Structure of human luteinizing hormone alpha subunit. Structural studies have substantiated the concept that the glycoprotein hormones consist of a "common" alpha subunit and "hormone-specific" beta subunit. Despite this, consensus is still lacking concerning certain portions of the amino acid sequences, including alignment of residues 81--82 in the alpha subunit. We have carried out sequence analysis of the alpha subunit of human luteinizing hormone (hLH) to clarify the assignment of these residues and to examine further the nature of the amino-terminal heterogeneity found among the different alpha subunits. Our structure showed residues 80--84 to be -His-Cys-Ser-Thy-Cys-, consistent with findings of others for human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). The extensive degree of heterogeneity found in the amino terminal region of hFSH and hCG is present to only a minor extent in hLH. The differences in the pattern of amino-terminal heterogeneity among the various hormones may result from differences in the nature of cleavage of subunit from a larger intracellular precursor peptide.


The occurrence of 11-deoxycorticosterone (DOC)-induced hypertension in the Long-Evans rat. The sensitivity of two strains of rat to the hypertensinogenic action of DOC was studied. Hypertensive cardiovascular disease was evident within 3 weeks of implantation of DOC  pellets in sensitized female rats of the Sprague-Dawley and Long-Evans strains. Cardiac and renal hypertrophy due to DOC treatment was evident in both strains of rat. The DOC treatment also resulted in a significant decrease in absolute adrenal weight. These results, which indicate that Long-Evans rats are not resistant to DOC-induced hypertension, contrast with previous reports by others. An explanation of the discrepancy may be the use of free DOC rather than DOC acetate in the present study.


Electrophoretic and histochemical studies on hepatic 3 alpha-hydroxysteroid dehydrogenase in the rat. Four isozymes of 3 alpha-hydroxysteroid dehydrogenase (3alpha-HSD) appeared in rat livers to be classified into three categories concerned with the requirement of coenzyme. Two isozymes in the first group had affinity for both NAD and NADP. One of the other isozymes classified in the second was linked with NADP to show specificity for 5beta-androstan-3alpha-ol-17-one (etiocholanolone) as the steroid substrate. An isozyme belonging to the third required only NAD as cofactor. This has the same migration rate of a lactate-dehydrogenase isozyme. In the histochemical observation, the maximal activity of the enzyme was demonstrated with 5-alpha androstan-3alpha-ol-17 one (androsterone) but not with etiocholanolone as a substrate. On the other hand, all 3 alpha-HSD isozymes revealed by electrophoresis showed a higher affinity for etiocholanolone than androsterone. It is worthwhile to note that the zymogram of 3alpha-HSD in the cold acetone-treated section was essentially the same as the zymogram in the intact liver. All isozymes in the section were highest in activity when etiocholanolone was used as a substrate. These findings indicate that in the cold acetone-treated section the enzyme still has affinity for etiocholanolone to resist the histochemical procedure employed.


Histometric study of the pituitary in mice treated neonatally with steroids and the relationship between prolactin cells and mammary tumorigenesis. Neonatal female mice of the BALB/cC3H/Crgl strain were given daily injections of 17 beta-estradiol, progesterone and prolactin, singly and in some combinations, for 5 days beginning within 36 hr after birth. Mice were killed at tumor age or by 12 months of age. Differential cell counts of the anterior pituitary showed that prolactin cells were more numerous in neomatally estrogen-treated mice and progesterone-treated intact mice than in control mice. Paired analysis of tumor-bearing and non-tumor-bearing mice of all groups revealed that the occurrence of prolactin cells was greater in the former than the latter. Counts of gonadotropes and thyrotropes did not show any significant correlation with mammary tumorigenesis. However, neonatal estrogen and/or progesterone treatment resulted in significantly decreased numbers of gonadotropes in intact mice. In ovariectomized mice, gonadotropes were significantly increased regardless of neonatal treatment. The present results support the suggestion that the stimulatory effects of neonatal steroid treatment of mammary tumorigenesis may be a consequence of increased prolactin secretion, resulting from sustained minimal estrogen secretion by the ovary.


Inhibition of fetal adrenal 21-hydroxylase by naturally occurring steroids. Inhibitory effects of naturally occurring steroids on 21-hydroxylase activity were determined by measuring the initial conversion rate of 17-hydroxyprogesterone (17alpha-hydroxy-4-pregnene-3, 20-dione) to 11-deoxycortisol (17alpha, 21-dihydroxy-4-pregnene-3, 20-dione) in incubation experiments with the microsomal fraction (10,000--105,000 X g percipitate) of the human fetal adrenal gland. The apparent Michaelis constant (Km) for conversion of 17-hydroxyprogesterone was 13.3 X 10(-6)M. Human fetal adrenal 21-hydroxylase was inhibited by some of the steroids produced in the feto-placental unit. The following steroids acted as competitive inhibitors of the reaction; progesterone (Ki = 20.0 X 10(-6)M), 11-deoxycortisol (Ki = 87.5 X 10(-6)M) and estradiol-17beta (Ki = 87.5 X 10(-6)M). The most potent inhibitor among the estrogens was estradiol-17beta.


Effect of CB-154 therapy on the glucose tolerance, IRI and GH response to glucose administration in acromegaly. Effects of CB-154 therapy on the glucose tolerance, IRI and GH responses to the oral glucose administration in 5 acromegalic patients with impaired glucose tolerance were examined. Glucose tolerance improved in all patients with a reduction in GH levels. Insulin secretion in response to the oral glucose administration was exaggerated in 3 patients, while it was supressed in 2 patients who had severely impaired glucose tolerance. After receiving CB-154 therapy, hyperresponsiveness of insulin observed in the 3 patients and a suppressed response in one patient were corrected to normal, while a low response in another stayed unchanged. This observation suggest that variable abnormalities in insulin secretion induced by the excess of GH improve with a decrease in plasma GH levels. However, the abnormal GH secretion in response to hyperglycemia persisted still after administration of CB-154 in spite of decreased basal plasma GH levels.


Plasma TSH and prolactin response to insulin induced hypoglycemia in patients with hypothalamic pituitary disorders. Plasma TSH and prolactin (PRL) responses to insulin-induced hypoglycemia (ITT) were examined in 37 patients with various hypothalamic pituitary disorders. Four patients showed a definite TSH response to ITT. They were 2 patients with suspected pinealoma with diabetes insipidus (DI), a patient with pituitary adenoma and a patient with acute prophyria. A suspected hypothalamic dysfunction and a slightly impaired pituitary function manifested as GH deficiency were their common endocrinological features. On the contrary, plasma PRL response to ITT was noted in about one third of the patients examined, but no concomitant PRL response was observed in any of the patients with a definite TSH response. This suggested a different mechanism of TSH and PRL secretion may be different under this circumstance and a certain factor related to the hypothalamic disorder may play a modulating role in an unusual TSH response to hypoglycemia in these particular patients.


Effect of thyroidectomy on the secretory profiles of growth hormone, thyrotropin and corticosterone in the rat. Changes in pituitary hormone secretions following thyroidectomy were examined in sequentially collected blood samples every 15 min day and night for 7 hr from male rats via cannulae into the right atria. Pulsatile growth hormone secretions were observed every 2.5 or 3.5 hr in normal rats, while thyroidectomy 3 weeks prior to experiments abolished the growth hormone burst. Corticosterone profiles from normal rats showed also episodic patterns which were more often observed in the night experiment. The eisodic secretion of corticosterone seemed to inhibit the subsequent growth hormone burst but not vice versa. The interrelation between integrated concentrations of growth hormone and corticosterone showed a negative correlation although the coefficient was not statistically significant. After thyroidectomy, the basal level of corticosterone decreased and the episodic secretion was blunted. There was no nyctohemeral variation in the plasma thyrotropin level in normal rats but small and slow fluctuations. Thyroidectomy caused about ten-fold elevation in the basal thyrotropin level and, in addition to this, a decrease at 1500 hr and an increase during the night, followed thereafter by a decreasing tendency to 0400 hr. Power spectral analysis revealed smaller components in growth hormone secretion of normal rats, which had periods of 60, 40 and 36 min, besides the hormone burst. The spectral analysis also made it clear that there were small and frequent components (40 or 36 min period) in corticosterone secretion.


Methods of electrophoretical application of estrogen to a single neuron of the brain under in vivo and in vitro conditions. The techniques of the electrophoretical study of hormone sensitivities of neurons were examined in vivo and in vitro. For the in vivo study, estrogen and other hormones were applied electrophoretically to neurons of the rat brain of both sexes under urethane anesthesia. Some neurons were sensitive to estrogen and their activities were all inhibited. These neurons were distributed in the hypothalamus and the septal nucleus of the limbic system. So the in vivo method affords direct evidence for the presence of estrogen-sensitive neurons in the brain. Then a useful method was found to study the similar effects under the microscopic observations of a single neuron instead of stereotaxic insertion of the microelectrode. By this in vitro method the chemicals and hormones are able to be applied to any point on a single neuron through the micropipette and the medium composition around the neuron can be variously changed at the same time.


Reaction of 4-methylumbelliferylguanidinobenzoate with proteases in plasma of patients with cystic fibrosis. Protease activity, assayed using 4-methylumbelliferylguanidinobenzoate, an active site titrant of certain proteases, is significantly deficient in plasma of patients with cystic fibrosis. The deficiency can be demonstrated with both chloroform-ellagic acid activated plasma in which the proteases can hydrolyze esters of arginine and unactivated plasma in which the proteases have negligible activity towards these esters. The deficiency can also be demonstrated by separation of the proteases by isoelectric focusing on polyacrylamide gels or by chromatography on agarose columsn. Since protease deficiency can be demonstrated with unactivated plasma, the deficiency in cystic fibrosis is probably due to a reduced number of protease molecules rather than their decreased catalytic efficiency.


Dysregulation of protein synthesis in injured liver. A comparative study on microsomal and cytosole enzyme activities, microsomal lipoperoxidation and polysomal pattern in D-galactosamine and carbon tetrachloride-injured livers. Despite the presence of a marked decrease in liver protein content 48 h after a single injection of D-galactosamine, increased activities of glucose-6-phosphate dehydrogenase, low-Km hexokinase and pyruvate kinase type M2 were observed in the injured liver. Microsomal aniline hydroxylase activity and cytochrome P-450 content in liver decreased significantly in 48 h of galactosamine treatment but not in the first 2 h in contrast with carbon tetrachloride (CCL4) intoxication. The extents of those changes were not so great as in CCl4-treated rats. The disaggreation of polyribosomes in liver was observed in 24 h of galactosamine treatment. However, the formation of microsomal lipoperoxidation did not increase in the entire course of acute liver injury by the amino sugar. These results taken together with our previous observations indicate that the dysregulation of protein synthesis is an essential biochemical event of hepatocyte injury induced by treatment of rats with galactosamine as well as CCl4.


Assay of leucine aminotransferase in rat tissues and tumors. A survey of leucine aminotransferase activities in an extensive variety of rat tissues further defined the unique distribution of this enzyme. Its reaction was measured in cell-free extracts under optimal conditions, and its product assayed both colorimetrically and with 14CO2 formed from it by decarboxylation with H2O2. The two methods agreed, though the second was more precise and dependable. In all the tissues and cell fractions examined, except adult liver, the activities with valine and isoleucine were parallel and similar to that with leucine. In adult liver there was low activity only with leucine, which is referable to a known isozyme (type II). Although the fraction of enzyme activity present in mitochondria has often been neglected, these studies showed that this portion in all tissues is about equal, more or less, to that of the more familiar soluble forms. There was good agreement between the relative concentrations of this enzyme in the commonly measured rat tissue preparations as determined here and in the more recently published studies. The unique occurrence of high levels of this enzyme in heart, kidney, brain and muscle, in that order, was confirmed. The unusual distribution of this enzyme was emphasized by the recognition that equally high or even higher levels also occurred in pancreas, lactating mammary gland and salivary gland. The relative amounts were less in normal, undifferentiated and in neoplastic tissues.


Dihydropteridine reductase activity of adult, fetal and neoplastic tissues. Optimal conditions for the assay of dihydropteridine reductase in crude tissue extracts have been developed. Among adult rat tissues, liver and kidney had the highest activity followed by thymus, lung, cerebellum and cerebrum. Fetal tissues and neoplasms exhibit lower activities than the cognate adult tissues. In both liver and kidney the developmental upsurge of dihydropteridine reductase, which produces the cofactor necessary for the hydroxylation of aromatic amino acids, begins earlier than does that of phenylalanine hydroxylase. In human liver and lung also, the dihydropteridine reductase activity doubles between midgestation and adult life. The undifferentiated enzymic composition of neoplasms is reflected by the lower dihydropteridine reductase content of pulmonary tumors than of nonneoplastic human lung.


Postnatal behaviour of guanase activity in rat tissues. The behaviour of guanase activity and the 8-azaguanine/guanine deamination ratio of rat tissues during postnatal development were studied. In the liver and brain, the enzymatic activity, present from birth, gradually increases up to the 4th week. In the kidney, guanase activity appears after the 1st week and reaches the level found in adult tissues after the 6th week. The value of the deamination ratio of tissues at birth is higher than that of adult animal tissues.


Validity of the relative percent concept for equating training intensity. The validity of the relative percent concept for equating training intensity was investigated using the point of metabolic acidosis (anaerobic threshold) as the criterion variable. Percent oxygen uptake, heart rate and metabolic acidosis were determined at 60, 70, and 80% of max heart rate on a bicycle ergometer test(N = 31). At 60 and 70% of max heart rate only one individual was definitely in metabolic acidosis. At 80% max heart rate 17 subjects were working at a level above the point of metabolic acidosis while 14 were working at or below this point. Thus, it was suggested that even if subjects are exercising at the same relative percent miximum HR, the metabolic stress using metabolic acidosis as the criterion is not constant across subjects. It was further shown that the regression of percent VO2 max on percent max HR was a spurious one resulting in poor prediction of individual VO2 values. The data presented suggest that the relative percent concept for equating exercise or subsequent training intensity needs careful re-evaluation.


Cardiopulmonary adjustment and metabolic response to maximal and submaximal physical exercise of boys and girls at different stages of maturity. Cardiopulmonary and metabolic variables were investigated at maximal and submaximal bicycle ergometer exercises in 41 swimmers of both sexes, 8--18 years old. VO2 max and VO2 max . HR-1 were higher in boys than in girls and increased with maturity, while VO2 max. kg-1 and HVE were not influenced by this. The HV increased clearly during this growth period, the pubertal and postpubertal subjects showing 16 and 17% higher values for HV and HV . kg-1 than those reported in normal schoolchildren populations. During the submaximal exercise at 70% VO2 max the highest HR values were found in the prepubertal group, whilst the lowest were observed in the postpubertal subjects. These findings suggest that a given percentage of VO2 max as a reference unit, is more reliable than a certain HR to obtain comparable results in subjects with different ages. Blood samples were collected before, during, and after the submaximal exercise. Blood glucose and FFA did not differ in relation to the stages of maturity. During exercise, insulin decreased in prepubertal children, did not alter in pubertal adolescents, and increased in postpubertal subjects. The lactate concentration, during exercise, increased in relation to maturity. The same results were found for HGH, but no differences were found with regard to sex. Since the pattern of HGH secretion during exercise is similar to that found after arginine and insulin administration it is assumed that the same mechanism (i.e., sex hormones) triggers the HGH release.


Specificity of metabolic and circulatory responses to arm or leg interval training. The purpose of this study was to evaluate metabolic and circulatory responses to interval training of legs or arms during steady-state, submaximal cycling. 15 college males cycled on a bicycle ergometer twice with arms (63 and 83 W) and twice with legs (100 and 125 W) before and following 5 weeks of daily interval training. Seven subjects trained with arm cycling and eight with leg cycling. Significant post-training decreases in submaximal oxygen consumption (VO2), heart rate (HR), and venous blood lactate (LA(V)) were noted when cycling with trained and untrained muscles with the magnitude of change significantly greater with trained muscles. These results indicate metabolic and circulatory adaptations to interval training that are mediated centrally and peripherally. With respect to HR, but not VO2, training a larger muscle mass (legs) produced a greater central but lesser peripheral effect whereas the opposite was true for the smaller arm muscles. The data also suggested that the peripheral adaptation involves a common mechanism controlling both HR and LA(V)) changes with a separate mechanism controlling VO2.


[Performance- and strain-compensation with informatory work]. With results from literature it is shown that eye-blinking-rate is negative significant correlated with physiologic performance ability. Experiments with an abstract tracking task presented in this paper confirm that eye-blinking-rate is an indicator for activation. Taking in the indicator for activation (blinking-rate), tracking performance and central-physiological strain, phases of performance- and strain-compensation (stability, effort, fatigue, performance break-down) are defined in an hierarchical concept of stability. The compensatory processes of the human operator are discussed on the background of a stress-strain-concept taking account of the principle of feed-back.


Maximal oxygen intake and body composition of female dancers. Complete paucity of any data regarding the influence of habitual dance on cardiovascular-pulmonary fitness and body composition of female dancers prompted this study in which 12 female dancers and 12 sedentary female students of the same age range participated as subjects. Maximal oxygen intake was determined on the treadmill, body composition was calculated from skinfolds and vital signs were determined besides measurements of height and weight. Dancers had significantly lower weight, lower resting heart rate, and lower diastolic blood pressure. Systolic blood pressure was also lower in dancers but the difference between the means did not reach statistical significance. Maximal oxygen intake was higher in dancers when expressed in relative terms. Dancers had also significantly lower total body fat. In conclusion, it seems tha, dance with all the variations, should be encouraged in schools as a suitable behavioral modifier of sedentary habits and for maintenance of physical fitness and ideal weight.


Hormonal changes in serum in young men during prolonged physical strain. The endocrine response to severe physical strain including lack of sleep has been investigated in army personnel during a combat course of 5 days' duration. The thyroxine (T4) concentration in serum increased during the first 24 h, and then declined at a rate corresponding to a halflife of 7.6 days and on day 6 had reached the lowest level, 55 ng/ml. Triiodothyronine (T3) displayed a similar pattern, although an increase during the first 24 h could not be demonstrated. Within 48 h after the course T4 had returned to normal, whereas the serum level of T3 was significantly below the level before the course (p less than 0.05). The serum level of TSH was suppressed during the course. The serum level of prolactin was significantly suppressed and growth hormone was markedly elevated during the course with a significant negative correlation (r=-0.6) between the two. In agreement with a previous report, there was a rapid and sustained suppression of the serum level of testosterone to a mean level of 1.1 ng/ml on day 5. Short periods of sleep (3--6 h) were shown to be effective in reversing the changes described in this paper, especially for growth hormone, prolactin, and testosterone.


Subcellular localization of two long-chain acyl-coenzyme-A synthetases in Candida lipolytica. Studies have been made on the subcellular localization of two long-chain acyl-coenzyme-A synthetases as well as glycerolphosphate acyltransferase and the acyl-CoA-oxidizing system in Candida lipolytica grown on oleic acid. Acyl-CoA synthetase I is distributed among different subcellular fractions, including microsomes and mitochondria where glycerolphosphate acyltransferase is located. On the other hand, acyl-CoA synthetase II is localized in microbodies where the acyl-CoA-oxidizing system is located. These results support our previous conclusion that acyl-CoA synthetase I is responsible for the production of acyl-CoA to be utilized for the synthesis of cellular lipids, while acyl-CoA synthetase II provides acyl-CoA that is exclusively degraded via beta-oxidation.


Adaptive responses of enzymes of carbohydrate and lipid metabolism to dietary alteration in genetically obese Zucker rats (fa/fa). 1. Measurements have been made of the activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis in liver and adipose tissue from genetically obese (fa/fa) rats and their lean litter mates (fa/ --). The effect of food restriction for a period of three weeks on the enzyme profile of liver and adipose tissue of the obese rat was also studied. 2. The most striking increases in enzyme activity in livers from obese rats were: (a) among enzymes of lipogenesis; ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase, malate dehydrogenase (decarboxylating) and cytoplasmic glycerolphosphate dehydrogenase; (b) within the pentose phosphate pathway; glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase; (c) within the glycolytic pathway; glucokinase, pyruvate kinase and lactate dehydrogenase. All of these enzymes showed a significant increase in activity on the basis of U/g liver and U/mg DNA. In adipose tissue all the enzymes of lipogenesis, of the glycolytic route, of the oxidative segment of the pentose phosphate pathway and of the tricarboxylic acid cycle were increased when expressed as U/2 fat pads or as U/mg DNA. 3. The restriction of the food intake of obese rats to that consumed by their lean litter mates for periods of three weeks did not produce the expected adaptive decrease in enzymes of lipogenesis; in adipose tissue, only ATP-citrate lyase and malate dehydrogenase (decarboxylating) showed a marked decrease; no significant change was found in adipose tissue or liver of the activities of acetyl-CoA carboxylase and fatty acid synthetase, when expressed on a cell basis (U/mg DNA). The non-oxidative enzymes of the pentose phosphate pathway and enzymes involved in glycerogenesis (pyruvate carboxylase, malate dehydrogenase and phosphoenolpyruvate carboxykinase) all increased in adipose tissue from limit-fed obese rats. 4. The rate of conversion of specifically labelled glucose to (14C)O2 and 14C-labelled lipid by pieces of adipose tissue and by liver slices was also measured. Insulin caused an increase in the conversion of (1-14C)glucose to (14C)O2 and 14C-labelled lipid in obese rats fed ad libitum, limit-fed rats and in their lean litter mates. 5. The results are discussed in relation to the raised insulin and hypothyroid state of the obese rat. The effect of this altered hormonal status on the activity of cyclic nucleotide phosphodiesterases and cellular levels of adenosine 3' :5'-monophosphate and guanosine 3' :5'-monophosphate and guanosine 3' :5'-monophosphate in relation to the obese syndrome is considered.


Interaction between membrane functions and protein synthesis in reticulocytes. Isolation of RNase M, a membrane component inhibiting protein synthesis through specific endonucleolytic activity. An inhibitor of protein synthesis has been isolated from reticulocyte membranes by solubilization with Triton X-100; it has been purified using heat treatment, filtration on Amicon filters, DEAE-cellulose ion-exchange chromatography and Sephadex G-75 gel chromatography. A final purification of 120-fold was achieved. The purified inhibitor was found to be 95% homogenous when run on a dodecylsulfate/polyacrylamide gel system. Three independent methods were used to estimate the molecular weight of the purified inhibitor: Sephadex G-75 gel chromatography, dodecylsulfate/polyacrylamide gel electrophoresis and sucrose gradient all confirmed that the purified inhibitor was a small molecule with a sedimentation coefficient of 0.7 S and a molecular weight ranging between 5000 and 8000. The purified inhibitor was shown to possess a specific endonucleolytic activity, degrading the 28-S species of ribosomal RNA to species sedimenting between 10 and 14 S. Due to its membrane localisation the name RNase M is proposed. The purified inhibitor's endonucleolytic activity was characterized with regard to its kinetics, concentration dependence, pH optimum and its requirements for divalent cations. Kinetics showed that RNase M retained its specificity after 60 min of incubation with the RNA substrate. Specificity was also demonstrated by incubating the polysomal RNA with high concentrations of purified enzyme. The pH optimum was found to be between pH 6 and pH 7, and the enzyme did not require divalent cations for its activity. Pancreatic RNase B used at a similar protein synthesis inhibitory concentration as the RNase M caused a complete breakdown of ribosomal RNA to oligonucleotides and mononucleotides. The possible biological significance of the purified inhibitor in regulating protein synthesis in the maturing reticulocyte is discussed.


Characteristics of the active transport of Ca2+ by submitochondrial vesicles. Inner membrane vesicles have been prepared by cholate treatment of rat liver mitoplasts. The vesicles can actively accumulate Ca2+ in the absence or presence of inorganic phosphate. The uptake is inhibited by ruthenium red and uncouplers of oxidative phosphorylation. Like in intact mitochondria the driving force for the uptake reaction seems to be the negative inside membrane potential generated during the oxidation of substrates. The level of antimycin-A-sensitive reduction of ferricyanide by succinate indicates that the cholate inner membrane vesicles are about 70% right side out. Using cytochrome-c-extracted inner membrane vesicles it can be shown that only those which have the same right-side-out polarity as intact mitochondria can actively accumulate Ca2+.


A 1H nuclear-magnetic-resonance study of the solution conformation of the isoinhibitor K from Helix pomatia. The isoinhibitor K from Helix pomatia was investigated by high-resolution 1H nuclear magnetic resonance at 360 MHZ. Detailed studies of the labile protons and the resonances of the aromatic residues indicated extensive homologies between the spatial structures of the snail inhibitor, the basic pancreatic trypsin inhibitor (Kunitz) from bovine tissue and the cos colostrum trypsin inhibitor. Comparison of these three homologous inhibitors indicated that the overall flexibility of the globular protein conformation is reduced and its stability with respect to thermal denaturation raised when the content of amino acids with charged side chains is increased. It is suggested that this relation between amino acid composition and stability of the globular solution structure might be valid also for other classes of homologous proteins.


The amino-acid sequence of S-100 protein (PAP I-b protein) and its relation to the calcium-binding proteins. The amino acid sequence of a brain-specific protein, S-100, has been determined by the analysis of four cyanogen bromide peptides and fourteen tryptic peptides derived from the protein. The protein chain consists of 91 amino acid residues and has a molecular weight of 10507. The sequence shows regions of stong clustering of hydrophobic, basic and acidic amino acids, with an apparent calcium-binding site in the acidic cluster.


Pyrimidine metabolism in microplasmodia of Physarum polycephalum. If microplasmodia of Physarum polycephalum are exposed to 14C-labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2-14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2-14C]thymidine. Due to this a significant part of the trichloroacetic-acid-insoluble radioactivity from exogenously supplied [2-14C]thymidine is not associated with DNA. The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell-free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases. Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic. The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.


Conformation of erabutoxins a and b in aqueous solution as studied by nuclear magnetic resonance and circular dichroism. The 270-MHZ nuclear magnetic resonance (NMR) spectra of erabutoxins a and b have been observed in 2H2O solution. By the use of convolution difference and double resonance techniques, proton signals in the aromatic and methyl regions have been assigned. From the pH dependence of NMR chemical shifts, the pKa value of His-26 of erabutoxin b is found to be 5.8, whereas His-7 of erabutoxins a and b is not protonated at pH above 3. The imidazole ring of His-7 is protonated upon the denaturation at pH 2.85. The acid denaturation process has been followed by the His-26 and methyl proton signals and is found to be reversible but is slow as compared with NMR chemical shift time scale. The circular dichroism (CD) of erabutoxin b has also been observed. The denaturation is found to involve a major change from the beta-rich conformation to a disordered one. The NMR and CD changes upon acid denaturation are satisfactorily explained by the two-state process. The deuterium exchange rates of the C-2 protons of His-26 and His-7 of erabutoxin b indicate that His-26 is exposed to the solvent whereas His-7 is tightly buried in the interior of the protein globule. The pKa value of Tyr-25 is as high as about 12.0, possibly due to the hydrogen bond formation between the hydroxyl group of Tyr-25 and a carboxylate group. The hydroxyl group of Tyr-25 is reversibly titrated so that this group is not buried tightly in the interior of the protein globule. The line width of the aromatic proton signals of Tyr-25 is significantly broad at room temperature, suggesting a restricted rotation of the aromatic ring. The aromatic proton signals of Trp-29 are fairly sharp; this aromatic ring is exposed and mobile. Except for His-7, the micro-environments of Tyr-25, His-26, and Trp-29 residues and methyl proton signals of valine and isoleucine are consistent with the locations of alpha carbon atoms as elucidated by X-ray crystal analyses.


The complete amino-acid sequence of histone H2B(3) from sperm of the sea urchin Parechinus angulosus. The primary structure of a third H2B histone isolated from sperm of the sea urchin Parechinus angulosus has been determined. H2B(3) consists of a polypeptide chain of the following 148 amino acid residues: Pro-Arg-Ser-Pro-Ala-Lys-Thr-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Ser-Arg-Lys-Ala-Ser-Pro-Lys-Arg-Gly-Gly-Lys-Gly-Ala-Lys-Arg-Ala-Gly-Lys-Gly-Gly-Arg-Arg-Arg-Arg-Val-Val-Lys-Arg-Arg-Arg-Arg-Arg-Arg-Glu-Ser-Tyr-Gly-Ile-Tyr-Ile-Tyr-Lys-Val-Leu-Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Arg-Ala-Met-Ser-Val-Met-Asn-Ser-Phe-Val-Asn-Asp-Val-Phe-Glu-Arg-Ile-Ala-Ser-Glu-Ala-Ser-Arg-Leu-Thr-Ser-Ala-Asn-Arg-Arg-Ser-Thr-Val-Ser-Ser-Arg-Glu-Ile-gln-Thr-Ala-Val-Arg-Leu-Leu-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His-Ala-Val-Ser-Glu-Gly-Thr-Lys-Ala-Val-Thr-Lys-Tyr-Thr-Thr-Ser-Arg. H2B(3) Parechinus closely resembles HIB(2) Parechinus but has one additional repeating pentapeptide in the amino-terminal region and a serine replacing glycine at position 98.


Ecdysone oxidase: reaction and specificity. Ecdysone oxidase oxidizes 3-hydroxysteroids of the excysteroid type irreversibly to 3-dehydro derivatives. The hydrogen of the steroid is transferred by the enzyme to oxygen which is reduced to hydrogen peroxide. Ecdysone oxidase is relatively specific for ecdysone and ecdysterone. Apparent Michaelis constants for these two physiological substrates are 98 and 31 micron, respectively. Cholesterol oxidase and 3alpha-hydroxysteroid and 3beta-hydroxysteroid dehydrogenases which catalyze similar reactions are unable to oxidize ecdysteroids.


D-Glucose transport in Trypanosoma brucei. D-Glucose transport is the rate-limiting step of its metabolism. D-Glucose and 2-deoxyglucose enter the 'long-slender' bloodstream form of Trypanosoma brucei only by means of a carrier-mediated process; no free diffusion can be observed. Permeation is not energy-dependent. The uptake is driven by the downhill concentration gradient of free substrate. The latter is maintained by the continual removal of sugar, due to the extremely high activities of the glycolytic enzymes. The permeation process is the rate-limiting step of glucose consumption, because permeation proceeds at a rate slower than metabolism. The inhibition of sugar uptake by glycerol was tested. Interactions at the carrier site can be ruled out since glucose and its 2-deoxy analog exhibit different inhibition kinetics.


Structure of an (A-blood-group)-active glycolipid isolated from human erythrocytes. An (A-blood-group)-active glycolipid possessing 23 sugars has been isolated, from human erythrocyte membrane. Smith degradation and methylation studies allow a structure to be proposed of the nonreducing end of the oligosaccharide chain.


High-resolution proton-magnetic-resonance studies of chromatin core particles. The binding of histones in chromatin core particles and in core particles depleted of histones H2A and H2B has been studied by high-resolution proton nuclear magnetic resonance (NMR) at 270 MHZ. At low ionic strengths it is shown that histones H3 and H4 are bound in the core particle. Further, whereas the apolar regions of H2A and H2B are also bound to the core particle, the basic N-terminal and C-terminal regions are more mobile and give rise to sharp resonances in the NMR spectrum of the core particle. Between 0.3 and 0.6 M NaCl there is further release of basic regions of histones H3 and H4 from the complex. The dissociation of the core particle between 0.6 and 2.0 M NaCl is accompanied by the release of the structured apolar regions of the histones as evidenced by the appearance of a complex aromatic spectrum and perturbed upfield ring-current-shifted methyl resonances. Arginine residues are implicated in the binding between histones and DNA and 69% of these residues are found in the apolar regions of the histones. The interactions between histones and DNA in the core particle thus involves H3 and H4 and the apolar regions of H2A and H2B. It is suggested that these basic regions of H2A and H2B have binding sites outside the core particle.


Analysis of phosphorylation of protein synthesis initiation factor eIF-2 by two-dimensional gel electrophoresis. We describe a method for examining the state of phosphorylation of initiation factor eIF-2 in reticulocyte lysates. The procedure involves incubation of the lysate with iodo[1-14C]acetate in 8.5 M urea, and fractionation of the labelled proteins by two-dimensional acrylamide gel electrophoresis. This approach has been used to show that haem deficiency, double-stranded RNA, and oxidised glutathione, which all inhibit the initiation of protein synthesis in an analogous manner, all cause a net increase in the level of phosphorylated eIF-2 in the complete lysate protein synthesis system.


Biochemical characterization of the restriction-modification system of Bacillus sphaericus. A type II restriction endonuclease (endo R . Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg2+ for optimal activity and it is inhibited by Zn2+. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported. The enzyme also cleaves single-stranded DNA, albeit at a slower rate. It seems likely that single-stranded DNA is cleaved at the same sequences as double-stranded DNA. Bacillus sphaericus also contains a modification methylase (meth M . Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg2+, it transfers the methyl group of S-adenosyl-methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R . Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG-dG-dC-dC) as the nuclease.


The structure of chromatin replicated in vitro. Nuclei from concanavalin-A-activated lymphocytes were used to study the replication of chromatin in vitro. Micrococcal nuclease was employed to obtain information about the structure of the replicated chromatin. The nuclease digestion products were examined by sucrose gradient sedimentation and by gel electrophoresis. Experiments are presented which indicate that DNA replicated in vitro is organized into chromatin whose structure is similar to that of bulk chromatin. This conclusion is based on the following observations: (a) DNA replicated in vitro is associated with typical chromatin subunits (nucleosomes) even after short replication times, when the newly replicated DNA consists almost entirely of Okazaki fragments; (b) the length of internucleosomal spacer DNA in part of the replicated chromatin corresponds to that in bulk chromatin. Evidence which suggests that the structure of nucleosomes is transiently altered in the vicinity of the replication fork is presented.


Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA. An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.


Mannosylation of endogenous proteins of rough and smooth endoplasmic reticulum and of Golgi membranes. Mannosylation of the proteins of microsomal and Golgi membranes was investigated both after incubation in vitro of the isolated subfractions with GDP-[14C]mannose and after injection of [3H]mannose into rats followed by separation of these subfractions. Mannosylation of endogenous and added exogenous dolichol phosphate and also of dolichol pyrophosphate-oligosaccharide occurs in all three fractions. It was essential to inhibit antagonistic enzymes during incubation and to centrifuge after incubation. The presence of detergent in the incubation mixture influences the incorporation pattern of the different fractions in very different ways. In a system in vitro predominantly membrane proteins and not secretory proteins are mannosylated. Trypsin treatment of intact vesicles removes components from the outer surface only; such treatment liberates about one third of the radioactive mannose associated with lipid, releases radioactivity from the protein acceptor to the same extent and causes some inactivation of the transferase activities. It appears that a part of the mannosyl transferase system in rough and smooth endoplasmic reticulum and in Golgi membranes is localized at the cytoplasmic side of these membranes. This activity is probably involved in the glycosylation of proteins localized at the cytoplasmic surface of the endoplasmic reticulum.


The binding of low-affinity and high-affinity heparin to antithrombin. Fluorescence studies. The interaction between antithrombin and two forms of heparin, differing in their affinity for the matrix-linked protein, has been studied by fluorescence. The binding of the high-affinity heparin fraction to antithrombin leads to activation of the inhibitor, allowing it to react more rapidly with a number of serine proteases of the coagulation cascade. The interaction with the low-affinity heparin fraction, however, has considerably less influence on this inhibition rate. The binding of either fraction to antithrombin was found to result in an increase of the tryptophan fluorescence of the protein. This increase was much larger for high-affinity heparin than for low-affinity heparin, suggesting a different mode of binding of the two fractions to the protein. The fluorescence enhancement caused by high-affinity heparin is consistent with a conformational change of antithrombin related to its activation. Only the fluorescence enhancement observed on the binding of high-affinity heparin was of a sufficient magnitude to allow quantitative studies. These showed high-affinity heparin to bind to antithrombin with a stoichiometry of about one and with a binding constant at physiological ionic strength of about 8 X 10(7) M-1. At higher ionic strengths, however, the affinity decreased markedly.


Birefringence of oriented thin filaments in the I-bands of crab striated muscle and comparison with the flow birefringence of reconstituted thin filaments. Birefringence of the I-band of glycerinated myofibrils of the walking legs of the crab, Plagusia dentipes was examined by rectified polarization optics. The coefficient of birefringence of the I-band was estimated to be 1.64 X 10(-3), which attained about 60% of the birefringence of the H-zone. The number ratio of thin filaments to thick filaments in the A-band was six according to electron microscopy and the spacing between neighbouring thick filaments was about 56 nm according to X-ray diffraction. From these values, the protein concentration in the I-band was estimated to be 91 mg/ml. Thus, the birefringence of the I-band was found to be of the same order as the birefringence of the reconstituted thin filaments oriented by flow at the same protein concentration. The ratio of protein concentration in the I-band to that in the H-zone was determined by interferometry. The ratio of birefringence of the I-band to that of the H-zone per unit protein concentration was found to be about 0.9, which was comparable to the ratio of birefringence of the thin filaments and thick filaments in solution. The imbibition experiment showed that 70% of the total birefringence of the I-band is form birefringence and remaining 30% comes from intrinsic birefringence.


Mechanism of spermine stimulation of rat-liver isoleucyl-tRNA formation. The mechanism of spermine stimulation of rat-liver isoleucyl-tRNA formation has been studied by Cleland's method [Biochim. Biophys. Acta, 67, 104--137 & 173--197 (1963)]. It was demonstrated that isoleucyl-tRNA formation proceeded through a ping-pong mechanism in the presence of spermine and magnesium. From a study of the effect of spermine on both the formation of isoleucyl-AMP . enzyme complex and on the subsequent formation of isoleucyl-tRNA from the isoleucyl-AMP . enzyme complex, evidence is presented that the stimulatory effect of spermine functions mainly through the spermine preventing the tRNA inhibition of isoleucyl-AMP . enzyme complex formation.


A microsomal (cytochrome P-450)-linked lauric-acid-monooxygenase from aged Jerusalem-artichoke-tuber tissues. A lauric acid monooxygenase which catalyzes the formation of hydroxylaurate from lauric acid has been characterized in ageing tissues of Jerusalem artichoke (Helianthus tuberosus L.) tuber. Three reaction products have been identified from the mass fragmentation pattern of their methyltrimethylsilyl derivatives: 10-hydroxylauric acid, 9-hydroxylauric acid and 8-hydroxylauric acid. Enzyme activity is located on the microsomal fraction which also carries cytochrome P-450 and NADPH cytochrome-c reductase. The apparent Km of the enzyme for lauric acid is 0.97 micronM. Laurate  monooxygenation is dependent upon O2 and inhibited by CO. The latter effect is light reversible. NADPH is the preferred electron donor although appreciable NADH-sustained activity was observed. NADPH cytochrome c reductase is involved in electron transfer as evidenced by the inhibitory effects of NADP+ and oxidized cytochrome c on laurate monooxygenation. Thus, the enzyme catalyzing laurate oxidation in Jerusalem artichoke tuber tissues appears to be a typical (cytochrome P-450)-linked monooxygenase.


Cellobiose oxidase, purification and partial characterization of a hemoprotein from Sporotrichum pulverulentum. An extracellular enzyme which utilizes molecular oxygen to oxidize cellodextrins to the corresponding aldonic acids has been isolated from culture filtrates of the white-rot fungus Sporotrichum pulverulentum. This enzyme, tentatively named cellobiose oxidase, has been highly purified by classical techniques and has been demonstrated to be a glycoprotein with a molecular weight of approximately 93000. Ultraviolet spectra of the enzyme in the presence and absence of substrate are characteristic of a hemoprotein. Acidic hydrolyses of the enzyme followed by a spectrofluorimetric investigation of the hydrolysate has demonstrated the presence of approximately one flavin component per enzyme molecule. The possible role of this complex enzyme in cellulose degradation is discussed.


Blood-group-(A + H) complex fucolipids of hog gastric mucosa. A new group complex glycosphingolipids exhibiting blood-group-(A + H) activity was isolated from the lipid extract of hog gastric mucosa. Four components of this group have been purified to homogeneity and their structures and immunological properties were studied. The branched structures of glycolipids were identified by partial acid hydrolysis, sequential degradation with specific glycosidases, oxidation with periodate and chromium trioxide, and comparison of the permethylation products of the native and defucosylated compounds. The structures of these fucolipids are proposed to be as follows.


Regulation of translation of ovalbumin messenger RNA by estrogens and progesterone in oviduct of withdrawn chicks. In the oviduct of chicks withdrawn from previous treatment with estrogens, no ovalbumin synthesis can be detected, although there are a limited number of ovalbumin mRNA sequences. These sequences are predominately associated with membrane-bound ribosomes. However, the size of the polysomes is small compared to those from the laying hen, suggesting that the inability to detect ovalbumin synthesis is the result of inefficient initiation of ovalbumin synthesis. When the rate of peptide chain elongation is reduced by treatment of chicks with cycloheximide, there is an increase in the average size of polysomes and a shift of ovalbumin mRNA sequences from small to large-sized polysomes. Readministration of estrogen to withdrawn chicks results in a time-dependent shift of monosomes to polysomes and a proportional shift of ovalbumin mRNA sequences between the two fractions, indicating that estrogen stimulates the rate of initiation of all mRNA species in the oviduct to essentially the same extent. In contrast, progesterone administration results in a preferential shift of ovalbumin mRNA relative to total RNA, suggesting a preferential effect of progesterone on initiation of protein synthesis with ovalbumin mRNA.


The composition, structure and origin of proteose-peptone component 5 of bovine milk. Proteose-peptone component 5 has been isolated from bovine milk. Molecular weight values within the range 12000--13500 were obtained by sedimentation equilibrium, dodecylsulphate/polyacrylamide gel electrophoresis and gel filtration in urea-containing buffers. A dansylation procedure showed that the sequence Arg-Glu occupied the N-terminal position while hydrazinolysis revealed C-terminal lysine. The latter was confirmed by experiments with carboxypeptidases B and C which indicated that a mixture of molecules was present, about 80% of which had a C-terminal sequence -(Ala-Met)-Ala-Pro-Lys while about 20% had an additional -His-Lys in the terminal position. These results, together with data on the overall composition, showed that this component of the proteose-peptone fraction of milk corresponded to a mixture of molecules representing residues 1--105 and 1--107 of the beta-casein molecule, a finding that was confirmed by peptide mapping. This demonstration that proteose-peptone components correspond to the N-terminal portions of the beta-casein molecule while the gamma-caseins represent the matching C-terminal portions provides strong evidence in favour of a proteolytic mechanism for the formation of these substances in vivo and in vitro.


The composition, structure and origin of proteose-peptone component 8F of bovine milk. Proteose-peptone component 8F (or '8-fast') has been prepared from bovine milk. Sedimentation equilibrium analysis, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and gel filtration in urea-containing buffers all gave molecular weight values between 3300 and 3900. The N-terminal sequence was found to be Arg-Glu- by dansylation and Edman degradation. Hydrazinolysis released lysine from the C-terminus. A mixture of carboxypeptidases A and B showed that the C-terminal sequence was -Thr-(Arg,Ile,Asn)-Lys. The phosphate content was 3.8 mol/mol and was completely released by a short alkaline hydrolysis indicating linkage to serine. This and all other aspects of the composition were entirely consistent with the identification of this proteosepeptone as residues 1--28 of the beta-casein molecule. This identity was confirmed by a peptide mapping procedure. Thus proteose-peptone component 8F represents the N-terminal fragment when the gamma1-caseins are formed by proteolysis of beta-casein.


Evidence against the existence of real isozymes of hypoxanthine phosphoribosyltransferase. A method for reducing the degree of heterogeneity in the electrophoretic enzyme activity pattern of hypoxanthine phosphoribosyltransferase preparations by incubation with a (magnesium) phosphoribosyl diphosphate substrate is described. Hypoxanthine phosphoribosyltransferase was isolated from human erythrocytes and Chinese hamster livers. A subunit molecular weight of 26000--27000 as reported by other authors was obtained for both enzymes by gel electrophoresis in the presence of dodecylsulfate. Gradient gel electrophoresis revealed that the native enzymes mainly have a molecular weight of 105000--110000 and are thus apparently tetrameric, when held in the active state by the presence of phosphoribosyl diphosphate. The dimeric enzyme with a molecular weight of 52000--55000, was also found under other conditions. The trimer occurred only in the absence of phosphoribosyl diphosphate, for instance by glycerol gradient centrifugation. The enzyme from human erythrocytes was partly degraded during purification in the absence of a protease inhibitor. The purified enzyme has a very low protease contamination level. Proteolysis is an additional cause of heterogeneity and might therefore explain earlier conflicting results. Since the heterogeneous nature of hypoxanthine phosphoribosyltransferase is caused only by the secondary processes of dissociation/association and, in the case of the human erythrocyte enzyme, degradation, we suggest that the use of the term 'isozyme' to describe the different forms should be avoided.


Human serum galactosyltransferase: distinction, separation and product identification of two galactosyltransferase activities. Two different galactosyltransferase activities have been found in normal sera from A and O donors. Galactosyltransferase A incorporated galactose from UDP-Gal into sialic-acid-free ovine submaxillary mucin (asialo-mucin), whereas galactosyltransferase B transferred galactose from UDP-Gal to free N-acetylglucosamine or N-acetylglucosamine-glycoproteins. Specificity, kinetic and stability differences permitted the distinction of the activity of galactosyltransferase A from that of galactosyltransferase B; the only substrate found for galactosyltransferase A was asialo-mucin, whereas galactosyltransferase B showed only low activity towards asialo-mucin and free N-acetyl-galactosamine, but had a main specificity for either free N-acetylglucosamine or N-acetylglucosamine-protein. Galactosyltransferase B was more stable on heat inactivation than galactosyltransferase A; galactosyltransferase B could be separated from galactosyltransferase A by affinity chromatography on N-acetylglucosamine-derivatized agarose. The products of both enzyme activities have been analyzed. The galactosyltransferase A product was cleaved from asialo-mucin by alkaline-borohydride treatment. The acceptor used to identify the galactosyltransferase B product was free N-acetylglucosamine. Periodate oxidation studies performed on the reduced disaccharides indicated the linkage type of the products. The anomeric configuration of the respective galactosyltransferase products were determined with specific galactosidases. Using these methods, galactosyltransferase A was found to form a Galbeta (1 leads to 3)GalNAc-protein linkage and galactosyltransferase B was found to form a Galbeta(1 leads to 4)GlcNAc-linkage.


The biosynthesis of linoleate from oleoyl-CoA via oleoyl-phosphatidylcholine in microsomes of developing safflower seeds. Microsomes prepared from developing safflower seeds rapidly desaturated added [14C]oleoyl-CoA in the presence of NADH. The distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine was labelled with [14C]linoleate before any other lipid class investigated. Considerable desaturation continued after the disappearance of [14C]oleoyl-CoA in the reaction mixture. No [14C]linoleoyl-CoA could be detected. Incubation in the absence of added NADH caused a rapid disappearance of [14C]oleoyl-CoA by incorporation into triacylglycerols and phosphatidylcholine and also by release as [14C]oleic acid. Upon subsequent addition of NADH, [14C]linoleate was formed to the same extent as when NADH was present at the onset of the incubation. These data are contradictory to the previously assumed pathway for linoleic acid biosynthesis in developing safflower seeds, claiming oleoyl-CoA as the direct substrate and linoleoyl-CoA as the primary product. However, our data corroborate published results on Chlorella, Candida, Torulopsis and on developing pea leaves, which strongly suggest that oleoyl-CoA is first incorporated into phosphatidylcholine and then desaturated to linoleoyl-phosphatidylcholine.


Primary structure of histone H2A from gonad of the sea urchin Psammechinus miliaris. The complete amino acid sequence (125 residues) of sea urchin histone H2A has been established by structural studies of peptides derived from tryptic and chymotryptic cleavage of the maleylated protein and from thermolysin cleavage of the intact protein. By comparison with calf homologous histone, the basic amino-terminal and carboxy-terminal parts of the protein show 11 substitutions and 4 deletions. The remainder of the sequence, mostly hydrophobic, is almost completely unchanged.


Isolation and characterization of the lipopolysaccharide of Thiocapsa roseopersicina. The lipopolysaccharide from Thiocapsa roseopersicina was isolated by phenol/water, being found in the water phase. It is cleaved into a polysaccharide moiety (degraded polysaccharide) and lipid A by hydrolysis with 10% acetic acid (100 degree C, 3 h). D-Mannose, L-rhamnose, 3-amino-3, 6-dideoxy-D-galactose and D-glucose are the major constituents of the degraded polysaccharide. 2-O-Methyl-L-rhamnose, 3-O-methyl-D-mannose, D-galactose, glucosamine and quinovosamine are minor constituents. D-Glycer-D-manno-heptose (tentatively identified) and 3-deoxy-D-manno-octulosonic acid were detected in only small amounts. Conspicuously, lipid A from T. roseopersicina contains a neutral sugar, D-mannose, in addition to D-glucosamine, as had been observed with lipid A from Chromatium vinosum D. Major fatty acids are beta-hydroxymyristic and lauric acids. Only trace amounts of phosphorus were found indicating this lipid A to be free of phosphate. The lipopolysaccharide of T. roseopersicina represents the O-antigen of the strain. It reacts with antisera prepared against living or heat-killed cells in passive hemagglutination.


Anthracycline antitumor antibiotic nucleic-acid interactions. Structural aspects of the daunomycin poly(dA-dT) complex in solution. The helix-to-coil transition of the synthetic DNA poly(dA-dT) in the presence of the anthracycline antitumor antibiotic daunomycin has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectrocopy in 1 M salt solution. The dissociation of the complex, containing molar ratios of phosphate to daunomycin (Pi/drug) of 50, 25, 9 and 5, with increasing temperature can be monitored independently at the nucleic acid and the antibiotic resonances under conditions of fast exchange. The antibiotic complex formation shifts suggest that either ring B and/or C of the intercalated anthracycline chromophore of daunomycin overlaps with adjacent nucleic acid base pairs. Ultraviolet/visible melting studies of daunomycin complexes with a series of synthetic DNAs substituted with halogen atoms (Br, I) at position 5 of the pyrimidine ring suggest that intercalation of the antibiotic into poly(dA-dU) is not perturbed by bulky substituents at this position. A comparison of the melting curves for the daunomycin . poly(dA-dT) complex with an analog of the antibiotic where the NH3 + group is replaced by dimethylglycine demonstrates the important contributions of electrostatic interactions between the amino sugar and backbone phosphates to the stability of the complex in low salt solution. The ultraviolet/visible and NMR studies monitor biphasic melting transitions at the nucleic acid markers in the daunomycin . poly(dA-dT) complexes, Pi/drug = 50--9, so that antibiotic-free base-pair regions and those centered about bound daunomycin can be independently studied at the synthetic DNA level in solution.


Studies on the occurrence of disialosyl groups in glycoproteins of salivary glands. The major glycoprotein of pig submandibular and rat sublingual glands were investigated for the presence of disialosyl groups. The content of (C-8)-substituted sialic acid in the glycoproteins was determined by three different procedures: colorimetric, periodate oxidation and permethylation. Approximately 13% of sialic acid in pig submandibular glycoprotein, but none in rat sublingual glycoprotein, contained a substituent at C-8. This substituent was identified as sialic acid from its susceptibility to neuraminidase treatment. From our estimation, it appears that one out of four sialic acid residues in pig submandibular glycoprotein occurs in an N-acylneuraminyl-alpha(2 leads to 8)-N-acylneuraminyl group.


A nuclear-magnetic-resonance study of the globular structure of the H5 histone. The structure of the globular region of the chicken erythrocyte H5 histone has been studied by 270-MHz proton magnetic resonance. The aromatic resonances have been partially assigned by a combination of selective deuteration and iodination with the nuclear magnetic resonance spectroscopy. Detailed titration studies have revealed interactions between residues in the structure. A technique involving the measurement of small nuclear Overhauser effects has enabled  the assignment of the aromatic residues causing the perturbation of the ring-current-shifted methyl resonances occurring in the upfield region of the spectrum. Spin-decoupling experiments on these peaks has enabled a partial assignment of shifted methyl resonances. The results support the notion that the histone H5 globular structure is different from that of the homologous histone H1 molecule.


Manipulation of phospholipid composition of membranes with the aid of lipid exchange proteins. Incorporation  of phosphatidylcholine into protoplasts of Micrococcus lysodeikticus. Incubation of Micrococcus lysodeikticus protoplasts with phosphatidylcholine liposomes and rat liver exchange proteins (pH 5.1 supernatant fraction) resulted in replacement of about one half of the bacterial total phospholipids by phosphatidylcholine. Protoplasts modified by phosphatidylcholine showed a decreased rate of oxidation of exogenous substrates (NADH, malate) and decreased ferricyanide reductase activity as compared to the initial protoplasts. At the same time incorporation of phosphatidylcholine had no influence on the level of endogeneous respiration. Protoplasts modified by phosphatidylcholine were osmotically more stable than the initial protoplasts. After osmotic lysis of the phosphatidylcholine protoplasts their NADH (malate) oxidase and ferricyanide reductase activities were restored. Incorporation of phosphatidylcholine into membrane ghosts, obtained by osmotic rupture of the initial protoplasts had only small if any effect on the malate and NADH oxidase and dehydrogenase activities. It is concluded that phosphatidylcholine in incorporated predominantly into the outer part of cytoplasmic membrane and that proteinmediated transfer of phosphatidylcholine results in restoration of the permeability barrier due to repair of local defects in the initial protoplast membrane.


Naja haje haje (Egyptian cobra) venom. Some properties and the complete primary structure of three toxins (CM-2, CM-11 and CM-12). Three toxins (CM-2, CM-11 and CM-12) were purified from Naja haje haje (Egyptian cobra) venom. Whereas toxin CM-11 contains 65 amino acid residues and five intrachain disulphide bridges, toxin CM-2 and CM-12 comprise, respectively, 61 and 62 residues but both contain four disulphide bridges. The complete primary  structures of the three toxins have been established. The sequence and the invarient amino acid residues of CM-2 resemble those of part of a long neurotoxin, a short neurotoxin and a cytotoxin. The sequence of CM-11 reveals that it is a homologue of the neurotoxins and to some extent also a cytotoxin. The immunochemical properties and the sequences of CM-12 suggest that it is related to the cytotoxin group. Further, the sequences of CM-11 and CM-12 from Naja haje haje venom show a high degree of homology with those of the corresponding toxins isolated from NaJA annulifera or NaJA melanoleuca venoms.


Phosphorylase kinase isoenzymes in deficient ICR/IAn mice. ICR/IAn mice present a deficiency in phosphorylase kinase activity; the extent of this deficiency is less in some tissues [Lyon, S.B. Biochem. Genet. 4, 169--185 (1970)] than in skeletal muscle, where enzyme activity is 0.3% of normal [Cohen, P.T. W & Cohen, P. FEBS Lett. 29, 113--115 (1973)].  New-born mice of this strain were also reported (Lyon, 1970) to reveal a small amount of skeletal muscle enzyme activity. The properties of these residual phosphorylase kinases were compared to those of control C57 BL mice, with reference to control muscle and liver enzymes which were shown to be of different molecular species [Daegelen-Proux et al. Biochim. Biophys Acta, 452, 398--405 (1976)]. The properties investigated were the immunological reactivity against an antiserum raised against muscle phosphorylase kinase, the thermal stability and the Ca2+ dependency. The results suggest that the muscle enzyme from the new-born ICR/IAn mice and the heart enzyme from adult deficient mice are different to the muscle enzyme from adult normal mice, but they have properties in common with normal adult liver enzyme. These results lead to the conclusion that there exists in the muscle of I strain a "foetal form" of phosphorylase kinase, the activity of which decreases progressively after birth. Out work also confirmed the observations made by Cohen et al. [Eur. J. Biochem. 66, 347--356 (1976)] which showed that there is no evidence for the existence of a cross-reacting material in the muscle of adult deficient mice.


Degradation of the ribosomal genes by DNAse I in Physarum polycephalum. Treatment of nuclei from Physarum polycephalum with DNAse I leads to DNA fragments with a regular pattern of multiples of 10 nucleotides, when analyzed on gels under denaturing conditions as has been shown for other eukaryotes. Reports from Weintraub and Axel lead to the conclusion, that active genes are preferentially digested by DNAse I. When Physarum chromatin is degraded by DNAse I, the ribosomal genes are no longer available for hybridization with 19-S and 26-S rRNA and are thus preferentially destroyed. Degradation of chromatin from nuclei in mitosis, where no rRNA is synthesized and from nuclei in late G2 phase, where rRNA synthesis is maximal, leads to the same proportion of the ribosomal sequences being lost for hybridization. Therefore the preferential degradation of the ribosomal genes in Physarum by DNAse I probably does not reflect the actual momentary activity of these genes. This suggests that DNAse I treatment may distinguish between active chromatin and very strongly repressed chromatin.


Message sequences are not adjacent to poly(A) in heterogeneous nuclear RNA of Friend leukemic cells. Hybridization of labeled RNA with excess amounts of unlabeled complementary DNA (cDNA) was used to investigate the location of cytoplasmic mRNA sequences in heterogenous nuclear RNA (hnRNA) of noninduced Friend leukemic cells. A heterologous hybrid between hnRNA and cytoplasmic cDNA was formed. Two homologous hybrids were also formed, one between poly(A)-containing mRNA and cytoplasmic cDNA, and the other between poly-(A)-containing hnRNA and nuclear cDNA. All hybrids were selected on hydroxyapatite columns after RNase treatment. The hybrids were further investigated for the presence of poly(A). No poly(A) was found in the heterologous hybrid, while both homologous hybrids contained poly(A). From these results we conclude that there exists a spacer nucleotide sequence between the poly(A) and the message sequences in hnRNA.


Purification and properties of a ribonuclease induced during the early larval development of Artemia salina. Dormant gastrulae and developing embryos of the brine shrimp Artemia salina contain very low levels of nuclease activity. During early larval development, there is an induction of ribonuclease which has been partially purified and characterized. The enzyme catalyzes an endonucleolytic cleavage of RNA and has no detectable activity on native or denatured DNA. Among a series of synthetic polynucleotides, poly(U) is hydrolyzed with the highest efficiency and poly(G) is not cleaved by the enzyme. The activity on poly(U) is 100 times higher than on RNA. The enzyme requires Mg2+ or Mn2+ and in inactivated by treatment with chelating agent. The inactive preparations can be reactivated by Ca2+ and Mn2+ but not by Mg2+. The ribonuclease is thermosensitive and has maximal activity at pH 7.5. These properties distinguish the Artemia salina ribonuclease from other eukaryotic ribonucleases already reported. The high activity and specificity of this ribonuclease on poly(U) may suggest a role for this enzyme in the processing of the messenger RNA.


Photo-induced protein-RNA cross-linking in mammalian 40-S ribosomal subunits. RNA-protein interaction in the 40-S subunits of rat liver ribosomes were studied by measuring cross-linking of proteins to RNA induced by ultraviolet radiation. Under conditions which caused neither extensive degradation of the 40-S subunits (or 18-S RNA) nor biological inactivation, the total staining intensity of the proteins extracted from irradiated subunits was considerably reduced on the two-dimensional electrophoregrams. Convincing evidence was obtained that cross-linking of the proteins to 18-S RNA was the predominant reaction. The cross-linking extent of the individual proteins was studied as a function of the radiation dose. At 4 degree C, 13--15 proteins were found to cross-linked to RNA even at low doses of quanta. They generally correspond to proteins which have been previously shown to react poorly on the ribosomes with various chemical reagents. At 25 degree C, all the proteins became cross-linked to RNA using the same radiation doses.


Second-derivative spectroscopy of proteins. A method for the quantitative determination of aromatic amino acids in proteins. Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290-295-nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.


Identification and characterization of ribosomal proteins phosphorylated in vaccinia-virus-infected HeLa cells. Two-dimensional analysis of 32P-labelled ribosomal proteins revealed three proteins which are phosphorylated in vaccinia-virus-infected HeLa cells. All three proteins belong to the 40-S ribosomal subunits and were identified as S2, S6 and S16. The ribosomal protein S6 is phosphorylated also in uninfected HeLa cells. Phosphoserine was detected in all three proteins, phosphothreonine only in the protein S2. Phosphorylation of these ribosomal proteins in infected cells is dependent on the multiplicity of the viral infection and increases during the first six hours of infection. All three proteins are also phosphorylated in virus-infected cells treated with cycloheximide and in cells infected with ultraviolet-irradiated virus. This suggests that the phosphorylation reaction involves a vaccinia virion-associated protein kinase.


Chemical composition of cholinergic synaptic vesicles from Torpedo marmorata based on improved purification. Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata have been purified to a constant composition and a higher transmitter content than previously reported. By optimising the extraction conditions and using a two-step purification on discontinuous and continuous sucrose density gradients, 10-fold higher acetylcholine and ATP values per weight of protein were obtained. The purity of the vesicle preparation was confirmed by electronmicroscopy, absence of marker enzymes, behaviour in density gradient centrifugation, as well as by a specific and reproducible protein composition. Vesicles contain 6.9 mumol acetylcholine and 1.0 mumol ATP per mg protein. The lipid/protein ratio of 3.5 (w/w) indicates a lipid-rich membrane. The value suggests the absence of a proteinaceous core. Upon dodecylsulphate gel electrophoresis a distinct protein pattern is obtained with components ranging from 20000 to 160000 in molecular weight. Vesiculin, reported earlier to be a low-molecular-weight vesicle protein, is not detected. One of the major bands comigrates with muscle actin from the same animal. Further characterisation of this protein by two-dimensional gel electrophoresis suggested that it is an actin-like polypeptide. Evidence for a specific association of this actin-like protein with vesicles and its possible involvement in the neurosecretory process is discussed.


Physico-chemical evidence for the interaction between aldolase and glyceraldehyde-3-phosphate dehydrogenase. Polarization of fluorescence measurements of aldolase and D-glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocyanate have been used to detect the possible formation of a soluble complex between the proteins. The results suggest an interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase with an apparent dissociation constant 3 X 10(-7) M and an apparent stoichiometry of two aldolase tetramers bound per tetramer of D-glyceraldehyde-3-phosphate dehydrogenase.


RNA-binding proteins of rabbit reticulocytes. Isolation and electrophoretic characteristics. A complete set of RNA-binding proteins was isolated from the ribosome-free extract of rabbit reticulocytes using the method of affinity chromatography on RNA covalently coupled with Sepharose. The purity of the isolated proteins was no less than 90%. These proteins comprised about 1% of the total protein of the extract and included the main polypeptide chains of three sizes, with molecular weights of about 95000, 49000 and 36000, as well as numerous minor components. An analogous set of proteins was observed as a result of chromatography of the extract on the column with poly(U) covalently coupled with Sepharose. The protein with the molecular weight of 49000 had the highest affinity to RNA.


Studies on the structure of rat-liver polysomes by small-angle X-ray scattering. The spatial structure of rat liver polysomes in dilute solution has been studied by the small-angle X-ray scattering technique. The distance between the mass centres of neighbouring ribosomes on the mRNA molecule is 35 nm, and the distance from next-but-one neighbour, about 55 nm. The spatial arrangement of the ribosomes on the polysome is compact; stretched configurations can be ruled out. The best model is a solenoid with six ribosomes per turn and a pitch of about 33 nm. The greatest diameter of a polysome with eight ribosomes is 130 nm, the radius of gyration 34 nm.


Transcription in vitro of adenovirus-2 DNA by RNA polymerases class C purified from uninfected and adenovirus-infected HeLa cells. DNA-dependent RNA polymerase class C (or III) has been solubilized from either uninfected or adenovirus-2-infected HeLa cells and purified by chromatography on phosphocellulose, DNA-cellulose, CM-Sephadex and DEAE-Sephadex. The last column separated the enzyme into three forms CI, CII and CIII, which were completely free of RNA polymerases class A and B and of DNase and RNase. The total and the relative amount of these different enzyme C forms did not vary whether purified from uninfected or infected cells. Irrespective of the stage of purification, the three enzyme forms transcribed deproteinized adenovirus-2DNA very efficiently. This transcription was highly sensitive to elevated ionic strength (especially in the presence of Mg2+) and was accompanied by continuous reinitiation as shown by adding poly(rI), a potent inhibitor of initiation. In addition heparin-resistant initiation complexes could be formed at elevated temperature. The RNA synthesized in vitro on deproteinized intact adenovirus-2 DNA by the different forms of RNA polymerase class C, has been characterized. Analysis of the transcripts by gel electrophoresis, RNA self-annealing, hybridization to separated adenovirus-2 DNA strands and to restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that the various regions of the adenovirus-2 genome were randomly transcribed. In addition, hybridization of RNA transcripts labelled at their 5' end by either  [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected). The results are discussed in terms of the components which are possibly involved in specific transcription.


Subacute necrotizing encephalopathy (Leigh) in an adult. The case of a 31-year-old man who died after a 4 month illness of adult subacute necrotizing encephalomyelopathy (Leigh) is reported. The disease presented with visual disturbances and the principal symptoms were ptosis, a conjugate ophthalmoparesis and a slight tremor of the hands. The case was misdiagnosed as probable multiple sclerosis. Neuropathology disclosed characteristic symmetrical necrotizing lesions, mainly localized in the brain stem. The similarity of the lesions with Wernicke's disease is pointed out. Possible etiological and pathogenetic factors are discussed.


Steroid-CPK test. A new diagnostic aid for muscular dystrophy and its carriers? The response of serum creatine phosphokinase (SCPK) to intravenous hydrocortisone was studied in different neuromuscular diseases, in Duchenne carriers and relatives of various muscular dystrophy (MD) cases. SCPK activity increased significantly in MD cases, 50% of known and 18.7% of possible Duchenne carriers. No such increase was found in other neuromuscular disease, in other relatives of MD cases and in normal controls. An inverse correlation was observed between the grade of disability and post-steroid percentage increase of SCPK activity in X-linked severe (DMD) cases. Such an inverse correlation was also found between the duration of the disease and post-steroid percentage increase of SCPK activity in DMD cases. A possible explanation is given.


The dependence of cerebral blood flow on age. Cerebral circulation time was studied with angiography of the carotid artery within a group of 45 normal cases of different age. The results showed an approximately linear increase of the circulation time with age, the coefficient of which amounts to 1% per year. For an average age of 35 years the average circulation time was determined and found to be 3.2 sec with narcosis and 4.3 sec without narcosis. This indicates that narcosis is another important parameter for the cerebral circulation.


Nerve cell loss with aging in the putamen. A significant correlation between age and decrease in the number of nerve cells has been found in the putamen. Both small and large cells in this nucleus were proportionally involved. A possible relationship with the outfall of cells in other structures of the brain, with which the putamen is connected, in order to balance the activity of the extrapyramidal system has been suggested.


Motor nerve conduction velocity in experimental portal-systemic shunting. Preliminary communication. The influence of portal-systemic shunting on motor nerve conduction velocity (MCV) was analyzed in rats with portacaval shunts. At 3 and 8 weeks after the shunting there was a marked but transient fall in MCV. At 16 weeks the MCV had almost normalized, despite sustained portal-systemic shunting. The results favour hepatocellular failure as the more important pathophysiological mechanism in hepatic neuropathy.


Effect of a xanthine oxidase inhibitor on adenine nucleotide degradation in hemorrhagic shock. Biochemical effects of treatment with a xanthine oxidase inhibitor (allopurinol) were investigated in an experimental hemorrhagic shock procedure. Allopurinol pretreatment abolished the increase in plasma uric acid which occurs in untreated dogs during hemorrhagic hypotension and resulted in a much lesser increase in plasma allantoin. The pancreatic, liver and duodenal adenosine triphosphate (ATP) and total adenine nucleotides of untreated dogs were severely reduced, while those of allopurinol-pretreated dogs were essentially normal 2 h following reinfusion. Pretreatment with allopurinol resulted in a significantly lesser release of the lysosomal enzymes, acid phosphatase and beta-glucuronidase, following reinfusion. When treatment was delayed until after reinfusion, an infusion of hypoxanthine + allpurinol restored normal ATP concentrations. The role of adenine nucleotide breakdown in irreversible shock is discussed.


Effect of ex vivo perfusion of isolated canine stomach with fluorocarbon on the composition of gastric tissues. Isolated canine stomachs were perfused for 6 h with fluorocarbon emulsion suspended in a simulated physiological solution of electrolytes with glucose added. Gastric mucosa and extramucosal tissues of perfused stomachs were sampled for biochemical analysis of high energy phosphates, glucose and electrolytes. Comparable samples were also taken from normal canine stomachs, dissected under similar surgical conditions as the stomachs used for perfusion. Gastric tissue ATP, ADP and AMP were reduced but CP was increased in the tissues perfused with fluorocarbon as compared with controls. Gastric tissue water content and sodium were increased but potassium was reduced in a stomach so perfused. The biochemical tests performed were considered as viability tests of organs preserved-perfused in fluorocarbon prior to possible transplantation. Significance of the changes observed is discussed in the light of the current knowledge on energy metabolism.


Modulation of the prostaglandin-induced intestinal motility in humans through the transmitters of the vegetative nervous system. Research was done on the reciprocity between adrenergic and cholinergic stimulating or inhibiting pharmaceuticals and the prostaglandin-induced intestinal motility. By cholinergic activation, the amplitudes of the prostaglandin-dependent rhythmical contractions of human taenia coli were intensified. There was very little influence on the frequency of contractions, however. Simultaneous stimulation of adrenergic beta-receptors by increasing doses of adrenaline or noradrenaline caused the contraction amplitude and frequency to decrease continually until the contractions were completely eliminated. The cholinergic effect could be suppressed with atropine, the adrenergic-stimulated reaction was reversed by the blockage of beta-receptors. It was completely abolished by simultaneous addition of alpha- and beta-receptor-blocking drugs.


Release of inflammatory mediators into the gastric wall of rats with acute acetic acid induced ulcer. An acute, acetic acid induced gastric ulcer in rats is associated with increased capillary permeability and albumin leakage into the gastric wall surrounding the ulcer. This is an inflammatory reaction, and the present experiment was undertaken in an attempt to identify inflammatory mediators in the gastric wall around the ulcer. Rats were pretreated with inflammatory antagonists, and the protein leakage was estimated by means of 125I-labelled albumin 40--60 min and 4 h after ulcer induction. We found no effect on the protein leakage of mepyramine (antihistamine) deseril (antiserotonin) or indomethacin (antiprostaglandin) given separately, or of the combination of mepyramine and deseril. The combination of all three substances, mepyramine, deseril and indomethacin significantly decreased protein leakage at 4 h after ulcer induction. This indicates that prostaglandins are released in the tissues near the acute ulcer. Cimetidine (histamine-H2-antagonist) did not decrease the protein leakage either alone or in combination with mepyramine (histamine-H1-antagonist).


Ex situ surgery of the kidney: experience with 20 autotransplantations. The authors propose a classification of ex situ surgery of the kidney and present their clinical experience of 20 renal autotransplantations over a period of 6 years: 16 were performed for renovascular hypertension with 2 failures and 4 for recurrent staghorn calculi, with 1 stone recurrence. Based on their experience and on the literature they discuss the present status and the future implications of ex situ surgery of the kidney.


Primary hyperparathyroidism and stone formation in medullary sponge kidneys. The etiological role of primary hyperparathyroidism in stone formation in medullary sponge kidney was studied. The necessity of carrying out special biochemical tests for primary hyperparathyroidism has been shown. The combination of two diseases in cases of stones and nephrocalcinosis is explained by the genetic nature of sponge kidney and primary hyperparathyroidism. 4 patients have been under observation; all underwent successful parathyroidectomy.


Continuous determination of various enzymes and sodium concentration in urine. a usable method for diagnosis of kidney graft rejection. We investigated changes in urinary enzyme activity and sodium concentration of kidney transplant patients. We found that the increase of the activity of brush border enzymes is one of the earliest signs of tubular damage following rejection. The decrease in the urinary sodium concentration points also to rejection episodes. Despite the unspecificity of both variables, their continuous determination and combined analysis of the results could improve the differential diagnosis of rejection after transplantation and might give important information about the pathogenesis of the graft damage.


Radical surgery for renal cell carcinoma. A modified radical nephrectomy to cope with renal carcinoma presenting at advanced stages, is evaluated. A total of 50 cases were treated in this hospital from 1962 to 1976. The poor results from treatment of renal cell carcinoma in the earlier years, have prompted the modification of the conventional radical nephrectomy to cope with this disease which is still frequently seen at advanced stages. The modification includes exclusion of distant metastasis. The adherent segment of colon is resected along with the tumour. The renal vessels are then isolated. If the IVC is involved by a right-sided tumour, it is resected along with the tumour. The ureter is then ligated and the tumour with the adrenal gland, the resected colon segment, regional lymph nodes and the IVC are removed in one block. 2 such patients who otherwise would have been considered inoperable have survived for over 3 years.


Usefulness and limitation of estrogen receptor protein (ERP) assay in human renal cell carcinomas. The presence of estrogen-binding sites in human renal cell carcinomas appears certainly demonstrated. The authors analyze the clinical contributions which may be obtained from this finding and the possibility of using the ERP assay currently for the screening of patients with estrogen-dependent tumors.


Urinary fibrinogen degradation products (FDP) in bladder cancer. The value of urinary fibrinogen degradation products (EPD) as a biological marker of bladder cancer has been studied. An increase in FDP was found in 75% of bladder cancer patients, independent of the fact that they had active tumours or that they were disease free. A high correlation between the amount of the increase and the grade, local invasiveness, and risk of recurrence of the tumour has been established. The assessment of urinary FDP is considered as a valuable screening and follow-up test in patients with bladder neoplasms.


Characterization of bladder tumours by flow cytometry on bladder washings. The DNA content of individual nuclei in bladder washings was estimated by means of flow cytometry. A method of preparation using a detergent to produce single nuclei was used. By means of a differential count the DNA distribution was corrected for the content of leucocytes. The DNA histograms were divided into a 2c fraction containing nuclei with less than 3c DNA, a 4c fraction with 3c--6c DNA and an 8c fraction with more than 6c DNA. Three groups of patients were investigated: 17 patients with invasive bladder tumours, 38 patients with non-invasive bladder tumours, and 22 patients with normal bladders. The washings from patients with invasive tumours contained significantly larger 4c and 8c fractions than washings from normal bladders. There was a correlation between cell anaplasia and the DNA distribution: grade III bladder tumours had larger 4c and 8c fractions than grade II tumours.


On the etiology of cancer of the prostate. This paper demonstrates experimentally that the penetration of spermatozoa into the prostatic tissue can induce prostatic lesions such as abscesses, lithiasis and carcinoma.


Experimental and clinical investigations on the transfusion of autologous blood in urological operations. In view of the known risks in homologous blood transfusions, the procedures for transfusion of endogenous blood are gaining increasing importance. Using this fact as a basis, the feasibility of direct intraoperative autotransfusion in the area of urology was investigated. 6 mongrel dogs received blood-urine autotransfusions after cystotomy and cavotomy (n = 2) as well as after left-side nephrotomy (n = 4). Coagulation and hemolysis parameters were examined 1, 2, 24 h and 7 days after the autotransfusion, and showed transitory pathological changes which were, without exception, reversible within 7 days. Clinical effects were not observed. On the basis of favorable experience, the intraoperative, machine autotransfusion was also used clinically for renal traumas (n = 13), urinary bladder traumas (n = 4) and nephrotomies (n = 3). Postoperative checks showed pathological changes in the hemolysis and coagulation values only during the first 3 postoperative days. The blood-urine autotransfusion was well tolerated without exception. Indications and contraindications for direct intraoperative autotransfusions in the area of urological operations are discussed.


Alloplastic replacement of the canine ureter by expanded polytetrafluoroethylene (gore-tex) grafts. Preliminary report. In 12 female dogs, subtotal unilateral (8 dogs) or bilateral (4 dogs) ureteric resection and replacement by expanded polytetrafluoroethylene (Gore-Tex) prostheses were performed. Follow-up consisted of periodic excretory urographies. Short- and long-term results, consisting of periodic excretory urographies for up to 12 months, are promising and induce further studies with this new type of graft.


Cystic kidneys, renal dysplasia and microdissection data in 5 children with congenital valvular urethral obstruction. A survey of the findings in 5 children with urethral obstruction due to valves is presented. The microdissection data found in the 7 kidneys of this series were compared. The microdissection findings in all 7 kidneys are mostly the same with a continuum of processes ranging from relatively mild to severe derangements. We could not find two varieties of renal change designated type II or IV by potter. The characterization of the lesions by nephron dissection always approached those of the type II (multicystic, dysplastic kidney). Renal dysplasia was associated with extrarenal malformations in 2 of our cases and we therefore believe that the significance of the dysplasia lies rather in the period of the insult during organogenesis than in the obstruction.


Fungus ball formation of aspergillus in the bladder. an unusual case report. An unusual case of fungus ball formation of Aspergillus in the bladder without any evidence of disseminated and renal aspergillosis is presented. A 49-year-old man whose chief complaint was progressively worsening dysuria after a stomach operation was admitted. Cystoscopy revealed many ball-like masses on the retrotrigone and left wall. Histological studies showed that these masses were composed of many Aspergilli. The uniqueness of the case and the route of infection are discussed.


Variant forms of angina pectoris.

Variant forms of angina pectoris. 3 patients with different clinical and electrocardiographic manifestations of coronary artery spasm are discussed. All 3 patients had anginal attacks at rest. In addition, 2 of these patients, who did not have significant preexisting narrowing of their coronary arteries, also had anginal pain related to exercise. During pain, 1 patient showed ST-segment elevation, the other ST-segment depression, while the third showed ST-segment depression shortly followed by ST-elevation on the electrocardiogram. At coronary  angiography, spontaneous or induced spasm of one of the major coronary arteries could be demonstrated in all 3 patients. In 2 cases, sublingual nitroglycerin failed to completely relieve the spasm. This raises the question whether a residual stenosis after NTG conclusively proves a fixed organic narrowing. It is concluded that the clinical spectrum of spasm of the coronary arteries is wider than was originally reported by Prinzmetal and coworkers. Clinical and electrocardiographic manifestations are probably dependent on the site and severity of the spasm, which may cause different degrees of myocardial ischemia.


Complete heart block due to granulomatous giant cell myocarditis: report of 3 cases. 3 patients with chronic complete AV block were found at autopsy to have granulomatous giant cell myocarditis (GGCM). In 1 patient an unusual clinical course led to more extensive investigation including echocardiography which revealed ventricular septal abnormalities. A review of the literature is presented. Although GGCM is a rare disease echocardiography may be a useful screening procedure in patient with AV block especially in the presence of immunological disorders.


The electrocardiographic response to exercise in 44 patients with leaflet prolapse. The response to electrocardiographically monitored submaximal exercise stress testing has been studied in 44 patients with mitral leaflet prolapse (MLP). With exercise, ventricular premature contractions occurred in 7, ventricular tachycardia in 1, and atrial fibrillation in 1. Exercise was terminated short of target heart rate in 18 patients, because of chest pain (5), fatigue (7), ventricular arrhythmia (4), dizziness (1) or ST segment depression (1). 23 patients developed postexercise ST segment abnormalities, of whom 5 had 'ischemic' patterns and arteriographically proven coronary artery disease (CAD); among the 18 others, the ST segments were depressed and minimally downsloping in 2, slowly ascending from depressed J point in 3, horizontal for greater than or equal to 80 msec with J depression of less than 1 mm in 12, and cupped in 1. The incidence of arrhythmias provoked by submaximal exercise stress testing in patients with MLP was lower than suggested in previous reports. In all 5 cases where MLP and CAD coexisted, the classical 'ischemic' electrocardiographic response to exercise was not obscured. Even in the absence of CAD, postexercise ST segment abnormalities were common with MLP (18/39 = 46%) and differed from the progressively resolving ST segment deviation characteristic of CAD with angina. Exercise testing can safely be recommended, subject to standard contraindications, in patients with MLP and yields useful information.