuspid atresia by sealing of the valves, associated with an intact ventricular septum, a hypoplastic right ventricle and the absence of the pulmonary valve, is described. The foramen ovale and the ductus arteriosus were widely patent but there was no interventricular communication. The pulmonary flow was assured by the ductus which had a saccular continuation in the pulmonary trunk. It is postulated that the lesions probably occurred during the period between the XIX and XXII horizons and that the malformed right coronary artery may have played an important role in producing the lesions of the right ventricle.


Symptomatic atrial septal defect in the neonate and infant. Atrial septal defect, secundum type, is rarely diagnosed and seldom produces symptoms in infancy. 7 patients, aged between 2 weeks and 8 months, who developed congestive failure, are presented. 6 of them had an atrial LR-shunt documented at cardiac catheterization. 1 diagnosis was made at post mortem in the only infant who died. The clinical signs of an ASD do not vary from those normally found in the older child; the causes for early failure are probably rapidly decreasing pulmonary vascular reistance and disturbances of left ventricular compliance. 6 infants improved with conservative or no treatment, although spontaneous closure or significant diminution of the shunt did not occur. Surgical closure can be deferred to a later optimal age for operation. ASD secundum type should be considered as a cause of heart failure in the neonate and infant.


The radiological diagnosis of the fetal-face (= Robinow) syndrome (mesomelic dwarfism and small genitalia). Report of 3 cases. Report of 3 cases with Robinow syndrome. Analysis of the X-ray findings, including the formerly reported cases. On radiological grounds, the diagnosis of the Robinow syndrome is possible by the combined observation of mesomelic shortening of the extremities, hemivertebra formation and fusion anomalies of spine and of the ribs. The "splitting" of terminal (bifid) phalanges and toes is a facultative, but highly diagnostic radiological sign. In 2 cases, the pattern profiles were of considerable similarity. The practical importance of the correct diagnosis in this syndrome is emphasized.


Immunological decay in thymectomized infants. Four infants underwent radical thymectomy for a suspected malignancy of the thymus. Histology revealed no malignant cells. The patients did clinically well as long as 14 years after the operation. There was laboratory evidence for a cellular and humoral immunodeficiency. Affected were mainly the response to oral poliomyelitis vaccinations and the skin tests of delayed type hypersensitivity.


Development of meningoccal disease: could it depend on serum IgM levels? Immunoglobulins M, G and A were studied in 88 children with meningococcal meningitis and septicaemia. No difference in the level of immunoglobulins was noted between patients and age matched controls. Serial estimations of serum samples taken on admission to the hospital, 48 h after starting treatment, and on the day of discharge showed a marked increase of IgM levels in almost all patients at discharge irrespective of age or sex. Data of this study do not support the theory that IgM deficiency is the cause which favors the development of meningococcal disease.


Surgical approaches to certain long bones of the horse for application of tension band plates. In the horse, surgical approaches for plating fractures of the ulna, radius, metacarpus, tibia, and metatarsus involved a single curvilinear skin incision and incision of the superficial fascia in a manner to prevent postoperative dehiscence. The tension side of the bones, as well as location of nerves, arteries, veins, and muscles, were the major determinants in developing these approaches for atraumatic surgery and short operative time.


Effect of antiparasitic medication in ponies on pasture. Twenty Shetland ponies, 6 to 7 months old, were naturally infected with gastrointestinal nematodes and stomach bots. The ponies were allotted to 2 groups of 10 and were maintained on separate similar pastures that were free of infective larvae at the beginning of the study. The ponies in 1 group were treated monthly for 17 months with a therapeutic dose of a thiabendazole and piperazine mixture; those in the other group were not treated. During the 3rd and 5th months of the experiment, the ponies in the treated group were also given therapeutic doses of dichlorvos to remove bots. Various physical, hematologic, parasitologic, and blood chemical observations were made at weekly intervals. Each group of ponies was weighed at biweekly intervals. At the end of the experiment, the greatest differences between the treated and control ponies were in the mean number of worm eggs in fecal samples (0 vs 1,866 eggs/g), mean body weight (151.9 vs 117.0 kg), mean heart girth (126.5 vs 116.3 cm), mean packed cell volume (36.4 vs 30.8%), and mean serum protein content (8.47 vs 9.33 mg/100 ml), especially beta-globulin content (mean of 1.9 vs 3.4 mg/100 ml). The treated ponies remained clinically normal and were more spirited and more difficult to restrain for blood sampling and weighing than were the untreated controls. Parasitic nematode larvae were not recovered from grass samples from the pasture grazed by the treated ponies, but many such larvae (up to 500/300-g sample) were recovered from grass samples from the pasture grazed by the untreated ponies.


Squamous cell carcinoma in horses.

Squamous cell carcinoma in horses. In a review of neoplasm registry records at Kansas State University (1961 through 1971), 58 squamous cell carcinomas were reported in 10 breeds of horses. Mean age of the affected horses was 12.4 years. The head, eye and ocular adnexa accounted for 43.1%, the external male genitalia, 44.8%, and female perineal region, 12.0% of the squamous cell carcinomas, altogether representing 20.2% of 287 neoplasms recorded.


Treatment of fractures of the tibial tuberosity in the dog. Of 27 dogs with fractures of the tibial tuberosity, 24 were treated by various methods of open reduction. Follow-up examination was obtained in 21 cases, and it was found that all but one fracture healed in 8 weeks. Tension band wiring and single interfragmentary screws were most commonly used; healing was similar in both groups.


Bilateral optic nerve hypoplasia in a pup. A 6-week-old Beagle failed to respond to visual stimulation. Examination revealed fixed dilated pupils and hypoplasia of both optic disks. Results of electroretinography were normal. Radiography of the skull revealed small optic nerve foramina. Histologically, the outer layers of the retinas were normal, the retinal ganglion cells were scarce, and the nerve fiber layers, optic nerves, and optic chiasm were hypoplastic.


Aortic regurgitation in the dog.

Aortic regurgitation in the dog. In a review of aortic regurgitation in 12 dogs, breed or sex predilection was not found. Clinical signs included decreased exercise tolerance and diastolic murmur. Associated anomalies included ventricular septal defect and aortic stenosis. It was concluded that when aortic regurgitation develops secondary to a ventricular septal defect, the prognosis should be grave.


Isolation and description of a menaquinone mutant from Bacillus licheniformis. A menaquinone mutant (SG1) of Bacillus licheniformis has been isolated by selecting for colonies that are resistant to low levels of kanamycin (1.5 mug/ml) but sensitive to the same concentration of kanamycin in the presence of shikimate (25 mug/ml). The wild type (IU1) contained 0.38 +/- 0.02 nmol of menaquinone-7 (MK-7) per mg (dry weight) of cells when grown +/- shikimate, whereas SG1 had less than 0.01 nmol of MK-7 per mg (dry weight) of cells when grown in the presence of shikimate. SG1 had a generation time of 85 min, as compared to 24 min for IU1 grown +/- shikimate. SG1 doubled with a generation time of 28 min when grown in the presence of shikimate. IU1 consumed O2 at various rates depending on the stage of growth. A triphasic O2 consumption curve with maxima at mid-exponential phase, the transition from exponential to stationary phase, and early stationary phase was found for IU1 +/- shikimate and SG1 + shikimate. SG1 grown without shikimate consumed O2 at a low level (10 to 20% of IU1). Normal respiration could be restored to SG1 8.5 min after shikimate addition, whereas normal growth was not restored until 40 min after shikimate addition. Electron microscopic studies of SG1 and IU1 have indicated a morphological alteration in the mutant. SG1 is a dwarf cell as compared to IU1, when grown without shikimate. However, SG1 grown with shikimate became morphologically indistinguishable from IU1.


Substructure and in vitro assembly of the outer, structured layer of Spirillum serpens. Electron micrographs of disintegrating units of the outer, structured (HP) layer of Spirillum serpens and of the isolated protein obtained from the HP layer revealed V- and Y-shaped and linear profiles. Interpretation of these forms, influenced by the seemingly trimeric form of the isolated protein and by biochemical data, suggested that the protein subunits were identical and Y shaped. A model is proposed for the assembly of the Y-shaped subunits to form a hexagon composed of two triads (three Y-shaped subunits each). The isolated protein adsorbed to a template of wall fragments (basal layer) to the same degree (over 90%) in high concentrations of Na+, K+ (5 X 10(-2) M), Ca2+, Sr2+, and Mg2+ (10(-2) M). At a lower concentration (4 x 10(-5) M) of the cations there was differential adsorption of the protein. Adsorption to the template in the presence of each cation, followed by dilution, also led to differential release of the protein. The adsorption of the protein to the basal layer was correlated with reassembly of the HP layer on the template. The mechanisms seem to be: (i) an ionic strength-dependent reassembly, which results in an HP layer loosely attached to the template (this layer is easily dissociated by decreasing the ionic strength); and (ii) a cation-specific (Ca2+ or Sr2+, but not Mg2+, Na+, or K+) mechanism independent of ionic strength. In this latter case, the specific cations presumably form strong noncovalent "salt" linkages between triads and the basal layer, enabling stable hexagons and the HP layer to be formed.


Effect of cis-platinum(II)diamminodichloride on cell division of Hyphomicrobium and Caulobacter. Low concentrations of the radiomimetic agent cis-platinum(II)diamminodichloride (PDD) inhibited cell division in Caulobacter crescentus (0.1 mug/ml) and Hyphomicrobium sp. strain B-522 (1.0 mug/ml) without altering the length of prosthecae. After exposure, cells of C. crescentus appeared as long filaments, whereas only the bud portion of Hyphomicrobium underwent elongation. PDD-treated cells of both species were multinucleated. After the removal of PDD by washing, filaments of C. crescentus fragmented unequally and then normal growth resumed. In Hyphomicrobium (where division involves release of swarmer cells that arise as buds on the distal ends of hyphae), potential septation sites formed in the presence of PDD remained inactive after washing. Reinitiation of cell division in this species was dependent upon the synthesis of new hyphae that could arise from either end of the elongated bud. This finding suggests that the PDD-induced lesion at a given septation site is irreversible and, upon removal of this compound, alternate sites must be synthesized for the subsequent occurrence of cell division.


Genetic co-regulation of galactose and melibiose utilization in Saccharomyces. The gal3 mutation of Saccharomyces, which is associated with an impairment in the utilization of galactose, has been shown to be pleiotropic, causing similar impairments in the utilization of melibiose and maltose. Milibiose utilization and alpha-galactosidase production are directly controlled by the galactose regulatory elements i, c, and GAL4. The fermentation of maltose and the induction of alpha-glucosidase are regulated independently of the i, c, GAL4 system. The production of alpha-galactosidase and galactose-1-phosphate uridyl transferase is coordinate in galactokinaseless strains. Galactose serves as a nonmetabolized, gratuitous inducer of alpha-galactosidase in strains lacking the genes for one or more of the Leloir pathway enzymes.


Mutants of Saccharomycopsis lipolytica defective in lysine catabolism. Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.


Role and regulation of the ortho and meta pathways of catechol metabolism in pseudomonads metabolizing naphthalene and salicylate. The enzymes of naphthalene metabolism are induced in Pseudomonas putida ATCC 17484, PpG7, NCIB 9816, and PG and in Pseudomonas sp. ATCC 17483 during growth on naphthalene or salicylate; 2-aminobenzoate is a gratuitous inducer of these enzymes. The meta-pathway enzymes of catechol metabolism are induced in ATCC 17483 and PPG7 during growth on naphthalene or salicylate or during growth in the presence of 2-aminobenzoate, but in ATCC 17484 and NCIB 9816 the ortho-pathway enzymes of catechol metabolism are induced during growth on naphthalene or salicylate. 2-Aminobenzoate does not induce any enzymes of catechol metabolism in the latter two organisms. In Pseudomonas PG the meta-pathway enzymes are present at high levels under all conditions of growth, but this organism and PpG7 can induce ortho-pathway enzymes during naphthalene or salicylate metabolism. Salicylate appears to be the inducer of the enzymes of naphthalene metabolism in all of the organisms studied and, where they are inducible, of the meta-pathway enzymes, but the properties of Pseudomonas PG suggest that separate, regulatory systems may exist.


Rare occurrence of the tetratype tetrads in Saccharomycodes ludwigii. Genetic data suggesting the absence of crossover in Saccharomycodes ludwigii have been described. Tetrad data obtained from 888 asci from 60 pairs of genes with 22 genetic markers showed the absence of tetratype asci, except for 5 asci in which a single pair of alleles showed tetratype segregation to the other genetic markers in each ascus. Spore arrays in the linear asci showed that the + - + - and + - - + (or - + + -) asci occurred at almost equal frequencies. The two coherent spores at each end of an ascus were always marked with different alleles of a gene.


Properties of an inducible uptake system for beta-ketoadipate in Pseudomonas putida. Wild-type strains of Pseudomonas putida form an inducible uptake system that appears to act on beta-ketoadipate under normal physiological conditions. The system is induced by beta-ketoadipate and is represented by catabolites derived from it. Adipate is metabolized very slowly by wild-type P. putida cultures; [14C]adipate was used as an analogue of beta-ketoadipate to measure the transport activity in wild-type cells and in cells that constitutively produced the uptake system. Constitutive cells that contained high levels of the uptake system concentrated adipate to a level up to 200-fold above the concentration in the external medium. The process was energy dependent. The activity of the system with radioactive adipate was inhibited by beta-ketoadipate, by beta-ketoadipate analogues, and by some compounds (e.g., acetate, glucose) that are structurally unrelated to beta-ketoadipate; it is not known if the inhibitory effects are exerted directly by the compounds themselves or indirectly by catabolites derived from the compounds. The discovery of the beta-ketoadipate uptake system is surprising in view of earlier studies that had indicated that beta-ketoadipate does not permeate the membrane of wild-type P. putida cells. Contradictions between the former investigations and the present analysis are due primarily to the relatively high concentrations of substrate used in the earlier experiments. The existence of the beta-ketoadipate uptake system indicates that beta-ketoadipate may exist as a selective nutrient in the natural niche of P. putida and may play a determinative role in the evolution of induction mechanisms that are characteristic of fluorescent pseudomonads.


Peptidoglycans synthesized by a membrane preparation of Micrococcus luteus. By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsin-solubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala leads to L-Lys and D-Ala leads to L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.


Biosynthesis of peptidoglycan in Staphylococcus aureus: incorporation of the Nepsilon-Ala-Lys moiety into the peptide subunit of nascent peptidoglycan. UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla was isolated from extracts of Staphylococcus aureus Copenhagen. This nucleotide accumulated in media deficient in glycine. To establish its role in peptidoglycan biosynthesis, the nucleotide-hexapeptide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase and in the membrane-catalyzed nascent peptidoglycan-synthetizing system. In the exchange reaction catalyzed by the translocase, the Rmax and Rmax/Km are 1.79 muM/min and 4.47 X 10(-2)/min, respectively, for UDP-MurNAc-pentapeptide and 1.81 muM/min and 4.46 X 10(-2)/min, respectively, for UDP-Mur-NAc-hexapeptide. In the synthesis of nascent peptidoglycan, the Vmax is 1.8 muM/min X 10(-2) for both the nucleotide-hexapeptide and -pentapeptide. The Vmax/Km is 5.6 X 10(-4) and 4.3 X 10(-4)/min for the nucleotide-pentapeptide and -hexapeptide, respectively. Schleifer, Hammes, and Kandler (Adv. Microb. Physiol. in press) observed that growth of S. aureus Copenhagen on a glycine-poor medium results in a peptidoglycan structure in which 20% of the lysine residues are substituted at the epsilon-amino group by L-alanine residues that do not participate in interpeptide bridge information. The in vitro studies demonstrate that UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla is a possible precursor of the Nepsilon-Ala-Lys moiety.


Structure of plain and complex flagellar hooks of Pseudomonas rhodos. The proximal hooks of plain and complex flagella produced by a strain of Pseudomonas rhodos have been analyzed by electron microscopy and optical diffraction and filtering. Plain flagellar hooks are cone-shaped, 70 nm long, and 13 to 21.5 nm wide, and consist of helically arranged subunits. Complex flagellar hooks are cylinders, 180 to 190 nm long, and 15 to 16 nm wide, and are composed of globular subunits. The structure comprises four small-scale helical rows of subunits intersecting bewteen 10 and 11 large-scale helices of pitch angle 80 degrees. The axial and lateral dimensions of the unit cell, which define the surface lattice, are 4.9 and 4.7 nm, respectively. In addition, a core structure, approximately 5 nm wide, has been demonstrated inside the hook cylinder. Complex flagellar hooks were isolated and purified by gradient centrifugation after acid degradation of the attached filaments. Isolated hook particles have an average sedimentation constant of 130S and consist of a protein of molecular weight 43,000. A model of the complex flagellar hook is presented, and its possible role in flagellar assembly and rotation is discussed.


Uric acid degradation by Bacillus fastidiosus strains. Seven Bacillus strains including one of the original Bacillus fastidiosus strains of Den Dooren de Jong could grow on urate, allantoin, and, except one, on allantoate. No growth could be detected on adenine, guanine, hypoxanthine, xanthine, and on degradation products of allantoate. Some strains grew very slowly in complex media. The metabolic pathway from urate to glyoxylate involved uricase, S(+)-allantoinase, allantoate amidohydrolase, S(-)-ureidoglycolase, and, in some strains, urease.


Ultrastructure of superficial mycosidic integuments of Mycobacterium sp. Cells from pellicle growth of Mycobacterium sp. NQ are enveloped in a mycoside layer which extends outward as long filaments, 5 nm in diameter. Underneath this outer mycosidic casement, ramified ropelike structure, embedded in a dense matrix, overlay the rigid peptidoglycan of the cell wall.


Phosphorylation of mononucleotides and formation of cytidine 5'-diphosphate-choline and sugar nucleotides by respiration-deficient mutants of yeasts. Respiration-deficient mutants (Rho-, petite) of Saccharomyces carlsbergensis were obtained by treatment with trypaflavin (euflavine). Dried cells of these mutants phosphorylated mononucleotides to their triphosphates and further formed not only cytidine 5'-diphosphate-choline, but also sugar nucleotides, such as uridine 5'-diphosphate-glucose, guanosine 5'-diphosphate-mannose, etc. The activities were the same or slightly greater than those of the wild strain. These results showed that energy (adenosine 5'-triphosphate) necessary for phosphorylation of mononucleotides was sufficiently supplied by the glycolysis system.


Glycerol utilization by a mutant of Rhodopseudomonas capsulata. The glycerol-catabolizing enzymes of a mutant of Rhodopseudomonas capsulata were found to be constitutive and modulated coordinately, although apparently not functional in the presence of malate. No difference in glycerol permeation was found between the mutant and wild type.


Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles. Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high  energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.


Structure and function of initiation complexes which accumulate during inhibition of protein synthesis by fluoride ion. Incubation of the reticulocyte lysate cell-free system with KF results in the accumulation in polysomes of complexes containing deacylated tRNAMet and of complexes which can initiate globin chains in the presence of aurintricarboxylate. Degradation of these polysomes with T1RNase yields both 40 S and 80 S particles, and tRNAMet is found in both of these fractions. When the 80 S particles are reincubated with the soluble fraction of the lysate plus reagents for protein synthesis, short peptides which have the properties of the NH2-terminal regions of globin are synthesized de novo. These peptides are deficient in NH2-terminal methionine, but occur under conditions where nascent globin peptides of comparable length, containing NH2-terminal methionine, are completely protected from the methionine aminopeptidase.


Reaction of tetranitromethane with lutropin, oxytocin, and vasopressin. Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.


Effect of selective nitration of ovine lutropin on the subunit association and biological activity of the hormone. Different nitrated lutropin (LH) subunits were tested for their abilities to recombine with their counterpart subunits. (A detailed description of the preparations tested is presented in the preceding paper (Burleigh, B. D., Liu, W.K., and Ward, D.N. (1976) J. Biol. Chem. 251, 308-315).) The results indicated that the fully nitrated beta subunit of ovine LH (oLHBeta) (diatyrosyl oHbeta) can combine with the alpha subunit of ovine LH (oLHalpha) as wen which tyrosine residues alpha21, alpha41, alpha93 are modified) recombined partially with oLHbeta yielding about 40% as recombined product under specified recombination conditions. The fully nitrated oLHalpha (pentaatyrosyl oLH) combined poorly with oLHbeta and only 20 to 25% was obtained as the recombined product. The various nitrated LH and recombined LH derivatives containing separately nitrated subunits were tested for their potency in an in vitro radioligand assay and their in vivo biological potency in the ovarian ascorbic acid depletion assay. In the radioligand assay, the monoatyrosyl oLH and the oLHalpha + diatyrosyl oLHbeta were the most active derivatives and had about the same competitive inhibtion curves as that of oLHalpha + oLHbeta. Diatyrosyl oLH had intermediate native potency and the triatyrosyl oLH, tetraatyrosyl oLH + OLHbeta, and pentaatyrosyl oLHalpha + oLHbeta were the least active derivatives in this series. No tetranitromethane-modified LH derivative in our study was completely devoid of the specific binding activity in the radioligand assay. At a dosage of 1000-fold excess over the 125I-labeled oH, all the preparations tested in this series completely inhibited the specific binding of 125I-labeled oLH to the hormone receptors in rat testis preparations. In the in vivo ovarian ascorbic acid depletion assay, both the monoatyrosyl oLH and oLHalpha + diatyrosyl oLHbeta retained about 50% of its original activity; the diatyrosyl oLH and triatyrosyl oLH had about 10% of the activity and the recombined products of tetraatyrosyl oHalpha + oLHbeta and pentaatyrosyl oLHalpha + oLHbeta showed virtually no activity. Based on the results obtained from this paper and the preceding paper, the role of the individual tyrosine residues in oLH is assessed.


Rat liver pyruvate carboxylase. Inhibition by chromium nucleotide complexes. The effect of inert coordination complexes of chromium (III) with various nucleotides on the catalytic activity of rat liver pyruvate carboxylase was determined. The chromium nucleotides are effective initial inhibitors of pyruvate carboxylase and the inhibition becomes more severe with time. The initial rate decreases for several minutes, reaching a new slower rate that is then maintained until considerable net reaction occurs. Incubation of the enzyme with chromium nucleotides in the presence of Mg2+ and HCO3- causes maximal inhibition of the reaction and linear initial rates are then observed. This effect is similar to that found with yeast hexokinase (Dannenberg, K.D., and Cleland, W.W. (1975) Biochemistry 14, 28-39). The specificity of the carboxylase toward the nucleotide complexes suggests that the alpha and beta nucleotide phosphates are as important as the gamma phosphate in binding to the enzyme. A stable pyruvate carboxylase chromium nucleotide complex was not observed. These results are quite different from those found with yeast hexokinase where a stable complex between CrATP, sugar, and enzyme is found and hexokinase appears to be specific toward the beta, gamma phosphates of its nucleotide substrates.


A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. Four proteins active in blood coagulation have long been known to require vitamin K for their proper biosynthesis: factors II, VII, IX, and X. This paper describes the purification of a hitherto unrecognized vitamin K-dependent glycoprotein from bovine plasma. The biosynthesis of this protein is interfered with by the vitamin K antagonist Dicoumarol. The molecular weight of the protein is approximately 56,000 and, like factor X, it has two polypeptide chains. The light chain binds Ca2+. Its NH2-terminal amino acid sequence is homologous to the NH2-terminal sequences of the other vitamin K-dependent proteins and it contains vitamin K-dependent gamma-carboxyglutamic acid residues. The biological function of this protein is unknown.


Inhibition of pancreatic lipase B activity by taurodeoxycholate and its reversal by colipase. In our two-phase reaction system taurodexycholate prevents the adsorption of pancreatic lipase B to the nonaqueous phase. Our data are consistent with a mechanism for this reaction which involves the cooperative formation of an enzyme-(bile salt)4 complex in solution with a dissociation constant of 1.4 X 10(-15)M4. Whereas the free enzyme is readily adsorbed to a bile salt-substrate-covered surface, the complex is not. Thus, the "inhibition" of substrate hydrolysis occurs because enzyme and substrate are separated physically. The protein cofactor, colipase, reverses the inhibitory effects of bile salt by providing a high affinity binding site at the interface for the lipase-(bile salt)4 complex. Steady state and presteady state kinetic data are consistent with the formation of a complex with a 1/1, lipase/colipase, ratio, and a dissociation constant of 0.4 to 2.8 X 10(-9)M. The rate of adsorption of lipase to adsorbed colipase appears to be controlled by diffusion through the unstirred layer with a second order rate constant of 1.3 X 10(6)M-1S-1.


Metabolism and biologica.

Metabolism and biologica. 24R,24,25-Dihydroxyvitamin D3 is capable of inducing a minimal intestinal calcium transport response in chicks when compared to an equal amount of 25-hydroxyvitamin D3. 1,24,25-Trihydroxyvitamin D3 is also less active than 1,25-dihydroxyvitamin D3, and its activity is much shorter lived than that of 1,25-dihydroxyvitamin D3. A comparison of the metabolism of 25-hydroxy[26,27-3H]vitamin D3 and 24,25-dihydroxy[26,27-3H]vitamin D3 in the rat and chick shows that 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 disappear at least 10 times more rapidly from the blood and intestine of chicks. Furthermore, examination of the excretory products from both of these species demonstrates that chicks receiving a single dose of 24,25-dihydroxy[26,27-3H]vitamin D3 excrete 66% of the total radioactivity by 48 hours, whereas rats receiving the same dose excrete less than one-half that amount. These results demonstrate that 24,25-dihydroxyvitamin D3 is considerably less biologically active in the chick than in the rat, probably due to more rapid metabolism and excretion.


Acetylation of 13-sophorosyloxydocosanoic acid by an acetyltransferase purified from Candida bogoriensis. Acetyl-coenzyme A: 13-sophorosyloxydocosanoic acid (Glc2HDA) acetyltransferase was purified 14-fold in low yield from Candida bogoriensis cells. The enzyme catalyzes acetylation of the 6' and 6" positions of the sophorosyl group, producing the 13-[2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyloxy]-docosanoic acid 6',6"-diacetate (Ac2Glc2HDA) and monoacetate (AcGlc2HDA) in a product ratio of 5:1. Neither the purification steps nor heat denaturation studies indicated separation of the first and second acetylation steps. The acetyltransferase has a molecular weight of about 500,000 as determined by gel filtration on a Sepharose 4-B column. It shows a pH optimum range from 7 to 9, is strongly inhibited by 1 mM concentrations of the sulfhydryl reagents N-ethylmaleimide, p-hydroxymercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid), but only partly inhibited by 10 mM iodoacetamide. It has an apparent Km of 30 muM for acetyl-CoA, utilizes propionyl-CoA at 45% the rate of acetyl-CoA, and utilizes longer chain acyl-CoA derivatives much less efficiently. The critical micelle concentrations of the C. bogoriensis glycolipids in pH 7.7 phosphate buffer were estimated by pinacyanol chloride binding as follows: Glc2HDA, 50 mum; AcGlc2HDA, 30 muM; Ac2Glc2HDA, 12 muM. The Stokes radius of Ac2Glc2HDA micelles was 22 A as estimated by gel filtration on Bio-Gel P-150. Glc2HDA was a much better acceptor than its methyl ester in the acetyltransferase assay. A plateau in the Glc2HDA saturation curve at 50 muM and a corresponding break in the reciprocal plot at this concentration indicate the enzyme utilizes the monomeric form of this lipid as substrate.


Hydrolysis of 13-sophorosyloxydocosanoic acid esters by acetyl- and carboxylesterases isolated from Candida bororiensis. Two enzymes have been isolated from Candida bogoriensis which catalyze the hydrolysis of 13-sophorosyloxydocosanoic acid (Glc2HDA) esters obtained from this organism. The 6',6"-diacetyl derivative of Glc2HDA (Ac2Glc2HDA) is hydrolyzed by an acetylesterase (EC 3.1.1.6) which has been purified 1300-fold. The acetylesterase has a molecular weight of 35,000 estimated from gel filtration, and is much more active with p-nitrophenyl acetate than with the acetylated glycolipid. The rate of hydrolysis increases with Ac2Glc2HDA concentration until a plateau is reached at a concentration of about 40 muM, near the critical micelle concentration of this glycolipid. These kinetic data are interpreted as an enzyme specificity for the monomeric, but not the micellar form of the glycolipid. The acetylesterase is inhibited by 0.1 to 10 mM diisopropyl fluorophosphate, 5 mM p-hydroxymercuribenzoate, and 5 mM N-ethylmaleimide, but only slightly by 5 mM iodoacetamide. The methyl ester of Ac2Glc2HDA is hydrolyzed by at least two carboxylesterases (EC 3.1.1) which differ in size according to gel filtration. Their molecular weights are estimated as 140,000 for carboxyesterase A and 40,000 for carboxyesterase B. Both carboxylesterases were purified over 20-fold, and carboxylesterase A was characterized further. Carboxylesterase A activity was inhibited completely by 0.1 to 10 mM diisopropyl fluorophosphate and by 10 mM p-hydroxymercuribenzoate, but only slightly by lower concentrations of p-hydroxymercuribenzoate or by N-ethylmaleimide or iodoacetamide. The carboxylesterase A preparation also acted as a thioesterase with palmityl-CoA (palmityl-CoA hydrolase, EC 3.1.2.2), showing the following approximate relative activities: palmityl-CoA, 100; octanoyl-CoA, 90; methyl Glc2HD, 22; butyryl-CoA, 18; methyl AcGlc2HD, 15; methyl Ac2Glc2HD, 10; and acetyl-CoA, O. Methyl Ac2Glc2HD showed some substrate inhibition at higher concentrations, but neither methyl Ac2Glc2HD nor palmityl-CoA approached enzyme saturation until well above their critical micelle concentrations, indicating hydrolysis of the micellar substrate was occurring. The carboxylesterase and palmityl-CoA hydrolase activities were destroyed in a parallel fashion by heat denaturation, and each substrate inhibited the action of the preparation on the other substrate, but the preparation has not been purified sufficiently to establish with certainty that both activities reside in the same protein.


Reaction of haptoglobin with hemoglobin covalently cross-linked between the alpha beta dimers. Hemoglobin tetramers which cannot split into alphabeta dimers, because they are covalently cross-linked between the beta chains across the polyphosphate binding site, form complexes with haptoglobin. The reaction is biphasic as measured by fluorescence quenching and peroxidase activity. A complex in which one of the alpha beta dimers of the cross-linked hemoglobin is bound to one of the sites in the divalent haptoglobin molecule, is formed reversibly during the initial fast phase. In the subsequent slower step, this product then either polymerizes, adds another cross-linked hemoglobin molecule or, in the presence of excess haptoglobin, combines with a second haptoglobin molecule. This latter complex, in which two haptoglobin molecules are bridged by a cross-linked hemoglobin tetramer, can still combine with normal alpha beta dimers at the vacant haptoglobin combining sites. In spite of the very low oxygen affinity of the cross-linked hemoglobin, combination with haptoglobin shifts if oxygen affinity to the very high value of the normal hemoglobin-haptoglobin complex.


Linear structure of the oligosaccharide chains in alpha1-protease inhibitor isolated from human plasma. Two glycopeptides present in equal amounts were isolated from a pronase digest of alpha1-protease inhibitor of human plasma by gel filtration on Sephadex G-50 and chromatography on DEAE-cellulose. The carbohydrate side chains in both glycopeptides are linked through asparaginyl residues. The glycopeptides were digested sequentially with specific glycosidases; and after each step, the released sugars as well as the composition of the residual peptides were determined. The linear structures of these glycopeptides deduced from these data are shown below. Based on the total carbohydrate content of the intact protein and with these structural data, it is postulated that 4 oligosaccharide units are attached to 1 molecule of the protein; 2 of these were represented as in Equation 1, the other 2 as in Equation 2.


Properties of ecto-(inoganic) pyrophosphatase of nervous system cells in culture. Activation upon partial release of sialic acid from the cell surface. Clonal line NN hamster astroblasts and clonal line N18 neuroblasts were treated with phospholipase C-free, protease-free, and hemolysin-free Clostridium perfringens sialidase, at a low level (5 X 10(-3) units/ml) so as to maintain cell intactness and to avoid spurious protein effects. A rapid, regular release of sialic acid was achieved. An approximately 9-fold increase in ecto-pyrophosphatase activity could be brought about by action of C. perfringens sialidase for 10 min. Since the sialidase preparations were employed at a level which gave a very low concentration of extraneous protein, and the preparations were free of demonstrable phospholipase C and protease activities, these effects appear to relate specifically to removal of cell surface sialic acid. Neutral p-nitrophenyl phosphatase was activated under the same conditions, but activity remained low compared with pyrophosphatase. Progress curves for activation of the two enzymes were dissimilar. Ecto-pyrophosphatase of NN and N18 cells had an absolute requirement for Mg2+ both before and after removal of cell surface sialic acid. In the presence of near optimum Mg2+ (5 mM), other divalent cations were inhibitory at a low level (10(-1)mM). The effect  of Mg2+ concentration, as well as inorganic pyrophosphate concentration, upon ecto-pyrophosphatase activity was shown to obey Michaelis-Menten kinetics for the control activity and for the sialidase-enhanced activity of both cell types. Km for Mg2+ and for pyrophosphate remained constant upon ecto-pyrophosphatase enhancement by sialic acid removal; increase in enzymatic activity was accounted for entirely by an increase in Vmax.


External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography. Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor  components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.


Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene in chromatin. RNA was transcribed from chromatin isolated from chick oviduct and spleen by using RNA polymerase from Escherichia coli. RNA was also transcribed from whole chick DNA using E. coli RNA polymerase. DNA complementary to ovalbumin messenger RNA (cDNAov) was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences (mRNAov) in these in vitro transcripts. Although chromatin from unstimulated chick oviduct was capable of substantial RNA synthesis, no detectable mRNAov sequences could be found in the transcript. Likewise, mRNAov sequences could not be found in RNA synthesized from spleen chromatin using E. coli RNA polymerase. However, chromatin from estrogen-stimulated chick oviducts was capable of supporting synthesis of ovalbumin mRNA. We estimate that approximately 0.01% of the RNA synthesized from estrogen-stimulated chromatin was mRNAov sequences. When RNA synthesized from chick DNA was tested with the cDNAov probe, mRNAov sequences could be detected in a concentration of approximately 10% that found in the RNA transcript from estrogen-stimulated chromatin. This was as expected if the ovalbumin gene is considered to be in the "open or derepressed" region of the estrogen-stimulated oviduct chromatin. Chromatin isolated from chicks withdrawn from hormone for 12 days was only capable of supporting mRNAov synthesis in vitro at a level of 5 to 10% of that observed in chromatin prepared from estrogen-stimulated chicks, thus indicating the requirement for estrogen to maintain the ovalbumin gene in the available or "open" state in the majority of oviduct cells. These data militate against post-transcriptional control as the primary mechanism of steroid hormone regulation of specific mRNA synthesis in the chick oviduct system, and favor primary gene derepression as the most likely mechanism for estrogen induction of ovalbumin synthesis.


The physical mapping of bacteriophage T5 transfer tRNAs. Transfer RNAs, isolated from Escherichia coli F cells infected with T5 bacteriophage, were charged with radioactive amino acids and used in RNA-DNA hybridization studies to detect and locate T5 tRNA cistrons in the T5 DNA chromosome. Hybridization of 14 3H-aminoacyl-tRNA species, including purified T5 [35S]Met-tRNAm and [35S]Met-tRNAf, to the separated strands of T5+ DNA indicates that most, if not all, of the T5 tRNAs are transcribed from the continuous heavy strand of T5 DNA. Heteroduplex mapping of eight mutant T5 DNA deletions has enabled us to locate and determine the size of these deleted segments. By correlating this information with the presence and absence of specific tDNA sequences in these mutants, as determined by tRNA-DNA hybridization, we were able to define the physical limits of four tDNA-containing loci along the T5 DNA molecule. A physical map for 15 tRNA species examined indicates that the structural genes for these tRNAs are clustered within a segment length of T5 DNA that represents approximately 11.2% of the total wild type T5 DNA. The existence of the deletion mutants indicates that T5 tRNAs are dispensable for T5 replication under the growth conditions and for the host employed.


Proteins of Friend leukemia cells. Comparison of hemoglobin-synthesizing and noninduced populations. Proteins of Friend leukemia cells induced to form large amounts of hemoglobin by dimethylsulfoxide treatment were compared with proteins from noninduced cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Approximately 98% of more than 500 proteins separated by this technique were qualitatively and quantitatively the same in both cell populations. Changes representing more than 50% of the control cell amount were detected in six non-histone chromosomal proteins, two nucleoplasmic proteins, and three cytoplasmic proteins. It is concluded that dimethylsulfoxide induces an extremely specific pattern of erythroid differentiation in these cells, which should be susceptible to detailed analysis. Comparison of protein patterns from Friend leukemia cells and HeLa cells revealed electrophoretic identity of approximately 20% of cytoplasmic proteins and 50% of non-histone chromosomal proteins.


Total drainage of the right lung into the right hepatic vein. Report of a case oa anomalous venous drainage of the right lung into the right hepatic vein treated by disconnection of the anomalous pulmonary vein and end-to-side anastomosis to the left atrium. Following a review of 25 surgically treated patients with infracardiac drainage of the right lung the different surgical approaches are discussed.


Blood and tissue silicone in extracorporeal circulation. A new  method for the determination of blood and tissue contents of silicone by atomic absorption spectrophotometry is described. Silicone was found in the blood, probably as droplets, in each of 14 patients undergoing open-heart surgery with the aid of a bubble oxygenator. In five fatal cases the brains and kidneys contained silicone. Although the clinical significance of the silicone embolization is unclear precautions should be taken to reduce the release of silicone from the extracorporealoxygenating devices.


Counterpulsation in experimental cardiogenic shock with ventricular arrhythmias. The antiarrhythmic effect of morphine. In two groups of dogs a cardiogenic shock was induced and their circulation assisted by counterpulsation. In the second group aside from the counterpulsation, 4 mgrs/Kg of morphine was administered when stable ventricular arrhythmias appeared. Morphine showed to eliminate the ventricular arrhythmias in three cases and lessened them considerably in one, permitting better assistance with a counterpulsator.


Abdominal aortic aneurysms.

Abdominal aortic aneurysms. The dangers of any abdominal aortic aneurysm are discussed, and the disastrous combination of an aneurysm and abdominal pain emphasised. These anerysms can be divided into 4 groups. The presentation and operative mortality for each group is discussed, as is the long term survival, state of the peripheral circulation and general health after operation. How results can be improved is considered. It is concluded that all such aneyrysms should be operated upon because of the good immediate and long term results in comparison with those not operated upon.


Surgical treatment of aorto-iliac occlusive disease. The results of 177 reconstructions for aorto-iliac occlusive disease are presented. All operation, and evaluations of immediate and long term results obtained, were performed by one surgeon. Two basic techniques, endarterectomy and synthetic grafts were used. The results were assessed by the life table method. At ten years there was no statistical difference between the two groups. Endarterectomy, and graft insertion appear to be complementing each other, rather than representing two different modalities of therapy. The importance of thorough evaluation of the cerebral and coronary circulation prior to reconstruction of the aorto-iliac segment is emphasized.


Surgical indication and results in ventricular septal defect associated with severe pulmonary hypertension. Sixty-four patients with large ventricular septal defects and severe pulmonary hypertension form the basis of this report. Prior to the end of 1970, 35 patients with a ratio of pulmonary to systemic artery pressure (pp/ps) of 0.8 or more were subjected to primary closure of the defect, resulting in 11 operative deaths Since the beginning of 1971, until March, 1974, however, the surgical results have markedly improved and 29 such patients were operated on without death. It has been our principle to perform elective  closure of the defect at the age of 1 to 3 years. An earlier operation was planned when a decrease in the apical diastolic rumble and the size of the enlarged left ventricle on the chest x-ray were found on the periodical observation since infancey. If cardiac failure could not be controlled with medical treatment, primary closure of the defect has been performed even under one year of age.


The value of arteriography of the hand in the Raynaud's syndrome. The hand arteriograms of 18 patients with Raynaud's syndrome are reviewed. In 17 of the patients the arteriographic abnormality consists of digital artery occlusions, collaterals and diminished vascularity of the tips of the affected fingers. These changes are non-specific and do not correlate with the clinically diagnosed underlying arterial disease. The results of upper thoracie sympathectomy are unrelated to the type or severity of the angiographic changes. It is suggested that hand arteriography has little place in the diagnosis or management  of patients with Raynaud's phenomenon. Arteriography is only of value to demonstrate a proximal source of digital emboli.


DNA denaturation in situ. Effect of divalent cations and alcohols. Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.


Compositional studies of myofibrils from rabbit striated muscle. The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.


Role of creatine in the regulation of cardiac protein synthesis. The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.


Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion. To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion.


Mating in Chlamydomonas: a system for the study of specific cell adhesion. II. A radioactive flagella-binding assay for quantitation of adhesion. To measure the flagellar adhesiveness of Chlamydomonas gametes in a more quantitative manner than agglutination assays permit, a binding assay was developed which measured the binding of radioactive flagella of one mating type to unlabeled gametes of the opposite mating type. With the appropriate assay conditions, the number of [3H] flagella specifically bound was shown to be proportional to the number of cells in the incubation mixture and, therefore, to the number of binding sites that were present. The assay was used to study the effects of trypsin treatment on the loss and development of flagellar binding sites. It was shown that after trypsin treatment at least 9 h were required for the return of a full complement of binding sites to the flagellar surface; moreover, the results indicated that these sites reappeared on existing, extended flagella.


Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation. Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.


Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study. New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae.


Sorting out of normal and virus-transformed cells in cellular aggregates. The sorting-out behavior (self-segregation of two cell types from mixtures of the two) of five different established cell lines was studied. Eight of the ten possible binary combinations of these lines, cultured as cellular aggregates, were examined. Mouse BALB/c 3T3 cells sorted out internally to the corresponding malignant SV40 virus-transformed 3T3 cells. The transformed 3T3 line (SVT-2) did not sort out from a revertant line selected from SVT-2 cells by resistance to concanavalin A (con A). The revertant cells sorted out externally to the parent BALB/c 3T3 cells, although segregation was generally incomplete. BALB/c 3T3 cells did not sort out from another contact-inhibited line of 3T3 cells derived from Swiss albino mice (Swiss 3T3). Both BALB/c 3T3 and Swiss 3T3 cells sorted out from cells of the contact-inhibited hamster line, NIL B. Instead of a two-layered sphere, however, a three-layered structure was observed with most of the NIL B cells external to the 3T3 cells, and a few NIL B cells comprising the center of the sphere. On the other hand, NIL B cells did not consistently sort out from either the SVT-2 or con A cells. In general, sorting out between pairs of these five lines are slower and less complete than is generally observed between the more extensively studied chick embryonic tissue cells, suggesting that the cultured cells may be more closely related in their adhesive properties. The internal segregation of BALB/c 3T3 cells relative to SVT-2 cells is consistent with the hypothesis that transformed cells are less adhesive than their nontransformed counterparts.


In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates. Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro. As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by 10 min of incubation at 30 degrees C, and no rupture of the vacuoles was detected during this time. After 10 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system.


Fate of ganglionic synapses and ganglion cell axons during normal and induced cell death. In order to understand the significance of cell death in the formation of neural circuits, it is necessary to determine whether before cell death neurons have (a) sent axons to the periphery; (b) reached the proper target organs; and (c) have established synaptic connections with them. Axon counts demonstrated that, after sending out initial axons, ciliary cells sprouted numerous collaterals at the time of peripheral synapse formation. Subsequently, large numbers of axons were lost from the nerves, slightly later than the onset of ganglion cell death. A secondary loss of collaterals later occurred unaccompanied by cell death. Measurements of conduction velocity and axon diameters indicated that all ganglion cell axons grew down the proper pathways from the start, but it was not possible to determine whether all axons had actually formed proper synapses. This was ascertained, however, in the ganglion itself where preganglionic fibres were shown to synapse selectively with all ganglion cells before cell death. During this period, degenerating preganglionic synapses were observed on normal cells. It can therefore be inferred that at least some preganglionics established proper synapses before dying and that a single synapse is not sufficient to prevent cell death. In this system neither preganglionic nor ganglionic cell death seems designed to remove improper connections but rather to remove cells that have not competed effectively for a sufficient number of synapses, resulting in a quantitative matching up of neuron numbers.


Glycine-specific synapses in rat spinal cord. Identification by electron microscope autoradiography. Glycine, an inhibitory transmitter in spinal cord, is taken up into specific nerve terminals by means of a unique high-affinity uptake system. In this study, [3H]glycine was directly microinjected into rat ventral horn in vivo and electron microscope autoradiography used to localize the label in various anatomic compartments. Quantiative analysis showed that [3H]glycine labeled a high proportion of axosomatic and axodendritic synapses which presumably act to inhibit spinal motor neurons.


Glucose-6-phosphate dehydrogenase activity in a hepatoma cell line: preliminary evidence for negative genetic control. A line of cells, positive for glucose-6-phosphate dehydrogenase activity, has been separated from a negative parental stock derived from the H4-II-E-3C rat hepatoma line. Cytogenetic analyses showed specific marker chromsomes of the parent line were present in the variant. On the other hand, cells of the variant line contained three chromosomes less, on the average, than the total present in parent cells. Karyotypic analyses indicated the diminution in chromosome numbers to be in the subacrocentric group. The appearance of glucose-6-phosphate dehydrogenase associated with loss of chromosomes suggests this enzyme is suppressed or under negative control in the parental stock. The consistent loss of chromosomes of the subacrocentric group suggests that the locus bearing regulatory activity is in this specific group.


Effects of local anesthetics on cell morphology and membrane-associated cytoskeletal organization in BALB/3T3 cells. Tertiary amine local anesthetics (dibucaine, tetracaine, procaine) reversibly affect the morphology of untransformed BALB/3T3 cells and the organization of membrane-associated cytoskeletal elements. In the presence of these drugs cells contract and become rounded in shape with the appearance of numerous surface "blebs." Electron microscope examination of anesthetic-treated cells revealed significant reductions in plasma membrane-associated microtubules and microfilaments and/or their plasma membrane attachment. The relationship of the findings on local anesthetic-induced changes in cellular cytoskeletal systems is discussed in relation to previous proposals on plasma membrane organization and control of cell surface receptor topography and mobility.


Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA). During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.


Evidence contrary to the protein error hypothesis for in vitro senescence. A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucose-6-phosphate dehydrogenase (G-6-PD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 +/- 1%, S.E.), in comparison with that in 19 old cultures (4.9 +/- 1%, S.E.). Old cultures had ceased active cell division (49-60 doublings); DNA replication, measured by [3H]thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5-22 doublings) had a [3H]thymidine labeling index of 75-85%. Titration of G-6-PD activity in extracts of young and old cells with neutralizing antibody directes specifically against G-6-PD failed to detect an increment of enzymatically defective G-6-PD in old cells. The thermostability studies were capable of detecting altered G-6-PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G-6-PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G-6-PD from the thermolabile mutant and G-6-PD inactivated with N-ethylameimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.


The effect of lanthanum on electrophoretic mobility and passive cation movements of the Ehrlich ascites tumor cell. We have investigated the effects of La+3 binding to the surface of Ehrlich ascites tumor cells on cell electrophoretic mobility and passive movements of Na+ and K+. Incubation of tumor cells in La+3-containing media results in a La+3 concentration-dependent decrease in net surface charge negativity. At [La+3] greater than 0.5 mM, the net surface charge becomes positive with maximum positivity occurring at [La+3] = 0.9 mM. The effects of La+3 binding on passive Na+ and K+ movements were investigated by following 22Na and K+ losses from ouabain-inhibited cells. Neither low (0.02) nor high (1.0 mM) [La+3] had any effect on the K+ efflux rate coefficient. 22Na losses from control and La+3-treated cells were consistent with washouts from two cellular compartments. Low [La+3] (0.02 mM) was without effect on Na+ losses from the cells. However, higher [La+3] (1.0 mM) resulted in a 48% inhibition of Na+ loss from the more slowly exchanging compartment. These results are not consistent with simple electrostatic interactions exerting a major influence on the passive movements of Na+ and K+. It is suggested that La+3 interacts with sites specific for Na+, perhaps involved in a carrier-mediated exchange system.


Passive cation movements in the Ehrlich ascites tumor cell: the effects of 2,4,6-trinitrobenzene sulfonic acid. We have investigated the effects of 2,4,6-trinitrobenzene sulfonic acid (TNBS), an amino reactive reagent, on passive cation movements in Ehrlich ascites tumor cells. Incubation of tumor cells with TNBS (3 mM) results in a two phase association of TNBS with the cells. An initial, rapid phase, presumably at the level of the membrane, is independent of temperature, while the second phase increases linearly in time and is temperature dependent. Kinetic analyses of Na+ movements indicate that TNBS: (1) inhibits Na+ movement from a slowly exchanging cellular compartment, but is without effect on a more rapidly exchanging compartment; (2) does not alter net Na+ accumulation in transport-inhibited cells; and (3) is without effect on non-exchange Na+ efflux at 0 degrees C. The actions of TNBS on K+ movements depend upon temperature and the continued presence of TNBS in the environment. At 22 degrees C two minute exposure of the cells to TNBS leads to 77% inhibition of K+ efflux. With continued exposure to TNBS, the inhibition is only 42%. Reduction of the temperature to 0 degrees C decreases K+ efflux in control cells by 82%. Two minute exposure to TNBS enhances K+ efflux by 50%, while continuous exposure increases it by 144%. These results suggest: (1) TNBS interacts with several classes of membrane sites which are involved with the regulation of passive cation movements; and (2) passive Na+ and K+ movements across the cell membrane proceed by different pathways.


Enhancement of phenotypic expression in cultured malignant liver epithelial cells by a complex medium. The ability of two media identically supplemented with serum to maintain functional and structural differential properties of cultured malignant liver epithelial cells was compared. Reuber H35 hepatoma cells grown in a complex medium, Medium E, possessed the gluconeogenic enzyme phosphoenolpyruvate carboxykinase and bile canaliculi. When grown in Eagle's Minimal Essential Medium, the activity of phosphoenolpyruvate carboxykinase was reduced and bile canaliculi were not formed. This decrease in properties was restored when cells maintained in Minimal Essential Medium was returned to Medium E. It was concluded that the enhanced phenotypic expression was probably due to enrichment of the nutrient environment.


Chromosome balance and the control of malignancy. The Giemsa banding pattern of the chromosomes has been analyzed in a line of transformed golden hamster cells, revertant and re-revertant cells and their tumors. The transformed and re-revertant cells were malignant in vivo and had gained an additional chromosome 5(7). Revertants with a suppression of malignancy lost this additional chromosome 5(7) and gained an additional chromosome 7(2). The tumors produced by segregants from the revertant cells were malignant, although to a lower degree than transformed and re-revertant cells. These tumors had lost the additional chromosome 7(2) found in revertants and gained one or two 5(12) chromosomes. The results support the hypothesis that the balance between genes for expression and suppression controls malignancy. The data indicate that chromosome 7(2) carries genes for suppression and that chromosomes 5(7) and 5(12) carry genes for expression of malignancy. The genes on chromosome 5(7) seem to result in a greater degree of expression than the genes on chromosome 5(12). The chromosome balance that controlled malignancy in these cells, also controlled the expression and suppression of transformed properties in vitro.


Systematic analysis of mycotoxins by thin-layer chromatography. The analysis of 37 mycotoxins and other fungal metabolites by thin-layer chromatography is described. Their R(F) values in eight solvent systems, colour reactions after chemical detection and detection under UV light are presented. From the R(F) values, "chromatographic spectra" which can be used as one of the characteristics for the characterization of unknown mycotoxins were constructed.


Bioautographic detection of mycotoxins on thin-layer chromatograms. A method for the bioautographic detection of mycotoxins on thin-layer chromatograms by using Artemia salina larvae is described. The method was tested on standard samples of mycotoxins (aflatoxin B1 kojic acid and sterigmatocystin) and on the extracts from toxicogenic fungi isolated from different sources.


Mass fragmentographic determination of diphenylhydantoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, in human plasma. A method is described for the mass fragmentographic determination of diphenylhydantoin and its main metabolite, 5-(-4-hydroxyphenyl)-5-phenylhydantoin (4-OH-DPH), in human plasma as their dimethyl and trimethyl derivatives, respectively. The derivatives are formed by using the recently described extractive alkylation technique. Pentadeuterated 4-OH-DPH is used as the internal standard. Following acidic hydrolysis of the plasma sample, conjugated 4-OH-DPH and, indirectly, the dihydrodiol metabolite, 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin, are measured. Using 100-mul plasma samples, the lower limit of detection is about 10 ng/ml 00.03 nmole/ml).


Computerized data analysis of amino acids in physiologic fluids. A Fortran IV computer program is described which identifies, computes, and statistically evaluates amino acid concentrations determined by an automatic amino acid analyzer in physiologic fluids. The program accepts retention time and intergrated peak areas from two calibration standards for identification and computational reference. Two internal standards are used to compensate for variations in injector performance and ninhydrin decay. Calibration standard responses are statistically treated to assist in detection of equipment malfunction. The statistical data base is automatically explanded through inclusion of data from normal patients. Amino acid concentrations are printed with appropriate mean and standard deviation values in a format acceptable as a final report.


Applications of gas-liquid chromatography in protein chemistry. Determination of C-terminal sequences on nanomolar amounts of proteins. A method for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins is described, based on carboxypeptidase A digestion of the protein, followed by removal of the partially digested protein and quantitative gas-liquid chromatographic determination of the amino acids released after known time intervals. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins.


Methylation artifacts in the gas chromatography of serum extracts. Competitive suppression with trimethyl-d9 anilinium hydroxide. In the course of the identification of drugs in body fluids by gas chromatography-mass spectrometry we have observed the appearance of N-methyl derivatives of certain compounds such as phenobarbital and diphenylhydantoin, especially in extracts of serum. With the aid of deuterium labelled phenobarbital it could, however, be shown that the N-methyl derivatives are not endogenous metabolites but artifacts generated upon injection of the serum extract into the gas chromatograph. Conversely, derivatization with trimethyl-d9 anilinium hydroxide (TMAH-d9) demonstrated the absence of any undeuterated methyl derivative and this reagent should be useful for the detection of endogenous N-methyl derivative which would, of course, remain undetected when using unlabelled TMAH in the course of the conventional methylation technique. There are good indications that lecithin is the methylating agent in serum responsible for the formation of these artifacts.


The formation of trimethylsilyl ethers of ecdysones. A reappraisal. The hydroxyl groups of 20-hydroxyecdysone react with trimethylsilylimidazole with varying ease, in the positional order 2,3,22,25 greater than 20 greater than 14. The 14alpha-hydroxyl group can only be silylated under forcing conditions. Confusion in silylation procedures has been caused by failure to recognize incomplete reaction. The conclusions are supported by mass spectra. In the presence of a catalyst, and absence of a 14alpha-oxy substituent, enol ethers are readily formed, but the rate is considerably reduced with a C-14 substituent present.


Liquid chromatography of dansyl derivatives of some alkaloids and the application to the analysis of pharmaceuticals. Derivatization of the alkaloids cephaeline, codeine, emetine, ephedrine, morphine, narcotine and others with dansyl chloride has been studied with the aim of developing a sensitive and specific liquid chromatographic method for these substances in complex pharmaceutical dosage forms. While codeine and narcotine do not react, the other compounds form completely substituted derivatives which possess maxima in their fluorescence emission spectra between 470 and 500 nm. The structure of the derivatives has been confirmed by nuclear magnetic resonance spectroscopy. The dansylated compounds have been separated by thin-layer chromatography and high-pressure liquid chromatography. The improved selectivity and sensitivity have permitted an analysis of these substances present in low concentrations in 10- 100-fold excesses of other drugs. Direct derivatization of syrups and aqueous slurries of capsules having a complex excipient and drug composition is feasible and time saving and serves as a pre-clean-up step. Detection limits are in the 1-10-ng range or better, depending on the efficiency of the detection device. The reproducibility of the method is limited by the derivatization step, but a relative standard deviation of less than 2% can be obtained. The analysis time for these pharmaceuticals may be reduced by at least one fifth of that required by conventional techniques.


Preparation, separation and degradation of oligouronides produced by the polygalacturonic acid transeliminase of Bacillus pumilus. Production of the end products of polygalacturonic acid degradation on a large scale was done by reacting free galacturonic acid with Bacillus pumilus polygalacturonic acid transeliminase (PATE, EC 4.2.2.2) to obtain a mixture of the barium salts of several oligouronides. Small amounts of the unsaturated oligouronides were separated by paper chromatography. Large quantities of unsaturated oligouronides were separated on a AG-1-X8 (formate) column by applying a sample of mixed oligouronides and stepwise elution was carried out with sodium formate buffer (pH 4.7). The unsaturated oligouronides were identified on the basis of chromatographic mobilities, Sephadex gel filtration data, COOH/CHO ratio, thiobarbituric acid-reacting material, bromine uptake, and chemical and enzymatic degradation data as unsaturated tri-, tetra-, and hexagalacturonic acids. The chemical degradation of these unsaturated oligouronides, done with 6 N HCl by heating at 100 degrees for 30 min, gave qualitatively identical products of hydrolysis. These products compared with authentic standards, were identified as galacturonic acid, formic acid, 5-formyl-2-furancarboxylic acid, and 2-furancarboxylic acid. Analysis of the enzymatic breakdown products of the higher unsaturated uronides showed that a minimum of four galacturonic acid units was required for the action of purified endo-PATE from B. pumilus. The unsaturated trimer was not attacked, thus accounting for its accumulation as the major end product of polygalacturonate degradation by this enzyme.


Quantitative gas-liquid chromatography of non-protein amino acids. The quantitative gas--liquid chromatographic analysis of non-protein amino acids, in the presence of protein amino acids, is described. The amino acids were determined as their N-trifluoroacetyl n-butyl esters on an ethylene glycol adipate column. The relative molar responses of 38 amino acids are reported.


A simple and sensitive gas chromatographic method for the determination of clonazepam in human plasma. A simple gas chromatographic method for the determination of clonazepam in human plasma has been developed. After solvent extraction, the compound is measured by an electron capture detector on an OV-17 column. The electron-capture response is linear for 5-120 ng/ml of plasma. There is no interference from other commonly used anti-epileptic drugs or endogenous substrates. Preliminary data from routin monitoring of epileptic patients shows a 10-fold variation in their clonazepam plasma levels.


Gas chromatographic method for the determination of nomifensine in human plasma. An analytical method based on solvent extraction, formation of a fluorinated derivative and quantitation by gas-liquid chromatography using an electron capture detector has been developed for the determination of nomifensine in biological fluids. The specificity (controlled by mass spectrometry) and the sensitivity appear to be satisfactory for drug level measurements in human body fluids. Its relative simplicity in fact permits its use in serial analysis.


Application of Carbowax 20M as an open-tubular liquid phase in analyses of nutritionally important fats and oils. In the first part, the reasons for the choice of Carbowax 20M-terephthalic acid as a liquid phase are indicated and the technique for preparation of columns is descirbed. The ECL of 14 to 24 carbon fatty acids (branched and positional isomers of unsaturated acids) as well as the accuracy of determination of some fatty acids, are presented. In the second part, examples of chromatograms are given as well as tables concerning the composition of various fats: vegetable oils, depot fats, and fats from organs of monogastric species, polygastric species, and fish.


Fatty-acid composition of rat brain lipids. Determined by support-coated open-tubular gas chromatography. The nonhydroxy fatty acid composition of rat brain lipids (except gangliosides) was determined by support-coated open-tubular (SCOT) gas chromatography. Fatty acids of both odd and even chain lengths ranging from C14 to C26 were detected. Brain lipids contained 49% saturated, 29% monounsaturated, and 22% polyunsaturated fatty acids. Monoenoic fatty acids were mainly of the omega-9 and omega-7 series with minor amounts of omega-10 and amega-11 isomers. Dienes and trienes consisted of omega-6, amega-7, and omega-9 series. Tetraenes were of the omega-6 series. Small amounts of omega-6 and omega-3 pentaenes were detected. The most abundant polyunsaturated fatty acid was 22:6omega-3. The advantages of support-coated open-tubular columns over wall-coated open-tubular columns for the analysis of brain lipid fatty acids are discussed.


Chromatographic methods in the determination of absolute and relative configurations of fatty acids. Chromatographic techniques for determination of configurations of branched and substituted long-chain fatty acids, both absolute and relative, are reviewed. Gas-liquid chromatographic and thin-layer chromatographic procedures are emphasized.


Automated fatty acid analysis.

Automated fatty acid analysis. An automated system for the analysis of fatty acids is described. Samples dissolved in CS2 are automatically injected and separated by temperature programmed gas chromatography. Peak areas and retention times were measured by an electronic integrator, and recorded on punched paper tape. Peaks are identified and amounts calculated by an off-line computer program based on one or more internal standards. The system is designed for use with samples of at least 10 mug. Some of the problems adn limitations are discussed.


An all-purpose injection system for gas chromatographic analyses of biological samples. A versatile injection system for the direct introduction of samples into the gas chromatograph (GC) is described. Several years of experience in biological and pharmaceutical chemistry have led to the construction and final modification of this device. No modification of the injection port of the GC is necessary.


Lung collagen heterogeneity. Synthesis of type I and type III collagen by rabbit and human lung cells in culture. The fetal and adult lung have a constant level of collagen synthesis that represents 4-5% of the total amino acids incorporated into lung protein. Prior studies have demonstrated that this collagen is not homogeneous but rather is composed of at least two collagen types, I and II, each localized to specific lung structures. Although it is known that explants of rabit lung parenchyma and blood vessels synthesize type I collagen and that rabbit lung tracheobronchial tree synthesizes type II collagen, it has been suggested that other collagen types are present in lung. It is not known which cells are responsible for the synthesis of any lung collagen type. To approach the problem of additional lung collagen heterogeneity and the identification of the cells responsible for lung collagen synthesis, techniques were developed to examine collagen synthesized by lung cells in culture. 10-15% of the proteins synthesized by confluent cultures of rabbit lung cells and fetal human lung fibroblasts are collagen. Separation and purification of this collagen by ion-exchange chromatography and cyanogen bromide (CNBr) peptide mapping techniques indicate that collagen secreted by these cells is composed of two collagen types, I and III. The CNBr peptides of type I collagen secreted by these cells are identical to the CNBr peptides of type I collagen synthesized by lung parenchyma and blood vessels. The peptides of type III collagen secreted by these cells are identical to fetal skin type III collagen CNBr peptides. The existence of 40 cell types and the insolubility of lung collagen increase the complexity of identifying the types of collagen in lung and the cells responsible for the synthesis of each type. The techniques described here should eventually lead to a complete description of the synthesis and composition of lung collagen, thus providing a probe to understand the role of collagen in lung development and structure in health and disease.