P-Azidophenacyl bromide, a versatile photolabile bifunctional reagent. Reaction with glyceraldehyde-3-phosphate dehydrogenase. The synthesis of the photochemically labile bifunctional reagent p-azidophenacyl bromide (1) is described. This compound may be covalently attached to a known site of an enzyme or other macromolecule by nucleophilic displacement at the alpha-bromo ketone moiety. Subsequent irradiation of the bound reagent gives a nitrene which may insert into a second portion of the enzyme forming a cross-link. Regeant 1 proved to be an excellent inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.


The effect of Mn2+ and Co2+ on the activities of a zinc metallodipeptidase from a mouse ascites tumor. Kinetic studies of the effect of addition of Co2+ or Mn2+ to a highly purified dipeptidase from Ehrlich-Lettré mouse ascites tumor cells show that these metals specifically activate the hydrolyses of certain classes of dipeptides. This enzyme was previously (S. Hayman and E. K. Patterson, 1971, J. Biol. Chem. 246, 660) reported to be a Zn-metalloenzyme. It is now shown that Zn is the only metal that can partially restore the activity of the EDTA-inhibited dipeptidase in cleaving Ala-Gly. Addition of Co2+ increases the Vmax of N-terminal Gly-dipeptides with increase in Km while addition of Mn2+ primarily activates the hydrolysis of Pro-Gly, again with increases in both Vmax and Km. Prior incubation (5 min, 30 degrees) of the dipeptidase with the appropriate metal ions causes decrease in initial lag time in the Co2+-activated hydrolysis of Gly-Gly and the Mn2+-activated hydrolysis of Pro-Gly. Long-term (6-19 hr, 0 degrees) incubation of the enzyme with Co2+ results in loss of activity toward Ala-Gly with a concomitant 13-fold increase in the rate of Gly-Gly hydrolysis and loss of 70% of the Zn2+ from the dipeptidase; these effects can be partially reversed by addition of Zn2+. In contrast, long-term incubation of the enzyme with Mn2+ results in no loss of Zn2+ and a twofold increase in activity toward Pro-Gly. One affinity constant of 1.4 muM for Co2+ and two constants of 0.23 and 27 muM for Mn2+ were determined by kinetic experiments. Comparison of the properties of this tumor enzyme with a dipeptidase purified in our laboratory from Escherichia coli B, and with mammalian dipeptidases highly purified by others, shows remarkable similarities in molecular weights and molecular activities toward the preferred substrates but in the case of bacterial dipeptidase, differences in substrate specificities and in the effect of metal ions.


Fragments of bovine serum albumin produced by limited proteolysis. Conformation and ligand binding. Twelve fragments of bovine serum albumin, isolated following limited tryptic or peptic hydrolysis, have been studied to define secondary structure and locate ligand-binding sites. Based on circular dichroism, the conformational pattern of albumin (68% alpha helix and 18% beta structure) is substantially retained by individual fragments, indicating that secondary configuration is locally determined and is not destroyed during the cleavage process nor during fragment purification. The strong bilirubin-binding site of bovine serum albumin is present in 3 of the 12 fragments. Residues 186-238 are common to the three fragments and absent from those fragments which do not bind bilirubin; consequently the strong bilirubin-binding site is suggested to involve this region. By similar reasoning, the presence of palmitate-binding sites in some fragments and not in others indicates that the three strongest sites for the binding of palmitate are located in the carboxyl-terminal two-thirds of the molecule. The first site (KA approximately 2 X 10(7) M-1) is suggested as residues 377-503; the second site (KA approximately 8 X 10(6) M-1), residues 239-306; the third site (KA approximately 2 X 10(6) M-1), residues 307-377. Bromocresol Green, a reagent used in the assay of ablumin, was bound by fragments rougly in proportion to their size but showed particular affinity for the region of the strong bilirubin-binding site. The fluorescent probe, 8-anilino-1-naphthalensulfonate, was in general bound by large fragments, supporting the concept that this ligand is held principally in clefts between domains of the macromolecule.


An essential arginyl residue at the nucleotide binding site of creatine kinase. Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.


Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites. Inactivation of bovine brain mitochondrial hexokinase by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a sulfhydryl specific reagent, has been investigated. The study shows that the inactivation of the enzyme by DTNB proceeds by way of prior binding of the reagent to the enzyme and involves the reaction of 1 mol of DTNB with a mol of enzyme. At stoichiometric levels of DTNB, the inactivation of the enzyme is accompanied by the formation of a disulfide bond. But it is not clear whether the disulfide bond or the mixed disulfide intermediate formed prior to it causes inactivation. On the basis of considerable protection afforded by glucose against this inactivation it is tentatively concluded that the sulfhydryl residues involved in this inactivation are at the glucose binding site of the enzyme, although other possibilities are not ruled out. An analysis of effects of various substrates and inhibitors on the kinetics of inactivation and sulfhydryl modification by DTNB has led to the proposal that the binding of substrates to the enzyme is interdependent and that glucose and glucose 6-phosphate produce slow conformational changes in the enzyme. Protective effects by ligands have been employed to calculate their dissociation constant with respect to the enzyme. The data also indicate that glucose 6-phosphate and inorganic phosphate share the same locus on the enzyme as the gamma phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+.


The location of the calcium ion binding site in bovine alpha-trypsin and beta-trypsin using lanthanide ion probes. The effect of Gd3+ on the nuclear magnetic resonance (NMR) relaxation rates, T1m-1 and T2m-1, of inhibitor protons in metal-inhibitor-trypsin ternary complexes has been measured. The Solomon-Bloembergen equations have been used to calculate distances of 10.0 +/- 0.5, 8.8 +/- 0.5, and 9.5 +/- 0.5 A between the metal ion and the methyl and ortho protons of p-toluamidine, and the methyl protons of acetamidine, respectively. Essentially the same results are obtained for both alpha-trypsin and beta-trypsin. Binding constants of 3.3 x 10(3) and 4.1 x 10(3) M-1 for the association of Gd(III) with alpha-trypsin and beta-trypsin, respectively, in the presence of p-toluamidine at pH 6.0 have been obtained by equilibrium dialysis. Calcium binding constants of 260 and 3700 M-1 at pH 6.0 and 8.0, respectively, with beta-trypsin have also been obtained. Calcium ion and gadolinium ion compete for the same site on the protein. Calcium has been shown to protect alpha-trypsin from further autolytic degradation to psi-trypsin. From examination of the crystal structure of the enzyme we propose that the calcium ion binding site of bovine trypsin is comprised of the side chains of Asp-194 and Ser-190 (based on the chymotrypsin sequence numbering system). This seems to be the only site which is comprised of at least one carboxyl group; which fits our distance requirements and which is conisistent with other chemical data.


Studies on lysophospholipases. IV. The subcellular distribution of two lysolecithin-hydrolyzing enzymes in beef liver. 1. In a previous paper (Biochim. Biophys. Acta (1974) 369, 50-63) the purification of two proteins with lysophospholipase activity (EC 3.1.1.5), provisionally denoted lysophospholipase I and lysophospholipase II, has been described. The subcellular localization of both enzymes was investigated by cell fractionation studies. 2. For each subcellular fraction the total lysophospholipase activity, after solubilization by n-butanol treatment, was separated into a lysophospholipase I and II contribution by DEAE-Sephadex ion exchange chromatography. 3. Lysophospholipase I was found to be a soluble enzyme with a bimodal distribution. Highest relative specific activities were measured in the mitochondrial and the cytoplasmic fraction. Evidence is presented indicating that this enzyme is present in the mitochondrial matrix fraction. 4. Lysophospholipase II appeared to be a membrane-bound enzyme with highest relative specific activity in the microsomal fraction.


The interaction of various control mechanisms in determining the rate of hepatic cholesterogenesis in the rat. In these studies rats were subjected to diurnal light-cycling, stress, fasting and the feeding of cholestyramine, beta-sitosterol and cholesterol in various combinations. In control animals exposed to light cycling for 2 weeks the rate of hepatic cholesterogenesis was 3.7 fold higher in the mid-dark than in the mid-light phase of the light cycle. The magnitude of this difference varied with the duration of light cycling and the size of the animals. Similarly, enhanced rates of cholesterol synthesis were seen in the mid-dark phase relative to the mid-light phase of the light cycle in rats where the base-line level of hepatic cholesterogenesis was increased by feeding cholestyramine (1.6-fold) or beta-sitosterol (2.9-fold) or was depressed by fasting (19-fold) or cholesterol feeding (2.1-fold). Restraining animals for 48 h also increased the rate of cholesterol synthesis in the liver; in control animals, this stress enhanced the level of cholesterogenesis seen at both the mid-light and mid-dark phases of the light cycle. In addition, both the effects of stress and of diurnal light cycling could be identified in groups of animals where base-line cholesterogenic activity was varied by fasting or by feeding cholestyramine, beta-sitosterol or cholesterol. These studies illustrate the complexity of the control of hepatic cholesterol synthesis and suggest that the final rate of cholesterogenesis may be the result of several different effectors modifying by different mechanisms the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase.


Circular dichroism of soybean leghemoglobin. Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.


Calculation of the energy difference between the quaternary structures of deoxy- and oxyhemoglobin. The energy difference between the quaternary structures of deoxy- and oxyhemoglobin is evaluated on the basis of the atomic coordinates determined by X-ray diffraction analysis. Calculation of the van der Waals interaction between subunits shows that in a hemoglobin molecule as a whole, the interaction is more attractive in the oxy form than in the deoxy form by about 8 kcal/mol, and that in each pair of two subunits except the pair alpha1alpha2, the interaction energy varies by about 15 kcal/mol. The electrostatic interactions originating in the partial charges on all constituent atoms of hemoglobin and in the polar residues on the surface of hemoglobin make only a small contribution to the energy difference between the quaternary structures of deoxy- and oxyhemoglobin. Thus, the contribution of the clusters of the polar residues in the internal cavity between like subunits and also of the freedom of rotation of the C-terminal of each subunit in oxyhemoglobin may be important energetically in the transition from deoxy to oxy quaternary structure. In this point, the present calculation supports Perutz' model, but suggests necessity of further investigations on the transitional characteristics of the quaternary structure in the intermediate steps of oxygenation. The discussion on the transitional characteristics is given in the last section.


Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles. The temperature dependence of the binding of PhNapNH2 (N-phenyl-1-naphthylamine) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 degrees C, some 30 degrees C above the broad phase transition region of the phospholipid. In the temperature range above 20 degrees C, deltaH0 =--6100 cal-mol-1 and deltaS0 = 9.7 e.u.; in the temperature range below 20 degrees C, deltaH0 = 0 cal-mol-1 and deltaS0 = 30.4 e.u. These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 degrees C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 degrees C, though significantly greater than that above 20 degrees C, is still significiantly less than that in the crystalline state.


Isolation of the outer acrosomal membrane from bull sperm. A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction. Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both alpha-fucosidase and beta-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.


Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin. The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.


Premature replication of late S period DNA regions in early S nuclei transferred to late S cytoplasm by fusion in Physarum polycephalum. Fusion of a late S period plasmodium of Physarum polycephalum to an early S period plasmodium causes premature replication of late S replicating regions in the nuclei of the early S plasmodium. The extent of ahead-of-schedule replication of late S replicating regions in early S period nuclei increases to a plateau of 16-20% for fusions with 40-70 min of phase difference, then declines for larger phase differences. The stimulatory factors for late S replicative units are present only in late S plasmodia and appear to act only on late S regions. Once replicated, early S replicating regions are not stimulated to replicate again by fusion to a plasmodium entering the S period. Our data do not discriminate between anti-termination of replication by factors of stop sites on long replicons, and a sequential initiation of replication on new, possibly non-adjacent regions, but does provide evidence that the stimulatory factors are distinct from one another and specific for certain target replicative units.


A solvent-partition method for measuring the binding of drugs to DNA. Application to the quinoxaline antibiotics echinomycin and triostin A. The development of a novel solvent-partition method for measuring the interaction between nucleic acids and drugs of limited water solubility is described. Factors relevant to the choice of a suitable water-immiscible solvent are summarised. i-Amyl acetate was selected for studying the binding of echinomycin and triostin A to DNA. Details of the experimental determination of extinction and partition coefficients are given; in the i-amyl acetate/buffer system employed for most experiments, the partition coefficients for echinomycin and triostin A were 111 +/- 4 and 943 +/- 23, respectively. Equilibration of echinomycin between the organic and aqueous phases was 90% complete within a few minutes, and a period of 2 h shaking was found satisfactory to ensure full attainment of equilibrium. Representative results are presented showing specific binding of the quinoxaline antibiotics to DNA, strong preference for double-helical as opposed to heat-denatured or single-stranded DNA, and restricted uptake by closed circular duplex PM2 DNA. The method is potentially applicable, with appropriate modifications, to the study of interactions between other ligands and DNA.


Inhibition of enzymatic hydrolysis of end-phosphate DNA by iridium chlorocomplexes. The enzymatic hydrolysis of end-phosphate DNA by calf intestine alkaline phosphatase is inhibited by iridium chlorocomplexes. The inhibitory effect is strongly influenced by the chemical form of iridium. While hexachloroiridate (IV) and hexachloroiridite (III) strongly inhibit the enzymatic activity of calf intestine alkaline phosphatase no inhibitory effect was observed when these chlorocomplexes were previously irradiated by light and transformed to their photochemical reaction products. Evidence is presented which suggests that the noncompetitive and irreversible inhibitory effect was due to an effective interaction of iridium with the protein without release of its metal constituent at a very low concentration of enzyme. The protective effects of various chemicals on the inhibitory action was also briefly investigated.


Modification of morphine-withdrawal syndrome in rats following transauricular electrostimulation: an experimental paradigm for auricular electroacupuncture. The experimental paradigm reported here provides a useful animal model with which to further study the effects of regional electrostimulation and its possible relationship with electrical acupuncture. What similarities there may be between electrical acupuncture and the technique of regional electrocerebral stimulation used for production of electrosleep and electronarcosis in animals and man (Wageneder et al., 1966) remain to be elucidated. Further research studies, both clinical and basic, are clearly needed to answer some of the questions relating to the safety, efficacy, and possible mechanisms by which a technique such as electrical acupuncture may exert its effects.


Permselectivity of the glomerular capillary wall to macromolecules. I. Theoretical considerations. The transport of macromolecules across the renal glomerular capillary wall has been described theoretically using flux equations based on (a) restricted transport through small pores, and (b) the Kedem-Katchalsky formulation. The various assumptions and limitations inherent in these two approaches are discussed. To examine the coupling between macromolecular solute transport and the determinants of glomerular filtration rate, these flux equations were combined with mass balance relations which allow for variations in the transmembrane driving forces along a glomerular capillary. It was predicted, using both pore theory and the Kedem-Katchalsky equations, that fractional solute clearance should be strongly dependent on the determinants of glomerular filtration rate when convection and diffusion both contribute to solute transport. When convection becomes the sole mechanism for transcapillary solute transport, however, fractional solute clearance is essentially independent of changes in the determinants of glomerular filtration rate. Consequently, unless diffusion is absent, fractional solute clearances alone are insufficient to characterize the permselective properties of the glomerular capillary wall, since these values may be altered by changes in glomerular pressures and flows as well as changes in the properties of the capillary wall per se.


Timid singly-housed mice: their value in prediction of psychotropic activity of drugs. 1 About 45% of singly-housed male mice showed timidity (alert postures, running away, defensive postures) instead of aggression on interactions in pairs with group-housed male mice, though their partners did not show any aggression. The isolation-induced timidity was stable in repeated interactions. Timid mice also showed locomotion (walking across cage and rearing) and a small amount of sociable activity (sniffing, following partners and climbing over them). 2 Diazepam (5 mg/kg), chlordiazepoxide (20 mg/kg), chlorpromazine (7.5 mg/kg) and barbitone (60 mg/kg) given orally inhibited the isolation-induced timidity without reducing other motor activities in the timid mice. Imipramine lessened timidith only in a dose (80 mg/kg) which also decreased other components of behaviour in the timid isolates. (+)-Amphetamine and lysergic acid diethylamide (LSD) increased the timid response. 3 Comparison of the inhibition of timid activities with changes in other behaviour occurring at the same time seems a better measure of selective timidity-reducing effects of drugs than the rota-rod test. 4 Diazepam (5 mg/kg) increased sociable and locomotor activities. Barbitone (20 and 60 mg/kg) increased sociable activities; however, the higher dose also evoked some aggression in timid mice. 5 Behaviour of timid singly housed male mice seems to be a good measure for prediction of activity of drugs in relieving anxiety as well as for detection of aggression-evoking and sociability-increasing effects of drugs.


Reduction in the cholinesterase activity of the rat anococcygeus muscle produced by corticosterone. 1 Administration of corticosterone caused a 47% reduction in the cholinesterase (ChE) activity of homogenates of the rat anococcygeus muscle. 2 ChE activity was also reduced by morphine withdrawal and this effect was abolished by the corticosteroid synthesis inhibitor, metyrapone. 3 A single dose of reserpine reduced ChE activity in normal but not in adrenalectomized rats. 4 ChE activity was increased by adrenalectomy or by metyrapone treatment. 5 The mechanism of the corticosteroid-induced reduction in ChE activity is discussed. 6 The reduction in ChE activity produced by corticosterone, morphine withdrawal, or a single dose of reserpine might explain the leftward shift of the dose-% response curve to acetylcholine produced by these procedures in the isolated anococcygeus muscle of the rat.


Premenstrual affective syndrome and affective disorder. Sixty-four per cent of 874 freshmen and sophomore women sent questionnaires about premenstrual and menstrual symptoms returned them. They differed from those not returning the questionnaires only in year of school. As predicted, women reporting premenstrual affective symptoms were more likely than those who did not report them to seek psychiatric care at the Student Health Service and to be diagnosed as affective disorder at the service.


Attitudes towards self-poisoning among physicians and nurses in a general hospital. Twenty-five doctors and 50 nurses working in the medical wards of a general hospital were interviewed. They were asked to indicate typical motives for self-poisoning and were then shown brief accounts of four typical case histories. They were asked to choose from a list of motives those they considered applied to each case. They then rated each motive for acceptability and understandability, and each case for sympathy and readiness to help. Motives were of two principal types--"manipulative' (i.e. aimed at eliciting a response from others) and "depressive' (i.e. communicating despair and aimed at withdrawal, escape or death). "Depressive' motives were more acceptable and evoked more sympathy or readiness to help in both doctors and nurses than "manipulative' motives. Doctors and nurses differed in various ways. Nurses were generally more accepting, more sympathetic and more likely to seek professional help as an alternative way of coping. Doctors distinguished more clearly between "suicidal' motives, of which they were relatively accepting, and "manipulative' motives, which they accepted less. Attitudes to the four cases differed, primarily in the motives attributed. Those differences may also reflect differing severity of problems, personal responsibility for problems, social class, sex or age.


A study of 65 impotent males.

A study of 65 impotent males. Sixty-five patients primarily referred for erectile impotence were investigated. Information was obtained from patients and their partners. The demographic data are compared with those from a non-impotent psychiatric out-patient group, matched for age. Results indicate that impotent cases do not form a homogeneous population and can be classified into three fairly distinct groups. These groups differ in age, marital status, sex drive, pre-marital and post-marital relationships, duration of illness, etc. Group 1 develop impotence because of anxiety in sexual situations. Group 2 react to the sexual response and personality of their partners, while Group 3 decline, perhaps from inherent constitutional causes. Factors such as religious restrictions, sexual taboos, alcoholism and homosexuality do not appear to be of any aetiological importance.


International dosimetry: an evaluation of treatment planning in clinical trials. A tissue-equivalent phantom containing thermoluminescent dosimeters was mailed in succession to Mount Vernon Hospital, Northwood, Middlesex, England, to Groote Schuur Hospital, Capetown, South Africa, and to Winnipeg General Hospital, Winnipeg, Canada, to determine the accuracy and consistency in treatment for carcinoma of the cervix under hyperbaric oxygen conditions. (Protocol of the Medical Research Council's Working Party on Radiotherapy and Hyperbaric Oxygen.) The data were analysed by the Radiological Physics Center, Houston, Texas, and substantiate uniformity at and between the participating institutions.


Urinary tract dilatation in pregnancy.

Urinary tract dilatation in pregnancy. Two radiologists individually studied 220 intravenous urograms done during pregnancy in patients with apparently normal urinary tracts, and an observer-agreement technique was used to obtain the results. The few cases seen in early pregnancy show that dilatation of the urinary tract is uncommon, or at least mild, before mid term. Shortly thereafter, however, dilatation appears abruptly and remains virtually unchanged both in incidence and degree until full term. After mid term, the right side is dilated in about three-quarters of cases and the left side in about one-third. In only 10 % was the left side fuller than the right; in 86%, the right side was the fuller. Severe dilatation is infrequent, especially on the left. The dilatation never extends into the pelvis. The abdominal ureters are displaced outwards in a minority of cases, more often on the left. Little or no association was found between the dilatation and fetal position, maternal gravidity or urinary infection. These statistically derived conclusions should provide a firmer basis for what constitutes the limits of normality in the intravenous urogram during pregnancy.


In-vivo determination of error in the measurement of urethral pressure by the method of Brown and Wickham. A method of determining the error caused by the infusion of water into the urethra during urethral wall pressure and urethral pressure profile measurements is described and discussed. The test is conducted in vivo and the measured pressure is related to the actual urethral wall pressure. The "intrinsic error" is found to be approximately 1-0 cm H2O for every ml/min of infusion rate. Incidental findings concerned with changes in urethral resistance are also reported.


Calcium excretion and deposition during sporogenesis in Physarella oblonga. Calcium excretion and deposition were studied during sporogenesis in the myxomycete, Physarella oblonga. Prior to fruiting, the migrating vegetative plasmodium contains numerous, large channels connected to the external environment. During early sporangial development, these channels become smaller and are isolated as channel remnants. Plasmodial microfilament bundles also disappear at this time. Internally, two types of spheres, each about 0.5-1.5 mum in diameter, are formed in the channel remnants. A dense sphere appears to originate from small particles initially secreted from the mitochondria. These particles then enter the channels and channel remnants by reverse pinocytosis and fuse to form the dense spheres. A second, less dense sphere has no detectable cytoplasmic origin but also appears to form in the channels and channel remnants. Both types of spheres are concentrated by the fusion of channel remnants to form enlarged regions (knots and spikes) of the developing capillitium. Some of the channel remnants also fuse with the outer surface of the sporangium, depositing both spheres to the outside. Both spheres have been studied by various light and electron microscopic techniques and with specific histochemical and analytical procedures. The less dense spheres appear to contain a major portion of calcium in the form of calcium carbonate. These results are compared to other recent studies on calcium deposition in the Myxomycetes and a different mechanism for calcium deposition is proposed.


Amino acid and protein metabolism during differentiation (sclerotization) of the myxomycete Physarum flavicomum. Protein synthesized by growing plasmodia of Physarum flavicomum was steadily degraded when the plasmodia were induced to differentiate (form sclerotia). Protein synthesis occurred during the initial one-fifth (9 h) of the 48 h differentiation period, but most of this protein was also degraded shortly after its synthesis. Amino acids were primary catabolites during the differentiation process, and catabolism was extensive, even in the presence of dextrose. Glutamic acid was catabolized at a rate about two and a half or three times greater, respectively, than that observed for valine and arginine. Active transport systems for amino acids appeared to be present and to remain functional in P. flavicomum during differentiation. Amino acids included in the sclerotization media were rapidly accumulated into the cell pool and protein fractions. Intracellular amino acids were actively retained and were not released into the medium during differentiation. Differentiation of this Myxomycete, therefore, is characterized by a change in the metabolism of the sclerotizing plasmodium to an autolytic type, as cellular proteins and amino acids are actively catabolized during the formation of the dormant sclerotia.


Milk yield in lines of mice selected for growth or maternal ability. Two experiments were conducted to measure milk yield as a correlated response to selection for increased 12-day litter weight (maternal lines) for 13 generations and increased and decreased body weight gain from 21 to 42 days of age (postweaning gain lines) for 12 to 14 generations. Milk yield was measured from day 6 to day 21 of lactation by separating litters from their dams for 6 h, then weighing litters before and after a 1.5 h suckling period. Average total milk production was 30.3 +/- 0.9 g and 28.6 +/- 1.1 g in maternal and control lines, respectively. This difference was not significant (P greater than 0.05). Differences among gain lines were significant (P less than 0.01) in the second experiment with average production of 44.1 +/- 1.4 g for increased gain lines, 22.5 +/- 0.9 g for the decreased gain line and 31.2 +/- 1.0 g for the control. These results indicated that response to selection for increased 12-day litter weight was not associated with increases in milk yield, but sizable changes inmilk yield accompanied bi-directional selection for early postweaning gain.


Diagnosis before treatment.

Diagnosis before treatment. The essence of a medical approach in psychiatry is the application of a structured diagnostic assessment. Treatment without diagnosis is a form of quackery. Psychiatric therapy, like any form of medical treatment, must be tailored to fit the individual patient.


Primary malignant germinal tumors of the mediastinum. A study of 24 cases. The malignant germinal tumors found in the testis (seminoma, teratocarcinoma, embryonal carcinoma, choriocarcinoma, and mixtures of these) can originate in the anterior mediastinum. An occult testicular tumor must be sought, but primary mediastinal germinal tumors constitute a true entity. The details of 24 such patients treated at Walter Reed General Hospital are presented. Of 19 patients who died, 2 were women; postmortem examination of the testes of 13 men failed to reveal a primary tumor. Five patients are alive and well with no testicular abnormality. It is important to distinguish the histopathologic types. Seminomas are readily controlled with modest doses of irradiation. Embryonal carcinomas are also radiocurable with somewhat higher doses; 2 long-term disease-free survivors are reported. Teratocarcinomas are highly malignant and require higher doses for control. Choriocarcinomas progress so rapidly that chemotherapy is necessary.


Intravascular angiomatosis: development and distinction from angiosarcoma. Histologic features similar to those present in angiosarcomas were found in organizing thrombi. Within the thrombi, freely anastomosing small channels, often lined by one or more layers of prominent, occasionally atypical, endothelial cells were noted. Papillary-like projections of organizing thrombus material lined by similar cells heightened the similarity of the process to angiosarcomas. The papillary-like structures appeared to develop due to a combination of endothelialization of thrombus fragments and of ingrowth of interlacing vessels. The peculiar process of thrombus organization, which has been called "intravascular angiomatosis," may be mistaken for true angiosarcoma and thus lead to unnecessary irradiation or radical surgery. The pseudoangiosarcoma differed from angiosarcoma in its confinement entirely within large vascular lumens and in its lack of mitoses, necrosis, and true solid cellular areas devoid of vascular differentiation.


Freeze-fracture replication of junctionsl complexes in unincubated and incubated chick embryos. Junctional complexes have been investigated in the epiblast of young chick embryos by examination of freeze-fracture replicas and of sections of comparable specimens stained with lanthanum nitrate. By means of freeze-fracture, tight junctions were shown to be present in the unincubated embryo (stage 1 of Hamburger and Hamilton). The number of ridges or grooves was found to vary between 2 and 10 near the dorsal border, whereas isolated ridges were found more ventrally. Lanthanum was unable to penetrate between the cells in the region of the dorsally situated tight junctions. Similar tight junctions were found in incubated embryos (stage 3) examined by both techniques. Tight junctions were also seen in cleavage (pre-laying) embryos examined in section. Gap junctions were extremely uncommon in unincubated embryos, though occasional aggregates of gap junction particles were seen on the lateral cell membranes close to the dorsal surface. In only one instance were associated pits visible. By contrast, gap junctions were more frequently encountered by stage 3, and these junctions possessed both pits and particles. Desmosomes were never seen in the freeze-fracture replicas at either stages 1 or 3, though structures which might be developing desmosomes were visible in sections. The functions of both the tight and gap junctions in the young chick embryo are discussed. The results are also considered in relation to recent theories about the way in which gap junctions are formed.


Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization. Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.


Properties of sea urchin embryo messenger RNA containing and lacking poly(A). Various properties of nonhistone messenger RNA species containing poly(A), [+A]mRNA, and lacking poly(A), [-A]mRNA, are described: the rates of turnover of these mRNA classes are not significantly different, as indicated by their similar rates of entry into and decay from the cytoplasm; each mRNA class is essentially entirely transcribed from unique DNA sequences; the ratio of [+A] to [-A] nonhistone mRNA increases with increase in size of free polyribosome, although the average molecular weights of these mRNAs are similar in each polysomal size class. These results indicate that the [+A]mRNA species tend to be more fully loaded with ribosomes than the nonhistone [-A]mRNA species.


Changes in the basic nuclear proteins of the cellular slime mould Dictyostelium discoideum during growth and differentiation. Nuclei isolated from myxamoebae and differentiating cells (slug stage) of Dictyostelium discoideum contain similar ratios of DNA, RNA and protein (1:8:29) and acid soluble proteins present in amounts equal in weight to the nuclear DNA can be extracted therefrom. On urea polyacrylamide gels these basic proteins were shown to be very similar with the exception of one band, present in the myxamoebae, which was virtually absent from the differentiating cells.


Evaluation of Trinder's glucose oxidase method for measuring glucose in serum and urine. Trinder's method for glucose has nearly all the attributes of an ideal automated colorimetric glucose oxidase procedure. The chemicals used in the color reaction with peroxidase are readily available, the solutions are stable and can be prepared by the user, the method is highly specific and largely free of interferences, the sensitivity can be adjusted by the user to cover a wide range of glucose concentrations, and the reagents are not hazardous. We found very good agreement between results by this method and by the hexokinase and Beckman Glucose Analyzer methods. The method has been modified and adapted to the AutoAnalyzer I and SMA 6/60 (Technicon) with manifolds that give very little interaction between specimens. A study of the method by the simplex technique revealed that the glucose oxidase activity in the reagent is the most critical variable.


Enhancing effect of surfactant and protein on hydrolysis of thymolphthalein monophosphate by purified prostatic acid phosphatase. Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.


[Radio-immunoassay method for determination of parathyroid hormone. Use of a commercially available antiserum and an international standard (author's transl)]. A radio-immunoassay method of parathyroid hormone in peripheral circulation is described utilizing both commercially available antibody and bovine PTH preparation for iodination. As a standard, the bovine PTH delivered from the Medical Research Council (MRC) is used. Antibody-bound hormone is separated from free hormone by solid phase immunoprecipitation. Good cross reactivity of this antiserum to human PTH is shown, but because of the lack of human standard, it is demonstrated, that results may be expressed in reference to the MRC bovine hormone. The coefficient of interassay variation is 12% and the detection limit is 300 pg/ml. In every healthy subject, immunoreactive PTH is detected: the normal distribution range (n equals 31) was 400 to 1800 pg/ml. Two hypoparathyroid patients have undetectable levels. There is a slight overlap between normal and those from hyperparathyroid patients. The present method gives results expressed in international units (MRC standard). The reliability of the PTH radio-immunoassay is dependent on the characteristics of the antiserum. This method provides a basis for the establishment of PTH values with the possibility of comparing results in health and disease.


Moral and ethical problems of pre-natal diagnosis. The moral and ethical problems resulting from application of pre-natal diagnosis are identified: abortion, questions about euthanasia for defective newborns, and the concept of genetic health. A holistic approach to ethical problems is presented that includes attention to both principles and consequences.


Transmission and scanning electron microscopy of changes associated with microcyst formation in the myxamoebae of Physarum polycephalum. Surface and internal changes associated with transition from myxamoebal to microcyst stages in Physarum polycephalum have been observed with transmission and scanning electron microscopy. Nuclear morphology, granules studded with ribosomal-like particles, synthesis of polysaccharides which are deposited exterior to the cell, and origin of material for the cell wall appear to be similar in the amoeba and in the plasmodium. In addition, the viability of the strain used in this research after being allowed to remain in a quiescent or encysted stage was observed to be approximately six months.


A case of limbic system dysfunction with hypersexualith and fugue state. This paper describes a case of limbic dysfunction in a 28-year-old female veteran who presented with psychoses and sexual promiscuity. She had EEG abnormalities and showed dramaic improvement with anti-convulsant medication. Limbic system dysfunction in general may present as "functional psychoses." The literature is briefly reviewed.


Prolactin and growth hormone levels in different inbred strains of mice: patterns in association with estrous cycle, time of day, and perphenazine stimulation. These experiments were designed to compare prolactin (PRL) and growth hormone (GH) secretion in strains of mice with varying incidences of mammary tumors. In addition to the basal levels, PRL concentrations were compared after stimulation with perphenazine. Although pituitary concentrations of PRL and pituitary serum concentrations of GH appeared to be generally higher in strains with high incidence of mammary tumors, basal PRL levels in single decapitate serum samples seemed to have little correlation with the incidence of mammary tumors in different strains. However, PRL concentrations in sera after perphenazine injection followed a pattern characteristic of the mammary tumor incidence rate of the strain: C3H/St and CBA/St--the two high-incidence strains--had lower levels of PRL; C57BL/St and BALB/cST--the two low-incidence strains--had higher levels; and DBA/2St--the medium-incidence strain--had an intermediate level. PRL contents of the pituitary glands were depleted virtually equally in all strains except the BALB/cSt. These results suggested that the rate of metabolism of PRL in strains with high incidence of mammary tumors may be faster than in those with low incidences. Perphenazine had no influence on GH secretion in most mice. The strain-specific differences in PRL and GH concentrations were usually present even during cyclical and diurnal fluctuations. Serum PRL and GH levels were generally higher during the follicular phase and lower during the luteal phase of the estrous cycle in both C3H/St and C57BL/St strains. There were signs of episodic secretion of PRL together with evidences of circadian periodicity in the secretion of PRL and GH in mice of both strains. The levels of GH were usually high during the morning hours of those of PRL high during the evening hours. The data show a high degree of specificity in the secretion of PRL and GH im mice of different inbred strains, and it is possible that these strain-specific differences may be an important factor in the development of mammary tumors.


Electron microscopic-immunocytochemical studies of rat pituitary gonadotrophs: a sex difference in morphology and cytochemistry of LH cells. Morphological criteria have been used by Kurosumi and Oota to distinguish follicle-stimulating hormone (FSH) from luteinizing hormone (LH) cells in the rat pituitary. In this study the techniques of ultrastructural immunocytochemistry were used to determine if these cell types could be distinguished on the basis of their LH content. In female rats in diestrus a 1:125,000 dilution of anti-bLH-beta stained Kurosumi-Oota "LH cells," "FSH cells," and cells with some morphological characteristics of both types. Absorption with 10 ng of LH abolished the staining in all cell types. In pituitaries taken from rats in proestrus and estrus, the stained cells were mostly of the "LH" cell type. Such cells were generally poorly granulated in some of the estrous females. In males, predominantly one gonadotrophic cell type, the classical Kurosumi-Oota "FSH cell," reacted with anti-bLH-beta. Ten nanograms of LH was effective in abolishing the staining in the largest granules (larger than 400 nm). Both LH and TSH were equally effective in the neutralization of the staining of small granules. Our results suggest that female rats have two distinctive LH cell types and males have one. We also confirm other immunocytochemical reports which show that the classical morphological definition of a "FSH cell" (5,6) is not completely accurate since some of these cells contain LH. The small granules appear to contain an immunoreactive site common to both TSH and LH which is only weakly reactive with the anti-bLH-beta.


Effects of polluted and non-polluted sediments on the growth of Gonyaulax tamarensis. Cultures of Gonyaulax tamarensis were grown on Hastings medium based on 100%, 10%, 1%, 0.1% and 0% (control) of seawater elutrients of bottom sediments. Both "clean" and heavily polluted sediments were used. Very low concentrations of polluted elutrients (0.01% v/v) slightly enhanced the growth of Gonvaulax tamarensis; greater concentrations of polluted elutrient depressed growth. Elutrient from "clean" sediments did not enhance growth; the depression of growth at any given elutrient concentrate was not  as great as that seen with the polluted elutrients.


Suppressant effects of alcohol on audiogenic seizures. Genetically seizure susceptible DBA/1/Bg mice fed 10% ethanol in their drinking water exhibited a marked diurnal variation in blood alcohol levels. At peak levels, sound-induced seizures were significantly reduced. At trough levels, seizures remained unaffected. Ethanol administered during early development enhanced seizures at post-weaning age. Such alcohol-augmented seizures were suppressed by ethanol feeding during the testing period, to the same base level as in animals not pretreated with ethanol in early life.


Unconsciousness, automatisms, and myoclonic jerks with diffuse rhythmic 9-Hz discharges. A 25-year-old man presented with seizures characterized by unconsciousness, automatic behavior, and myoclonic jerks lasting 0.5 to 8 sec. Videotape analysis of 300 simultaneously recorded electroencephalographic and clinical attacks revealed 8- to 9-Hz 150 to 200 muV sharp waves beginning in both medial temporal areas during eyelid flutters. When discharges spread diffusely on both sides and lasted 2 to 3 sec, 20 degrees forward flexion of the head, unconsciousness, pupil dilation, and staring became evident. Asymmetrical myoclonic jerks invariably appeared. When paroxysms were greater than 3 sec, automatisms and tonic posturing occurred. The electroencephalographic and clinical seizures appeared during the awake and relaxed states, i.e., with normal alpha rhythm. They were suppressed during physical and mental activities and during sleep, i.e., in the absence of alpha rhythm. Our studies suggest that these seizures associated with diffuse electroencephalographic alpha-like activities are subcortical in origin and should be tested in their responsivity to alpha suppressants and inducers.


Carboxymethylation of horse-liver alcohol dehydrogenase in the crystalline state. The active-site zinc region and general anion-binding site of the enzyme correlated in primary and teritiary structures. Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)2-/4 , 1,10-phenanthroline, Au(CN)-/2 and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboyxmethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN2-/4 to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN2-/4 and to a lesser extent Au(CN)-/2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identificationof this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.


Non-histone chromosomal proteins. Their isolation and role in determining specificity of transcription in vitro. We describe a method for fractionation of chromatin components by selective dissociation with salt in buffers containing 5 M urea in combination with cromatography on hydroxyapatite at 4 degrees C. This results in two histone and four non-histone fractions which are recovered in high yield and with minimal proteolytic contamination. Template capacity measurements of the isolated chromatins and pre-saturation competition hybridization experiments support the idea that a group of non-histone proteins activate the transcription of specific DNA sequences which were not transcribed from purified DNA to the same extent. In reconstitution experiments a non-histone protein fraction, NH4, prepared from lymphocyte chromatin by hydroxyapatite chromatography is shown to cause transcription in vitro of lymphocyte-specific RNA sequences. A subfraction with a molecular weight of 30 000 comprising 40% of the NH4 fraction protein is characteristic for this tissue and not found in liver chromatin.


Glucosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate. Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.


Studies concerning the mechanism by which translational-control RNA regulates protein synthesis in embryonic muscle. Muscle translational-control RNA (tcRNA) has been separated into two classes, polysomal and messenger ribonuclear protein (mRNA - protein), which have different sizes as determined by acrylamide gel electrophoresis. While normally translation of mRNA - protein mRNA is inhibited by tcRNA derived from the same mRNA - proteins, this inhibition does not occur if the messenger is previously de-adenylated. This suggests that the poly(A) segment of mRNA is required for the tcRNA activity. Utilizing different mRNA - protein fractions from muscle, myosin mRNA - protein and small mRNA - proteins ( less than 30 S), we have been able to demonstrate that a degree of specificity exists in the interaction of tcRNA and mRNA derived from the same mRNA - proteins. This is illustrated by the facts that (a) each tcRNA only inhibits the translation of its respective mRNA and (b) the highest percentage of structural change occurs when each tcRNA is hybridized to its respective mRNA as measured by its resistance to T1 and T2 RNase.


Two-stage occulsion of the protal vein in the rat:survival related to weight variation and the interval between partial and total occulusion. A portal vein stenosis with a diameter of 1.1 mm was performed in female Wistar rats of four different weight groups. The mortality rate was found to increase with increasing weight of the animals, a near linear correlation between mortality rate and body weight being found. Severe circulatory disturbances of the small intestine with haemorrhage and necrosis of the bowel wall were a prominent finding at death. In the survivors total occulsion was carried out at varying times, from 48 h after the first operation, and the mortality rates were recorded. A major contributing cause of death appeared to be ischaemic infarction of the liver. Only a slight congestion of the duodenum was found and no circulatory disturbance of the small intestine. Thus by 48 h the porto-systemic collaterals are sifficiently developed to prevent subsequent venous congestion. The survival rates showed a significant increase when the time interval between partial and total occlusion was extended from 48 to 72 h. This may be due to an increased blood flow through the hepatic artery and collateral arteries to the liver.


Primitive ventricle with acquired subpulmonary stenosis. The course and natural history of two children aged 1 yr and 5 yr with primitive ventricle with outlet chamber and normally related great arteries are described. They initially presented as ventricular septal defect with increased pulmonary blood flow and subsequently developed 'cyanotic attacks', chronic cyanosis and diminished pulmonary flow by acquiring subpulmonary obstruction of the bulboventricular foramen. Both had successful surgical treatment utilizing their own normal pulmonary valves by connecting the right atrium to the right ventricular outflow, with a Dacron conduit in one, and in the other by detaching the pulmonary artery and valve from the rudimentary outflow chamber and anastomosing it directly to the right atrial appendage. Changing morphology and its effect on function are discussed.


In vitro Purkinje cell electrical behavior related to tissular environment. Cerebellar explants from newborn rats and kittens were cultivated in Maximow assemblies. The spontaneous electrical activity of Purkinje cells was recorded simultaneously by means of 3 extracellular micropipettes. These were placed near to cell bodies which appeared in the same microscopic field. Identification of these neurons was performed by intracellular staining with Procion Yellow. Mature Purkinje cells which had been deprived of their extracerebellar afferent connections tend to fire in grouped discharges (bursts). No correlation was ever observed between the firing pattern of a Purkinje cell and that of its neighbors. Purkinje cells which grew in connection with extracerebellar nuclei showed comparable burst discharges. In these explants synchronization of the burst pattern was obvious from the statistical analysis of the simultaneous recordings. Discharges in bursts were not observed in immature Purkinje cells from young cultures, from cells situated in peripheral thin areas of older explants or from cells grown with an antimitotic (MAM). These cells showed a rather regular frequency of discharge. Synchronized slow waves occurred in cerebellar which grew in the presence of MAM. These "inhibitory" waves accompanied the arrest of cell activity and may have analogues in vitro. The observed differences in the spontaneous firing of these neurons were discussed on the basis of different synaptic organizations of the explants.


Penfluridol blockade of apomorphine: dependence of duration on species and endpoint. Penfluridol, at relatively low doses, blocks apomorphine-elicited emesis in dogs and apomorphine-elicited floor pecking in pigeons for over a month. In mice tested for apomorphine-elicited hypothermia, and in rats tested for apomorphine-elicited chewing behavior, however, the anti-apomorphine activity of penfluridol does not persist for longer than 2-3 days even when high doses of penfluridol are given. In rabbits tested for apomorphine-induced hyperthermia and gnawing, on the other hand, penfluridol blocks apomorphine for about a week. Thus, of the 5 species tested, only in rabbits does the duration of penfluridol's anti-apomorphine action approximate the 1-week duration reported from human therapeutic trials. In mice given high-dose penfluridol apomorphine consistently elevates body temperatures, rather than exerts its usual hypothermic response. Conversely, in rabbits given penfluridol apomorphine tends slightly to decrease body temperatures, rather than exert its usual hyperthermic response.


Dopaminergic nature of amphetamine-induced pecking in pigeons. d-Amphetamine was found to induce a pecking response in pigeons. The pecking response induced by d-amphetamine was antagonized by chlorpromazine, haloperidol or bulbocapnine indicating that this pecking response was caused by dopaminergic receptor stimulation. Pretreatment of pigeons with alpha-methyltyrosine (alpha-MT) reduced d-amphetamine-induced pecking, while the combined treatment of pigeons with alpha-MT and L-dihydroxyphenylalanine (L-DOPA, 100 mg/kg) partially restored the pecking response. d-Amphetamine-induced pecking was not reduced by a dopamine-beta-hydroxylase inhibitor, 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624). Alpha-MT reduced brain dopamine but not norepinephrine level, whereas U-14,624 decreased brain norepinephrine but not dopamine. Thus there is a correlation between brain dopamine level and d-amphetamine-induced pecking response. It is concluded that d-amphetamine-induced pecking is mediated indirectly by the release of dopamine.


Further evidence that extrinsic acetylcholine acts preferentially on extrajunctional receptors in the chick biventer cervicis muscle. The specificity of action of extrinsic acetylcholine on extrajunctional and junctional receptors in the chick biventer cervicis muscle was studied by determining its ability to protect the responses evoked by acetylcholine and by tetanic nerve stimulation from the blockade by alpha-bungarotoxin, an irreversible binding agent of acetylcholine receptors. At concentrations of 50-100 mug/ml, acetylcholine caused a desensitization to extrinsic acetylcholine but not to nerve stimulation and protected only the contractile response to extrinsic acetylcholine from the toxin blockade whereas neither the response to tetanic nerve stimulation nor the endplate potentials were protected. For the protection of the latter, higher concentrations of acetylcholine were needed. In the presence of physostigmine, a concentration of acetylcholine as low as 10 mug/ml protected the endplate potentials from the toxin blockade. By contrast, d-tubocurarine protected the tetanic contraction and the endplate potentials induced by nerve stimulation at a concentration which produced the same protection of acetylcholine-induced contraction as that produced by 50-100 mug/ml acetylcholine. These results indicate that in contrast to d-tubocurarine, extrinsic acetylcholine at low concentrations acts preferentially on the extrajunctional receptors in the absence of an anticholinesterase.


The effect of vaginal lubricants on sperm motility in vitro. Apart from the documentation  of the spermicidal effects of KY Jelly and Surgilube, little information about the effect of vaginal lubricants on sperm motility has been available. Fifteen substances utilizable as vaginal lubricants were therefore tested for their effect on sperm motility in vitro. Petroleum jelly and glycerin had minimal detrimental effects on motility and apparently are the lubricants of choice when an infertility problem exists.


Anti-PP1Pk (anti-Tja) and habitual abortion. Blood group incompatibility as a cause of early or habitual abortion has been a matter of much debate. However, the abortion rate in such cases is much higher than that found in the general population. Two sisters having the rare genotype pp and anti-PP1Pk (anti-Tja) in their serum were reported as having habitual abortions; a third sister, with a normal P group, had a normal obstetric history. The relationship of anti-PP1Pk (anti-Tja) to the high rate of habitual abortion was discussed and added support was given to the existing evidence that certain maternal blood group antibodies can affect embryos early in uterine life.


[Effect of caffeine and phenamin on caudate inhibition of aggressive reactions in cats]. In chronic experiments conducted on cats it was shown that caffeine (10--30 mg/kg) failed to change agressive reactions developing in stimulation of the meso- or diencephalic structures. Phenamine (1--3 mg/kg) facilitated the appearance of emotional manifestations and lowered the threshold of the agressive response. Subliminal stimulation of the caudate nucleus in control experiments caused motor tranquilization and depressed the agressive behaviour to a lesser degree when practised against the background of the caffeine action. At the same time, phenamine abolished the influence not only of the threshold, but also of the subliminal stimulation of the caudate nucleus on the spontaneous motor activity and the rage behaviour.


[Studies of the relationship between renin secretion and the sympathetic nervous system responsiveness in man (author's transl)]. In recent years many investigators have reported the role of the sympathetic nervous system and the catecholamines in the regulation of renin secretion. It has been recently reported that plasma dopamine-beta-hydroxylase (DBH) excreted through the mechanism of the exocytosis with noradrenaline from the sympathetic postganglionic nervous endings is a mirror of the function of the sympathetic nervous system. In this investigation, blood pressure, urinary catecholamine excretion, plasma DBH activity and plasmsa renin activity (RPA) have been determined in normal individuals during the mecholyl test and insulin-induced hypoglycaemia. In the mecholyl test, plasma DBH activity and PRA markedly increased in five males who showed prominent responses of systolic blood pressure to mecholyl (S type) and changed slightly in eight males who showed minimal responses of systolic blood pressure to mecholyl (N type). It showed that there was a significant relationship between the maximal percent increases from their respective control values of plasma DBH activity and those of PRA following mecholyl. In insulin-induced hypoglycaemia, plasma DBH activity, blood pressure and PRA remarkably increased following a large dose of insulin administration (0.15 u/kg). In a dose of 0.1 u/kg, there was no significant increase of plasma DBH activity and blood pressure. PRA singificantly increased but was lower than a large dose of insulin. Also, it showed that their was a significant relationship between maximal percent increases from their respective control values of plasma DBH activity and those of PRA following insulin administration. These data indicate a close correlation between changes in plasma DBH activity and those of PRA during mecholyl and insulin-induced hypoglycaemia and lend additional support to the concept of sympathetic nervous system mechanism of renin secretion.


[Serum immunoreactive insulin after the oral administration of single dose of tolbutamide. I. Peripheral vein immunoreactive insulin in normal subjects and mild diabetics (author's transl)]. It has been confirmed in numerous studies that the hypoglycemic effect of sulfonyl-ureas is mainly owing to its insulinogenic action. Intravenous administrations of the drugs have been adopted in many of these studies. But the oral administrations of the drugs in a few studies lead to conflicting results concerning its insulinogenic action. In this study, the concentrations of blood glucose, serum immunoreactive insulin (IRI), serum free fatty acid (FFA) and serum tolbutamide were measured following the oral administration of single dose of tolbutamide in six normal and eleven maturity onset mild diabetic subjects. The same parameters were measured after the oral administration of tolbutamide plus sodium bicarbonate in six normal subjects. The changes of these parameters were compared with the changes following the intravenous administration of sodium tolbutamide in six normal subjects. The oral administration of three grams of tolbutamide alone caused a gradual but significant decrease of blood glucose level. Serum FFA response showed an initial decrease, followed by a rebound elevation. In spite of 21 per cent reduction of blood glucose level, serum IRI level did not show any significant change throughout the observation for five hours. Serum tolbutamide concentration rose gradually and reached to 24.4+/-3.9 mg per 100 ml (Mean+/-SEM) at the end of the observation. Almost identical results were obtained in diabetic subjects. Three grams of tolbutamide plus the same dose of sodium bicarbonate were administered orally to the normal subjects. A profound decrease of blood glucose level with a nadir (35 per cent reduction) at 45 minutes and a significant increase of serum IRI level with a peak (273 per cent increase) at 20 minutes were obtained, and associated with a comparatively rapid elevation of serum tolbutamide concentration reached to 31.9+/-3.3 mg per 100 ml after three hours. From these results, it is suggested that slow rise of serum tolbutamide concentration after the oral administration of tolbutamide alone might lead to moderate secretion of insulin and sooner rise might evoke larger secretion into the pancreatic vein, and that an existence of moderate hyperinsulinism in the pancreatic venous blood after the oral administration of tolbutamide alone might cause a decrease of gluconeogenesis in the liver. And the failure of serum IRI response in the peripheral blood in spite of hyperinsulinism in the pancreatic vein might be due to hepatic trapping of the secreted insulin.


[Serum immunoreactive insulin after the oral administration of single dose of tolbutamide. II. Pancreatic vein immunoreactive insulin in dogs (author's transl)]. The mode of action of sulfonylureas as hypoglycemic agent is not clarified yet. But it has been said in general to act by increasing the secretion of insulin from the beta cells. While, some believe that their effect is independent on insulin secretion. In the previous report, it was demonstrated that the oral administration of single dose of tolbutamide failed to increase immunoreactive insulin (IRI) in the peripheral vein in spite of significant decrease of blood glucose and free fatty acid levels both in normal and diabetic subjects. In order to observe changes of IRI level in the pancreatic vein following the oral administration of single dose of tolbutamide, the present study was carried out to determine serum IRI level of the pancreatic and peripheral vein in dogs. Mongrel dogs of both sexes, weighing 7-12 kg were anesthetized with sodium pentobarbital. A catheter was inserted into the superior pancreatico-duodenal vein through its duodenal branch after a laparotomy. Another catheter was inserted into the femoral vein for the collection of peripheral venous blood. The experiment was started one hour after the operation. Blood glucose level decreased gradually to 39 per cent reduction of the previous level at 180 minutes after intragastric administration of single dose of tolbutamide (0.1 g per kg). Serum IRI level in the peripheral vein showed only slight increase, similar to the response in man. While, pancreatic vein IRI level increased gradually and showed a fourfold increment after 180 minutes (from 143 muU per ml to 556 muU per ml), associated with a constant elevation of plasma tolbutamide concentration which showed 6.4 mg per 100 ml after 180 minutes. Simultaneous administration of 0.1 g per kg of tolbutamide and the same dose of sodium bicarbonate caused 49 per cent reduction of blood glucose level and a doubling of the basal IRI level in the peripheral vein. Serum IRI level in the pancreatic vein rose sharply and demonstrated a peak at 90 minutes (620 per cent increase). The elevation of plasma tolbutamide concentration was somewhat more rapid than that after tolbutamide alone. From these observations, it was confirmed that an apparent secretion of insulin into the pancreatic vein was induced by the oral administration of tolbutamide alone, in spite of the lack of increase in the peripheral vein IRI. And it is emphasized that the changes of insulin level in the pancreatic vein should be taken into account in a study of insulin dynamics.


Short-term treatment of alloxan-diabetic rats with intrajejunal administration of water-in-oil-in-water insulin emulsions. Alloxan-diabetic rats with fasting blood glucose levels above 300 mg./100 ml. were treated with intrajejunal administration of water-in-oil-in-water (W/O/W) insulin emulsions via an indwelling catheter at a dose of either 25 or 50 U./100 gm. body weight, three times daily for five to fourteen days. The course of diabetes was followed by determinations of glucose levels in blood and urine. During treatment a significant reduction in urinary glucose levels was observed in all rats studied. In two rats treated with 25 U./100 gm., fasting blood glucose levels did not change significantly. In four of five rats treated with 50 U./100 gm., W/O/W insulin emulsions significantly lessened hyperglycemia during treatment. Quantitative estimates suggested that the effectiveness of 50 U./100 gm. of intrajejunal W/O/W insulin emulsions was comparable to that after intramuscular regular insulin at doses between 1 and 2 U./100 gm. These results would indicate that diabetes can be controlled by enteral administration of insulin preparations.


Diurnal changes of bile lipid concentration in hepatic bile of cholecystectomized man. Diurnal variations of bile lipid concentration were studied in ten patients with a tube in the main bile duct following a cholecystectomy. 5-6 bile samples per 24 h were collected from each patient during 3-40 days. The enterohepatic cycle was not significantly modified since total bile samples did not exceed 40 ml/day. Significant diurnal variations were observed in cholesterol concentration. Changes in lecithin concentration seemed to be similar in seven patients but did not reach the level of significance in any individual patient. Maximal values were observed between 4 and 8 a.m. and minimal values at 4 p.m. Bile salt concentration varied without any circadian periodicity. Mean bile lipid concentration was calculated for each patient. The patients with highest cholesterol concentrations have also the highest mean lecithin concentration. Mean bile salt concentration does not differ much from one patient to another.


The sorting-out of thymidine-labelled chick hypoblast cells in mixed epiblast--hypoblast aggregates. Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa. The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.


Studies on maturation in Xenopus laevis oocytes. III. Energy production and requirements for protein synthesis. The following results were obtained: 1) The oxygen comsumption of progesterone-stimulated X. laevis oocytes increases at the time of germinal vesicle (GV) breakdown. 2) Continous treatment with 1 mM KCN, 1 mM and 0.1 mM DNP completely inhibits GV breakdown. 3) Pretreatment experiments with KCN, DNP and cycloheximide show that binding of progesterone to the plasma membrane and the specific hormone receptor requires neither energy, nor protein synthesis. 4) 1 h pulses of DNP (1mM) or cycloheximide (50 mug/ml) were applied, at various time intervals after progesterone treatment: early pulses strongly delay, but do not prevent GV breakdown; late pulses just before GV breakdown induce a number of cytological abnormalities, which ultimately lead to cytolysis. The significance of these results is discussed and the hypothesis that synthesis of the maturation producing factor (MPF) requires both energy and protein synthesis is proposed.


Removal of intact follicles from immature mice ovaries. A method was devised with the purpose of preparing significant amounts of isolated intact follicles suitable for "in vitro" studies on the protein synthesis machinery of mammalian oocytes. Ovaries of immature mice(8-14-days old), free of annexes and surrounding fat are squashed gently on a wire screen placed over the rim of a centrifuge tube. This step is followed by washing the screen in a saline-gelatin solution which is centrifuged twice at low speed to separate intact follicles from smaller ovarian components. At the mentioned ages about 170-190 follicles of 80 mu in diameter are found in the ovary. 20% of this population comes out intact from the screen which means that 30 ovaries may yield about 1,000 free follicles. The samples were examined under the light microscope to check their homogeneity and in the electron microscope to see whether squashing introduced changes in the cell cytoplasm. No significant changes were observed. This method is therefore considered useful as a previous step to studying, at the molecular level, the metabolic phenomena related to oocyte growth and further differentiation.


[A case of amniotic fluid embolism (author's transl)]. A case of lethal amniotic fluid embolism is reported. The microscopic findings are documented and the patho-physiologic course of events is reconstructed. The unsuccessful treatment is compared with recommendations in the literature and critically evaluated.


Weight gain and heterosis at different stages of development of the rat. Diallel crosses among four highly inbred lines of rats were made in order to evaluate their combining ability at 10 day intervals during postnatal growth. Analyses were undertaken to consider the relative magnitudes of different components of variation, including a heterotic effect, over a long developmental period. A general analysis of variance for least square means of body weights indicated that postnatal growth was influenced greatly by the maternal lines and to a lesser extent by the paternal line. Heterosis was measured as a statistically significant difference in body weight gain at 10 day intervals between the hybrid and either the averaged mean of the parent lines or the mean of the largest parent line. Of the effects analyzed, heterosis accounted for a major part of body weight variability in both sexes between 30 and 100 days of age but reached much higher levels in male than in female progeny. The heterotic response was most pronounced during the rapid growth phase; that is, between the ages of 20 to 50 days. Although interesting specific exceptions were observed, heterotic responses generally became more pronounced with increasing age through 100 days. Evidence for differential heterosis associated with particular lines was present in male progeny at growth stages immediately preceding and following the phase of most rapid growth. Specific combining ability, however, was generally lacking. Analyses of specific matings indicated that heterosis, in the lines tested, is a variable phenomenon which may be dependent upon the degree of genetic divergence between inbred parent lines and the combining ability of the genotype involved.


Carnosinase activity of human gastrointestinal mucosa. Carnosinase, the dipeptidase which hydrolyses carnosine and other histidine-containing dipeptides, was assayed in mucosal tissues of the human and of the rat gut. Kinetic properties of the intestinal enzyme were found to be similar to carnosinase of other animal tissues. Little or no activity was detected in human gastric or colonic mucosa, and the levels were lower in duodenal than jejunal mucosa. The distribution of carnosinase is similar to that of the disaccharidases. Mean carnosinase activity was 8-8 units/g weight in 15 patients with histologically normal mucosa compared with 5-7 units in five with villous atrophy. The enzyme levels increased with histological improvement of the mucosa in patients with coeliac disease on a gluten-free diet. Tolerance curves for carnosine and its constitutent amino acids showed malabsorption of the dipeptide in a patient with carnosinase deficiency. It is concluded that the intestinal mucosa has much less hydrolase activity for carnosine than for glycylglycine and other dipeptidases, and the relatively slow hydrolysis appears to be the rate-limiting step in the total absorptive process.


[Thrombophlebitis following intravenous injection--an experimental study]. Thrombophlebitis similar to that which is frequently observed following i.v. injection of irritative drugs in clinical treatment has been successfully reproduced in experimental animals by utilizing a newly devised technique. Thrombophlebitis at a high rate resulted within a few days by allowing the sample to remain in the sealed section of the V. retroauricularis in rabbits for 3 min after injection. The effects of several drug preparations on the thrombus as well as on the inflammation formed by means of this technique were examined, and the results were in good parallel with clinical cases reported in medical literature. Histopathologically, it was observed that each thrombus was formed by the irritative effect of drugs on the portion on the veneous wall damaged when needles had been inserted.


[Effects of linoleic acid hydroperoxide on the formation of lipid peroxide in the rat liver mitochondria]. An in vivo study was made on the effect of linoleic acid hydroperoxide on the formation of lipid peroxide in rat liver mitochondria, and results are as follows: There were no changes in contents of lipid peroxide and total sulfhydryl in rat liver mitochondria after daily administration of linoleic acid hydroperoxide (10 mg/kg, i.p.) for 3 or 7 days. Changes in the fatty acid composition in plasma and liver after i.p. administration of linoleic acid hydroperoxide for one week were not observed as compared to that of control animals. In addition, no appreciable change was found in the fatty acid composition of phosphatidylserine and phosphatidylinositol in liver mitochondria from rats treated with linoleic acid hydroperoxide. After treatment with linoleic acid hydroperoxide for 3 days, content of plasma triglycerides increased, but no effect was observed in liver triglycerides. However, plasma triglycerides returned to the control level and triglycerides in liver increased by treatment with linoleic acid hydroperoxide for one week. Free fatty acids decreased in plasma but not in the liver from rats treated with linoleic acid hydroperoxide for 3 or 7 days.


[A study on drug dependence using fast acting drugs]. Physical dependence on narcotics is induced in laboratory animals by intermittent parenteral administration (2 approximately 3 times daily). However, inducing of dependence on pethidine has been unsuccessful using the parenteral method. Recently, it has been reported that physical dependence on pethidine can be induced by continuous infusion methods (5.6). In the present experiment, pethidine was administered to rats (n=5 approximately 6) by ingestion of pethidine-admixed food preparations (0.5 approximately 4.0 mg/g of feed). The results indicated that (a) when rats are allowed free access to two food preparations (0.5 mg/g vs. 1 mg/g of food) for 7 weeks, spontaneous intake ratios of food (1 mg/g of food) gradually increased from 15% to 30% after 3 weeks. (b) Abrupt withdrawal for 48 hr after a 10 day administration period (2 mg/g of food on day 1 approximately 3 and 4 mg/g of food on day 4 approximately 10) resulted in a loss of body weight in the next 24 hr, and the prewithdrawal level of body weight was recovered in 48 hr. (c) The time course of body weight and food intake during the first 24 hr withdrawal period demonstrated the characteristic pattern of abstinence syndrome of pethidine, viz. early onset (12 approximately 13 hr) and rapid recovery (within 48 hr), as compared to morphine withdrawal. (d) Suppression of pethidine abstinence of both a single injection of morphine (10 mg/kg, s.c.) and substitution for morphine-admixed food was also realized. (e) When levallorphan (5 mg/kg, s.c.) was administered to both pethidine and morphine dependence rats, the maximal decrease in body weight was less than that in morphine dependent rats. These data indicate that pethidine possesses about one fifth the dependence liability of morphine and the maximal abstinence syndrome appears within 24 hr after withdrawal. Conclusively, application of a drug-admixed food preparation in drug dependence tests in rats has proven to be a useful method, particularly in the case of pethidine-like drugs which rapidly disappear from the blood.


[Application of animal mass psychology to pharmacology (XI). The role of light in the induction of convulsions in E1-mice]. In preliminary experiments to investigate the influence of masking the eyes of E1-mice on the rate of non-convulsions (RNC), it was suggested that the quantity of light as well as visibility itself played a role in inducing convulsions. The following experiments were thus carried out: (a) Both TSC (the threshold of number of shakings required for induction of convulsions) and RNC increased remarkably on the 5th day after enucleation of bilateral eyeballs from E1-mice, but 15 days later recovery to control level was observed. Moreover, TSC and RNC at 2 p.m. increased considerably compared with the control, however, the increase was rarely observed at 10 a.m.. (b) 2 groups of E1-mice reared intact under radiation of light plus different time schedules were shaken at 10 a.m. and 2 p.m., respectively. Consequently, TSC after radiation for 5 hours was less than that after radiation for 1 hour. When observed 9 hr after radiation, TSC could not be differentiated from TSC at 5 hr after the radiation. (c) E1-mice were reared under continuous lighting, continuous darkness or a normal lighting situation, after which TSC and RNC were examined. From our observations, the intensity of light appears to play a role in inducing convulsions. (d) When mice were shaken immediately after lighting, RNC decreased in inverse proportion to increase in the quantity of light. TSC was not influenced by the luminosity itself. One hour after lighting, however, the influence of lighting had disappeared. The intensity, quantity or irradiation time would not, however, influence the induction of E1-mice convulsions so much as the cognizance of light by the individual animals. Such a conception is derived from the "cognitive theory" in psychology.


[Formation of sulfhemoglobin using various drugs]. It is well known that aniline and hydroxylamine derivatives induce methemoglobinemia in humans as well as various animal species. It was noticed that sulfhemoglobinemia was induced by subchronic administration of N-(4-chlorophenyl)-beta-(4-hydroxymethylphenoxy)ethylurethane (HPU) to rabbits (p.o.), cats (i.p.) and mice (i.p.). Sulfhemoglobin (SHb) and methemoglobin (MHb) formation by various compounds structurally related to HPU were examined by a single and three consecutive intraperitoneal administrations to mice. It was found that by a single administration, methemoglobinemia was induced by phenylhydroxylamine (PHA), nitrobenzene (NB), aniline (A), 2-chloro-A (2-Cl-A), 3-Cl-A, 4-Cl-A, acetanilide (AA), phenacetin (PA), N-(4-chlorophenyl)ethylurethane (CPU), hydroxylamine (HA) and sodium nitrite (SN), and was not observed with phenylurethane, HPU, methylhydroxylamine (MHA), methylamine and nitromethane (NM). On the other hand, with sulfhamoglobinemia which appeared much more delayed than MHb, a single administration, was found to be induced by PHA, 3-Cl-A, 4-Cl-A, PA, CPU, MHA and SN. Furthermore, with three consecutive administrations, such was induced by NB, 2-Cl-A, AA, HPU and NM even though SHb was not demonstrated with a single administration. Structure-SHb forming property relationship and mechanism for SHb formation are discussed.


[Effects of 9-fluoro-11 beta, 21-dihydroxy-16 ampha-methylpregna-1, 4-diene-3, 20-dione (A41 304) and dexamethasone on the fetus of mice and rats]. A41 304 at 400 mug/kg produced depression of body weight gains in the rat dams but did not increase the number of the fetuses with gross malformations. At 400 mug/kg of dexamethasone, body weight gains of the rat dams were markedly depressed and fetuses presented cleft palate and depression of the placental weight, body weight and crown-rump length. As for the influence of both drugs upon the fetal skeleton, ossification of the odontoid process and metatarsus was delayed and lumbar ribs increased. Dexamethasone at 400 mug/kg, in addition to these findings, caused prominently delayed ossification to the caudal vertebrae. When A41 304 at 1600 mug/kg and dexamethasone at 400 mug/kg were given to pregnant mice, cleft palate occurred with high incidences, but neither visceral abnormalities nor skeletal malformations attributable to the drug administration were observed. The critical period of cleft palate which was noted in the mice given A41 304 was day 11 through day 15 of pregnancy, mainly 11 approximately 13 of pregnancy. Cleft palate was seen in a dose-related manner in either A41 304-treated groups or dexamethasone-treated groups, but the incidence at the same dosage level was higher in dexamethasome-treated groups than in A41 304-treated groups.


[Effects of drugs on cultured cells (III)]. Toxicity of drugs cultured cell line (RK12-, EL-cell line) were exposed to various concentrations/ml medium for 48 hours and for 14 days. The morphologic changes of both cell lines observed included granulation and shrinkage of the cytoplasm, formation of long and narrow cytoplasmic projection, and appearance of giant like cells. ID50 values of ibuprofen, naproxen, Y-5544, diclofenac and aminopyrine to RK13 cell were found to be 130, 290, 200, 140 and greater than 500 mug/ml respectively. On the other hand, ID50 values of each drug to FL-cell were found to be 105, greater than 500, 180, 170 and less than 500 mug/ml respectively. Minimum concentration, cuased by the detachment of the cell from the vessel wall, was as follows: to RK13 were ibuprofen 125 approximately 250, paproxen 250 approximately 500, Y-5554 125 approximately 250, diclofenac 62.5 approximately 125, aminopyrine 1000 approximately 2000 each mug/ml. On the other hand, to FL-cell were ibuprofen 62.5 approximately 125, naproxen 125 approximately 250, Y-5554 62.5 approximately 125, DIClofenac 62.5 approximately and aminopyrine 1000 approximately 2000 each mug/ml respectively.


[Synthesis of a [Glu5, Ala12, Ala18, Ala21]sheep insulin-A-chain by fragment condensation on a polymer support (author's transl)]. The synthesis of a [Glu5, Ala12, Ala18,-Ala21]sheep insulin-A-chain by condensation of 5 fragments on a polymer support is described. The 5 fragments Boc-Gly-Ile-Val-Glu(gammaOBut)-Glu(gammaOBut) (V), Boc-Cys(SiPr) (IV), Boc--Cys(SiPr)-Ala-Gly-Val (III), Boc-Cys(SiPr)-Ala-Leu-Tyr-Gln-Leu (II) and Boc-Glu(gammaOBut)-Ala--Tyr(Bzl)-Cys(SiPr)-N2H3 (I) were synthesized by conventional methods and coupled with dicyclohexylcarbodiimide/1-hydroxybenzotriazol (II, III, IV, V) and by the azide method (I) with coupling yields of 60-98% on an Ala-polymer. The failure sequence peptides were separated by ion exchange chromatography on DEAE-Sephadex and by chromatography on Biogel P4. The A-chain was obtained in 9% yield, which, after combinations with natural B-chain, gave insulin activities comparable to that obtained with natural A-chain. These results demonstrate that fragment condensation by the solid-phase method together with simple techniques for purification can be used for the synthesis of longer peptides.


Problems concerning the biochemical action of superoxide dismutase (erythrocuprein). The decay of the tetraperoxochromate- (V) complex (CrO83theta) was examined to study the substrate specificity of erythrocuprein (super-oxide dismutase). The decay of CrO83theta proved rather complex in aqueous solutions. Apart from the two known oxygen species O2theta and singlet oxygen (1 deltagO2), H2O2 and probably OH radicals were formed. No unequivocal evidence for the appearance of superoxide was obtained. The possible electron transfer from Cr5 to Fe3 (cytochrome c) was also discussed. In Tris buffer, pH 7.8, there were absolutely no signs of superoxide or OH radical formation. In fact, pulse radiolysis measurements employing a homogeneous OH source demonstrated that the Tris and OH radicals react with each other. One mol of H2O2 was generated from 1 mol of CrO83theta in Tris buffer. By contrast, only 0.5 mol H2O2 could be determined when the CrO83theta decay was carried out in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.8. The phenomenon of reducing oxidized cytochrome c could not fully be assigned to a superoxide-mediated reduction, since erythrocuprein was unable to inhibit this cytochrome c reduction efficiently. The energetic oxygen species (1deltag O2, OH etc.) appearing during the CrO83theta decay gave rise to a clearly detectable chemiluminescence. In this system, erythrocuprein was very active regardless of which buffer was used. Even in the absence of a chemiluminescent mediating agent, which might have interferred with the enzyme, erythrocuprein proved capable of inhibiting the CrO83theta-induced chemiluminescence in a rather specific way. No such specificity was seen in the presence of low molecular weight Cu-chelates including Cu(Tyr)2, Cu(Lys)2 and Cu(His)2. The ability to suppress chemiluminescence was approximately 3 orders of magnitude less pronounced than that of the native enzyme. It is presumed that erythrocuprein reacts with oxygen species other than the superoxide radical.


[Partial synthesis and properties of des-A1-glycine-insulin (author's transl)]. Des-Gly-A-chain-tetra-S-sulphonate was prepared by Edman degradation following two different routes. A) Via complete reaction of A-chain from bovine insulin with 150 equivalents of phenylisothiocyanate in pyridine/water and trifluoroacetic acid cleavage of the resulting phenylthiocarbamoyl A-chain. B) Via reaction of bovine insulin with about 20 equivalents of phenylisothiocyanate until a substitution degree of 2.3-2.5 was reached, trifluoroacetic acid cleavage of the crude derivatives and oxidative sulphitolysis of the resulting desaminoacyl insulins. Preparative electrophoresis (pH 2) or ion exchange chromatography using DEAE-Sephadex gave des-Gly-A-chain in a yield of 60-65% of theory according to method B, containing less than 1% of glycine. Des-GlyA1-insulin was prepared by combination with 0.67 equivalents of B-chain-bis-S-sulphonate and isolated in yields of 5-13%, based on B-chain, after gel filtration (pH 8) and ion exchange chromatography (CM-cellulose, pH 3-2). The electrophoretically (pH 2 and 8.6) homogeneous analogue did not crystallize in the presence of zinc ions. Its blood sugar lowering potency is 10-25%, its in vitro insulin activity (fat cell assay) only 1-2%. The immunoreactivity against anti-insulin sera in different test systems is markedly reduced. There are clear differences between the CD-spectra of des-Gly-insulin and insulin, indicating a loss of ordered secondary structure. From the results it is concluded that structure-stabilizing non covalent bonds are abolished by the removal of the invariant A1-glycine. This leads to conformational alterations which cause the far-going inactivation of the molecule.


[Lip prints--variability and genetics (author's transl)]. In a sample of 500 persons, including 76 families with 133 children, 22 mono- and 17 dizygote twins, lip prints were prepared for the study of variability and genetical basis of ridge-pattern in the region of mucous membrane lips. Taking 4 classes of pattern with different ridge-branching as a basis we observed more frequently branched pattern at the upper lip and mainly simple pattern at the lower lip. About 30% of the lip-prints showed whirling figures--at the upper lip simple and median, at the lower lip double and paramedian. Investigations during several months showed stability against environmental factors. The results of twins, families and mother(father)-child combinations proved a genetical basis of lip-prints. Applications of cheiloscopy to genetical investigations are reported.