Human psychophysical analysis of receptive field-like properties: V. Adaptation of stationary and moving windmill target characteristics to clinical populations. This paper describes the application of several key parameters of a windmill-shaped target (used in determinations of the psychophysical transient-like function) to clinical populations as a diagnostic tool for static perimetry. A technique for independently analyzing sustained- (Westheimer function) and transient-like retinally-based psychophysical responses is outlined, and stimulus characteristics, reliability and diagnostic potential of the tests are examined. Several particularly interesting clinical cases (one closure of a branch retinal arteriole) and two senile macular degeneration patients) are presented for illustrative purposes. In addition, a tentative 'working hypothesis' is presented as a basis for extensive future analysis of various clinical populations.


Early surgery and visual correction of an infant born with unilateral eye lens opacity. A healthy neonate born with unilateral cataract was operated upon on Life Day 4. Visual correction and testing were initiated promptly. Resolution showed continuous improvement in the normal eye, with no improvement noted in the operated eye until adequate visual correction was provided. Following correction there was prompt marked improvement of visual resolution.


Kinetic analysis of hormone-induced mitoses in epithelial cells of mouse uterus and vagina. The intracellular localization of 3H-estradiol-17beta and 3H-progesterone to the different types of cells in the mouse uterus was investigated using autoradiographic techniques. The kinetics of cell proliferation in the surface epithelium of the uterus and in the vaginal epithelium (basal layer) are analysed by means of cumulative labeling method and mitosis chase method using 3H-thymidine autoradiographic procedures. The results are as follows, (1) Epithelial cell population of the uterine lumen and basal cell population of the vaginal epithelium in the ovariectomized mouse are divided into a major subpopulation of GO cells and a minor subpopulation of proliferating cells. (2) Proliferative potencies of uterine surface epithelial cells and vaginal basal cells in the ovariectomized mouse are regulated by a steroid-independent mechanisms through which the proportion of the GO cell-compartment and Tc value of the proliferating cell-compartment are determined according to their age; as the castrated mouse grows older, Tc value becomes longer and the proportion of the Go cell-compartment becomes larger. (3) If the dose levels of estrogen administered exceed the threshold value, estrogen-dependent cell proliferation will be provoked by transferring the cells in the GO cell-compartment to the proliferating cell-compartment in all or none fashion, and by reducing the Tc value of proliferating cell to 1/2-1/3 of that in the castrated mouse. (4) It is suggested that proliferating cells in the uterine surface epithelium and in the vaginal epithelium turn the cell cycle at a constant Tc value during estrous cycle, and that the tissue growth during estrous cycle is dependent on the size of the proliferating cell-compartment but not on the Tc value. (5) The results obtained from autoradiography of tritiated steroids in the mouse uterus gave a supporting clue to the kinetic data.


Effects of clomiphene citrate and progesterone on resting and proliferative cell-populations in resting and proliferative cell-populations in mouse uterine epithelium. Resting cells (GO cells) of the uterine surface epithelium in castrated mice began to synthesize DNA with high synchrony from 10 hr after the injection of 50 ng of estradiol-17beta with or without 5.5 mug of clomiphene citrate. Highly synchronized DNA synthesis in GO cells elicited with estradiol was delayed approximately 5 hr when the simultaneous administration of 0.5 mg of progesterone was given. In GO cells of castrated mice which received 5.5 mug of clomiphene or 55 mug of clomiphene plus 50 ng of estradiol, DNA synthesis with partial synchrony began 15 hr after the injection. The effects of estradiol were completely suppressed by the administration of 55 mug of clomiphene. It is suggested that the inhibitory action of clomiphene may be due to the competitive blocking of estrogen binding, while progesterone suppresses the estrogen-induced DNA synthesis of the surface epithelium and transfers them to the GO cell-compartment.


No effect of pinealectomy on the parallel shift in circadain rhythms of adrenocortical activity and food intake in blinded rats. Twenty-four-hr patterns of plasma corticosterone levels were determined at 4-hr intervals every 3-4 weeks in sighted and blinded pinealectomized rats of adult age. Through the whole period of the experiment, 24-hr patterns of food intake were also measured weekly. The sighted rats manifested the same 24-hr patterns of plasma corticosterone levels and food intake for 15 weeks after pinealectomy as those observed in the intact control rats. The magnitude of peak levels of plasma corticosterone and the amount of food intake did not differ between the two groups. A phase shift in circadian rhythms of plasma corticosterone levels and food intake was observed in both groups of blinded rats, with and without pinealectomy. Between the two groups, the patterns of phase shift were essentially similar for 10 weeks examined after optic enucleation. The peak elevation of plasma levels took place at 11 p.m. at the end of the 4th week after optic enucleation. Thereafter, 4- to 8-hr delay of peak appearance was observed every 3 weeks. No significant differences were found in peak values between the two groups of blinded rats. Furthermore, the circadian rhythm of food intake shifted in parallel with that of plasma corticosterone levels. A phase reversal of these two activities was observed between the 8th and 10th week after the operation. These results indicate that the pineal gland does not play any important role either in the maintenance of normal circadian periodicities of adrenocortical activity and food intake or in the shift in circadian rhythms of the two activities in the blinded rats.


Failure of estradiol benzoate given after birth to prevent the adrenal glands from shrinking in newborn rats. Two hr after birth, newborn rats were given a subcutaneous injection of 0.01 mg estradiol benzoate dissolved in 0.05 ml sesame oil. Other siblings were given sesame oil alone. Autopsy was performed on days 1, 2 and 3 after birth. Gravimetric and histologic examinations of the adrenals from these animals and from normal littermate controls were made together with the determination of plasma corticosterone concentrations. In all the three groups of rats, estrogen-treated, oil-treated and non-treated, the weight of the adrenal glands declined with a concomitant decrease in the plasma corticosterone concentrations. The results indicate that estradiol benzoate, when given after birth, does not prevent the neonatal adrenal from shrinking.


Biological activities of tyrosine-containing somatostatin analogs on inhibition of secretion of thyrotropin and growth hormone. The following five tyrosine-containing analogs of somatostatin (GIF) were synthesized by the solid-phase method: Tyr-GIF: [Tyr6]-GIF; [Tyr7]-GIF; [Tyr8]-GIF; [Tyr11]-GIF. These analogs except [Tyr8]-GIF were demonstrated to possess almost the same potency to inhibit thyrotropin release stimulated by thyrotropin-releasing hormone as that of synthesized GIF in vivo. [Tyr8]-GIF had potencies less than 0.5% of GIF. They also had the activity to inhibit Nembutal-induced growth hormone rise. The structure-activity relationship and availability of these analogs for radioimmunoassay were discussed.


Effect of hypophysectomy on hypothalamic somatostatin content in rats. The effect of hypophysectomy on the hypothalamic somatostatin content was examined in rats. Somatostatin content in the acid extract of the pituitary stalk and the median eminence tissue (SME) was measured by specific radioimmunoassay. In young male rats, the mean somatostatin content in SME was 63.9+/-5.0 ng. Two weeks after hypophysectomy, it was reduced significantly to 34.4+/-3.3 ng. The result may indicate that the elimination of feedback actions of GH and/or TSH on the hypothalamus led to the decreased synthesis and/or the release of somatostatin. However, the possibility that structural changes in the pituitary stalk and the median eminence tissue ensued after hypophysectomy resulted in the depletion of somatostatin cannot be ruled out.


Effect of clomid, sexovid and prostaglandins on induction of ovulation and gonadotropin secretion in a freshwater catfish, Heteropneustes fossilis (Bloch). The effect of clomid, sexovid and some prostaglandins (PGE1 and PGF2alpha) on the induction of ovulation, ovarian activity and gonadotropin concentration in the pituitary and blood serum was studied in H. fossilis. Sham-hypophysectomized individuals recorded a very high percentage (90--100%) of ovulation on the 6th day of clomid (150 mug), sexovid (150 mug), PGE1 (100 mug) and PGF2alpha (100 mug) administration. Amongst hypophysectomized recipients only sexovid treated ones ovulated (78%). These drugs were very effective in promoting the ovarian activity in intact specimens but, except sexovid, failed to stimulate any response in hypophysectomized subjects. The gonadotropin concentration in the pituitary gland and blood serum was also elevated after the treatment with the above compounds. It is quite evident from the data that clomid, PGE1, and PGF2alpha, probably acting via hypothalamus, accelerate the synthesis and release of gonadotropin which in turn induces hightened ovarian activity resulting in ovulation. Sexovid seems to have twofold action-- the one indirect through hypothalamus leads to hypophysis leads to ovary like clomid and prostaglandins, and the other directly upon the ovary, possibly by increasing its response to the available gonadotropin, or by behaving like LH.


[Time course of thioacetamide-induced changes in estrogen metabolism in the rat liver]. Rats have been treated for 6 weeks with thioacetamide (0.1% in drinking water). The biochemical changes in liver microsomal metabolism of estradiol which are similar to those observed in human liver cirrhosis become already apparent after one week of treatment. These are diminishment of hepatic microsomal cytochromes P-450 and b5, comparable decrease of 2-hydroxylation and increase of formation of estrone from estradiol. The alterations of estrogen metabolism are reversible within two weeks after ending thioacetamide treatment. These data correspond to the already well established histological response of rat liver to thioacetamide, and its reversibility.


Testicular histology in toad (Bufo melanostictus) following prolonged heat exposure. A quantitative study of changes in the seminiferous tubule and the measurement of Leydig cell area were performed in the testis of toads exposed to 48 degrees C for 8 days. The frequency of occurrence of stages I and II was not affected whereas stages III and IV were influenced by the exposure to heat. The number of primary spermatocytes and secondary spermatocytes in stages III and IV were significantly decreased. The spermatocytes in stages III and IV were injured in the exposed toads. The area of Leydig cells including their nuclei was also decreased in the testes of heat exposed toads. It is concluded that the exposure to high temperature may have arrested spermatogenesis and affected Leydig cell function.


Effect of cyproterone acetate on the action and metabolism of testosterone in the mouse kidney. Cyproterone acetate given alone or in combination with testosterone propionate or 5alpha-dihydrotestosterone to castrated male mice does not exhibit an antiandrogenic action on the kidney. It functions rather as a slightly renotropic hormone, even potentiating the renotropic activity of androgens. The administered amount of cyproterone acetate to intact males or castrates supplemented with androgens, however, decreased dramatically the weight of seminal vesicles and of spleen. Cyproterone acetate treatment of the animals influences in vitro the testosterone to androstenedione conversion but not the activity of 5alpha-reductase in the mouse kidney slices.


Hyperkalemia unresponsive to massive doses of aldosterone in a patient with renal tubular acidosis. In a 53-year-old male patient aldosterone-refractory hyperkalemia was associated with renal tubular acidosis (RTA) due to chronic interstitial nephritis accompanied by peritubular hyaline deposits in the distal nephron. The hyperkalemia was not caused by an adrenal disorder or acidosis and could not be abolished by diuretics, cortisone, longacting synthetic ACTH, excessive doses of DOCA and aldosterone. The results of our experimental studies carried out on the hyperkalemic RTA patient as well as on various control subjects and patients suggested the presence of a specific defect in renal K excretion associated with a decreased aldosterone responsiveness of the renal tubules presumably due to the peritubular pathology.


The effect of calcitonin on glucose metabolism. The authors have studied the effect of calcitonin on glucose metabolism. Ten healthy volunteers were investigated. Calcitonin administered intramuscularly in one single dose inhibited the physiological decrease of blood glucose levels during the forenoon fast. Salmon calcitonin administered in an intravenous infusion in the dose of 0.15 U MRC/kg of body weight impaired the glucose tolerance test with the increase of blood glucose levels. The greatest difference was in the 120th minute after the administration of 100 gm of glucose. Calcitonin failed to produce any significant inhibition of immunoreactive insulin release following the glucose stimulus. However, further studies will be necessary to show whether this lack of the inhibitory beta cytotropic action of calcitonin was due to the low dose of calcitonin.


Hypertension in adult rats treated neonatally with angiotensin. Rats treated neonatally with angiotensin showed labile hypertension in adulthood. This finding may suggest a permanently changed responsiveness of a central nervous controller of the cardiocirculatory system caused by angiotensin-induced hypertension during brain differentiation.


Study of the antidiuretic effect of clofibrate in rat. The effect of clofibrate on the water metabolism was studied in rats with pituitary stalk lesion. It was found that this drug made the oliguria interphase much more marked and more prolonged. In rats anaesthetized with alcohol, clofibrate pre-treatment led to an enhancement of the effect of ADH. The experiments permit the conclusion that the presence of endogenous ADH is necessary for clofibrate antidiuresis. Its effect is presumably exerted via the mobilization of the ADH, but it can not be excluede that it also enhances the peripheral effect of ADH.


Effect of clomiphene citrate on PMSG-induced ovulation in the immature rat. The dose of 15 IU PMSG was found to be optimal for inducing ovulation within 74 hours in 27-day-old immature female rats. There was a significant  decrease in the mean number of ova per ovulating animal when the dose was raised beyond this (r = 0.6554, p less than 0.001 for the range from 15 to 100 IU), but the number of ovulating animals did not decrease unless more than 80 IU PMSG were injected. The PMSG-induced ovulation was not affected by treating the animals with 10 or 100 mug clomiphene citrate at 0, 24, 48, 54, 60, 66 hours or at 0, 24, and 48 hours.


A direct radioimmunoassay for free aldosterone in urine without extraction and chromatography. A direct radioimmunoassay for the determination of free (unconjugated) aldosterone in urine has been developed for the clinical laboratory. The method is based on a determination without extraction and chromatography. In comparison with a reference method including extraction and column chromatography on Sephadex LH 20 a highly significant correlation was found. The coefficient of variation was 5.6% for intraassay variability and 10.4% for interassay determinations. In normal adults the average value of the urinary free aldosterone was 190 ng/24 h (range: 60--320 ng/24 h). During sodium restriction, a 3 fold increase in free aldosterone was observed in normal subjects. In patients with adrenal insufficiency, the values were below 60 ng/24 h, and in two patients with Conn's syndrome, the excretion of free aldosterone was 890 and 1040 ng/24 h. The method was found suitable for routine clinical use.


Insulin secretion and glucose tolerance in acromegaly. Thirty patients with active acromegaly were divided into five groups according to the results of their IRI and blood glucose curves in the course of oral glucose tolerance test. Young acromegalics with short duration of acromegaly had grossly exaggerated IRI response with the peak in the first half-an-hour and the flat blood glucose curve. In the second group the peak of IRI response was in the first hour and the blood glucose curve rose to high values in the early stage after the glucose load. The IRI response in the third group was still exaggerated but delayed and the diabetic type of blood glucose curve was found in some of these patients. Acromegalics with overt diabetes mellitus lost their exaggerated IRI response and the IRI curves were flat in those with more advanced disturbance of glucose tolerance. It was suggested that the groups described might represent individual stages in the development of diabetes mellitus in acromegaly. Unclear position in this scheme is kept by the group of active acromegalics with IRI response in normal limits and glucose tolerance unimpaired.


[Relations between portal and peripheral venous insulin, proinsulin and glucagon concentrations after oral glucose in man]. During cholecystectomy a catheter was inserted into the V. porta via V. umbilicalis. 7 or 8 days later an oral glucose tolerance test was performed (100 g). Blood glucose, insulin (IRI), proinsulin-like material (PLM), and pancreatic glucagon (IRG) concentrations were determined in portal as well as in peripheral venous blood. 5 out of 6 patients showed a disturbed carbohydrate tolerance in comparison to 53 healthy subjects. The IRI increased promptly after the oral glucose load. The portal IRI-concentrations are significantly enhanced in comparison to the peripheral levels, but between both a positive correlation exists (r = 0.9447; p less than 0.01). PLM was slightly increased in the first 10 minutes. The basal IRG levels were not enhanced. Significant differences between the portal and peripheral IRG concentrations could not be observed. The correlation coefficient was 0.8902. Thus, the peripheral venous concentrations of IRI, PLM and IRG can be used as a diagnostic tool of pancreatic hormone secretion in man.


[Lipid mobilization in obesity without carbohydrate intolerance. 2. Caffeine-stimulated lipolysis]. The lipolysis induced by i.v. injection of caffeine (3 mg/kg body weight) has been investigated in 11 normal subjects and 18 obese patients with normal 50 g oral glucose tolerance test and normal thyroid-function. The pattern of blood-glucose and immunoreactive insulin (IRI) secretion were not influenced by caffeine in both groups. Both in the controls and obese group a significant response of FFA and glycerole were noted. In the obese group the caffeine-induced lipolysis was slightly higher as compared with controls. There are no differences in FFA-response between obese group and controls. The observations suggest that an impaired lipolysis is not a pathogenetic factor for development of obesity.


Further evidence of permanent behavioural changes in rats treated neonatally with neurodrugs. Pargyline or reserpine treatment of male rats during the first two weeks of life resulted in significantly increased exploratory activity (sniffing) associated with a significantly decreased emotional reactivity (defecation rate) in adult life. These findings suggest that nonphysiological concentrations and/or turnover rates of neurotransmitters during brain differentiation apparently produced by neurodrugs can lead to permanent behavioural changes.


Effect of cadmium on spermatogenesis in toad (Bufo melanostictus). A quantitative study of seminiferous tubules in toad treated with a single subcutaneous injection of cadmium chloride, showed that 7 days after cadmium injection some stages of spermatogenesis consisting of secondary spermatogonia and primary spermatocytes were decreased significantly.


Free aldosterone concentrations in the urine of newborns. In 1-day-old infants (24--48 hrs after birth), the free aldosterone excretion in urine was found to be significantly lower as compared to adults. The free aldosterone excretion increased then extensively by factor 11.4 at the age of 1 to 14 days and decreased at the age of 14 to 28 days.


Changes in energetic metabolism of rat myocardium during acclimation to high altitude. The results reported in this paper refer to the modifications of energetic equilibrium in the myocardium tissue, involved by altitude chronic hypoxia. Experiments have been done on adult rats (males and females) placed in the Jungfraujoch station, in the Alps, at 3,500 m. The obtained data were compared to control animals, bred in Geneva. On the first days of stay at high altitude, a significant fall of tissue energy charge, due to the non-modified enzyme levels in comparison with the controls, was observed. After several weeks of stay in chronic hypoxia, the metabolic modifications leading to the improvement of energy production and to the achievement of a new equilibrium state, take place. In acclimatized animals some good correlation, between the energy charge and the activity of glycolytic enzymes (PGK and PK) ensuring the ATP generation, is established.


Acid phosphatase heterogeneity in horse neutrophil and eosinophil leukocytes. Two distinct groups of acid phosphatase containing granules were characterized in neutrophils, each group displaying different multiple forms of the enzyme. The heavy granule acid phosphatase showed a lysosomal location. A second lighter group of particles contained a thermolabile, thiol-dependent acid p-nitrophenyl and alpha-naphtylphosphatase, an enzyme clearly different from lysosomal acid phosphatase. Acid phosphatase activity from eosinophil leukocytes appeared to be totally associated with the typical eosinophil granules. On mechanical disruption of these particles, an acid phosphatase was released which differed in substrate and inhibitor specificity, in electrophoretic pattern, and in thermosensitivity, from the remaining matrix-bound enzyme.


Interaction of rat-liver glucocorticoid receptor with DNA. The complex of [3H]dexamethasone and rat liver receptor binds to rat liver DNA. This interaction takes place only in the presence of hormone and is enhanced by 'activation'. No evidence of saturatability can be obtained with concentrations of steroid-receptor complexes corresponding to those observed physiologically in the intact liver cell. The binding is inhibited by high ionic strength and by millimolar concentrations of divalent cations. No species specificity has been observed: the complex binds equally well to prokaryotic and eukaryotic DNA'S. There was no difference between binding to native and denatured DNA. In comparable conditions twice as much [3H]dexamethasone-receptor complexes were bound by DNA than by rat liver nuclei. Thus, the interaction of steroid-receptor complexes with DNA probably does not correspond to the recognition of a few very specific sequences. It is however possible that this interaction is actually operating in vivo in the intact cell.


Parvalbumin from rabbit muscle. Isolation and primary structure. A parvalbumin, with its characteristic low molecular weight (approximately 12000) acidic isoelectric point (approximately 5.5), ultraviolet spectrum (maxm 259 nm) and Ca2+-binding capacity (2 mol/mol protein) has been isolated from rabbit (Oryctolagus cuniculus) muscle. Its primary structure has been determined from a study of its tryptic peptides and of overlapping peptides generated by limited tryptic digestion and by chymotryptic and thermolytic digestions of the protein. The amino acid sequence so obtained is considered in comparison with those known for other parvalbumin and for rabbit troponin C.


Complexes of aliphatic sulfates and human-serum albumin studied by 13C nuclear-magnetic-resonance spectroscopy. The interaction of human serum albumin and various long-chain sulfates has been studied. Binding of sodium octylsulfate to albumin increases in the concentration range studied as measured by equilibrium dialysis. In contrast, binding of the sodium salts of decylsulfate and dodecylsulfate is constant at a concentration of free ligand higher than 50 mM and 12 mM corresponding to binding of 110 and 83 sulfate molecules per albumin molecule, respectively. Viscosity measurements indicate that binding of decylsulfate and dodecylsulfate is associated with unfolding of the albumin molecule. In contrast, binding of octylsulfate does not cause gross conformation changes of albumin. However, the chemical shifts of bound octylsulfate obtained by natural abundance 13C nuclear magnetic resonance spectroscopy show significant changes at 80 mM and 150 mM free ligand. The spin-spin and spin-lattice relaxation times also show changes in the association between octylsulfate and albumin at 80 mM free sulfate. These observations indicate alterations in the binding properties at 10--11 and 20--21 bound ligand molecules, respectively. The relaxation times are considerably increased by binding to albumin, indicating less motional freedom of the molecules in the bound state. At high levels of sulfate binding the relaxation times of the terminal methyl group approach that of free ligand. The chemical shifts of all the bound carbon atoms studied, except the CH2 group nearest to the sulfate group (C1), are comparable to those observed in the micellar state indicating binding in a non-polar environment. However, the relaxation times indicate that the motional freedom of sulfates bound to albumin is much more restricted than in micelles. The shift of C1 indicates that this part of the ligand is situated in a polar environment. The following model for binding of high concentrations of aliphatic detergents is proposed. The sulfate group and the CH2 group nearest to it is situated in a polar medium caused by interaction between the sulfate group and a positive amino acid residue on albumin. The other CH2 groups interact with hydrophobic amino acid residues on albumin. The CH3 group does not interact with the albumin molecule but associates with other methyl groups of sulfates bound in the vicinity forming a hydrophobic medium.


Human-serum cholinesterase subunits and number of active sites of the major component. The major C4 component of human serum cholinesterase was highly purified by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The final product was about 8 000-fold purified with a yield of 64%. The subunit structure was determined by 8M urea polyacrylamide disc electrophoresis and by the sedimentation equilibrium centrifugation method in 5M guanidine hydrochloride. It was found that the C4 enzyme has a tetrameric structure. The subunits are equal in size and charge and a molecular weight comparable to that of the C1 enzyme from native serum. The major C4 enzyme and the minor C1 enzyme were subjected to an 'active enzyme centrifugation'. It was found that the C4 enzyme was a tetramer and the C1 enzyme was a monomer in the presence of substrate. The number of diisopropylphosphofluoridate-binding sites was measured from the molar ratio of bound diisopropylphosphate to protein. A value close to two binding sites was found for the C4 enzyme.


Studies on the mechanism of collagen glucosyltransferase reaction. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparations purified about 2500-5000-fold from extract of homogenate of whole chick embryos. Data obtained in experiments on initial velocity and inhibition kinetics of the reaction were consistent with an ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, UDP-glucose and collagen substrate, the addition of Mn2+ being at thermodynamic equilibrium and the binding site of the UDP-glucose to the enzyme not being the same as that for Mn2+ and collagen substrate. Only one metal co-factor seems to be involved in the reaction. The collagen substrate can probably also react in some conditions with enzyme-Mn2+ and with enzyme-Mn2+-UDP, and the UDP with the free enzyme, but in all these instances dead-end complexes are formed. Evidence is presented for an ordered release of the products in the following order: glucosylated collagen, UDP and Mn2+, in which Mn2+ need not leave the enzyme during each catalytic cycle.


Specific binding of a nonhistone chromosomal protein from lymphocyte to DNA. The isolation of a nonhistone chromosomal protein, NH30 000, from bovine lymphocytes by affinity chromatography on a DNA-agarose column is described. The procedure starts with nonhistone fraction NH4 obtained by hydroxyapatite chromatography as described previously [Blüthmann, H., Mrozek, S. & Gierer, A. (1975) Eur. J. Biochem. 58, 315-326]. Protein NH30 000 migrates as a single band with a molecular weight of 30 000 on sodium dodecylsulfate polyacrylamide gels. It contains, on a molar basis 25.3% acidic amino acids and 18.3% basic amino acid residues, 10.3% being lysine. End-group determination confirms that this protein is composed of one polypeptide chain with the NH2-terminal amino acid arginine. The binding of protein NH30 000 to native DNA of different molecular weights has been investigated by the nitrocellulose filter assay. The results suggest that in the presence of 0.2 M NaCl it binds about 30% of the DNA with binding sites every 1700 nucleotide pairs. Competition experiments show that protein NH30 000 exhibits a higher binding affinity for lymphocyte DNA in comparison to Escherichia coli DNA.


Endoribonuclease IV. A poly(A)-specific ribonuclease from chick oviduct. 1. Purification of the enzyme. A new endoribonuclease, termed endoribonuclease IV, has been described. This enzyme has been isolated from chick oviducts and purified 15 000-fold in a 25% yield nearly to homogeneity. The nuclease, which specifically degrades poly(A), forms oligonucleotides of an average chain length of 10. These (A)-10 fragments are terminated by 3'-hydroxyl and 5'-phosphate groups. The enzyme has a pH optimum at 8.7, requires Mn2+ or Mg2+ as a cofactor, and has a molecular weight of about 45 000.


Endoribonuclease IV. 2. Further investigation on the specificity. The poly(A)-specific endoribonuclease IV produces oligo(A) fragments of a chain length of 10 AMP nucleotides. One enzyme molecule performs 1700 cleavages per min; the cleavages occur randomly. The endoribonuclease IV is a nuclear enzyme which is present in the oviduct of quails in a concentration of 40 000 enzyme molecules per cell. Poly(A) segments on mRNA are selectively hydrolyzed by endoribonuclease IV; the poly(A)-free part of the RNA is not affected. After incubation with the enzyme, a residual oligo(A) stretch of 5 AMP nucleotides remains on poly(A)-rich RNA. The endoribonuclease IV does not disintegrate the polyribosomal complex after incubation in vitro and the enzyme has also no influence on the translational capacity of a cell-free protein-synthesizing system.


Resolution and purification of histones on homologous series of hydrocarbon-coated agaroses. Hydrophobic chromatography on alkyl-agarose columns has been applied to the fractionation of histones. This paper describes: (a) a two-column method for the resolution of whole histone from calf thymus into its five main components (H1, H2a, H2b, H3 and H4), (b) a rapid one-step procedure for the isolation of the H3 fraction from whole histone, (c) an alternative one-step procedure for the resolution of H3 and H2a (which co-elute during gel exclusion chromatography on Biogel P-60). These experiments are also used for gaining further insight into the mechanism of action of hydrocarbon-coated agaroses.


A novel endonuclease of human cells specific for single-stranded DNA. We have fractionated from human aneuploid cell cultures three different enzyme fractions degrading single-stranded DNA. We have purified and characterized one of these DNases; this is an endonuclease working at alkaline pH (around 9.5) and requiring Mg2+ for its activity. The enzyme degrades denatured DNA over 100 times more efficiently than native DNA in optimal conditions. The termini produced by the enzyme have 5'P and 3'OH ends. The enzyme can attack, though at reduced rate, the supertwisted circular molecule of Simian virus 40 DNA, whereas it is inactive on the nicked circular molecule. The ultraviolet irradiation of DNA, whether native or denatured, does not affect its efficiency as substrate of the DNase. The properties of this endonuclease distinguish it from those of the other DNases described previously in mammalian cells; the denomination DNase VI is therefore proposed. Its properties are similar to those of DNases described in Ustilago maydis and Bacillus subtilis, for which an essential role in recombination seems likely.


The primary structure of the parvalbumin II of pike (Esox lucius). The amino acid sequence of the parvalbumin II of the pike is reported. The protein has a molecular weight of 11 435. It consists of a single polypeptide chain of 107 amino acid residues with an acetyl group blocking the N-terminus and an alanine residue at the C-terminus. The molecule has been enzymically cleaved by trypsin, thermolysin and by the protease of the Staphylococcus aureus strain V8. Chemical cleavages make use of the CNBr reaction and of the sulfocyanoethylation method. The comparison of this amino acid sequence with that of the parvalbumin III of the pike indicates that these two homologous proteins belong respectively to two different subgroups derived from an early gene duplication of an ancestral gene at least prior to the differentiation of the Osteichthyes.


On the initiation of mammalian RNA polymerase at single-strand breaks in DNA. Mammalian RNA polymerase B is able to initiate at single-strand breaks in the DNA template. A 3'-OH end at a nick is required for initiation whereas the 5'-end may be either -- OH or phosphate. The 3'-OH group does not function as a primer. An appreciable part of the newly synthesized RNA started at a nick remains associated with the DNA in hydrid form. Initiation of exogeneous RNA polymerase B on chromatin exhibits similar requirements.


Maximal number of hormonogenic iodotyrosine residues in thyroglobulin iodinated by thyroid peroxidase. Almost non-iodinated human goiter thyroglobulin has been iodinated in vitro by thyroid peroxidase to levels as high as 75 iodine atoms per mol of protein. The following results were obtained. 1. The iodine distribution obtained in vitro with human thyroglobulin strongly ressembles that obtained in vivo for rat thyroglobulin. Thus the distribution of iodine seems to depend essentially on the structure of thyroglobulin and on the reactivity of the different tyrosine residues. 2. Although the number of hormone residues increased with iodination the highest efficiency of hormone synthesis was obtained in a very narrow range of iodination: in vitro (40%) between 25 and 30 iodine atoms, and in vivo (48%) between 10 and 20 atoms. This result suggests that the tyrosines which are coupled with a high efficiency are iodinated sequentially. 3. Maximal thyroxine content was found to be lower than approximately 3 mol/mol of thyroglobulin. This result might mean that the two 12-S subunits of thyroglobulin are not identical and that one of them is able to produce 2 mol of hormone while the second only 1 mol.


Transfer of the 1-pro-R and the 1-pro-S hydrogen atoms of ethanol in metabolic reductions in vivo. The transfer of deuterium from [1 R-2H]ethanol and [1 S-2H]-ethanol to reduced metabolites of administered compounds was measured in female rats provided with bile fistulas. Administered cyclohexanone was reduced to cyclohexanol, and in this reduction hydrogen was transferred only from the 1-pro-R position of the ethanol. The deuterium content in the cyclohexanol was about 67% of that in the ethanol. In the reduction of the 17-oxo group in 3beta-hydroxy-5alpha-androstan-17-one, hydrogen was transferred both from the 1-pro-R position and the 1-pro-S position, resulting in degrees of labelling that were about 25% and 2%, respectively, of those in the specific positions of the ethanols. The 1-pro-R and 1-pro-S positions of ethanol contributed about 9% and 5%, respectively, of the 3beta hydrogen in lithocholic acid formed from 3-oxo-5beta-cholanoic acid. The results indicate that alcohol dehydrogenase and aldehyde dehydrogenase do not share a common pool of NAD, and that NADH formed during acetaldehyde oxidation is utilized for reductions in the cytosol to a smaller extent than the NADH formed in the alcohol dehydrogenase reaction. This result supports the concept that aldehyde oxidation is mainly an intramitochondrial process. The relatively extensive utilization of the 1-pro-S hydrogen of ethanol in the reduction of 3-oxo-5beta-cholanoic acid, that is probably NADPH-dependent, indicates that cytosolic NADPH may be produced from malate or isocitrate formed intramitochondrially.


Template activity of synthetic deoxyribonucleotide polymers in the eukaryotic DNA-dependent RNA polymerase reaction. Template specificities of the eukaryotic DNA-dependent RNA polymerases A and B from rat liver, pea, and cauliflower have been investigated using synthetic polydeoxyribonucleotides. Polymerases A and B from the three species exhibit different specificities for single-stranded homopolymers: polymerase A preferentially reads poly(dT) and poly (dC). and polymerase B poly (dC). This preferential reading appears to be a property of eukaryotic DNA-dependent RNA polymerases. Polymerases A and B transcribe synthetic polyribonucleotides also, but at a reduced rate. The polyribonucleotides which can be read by DNA-dependent RNA polymerases have a base sequence similar to that of the polydeoxyribonucleotides, which are effeciently transcribed, suggesting that the base sequence of the template rather than its conformation is crucial in the template specificity for synthetic polymers. Competition experiments with polydeoxyribonucleotides indicate that the enzymes have different binding specificities, which are not the same as their template specificities.


Serine transhydroxymethylase from rabbit liver. Sequence of anonapeptide at the pyridoxal-5'-phosphate-binding site. The amino acid sequence of the coenzyme-binding site of serine transhydroxymethylase from rabbit liver has been determined. After reduction with NaBH4 and aminoethylation, a first sample of enzyme was digested with thermolysin and a single phosphopyridoxyl peptide was isolated. A second sample of similarly treated enzyme was digested with chymotrypsin and three phosphopyridoxyl peptides clearly originating from a unique coenzyme-binding site were isolated. Sequence analysis of these peptides indicate the following structure: Val-Val-Thr-Thr-His(Pxy)-Thr-Leu. Sequence homologies of the active site of various pyridoxalphosphate enzymes are discussed in terms of a possible catalytic role and of evolution of this class of proteins.


A study of calf-thymus histone H2B using 13C magnetic resonance. Calf thymus histone H2B has been studied by means of 13c magnetic resonance. Most sidechain resonances have been assigned. Analysis of 13c peak intensities by a simple computer-aided method proves that 13c magnetic resonance spectroscopy can be used to monitor aggregation phenomena more accurately than 1H magnetic resonance or other physico-chemical methods. Re-examination of 1H spectral simulations shows substantial agreement with 13C results. It is concluded that in 0.1M added NaCL the H2b aggregates include the chain segment from 50 (+/-5) to 108 (+/-5).


Kinetics of thyroglobulin iodination and of hormone synthesis catalysed by thyroid peroxidase. Role of iodide in the coupling reaction. The kinetics of tyrosine iodination and of thyroxine synthesis in thyroglobulin, different reactions catalyzed by the same enzyme (thyroid peroxidase), have been compared. Thyroxine synthesis always began after a lag period of 3-5 min. This lag was constant whatever the rate of iodination; this rate of iodination was increased either by increasing the concentration of iodide or enzyme or by decreasing the concentration of thyroglobulin. Increasing the rate of iodination resulted in increasing the number of iodine atoms incorporated during the lag period. Thus the lag observed for thyroxine synthesis was constant and did not depend on the fact that free iodide or non-iodinated tyrosine residues of thyroglobulin were exhausted before thyroxine synthesis occurred. Finally, it appeared that, whatever the explanation of the lag, the enzyme catlyzes thyroid hormone synthesis at a slower rate than iodination. The existence of a lag also allowed us to prepare thyroglobulin samples with different iodine contents but without thyroid hormones. Thus iodination and thyroxine synthesis could be studied independently and the following results were obtained. 1. Iodotyrosine residues which can couple to form thytoxine are made considerably before coupling occurs. 2. H2O2 is required for coupling of these hormonogenic residues; thus the coupling reaction requires enzymic oxidation of the iodotyrosine residues. 3. In addition a strict requirement for iodide was needed for coupling; the requirement was dependent on the concentration of iodide. Thus iodide, a substrate of the iodination reaction, may also have other effects on the activity of thyroid peroxidase.


Quantitative estimates of cytoplasmic and nuclear oestrogen receptors in chick oviduct. Effect of oestrogen on receptor concentration and subcellular distribution. (3H)Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with (3H)oestradiol during a 24-h incubation at 20 degrees C, 2 h at 30 degrees C or 45 min at 3 degrees C. Both "soluble" and "insoluble" nuclear receptors were stable for at least 6 h at 30 degrees C and 3 degrees C but a proportion (approx. 30%) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 degrees C. The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10x1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3-6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73-77%) of their oestrogen receptors sites in the receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82%) of receptor sites were located in the nucleus. A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 mug oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 mug oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 mug oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h.


Biochemical and electron-microscopic evidence that the subunit structure of Chinese-hamster-ovary interphase chromatin is conserved in mitotic chromosomes. Biochemical and electron microscopic studies demonstrate that the subunit structure of Chinese hamster ovary cell interphase chromatin is conserved in mititic chromosomes. Digestion of purified chromosomes or nuclei with micrococcal nuclease produces DNA in discrete size classes, as visualized by polyacrylamide gel electrophoresis, which are common to the two materials. Early in digestion the DNA fragments are integral multiples of a monomer approximately 177 base pairs in length, whereas after extensive digestion the remaining DNA fragments migrate ahead of the monomer position. The size of the repeating DNA unit was confirmed as being smaller than that produced by micrococcal nuclease digestion of rat liver nuclei by direct comparison. Electron microscopy of partially unravelled chromosomes at low ionic strength shows tightly packed spheres (nucleosomes) approximately 12 nm in diameter which are often arranged as linear chains. Chromosomal material prepared for electron microscopy after varying extents of micrococcal nuclease digestion is composed of fragments containing pregressively fewer nucleosomes, which parallels the loss of high DNA multimer bands in gel electrophoresis. Material unravelled from chromosomes in the presence of NaCl consists of nucleosomes packed packed in a different configuration which suggests the origin of higher order structures in chromosomes.


The translation in vitro of mRNA from developing cysts of Artemia salina. Successive stages in the development of the brine shrimp cyst were used as a model for studying differentiation at the level of mRNA transcription and translation. The poly (A)-containing mRNA from dormant cysts and free-swimming larvae (nauplii) was found to be efficiently translated in a wheat-germ cell-free system, and electrophoretic patterns of translation products in vitro resembled those of the endogenous proteins extracted from the equivalent developmental stages. Each stage, however, exhibits a characteristic protein pattern. Two low-molecular-weight proteins prominent in the cyst disappeared almost completely in the nauplius stage, whereas the proportion of actin increased 3-fold. Parallel patterns were observed upon translation in vitro of the respective mRNA preparations. The percentage of the acidic protein, tubulin, decreased somewhat during development.


Preparation and properties of S-cyano derivatives of creatine kinase. The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5'-dithio-bis(2-nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol-blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8 - 0.9-SH group per subunit. This mono-S-cyano derivative of creatine kinase was found 73% as active as the native unmodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted in the bis-S-cyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme.


Isolation and characterization of a Ca2+ -binding polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner. C-occolithophoridae, a group of mostly unicellular algae, possess a cell wall containing calcified plates, called coccoliths. The coccoliths from the species Emilania huxleyi (Lohmann) Kamptner contain a water-soluble acid polysaccharide. In this paper we describe the isolation and some characteristic properties of the polysaccharide, in particular its Ca2+ -binding capacity.  A large-scale cultivation of the Coccolithophoridae was worked out and a new procedure for isolating coccoliths was developed. The polysaccharide obtained from the coccoliths contained two types of monobasic acid groups in a total amount of 1.8 mumol/mg polysaccharide. One type consisted of weakly acid groups which were identified as uronic acids. The nature of the stronger acid groups remains to be established. The ratio between the respective groups was 1:0.8. Studies with 45Ca2+ demonstrated that the isolated polysaccharide is capable of binding Ca2+. Equilibrium dialysis revealed that the maximum amount of Ca2+ which can be bound in 0.92 +/- 0.05 mumol/mg polysaccharide. Flow-rate dialysis experiments strongly suggested the presence of two classes of Ca2+ -binding sites differing in affinity for Ca2+. High-affinity sites (dissociation constant Kd for Ca2+ :2.2 +/- 1.0 X 10(-5) M) were found to be present in amounts (0.38 +/- 0.04 mumol/mg polysaccharide) approximately equivalent to the strongly acid monovalent groups mentioned above (0.8 mumol/mg polysaccharide). Low-affinity sites (Kd for Ca2+: -11 +/- 39 X 10(-5) M) were estimated at 0.74 +/- 0.11 mumol/mg polysaccharide. Although this figure could be determined less accurately, it is suggested that the uronic acids (1.0 mumol/mg polysaccharide) are identical to the low-affinity sites. Preferential binding of Ca2+ occurred in a 100-fold excess of Na+ and Mg2+ as was shown by gel filtration. A 100-fold excess of Sr2+ inhibited Ca2+ binding to a great extent while no Ca2+ was bound in the presence of an equimolar amount of La3+. The dissociation constants of the high-affinity sites for Na+, Mg2+, Sr2+ and La3+ (in the presence of Ca2+) were determined with the flow-rate dialysis technique. They confirm the order of binding preference found with gel filtration. A polysaccharide with similar properties could be isolated from subfossil coccoliths of E. hyxleyi (about 1000 years old). The possible role of the polysaccharide as a heterogeneous matrix in coccolith formation is discussed.


Quantitative determination of N-acetylglucosamine residues at the non-reducing ends of peptidoglycan chains by enzymic attachment of [14C]-D-galactose. The ability of human milk galactosyltransferase to attach D-galactose residues quantitatively to the C-4 of N-acetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rod-shaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical 'mini' cells had chain lengths similar to those of the isogenic rod-like cells.


Replenishment and depletion of citric acid cycle intermediates in skeletal muscle. Indication of pyruvate carboxylation. The effects of various substrates on the concentrations of free amino acids, citric acid cycle intermediates and acylcarnitines were studies in perfused hindquarter of rat in presence of glucose and insulin in order to assess regulatory mechanisms of the level of citric acid cycle intermediates in skeletal muscle. 1. Acetate and acetoacetate effected a significant increase in the level of citrate cycle intermediates and accumulation of acetylcarnitine. These changes were accompanied by a reduction in the level of alanine. The concentration of AMP was significantly elevated. 2. Muscle mitochondria fixed 14CO2 in the presence of pyruvate. The products were identified as malate or citrate when whole and disintegrated mitochondria were used respectively. The fixation was greatly stimulated by acetylcarnitine. 3. Acetylcarnitine inhibited the production of pyruvate from malate by muscle mitochondria. 4. Perfusion with 2-oxoisocaproate and 2-oxoisovalerate promoted increases in the level of citric cycle intermediates, a drop in both alanine and glutamate, and accumulation of branched-chain acylcarnitines. 2-Oxoisocaproate also caused a reduction of alanine released from the muscle. 5. Perfusion with leucine and valine did not change the concentration of citric acid cycle intermediates, but elevated glutamate and still more the concentration of alanine. 6. It is concluded that citric cycle intermediate level in the perfused resting muscle is modified by a) conditions which change the concentration of acetyl-CoA and thereby modify the rate of pyruvate carboxylation and decarboxylation of malate via malic enzyme b) conditions which change the concentration of pyruvate cause changes in alanine and cycle intermediates in the same direction via transamination reactions c) conditions which change the concentrations of 2-oxoacids which are converted to cycle intermediates via oxidation.


Studies on the synthesis of casein messenger RNA during pregnancy in the rabbit. The quantity of casein mRNA in the rabbit mammary gland was assayed during the course of pregnancy, by translation of casein mRNA in a reticulocyte lysate and by hybridization to DNA complementary to purified casein mRNA. Both methods indicated that the major increases in the concentration of casein mRNA in both polysomal and total cellular RNA occurred between the 18th and 25th day of pregnancy. The change in casein mRNA concentration during this period coincided with a sharp rise in casein synthesis in mammary gland explants suggesting that the levels of casein mRNA determine the rate of casein synthesis in the mammary gland. The sensitivity of the hybridization assay made it possible to detect the presence of casein mRNA in virgin animals and during the very early stages of pregnancy. At day 5, casein mRNA was found associated with polysomes indication that there was probably some casein synthesis at this early stage of gestation. These results suggest that the hormones controlling lactogenesis in the rabbit may function by augmenting the rate of casein mRNA synthesis rather that initiating transcription of previously inactive genes.


A complex control circuit. Regulation of immunity in temperate bacteriophages. Temperate bacteriophages can display in a stable way two essentially different behaviours. In the immune state, a gene (cI) produces a repressor which prevents expression of all the other viral genes; in the non-immune state the typically viral functions are expressed. The choice between the two pathways and the establishment of one of them have much in common with cell determination and differentiation. This choice depends on a complex control system, in fact one of the most intricate nets of regulation known in some detail. Our paper provides a formal description and partial analysis of this regulatory net. It is shown that even for relatively simple known models, this kind of analysis uncovers predictions which had previously remained hidden. Some of these predictions were checked experimentally. The experimental part chiefly deals with the efficiency of lysogenization by thermoinducible lambda phage carrying mutations in one or more of the regulatory genes, N, cro and cII. Although N- mutations are widely known for preventing efficient integration, and both N- and cII mutations for preventing efficient establishment of immunity, it is shown that, as predicted by a simple model, both N- and cII- phage efficiently lysogenize at low temperature if they are in addition cro-. In contrast with lambda N- cro+, lambda N- cro- is not propagated as a plasmid at low temperature, precisely because it establishes immunity too efficiently. Genetic control circuits are described in terms of sets of logic equations, which relate the state of expression of genes or of chemical reactions (functions) to input (genetic and environmental) variables and to the presence of gene and reaction products (internal, or memorization varibles). From the set of equations, one derives a matrix which shows the stable stationary states (if any) of the system, and from which one can derive the pathways (temporal sequences of states) consistent with the model. This kind of analysis is complementary to the more widely used analysis based on differential equations; it allows one to analyze in less detail more complex systems. The language might be used as well, mutatis mutandis, in fields very different from genetics. The last part of the discussion deals with the role of positive feedback loops in our specific problem (establishment and maintenance of immunity in temperate bacteriophages) and in developmental genetics in general. As a generalization of an old idea, it is suggested that cell determination (for a given character) depends on a set of genes whose interaction constitutes a positive feedback loop. Such a system has two stable stationary states: which one is chosen will usually depend on additional controls grafted on the loop.


Studies on the influence of fatty acids on pyruvate dehydrogenase interconversion in rat-liver mitochondria. 1. The effect of fatty acids on the interconversion of pyruvate dehydrogenase between its active (nonphosphorylated) and inactive (phosphorylated) forms was measured in rat liver mitochondria respiring in state 3 with pyruvate plus malate and 2-oxoglutarate plus malate and during state 4 to state 3 transition in the presence of different substrates. The content of intramitochondrial adenine nucleotides was determined in the parallel experiments. 2. Decrease of the intramitochondrial ATP/ADP ratio with propionate and its increase with palmitoyl-L-carnitine in state 3 is accompanied by a shift of the steady-state of the pyruvate dehydrogenase system towards the active or the inactive form, respectively. 3. Transition from the high energy state (state4) to the active respiration (state3) in mitochondria oxidizing 2-oxoglutarate or plamitoyl-L-carnitine causes an increase of the amount of the active form of pyruvate dehydrogenase due to the decrease of ATP/ADP ratio in the matrix. 4. No change in ATP/ADP ratio can be observed in the presence of octanoate in mitochondria oxidizing pyruvate or 2-oxoglutarate in state 3 or during state 4 to state 3 transition. Simultanelusly, no significant change in phosphorylation state of pyruvate dehydrogenase occurs and a low amount of the enzyme in the active form is present with octanoate or octanoate plus 2-oxoglutarate. Pyruvate abolishes this effect of octanoate and shifts the steady-state of pyruvate dehydrogenase system towards the active form. 5. These results indicate that fatty acids influence the interconversion of pyruvate dehydrogenase mainly by changing intramitochondrial ATP/ADP ratio. However, the comparison of the steady-state level of the pyruvate dehydrogenase system in the presence of different substrates in various metabolic conditions provides some evidence that accumulation of acetyl-CoA and high level of NADH may promote the phosphorylation of pyruvate dehydrogenase. 6. Pyruvate exerts its protective effect against phosphorylation of pyruvate dehydrogenase in the presence of fatty acids of short, medium or long chain in a manner which depends on its concentration. It is suggested that in isolated mitochondria pyruvate counteracts the effect of acetyl-CoA and NADH on pyruvate dehydrogenase kinase.


Small-angle X-ray studies of the Fab and Fc fragments from the human immunoglobulin molecule Kol. The conformation of the Fab and Fc fragments from the human immunoglobulin molecule Kol [IgI I, chi2gamma2, Gm(f)+] was studied by small-angle x-ray scattering in solution. The fragments were studied in 0.02 M Tris-HCl buffer. For the Fab fragment the radius of gyration was found to be 3.15 +/- 0.15 nm, the volume to be 75 +/- 8 nm3. For the Fc fragment the respective values were 3.15 +/- 0.15 nm for the radius of gyration and 91 +/- 8 nm3 for the volume. A large number of models were calculated for both fragments to find models which fit these data and have the same scattering curve. The models with the best agreement were compared with the models found for the crystalline state by crystal x-ray studies.


A new concept of the function of elongation factor 1 in peptid chain elongation. An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments in which mixtures of [3H]EF-1, ribosomes from Drebs II ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a tRNA in their P-site. On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound.


5'-Capping structures of Artemia salina mRNA and the translational inhibition by cap analogs. The mRNA of the brain shrimp Artemia salina has two types of blocked methylated 5'-terminal structures (caps). About 75% of the mRNA molecules have the 5'-end structure of m7G5'ppp5'-AmpGp and about 25% have the structure of m7G5'ppp5'GmpGp. The only other type of methylated residue found in Artemia mRNA is N6-methyladenosine and which is located at internal positions along the mRNA chain. Translation of Artemia cyst or nauplius poly(A)-rich mRNA in wheat-germ extracts was found to be inhibited by 7-methylguanosine 5'-monophosphate, a chemical analog of the cap, as well as by snythetic caps such as m7G5'ppp5'Gm. On the other hand, the elongation activity on endonegous mRNA in an Artemia cell-free system was not sensitive to 7-methylguanosine 5'-monophosphate.


Evidence for the nucleotide sequence of 5-S rRNA from the flowering plant Secale cereale (Rye). Evidence for the sequence of rye 5-S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P-labelled molecule. The probable sequence of 5-S rRNA from four other flowering plants was deduced by aligning, with the homologous sequences in the rye 5-S rRNA, oligonucleotides produced by T1 and pancreatic A ribonuclease digestion. The most dissimilar differed in only seven positions. From a comparison of the sequence of rye 5-S rRNA with those known for other types of organism, it was possible to distinguish some structural features of the molecule which are common to all of them. Also, information was obtained about the possible phylogenetic relationship of the flowering plants to other organisms whose 5-S rRNA has been sequenced.


Ca2+, K+-regulated intramolecular crosslinking of S-100 protein via disulfide bond formation. Reaction of the thiol reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2) with the brain-specific protein S-100 favours stabilization of the quaternary structure of the protein via disulfide bond formation. This process is modulated by those cations (Ca2+ and K+) which are known to affect the conformation of the protein. Ca2+ markedly favours the reaction of S-100 with Nbs2 but inhibits subsequent disulfide bond formation; K+, on the contrary, is much less effective in promoting interaction with Nbs2 but strongly stimulates disulfide bond formation. These findings are interpreted assuming that in presence of Ca2+ the three subunits forming the native S-100 protein have two cysteine residues exposed to the solvent but mismatched to form disulfides while in presence of K+ the sulphydryl groups are in a less accessible position to Nbs2 but suitable for S-S bond formation. Crosslinking of S-100 subunits is characterized by the appearance in dodecylsulphate electrophoresis of two very close protein bands having a molecular weight almost identical to that of the native, undenatured protein but not of higher or lower-molecular weight components. This finding, and the demonstration that both the crosslinked and native S-100 proteins have identical profiles when analyzed by sucrose density centrifugation or gel chromatography indicate that disulfide bond formation occurs among subunits of the same molecule.


Five-coordinate iron-porphyrin as a model for the active site of hemoproteins. Characterization and coordination properties. Preparation of iron(III)-deuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) by coupling histidine methyl ester to deuterohemin has been performed using the mixed carboxylic/carbonic-acid-anhydride method. This compound, which is very soluble in various organic solvents, can be considered as a prosthetic group model for the active site of five-coordinate hemoproteins. In the oxidized state a basic, a neutral or an acid form can be isolated. The basic and acid forms are monomeric at all concentrations. The neutral form is found dimeric in concentrated solutions while it is monomeric at low concentration. The coordination state of iron in these various species is investigated. The neutral form reacts with ligands, such as nitrogenous organic bases, leading to six-coordinate well-known hemichromes which exhibit low-spin electron spin resonance (ESR) spectra. The reaction of anionic ligands, such as F-, CN-, NO-2 and N-3, with the neutral form model leads to unsymmetrical six-coordinate complexes whose optical and ESR spectra are similar to those of synthetic deuteromyoglobin. In benzene, toluene or dichloromethane solutions iron (II)-deuteroporphyrin 6(7)-methyl ester, 7(6)-histidine methyl ester), obtained from ferric forms by heterogeneous reduction with aqueous dithionite, exhibits an optical spectrum characteristic of a five-coordinate high-spin ferrous complex. At low temperature important spectral modifications are observed indicating a dimeric association. At room temperature it binds one nitrogenous base molecule leading to the well-known hemochrome. It reacts also with carbon monoxide with a very high affinity constant (4.5 X 10(8) M-1), comparable to that of the isolated human hemoglobin alpha and beta chains, but much higher than the values reported for other various models, suggesting that the side-chain length bearing the fifth ligand may have an important influence upon the reactivity of the sixth position of the iron ion. At low temperature in toluene or dichloromethane, this compound reversibly binds oxygen without oxidation of the iron ion while oxidation occurs at room temperature. The significance of these results is discussed in relation with the local environment, the electronic nature of the base and the immobilization of the heme group in hemoproteins.


Rat b/b anemia: translation of normal and anemic globin mRNA in wheat-germ cell-free system. Globin mRNAs were isolated from circulating reticulocytes both from rats carrying a homozygous, recessive mutation causing a severe thalassemia-like syndrome and from normal rats. After first identifying the rat globin chains as alpha or beta chains, the translational products primed by both polysomal and nonpolysomal mRNAs in wheat germ 30000 x g supernatant were analyzed: the ratio of alpha to beta globin mRNAs found in polysomes isolated from mutant rats is identical to the ratio of their products synthesized in vivo while the ratio of these mRNAs is quite different in the nonpolysomal fraction, the latter being enriched in alpha globin mRNA. No difference is found in the ratio of alpha and beta globin mRNAs in the polysomal and nonpolysomal RNA isolated from normal rats, both being identical to the ratio of their products synthesized in vivo. One third of the total amount of mRNA found in mutant cells is not in polysomes as compared to only 6 percent for the mRNA from normal lysates. These results suggest that a translational control mechanism is involved although the decreased globin synthesis in b/b anemia can not be fully accounted for by its operation.


Nucleolar DNA-dependent RNA polymerase from rat liver. 1. Purification and subunit structure. DNA-dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE-Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electrophoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%. The RNA polymerase I eluted as a single peak from DEAE-Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20% of the total RNA polymerase I activity and the IB enzyme comprised approximately 80%. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130000 (b), 65000 (c), 40000 (d), and 19000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme. Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme.


Nucleolar DNA-dependent RNA polymerase from rat liver. 2. Two forms and their physiological significance. RNA polymerase I (or A) was extracted from nuclear, nucleolar and nucleoplasmic fractions, and resolved into IA and IB forms on a phosphocellulose column. During the course of cycloheximide treatment, the activity of RNA polymerase IB decreased in the nucleoli with concomitant increase in the nucleoplasmic fraction, suggesting strongly that cycloheximide induced specific leakage of IB enzyme from the nucleolus. The activity of IA enzyme did not change in the nucleoli. When nucleoli were incubated in the presence of actinomycin D, all the IA enzyme activity and approximately 30% of the total IB enzyme activity were released in the incubation medium, whereaa 70% of IB activity remained associated with the nucleolar pellet where no IA activity was detected. The enzyme which was released into the incubation medium was tentatively designated as free or unbound RNA polymerase I and that which was associated with the nucleolar pellet as template-bound enzyme. During the treatment with cycloheximide, the activity of bound enzyme, which contained exclusively IB form, decreased rapidly, with kinetics almost identical to that of nucleolar RNA synthesis in vivo. The activity of free enzyme did not change appreciably. At 2 h after partial hepatectomy, IB enzyme activity in the free RNA polymerase fraction increased to almost twice the control, while the bound enzyme activity did not increase appreciably until 4h of regeneration. Enhancement of nucleolar RNA synthesis in vivo was not apparent at 2 h but became significant by 4 h after partial hepatectomy. These results strongly suggest that (a) the above-mentioned procedure is actually fractionating RNA polymerase I into free and bound forms, (b) RNA polymerase IB is the transcriptionally active form in vivo, (c) RNA polymerase IB exists in excess in the nucleoli, but the amount of bound IB molecules, which are engaged in transcription in vivo, must be determined by some other factor(s) than the mere concentration of IB enzyme in the nucleolus, and (d) IA form is not an artifact of isolation but is always present in vivo at a certain amount, although the exact nature of this molecule is not known at present.


Excretion of taurocholate from isolated hepatocytes. Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux. Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 muM. Ouabain increased the rate of efflux. The observations indicate that secretory functions are maintained in isolated liver cells.


Production and properties of vitamin-D-induced mRNA for chick calcium-binding protein. 1. Calcium-binding protein synthesis on chick intestinal polysomes is induced within 2 h of injecting vitamin-D-deficient birds with 1,25-dihydroxycholecalciferol. The induction is short-lived: the maximum output of the binding protein is reached by 13 h after hormone injection, and declines rapidly thereafter. 2. This induction of calcium-binding protein synthesis occurs by the production of active mRNA for the protein. The sedimentation coefficient of this mRNA in denaturing conditions is 18 S, equivalent to a molecular weight of approximately 700000, and the molecule contains a tract of polyadenylate. 3. Both polysomal and poly(A)-containing RNA extracted from intestinal polysomes stimulate the synthesis of a range of proteins (up to 70000 molecular weight) by the wheat germ cell-free system. Immunoprecipitable calcium-binding protein is translated from RNA obtained from 1,25-dihydroxycholecalciferol-dosed birds but not from control birds. This calcium-binding protein is the same size (27000 molecular weight) as authentic chick calcium-binding protein; No other proteins are specifically precipitated by the antiserum. Thus in chickens 1,25-dihydroxycholecalciferol-induced calcium-binding protein is not synthesised via any precursor molecule. The implications of this result are discussed.


Histone-hydrocarbon interaction. Partition of histones in aqueous two-phase systems containing poly(ethylene glycol)-bound hydrocarbons. The hydrophobic properties of histones have been examined with help of the two-phase partition technique using dextran-poly(ethylene glycol)-water systems. We have found that different fatty acid esters of poly(ethylene glycol) interact with total histones in a manner similar to proteins of the type beta-lactoglobulin and serum albumins. Thus the maximum interaction occurs when the fatty acid contains 16-18 carbon atoms. With less than eight carbon atoms in the polymer-bound fatty acid, no histone-hydrocarbon interaction is observed. The interaction of the five individual histone fractions with palmitate depends on the type of salt used and on its concentration. We suggest that the histones can be divided into three groups with decreasing hydrophobic properties: H3, H2a greater than H4, H2b greater than H1.


An assessment of some of the methods available for the determination of molecular weights of proteins as applied to aspartate aminotransferase from pig heart. The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g-1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g-1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non-empirical method based on quantitative N-terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights ans subunit compositions of proteins are discussed.


An analysis on the slope of Scatchard plots. The Scatchard plot, [X]b/[E]t[X] versus [X]b/[EP]t where [X]b denotes the concentration of bound ligand, [E]t the total concentration of the binding protein and [X] the free ligand concentration, was designed originally for plotting data of ligand binding to a macromolecule possessing identical non-interacting ligand binding sites. However, the plot is used for describing cooperative binding processes. In such cases, the slope of the Scatchard plot is not then equal to minus the intrinsic association constant any more. The meaning of the slope in such cases is a complex function of the binding parameters and its exact interpretation depends on the particular model used  to analyze the binding data. In this communication, the meaning of the slope of the Scatchard plot is analyzed in terms of the different allosteric models.


Studies on the adenine nucleotide translocase from rat liver mitochondria. Isolation, partial characterization and immunochemical properties of carboxyatractylate-binding protein. 1. Solubility of mitochondrial membranes in various solvent systems was determined quantitatively. The most effective agent was the anionic detergent, sodium dodecylsulphate, which solubilizes 90% of the protein at the concentration of 0.1% followed by Triton X-100 (70%), sodium deoxycholate (60%), Brij 56 (50%), and guanidine hydrochloride (40%) at a concentration of 2 M. 2. Affinity chromatography of a clear 0.1% sodium dodecylsulphate solution of digitonized mitochondria on Sepharose 4B containing carboxyatractylate always resulted in the separation of two fractions, one of which was not retained by the column and the other which could be obtained after elution with 2% sodium dodecylsulphate. 3. The retained protein showed a high binding specificity for ATP and [3H]atractylate when compared with the unretained fraction. The amount of bound [3H]atractylate or carboxyatractylate-sensitive binding of ATP was 10.5 +/- 4 nmol/mg protein, and 22 +/- 8 nmol/mg protein, respectively. 4. The major component within the retained fraction, comprising 85% of the total weight, was protein, followed by phospholipids (14%) and approximately 1% triglycerides. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a major (95%) and a minor (5%) component with an apparent molecular weight of 26000 +/- 1000 and 8300 +/- 400, respectively. The gels did not stain for carbohydrates. Ultracentrifugal analysis showed a single, symmetrical boundry. 5. Double immunodiffusion analysis gave a single precipitin line with the corresponding antiserum. [14C]ADP exchange of digitonin particles was completely inhibited by an antiserum to the carboxyatractylate binding protein fraction, whereas the adenine nucleotide transport of intact mitochondria remained unaffected. In the presence of specific immunoglobulins state-3 respiration rate of digitonin particles was prolonged and reduced by approximately 25%. State-4 respiration rate was unaffected.


Conformational analysis of a DNA triplet in aqueous solution. Thymidylyl-(3'-5')-thymidylyl-(3'-5')-2'-deoxyadenosine, d(T-T-A), studied by 1H nuclear magnetic resonance at 360 MHz. The proton signals of a 50 mM solution of thymidylyl-(3'-5')-thymidylyl-(3'-5')-2'-deoxyadenosine, d(T-T-A), in 2H2O at 65 degrees C and p2H=7.4, were detected by nuclear magnetic resonance at 360 MHz. Complete assignment of the 21 ribose proton signals was achieved by extensive decoupling experiments and computer simulations. The coupling constant data show that the ribose rings prefer to adopt the S (2'-endo) conformation. The presence of a substantial amount of a right handed helical structure of the d(-T-A) fragment is indicated from chemical shift considerations. The molecule displays a high degree of conformational purity along the sugar-phosphate backbone.


Use of methyl iodide for probing the polarity of the immediate environment of --SH groups in thiolenzymes. Reaction of methyl iodide with thiosubtilisin. A new approach is proposed for probing the polarity of the immediate environment of -SH groups in thiolenzymes, based on the alkylation of the -SH group with methyl iodide, a relatively small and non-polar molecule. Rate and activation parameters (delta H*, delta S*) for the reaction of the enzyme are compared to those of glutathione, a simple -SH compound alkylated in aqueous medium. The enzyme and model compound are also reacted with iodoacetamide, a polar counterpart of the non-polar methyl iodide. The above method was applied to thiolsubtilisin, an artificial thiolenzyme. 1. The ratio of the rates of alkylation of thiolsubtilisin and glutathione is about 20 times as high with methyl iodide as with iodoacetamide. 2. delta H* and delta S* for enzyme alkylation, as compared to those for glutathione, are remarkably lower with methyl iodide whereas they are slightly higher with iodoacetamide. 3. delta H* and delta S* for alkylation of thiolsubtilisin with methyl iodide are similar to those found with glutathione in 40% dioxane/water mixture. 4. The activation enthalpy and entropy values for the reaction of thiolsubtilisin with D-2-bromo-n-valeramide are lower than those for glutathione reaction. Consequently, in this respect, D-2-bromo-n-valeramide is similar to methyl iodide rather than to iodoacetamide. It is concluded that the -SH group of thiolsubtilisin is located in an environment less polar than water. The concentration of methyl iodide in this non-polar layer is higher than in the bulk solution, which results in an enhanced reaction rate.


Lipid requirement of membrane-bound 3-oxosteroid delta4-delta5-isomerase. Studies on beef adrenocortical microsomes. The role of phospholipid in the beef adrenal microsomal 3-oxosteroid delta4-delta5-isomerase (EC 5.3.1.1) has been investigated with the use of phospholipase A to alter the microsomal phospholipids. The byproducts of phospholipase A digestion have been removed with a wash solution containing bovine serum albumin. Removal of 80-85% of the phospholipid leads to loss of 80-90% of the 3-oxosteroid delta4-delta5-isomerase activity. Reconstitution experiments have been performed by introduction of lipid aqueous dispersions in the enzymatic assay. Asolectin, a commercially available preparation of soy phosphatides, is able to stimulate the enzymatic activity but does not restore the 3-oxosteroid delta4-delta5-isomerase activity in phospholipase-A-treated membranes. In contrast, the introduction of aqueous dispersions of microsomal total lipid mixtures in the enzymatic assay brings about a complete restoration of the 3-oxosteroid delta4-delta5-isomerase activity in the lipid-depleted membranes. It is concluded that the bovine adrenal microsomal 3-oxosteroid delta4-delta5-isomerase requires phospholipid(s) to exhibit its full catalytic activity.


An endonuclease from mouse cells specific for single-stranded DNA. An endonuclease with a molecular weight of about 70000 (5-6S) was extensively purified from mouse ascites cells. The enzyme specifically attacks single-stranded DNA which is degraded mainly to oligonucleotides, with 5-10 residues. Supercoiled covalently closed circular phage DNA is converted to the linear relaxed form. The enzyme activity is highly sensitive to salt and can be stimulated by reagents lowering the dielectric constant of the buffer such as dimethylsulfoxide and glycerol.


Renal cysts. I. Current pathogenic approach. Recent reviews on the genesis of cysts in the adult kidney lead to two major theories: mechanical and toxic. Obstruction indisputably plays a role in determining certain cysts in animals and examination in the human plus certain personal clinical data seems to confirm this. The role of chemical factors in the human has not yet been clearly established, although there is abundant evidence of its occurrence in animals.


Renal cysts. II. Chemical and dynamic study of cystic fluid. The chemical analysis of the fluid obtained by puncturing 100 simple renal cysts in adults show that the composition of cystic fluid resembles that of interstitial fluid. The chemical balance between cystic fluid and the interstitial space seems to occur passively. Antibiotics which were given orally (amoxycilline, minocycline, rifampicin) do not pass through the wall of a simple cyst. Study of the permeability of the cyst wall did not show any uptake of radioactive hippurate or radium pertechnetate.


Long-term results of surgical treatment of bladder exstrophy. A review of the long-term results of the treatment of 18 cases of bladder exstrophy is reported. Five cases have been operated on before 1961 by ureterosigmoidostomy; 4 (80%) patients have good renal function and have not had any complications, although 1 patient has anal incontinence; 1 patient presented bilateral renoureteral complications, requiring several corrective operations, and has anal incontinence too. Out of 13 cases operated on after 1961, 10 have been followed up from 3 to 14 years. In 1 patient an ileal conduit was performed because of the failure of an ureterosigmoidostomy performed at another hospital 10 years before. In 9 patients a personal technique based on the bladder substitution by a colonic loop plus creation of a sphincteric mechanism by trigonal tubulization was used (enterotrigonal urethroplasty). Morbidity has been high (66.6%), but the end results are very satisfactory: 7 patients (77.7%) are currently living a normal life, with normal renal function and good continence; 1 patient is incontinent and has bilateral reflux and a left retracted kidney; 1 patient has been secondarily diverted by means of a cutaneous Y transuretero-ureterostomy due to gross lreteral dilatation, renal damage and incontinence.


Voiding pressure in children.

Voiding pressure in children. A study of voiding pressure (VP) by a suprapubic catheter was performed in 100 children. The children were divided into two groups. Group I (n=96) comprised children with no evidence of clinical, radiological and endoscopic lower urinary tract obstruction. Group II (n=4) consisted of children with a lower urinary tract obstruction. 17 children of group I, judged to be free of uropathy, were considered as the control group. There was no significant difference in the VP of this group when compared with 79 children with uropathy (group IB=47 enuretic, 19 vesicoureteral reflux and 13 urinary tract infection). The VP was studied in each of these groups in relation to sex and age. Males had higher VP than females. There was a significant correlation between age and VP in enuretic children. It was concluded that the VP was only one of the parameters for investigating the urodynamics of the lower urinary tract and should always be accompanied by a measurement of urinary flow.


Unusual presentation of testicular torsion. A review of 40 cases. The symptomatology of testicular torsion was atypical in 16 patients among the 40 that we treated. The occurrence of fever together with the torsion (8 cases), the existence of intense local inflammatory signs (16 cases), its occurrence in adults (23 cases), ectopic testicle (2 cases), and the possibility of a traumatism (5 cases) are misleading and may delay its urgent surgical treatment. In most cases, testicular torsion forms a characteristic clinical picture with easy diagnosis, requiring most urgent operation. There are, however, cases where symptomatology is atypical and deceptive forms delay recognition of the torsion, leading to ischaemic necrosis.


New aspects of urinary tuberculosis.

New aspects of urinary tuberculosis. In urinary tuberculosis, during recent years, there has been remarkable progress in treatment but some new aspects are reported, which raise problems in diagnosis and therapeutics. There is an increase of silent forms, forms with rapid destructive development, even under treatment, and forms masked by association with other diseases. The percentage of elderly patients has increased, as has the number of cases presenting with advanced lesions with little clinical manifestation. This calls for more careful searching, assessment and survey of any suspected case.


Histological grading of renal cell carcinoma. The Erlangen system of histologic grading of the malignancy of renal cell cancers is described in detail. It takes into consideration the type of cell and pattern of the tumor and differentiates between grades 1, 2, and 3. Grade 1 was observed in 10%, grade 2 in 51%, and grade 3 in 39% of the cases. The difference in the 5-year survival rates between tumors of grade 3 and those of tumors of grades 1 and 2 is statistically significant.


Ureteric substitution with a tapered ileal loop. An experimental study. The authors present their experience in replacement of the ureter by a tapered ileal loop over a 24-month period, in a group of 10 dogs. With regard to the morphological and functional behaviour of the neo-ureter, the results on the whole were considered satisfactory. However, some problems such as pyelonephritis, associated with obstruction due to mucosal hyperexcretion, remain unsolved at the present time.


Urinary thyroxine in rats fed various diets and in renal calcium stone-forming patients. In rats fed with various diets as regard contents of magnesium, cholesterin, neutral fat, carbohydrates and proteins, urinary thyroxine (U-T4) appears low in general when compared with control chow, whereas renal calcifications and stones are absent only with the latter and the carbohydrate-rich diet. Normalization of U-T4 is hampered by differences in urinary creatinine excretion and in individual weight gain. In male renal calcium stone patients (20--40 years) U-T4 also is lower than in age-matched controls, but in other groups no abnormality could be detected.


Renal cysts in adults. IV. Therapeutic problems. The study of 1,342 renal cysts gathered from various centres emphasized the great diversity of the therapeutical approach. In this series, 1 patient out of 2 was operated on and 1 out of 6 operations was a nephrectomy. The mortality was 1.2% and complications occurred in 10% of the cases. Arguments in favour of systematic surgery are severely criticized and the risk of failure to recognize a cancer associated with a cyst is very small: when this association was encountered, there were warning signs or symptoms. Percutaneous puncture of the cyst may suffice for diagnosis and treatment of most cases. When surgery is indicated for a cyst as in doubtful diagnosis, symptoms from the swelling or in association with lithiasis, resection of the dome of the cyst is the operation of choice. Considering the extraordinary recovery of the renal parenchyma, the necessity for nephrectomy must be quite exceptional.


Staghorn calculi of the kidneys. A. Clinical review. A series of 91 staghorn calculi are reviewed. Possible aetiological factors are analysed. A high incidence of hypercalciuria was found, and the significance of this is discussed. The rates of complete stone clearance after conservative surgery are considered for different types of staghorn calculus and it is felt that local circulatory arrest with kidney cooling may be of value in removing stones due to Proteus. The place of elective nephrectomy in the management of staghorn calculi is discussed. Nephrectomy may be the treatment of choice in the management of large, unilateral stones in older patients.


Long-term survival after urinary diversion. A reappraisal of ureterosigmoidostomy. Long-term survival occurs following both ureterosigmoid and ileal diversions. Both manoeuvres carry a significant complication rate but where patients have good rectal continence, ureterosigmoid diversion offers the advantage of appliance free life. Success depends on initial good renal function and successful implantation of the ureters into bowel avoiding both reflux and stenosis. Where there is a likelihood of long survival as in such conditions as ectopia vesicae, this technique will provide very satisfactory management and three patients surviving more than a total of 100 years illustrate this approach.


Urethral injuries in Lagos.

Urethral injuries in Lagos. Good results have been obtained following the treatment of incomplete urethral rupture while the results of complete urethral rupture treated in this hospital by the conventional method have been very poor. This poor result is comparable to those from other centers using similar methods. A review of various treatment methods was undertaken and reasons are given for advocating the method of initial suprapubic cystostomy, followed by delay urethroplasty.


Electron microscopic and histological findings on urinary bladder epithelium in interstitial cystitis. Urinary bladder epithelium was studied with electron microscope in 50 patients with interstitial cystitis (IC) and 9 controls. In addition, biopsies of 14 patients with IC were studied with special stains for the presence of bacteria or viral inclusions in the epithelium. No bacteria or viral inclusions could be demonstrated. On the basis of these and earlier negative findings it appears that simple infection of the epithelium is ruled out as the aetiology in IC. Electron microscopy demonstrated an increase in large swollen epithelial cells with decreased amount of lateral processes in half of the IC samples and inflammatory changes in the mucosa. The similarity of the ultrastructure of epithelial cells in controls and IC patients makes it improbable that the disease process originates in the epithelium. In this study special attention was paid to large cytosomes (diameter 0.4--6.0 mum) in the epithelial cells. These contained small vesicular bodies (diameter 0.04--0.2 mum) and lipid droplets, and were found in controls as well as in IC patients.


Experimental study of synadelpho-ureterostomy (transuretero-ureterostomy). An experimental study with synadelpho-ureterostomy (transuretero-ureterostomy) was performed in 25 dogs. The conclusions reached are: (1) side-to-side anastomosis is to be preferred to end-to-side suture; (2) risk of disruption is very slight even where an unexpected obstruction is encountered (test with induced cellophanic periureteritis), and (3) segmental replacement of the lumbo-iliac ureter may apparently be achieved by means of inverted synadelpho-ureterostomy (the damaged ureter is the recipient ureter). This replacement requires a double synadelphostomy, but in our study the normal transposed ureter suffered no harm despite the presence of previous infection and ureteral dilatation.


An unusual ureteric anomaly: uretero-synadelphia with ectopic opening into the urachus. An extraordinary case is reported of "uretero-synadelphia" (a single Y-type ureteric tract for both kidneys) with ectopic opening into the bladder vertex. This case also showed an associated agenesis of the extrahepatic biliary ducts. The possible factors in the development of this anomaly are discussed. The term "synadelpho-ureterostomy" is proposed to describe the surgical techniques used to mimic these anomalies, in place of the more commonly accepted "trans-uretero-ureterostomy".


Idiopathic retroperitoneal fibrosis in multifocal fibrosclerosis. A case of rare combination of retroperitoneal fibrosis and Riedel's thyroiditis is presented. The term multifocal fibrosclerosis is used in the literature to denote such combination and others which include idiopathic retroperitoneal fibrosis, mediastinal fibrosis, sclerosing cholangitis, invasive (Riedel's) thyroiditis and pseudotumor of the orbit.


Further study on the excitation of pyramidal tract cells by intracortical microstimulation. The effective spread of stimulating current for pyramidal tract (PT) cells and fibers was studied using a method of cancelling the shock artifacts and the following results were obtained: 1. The excitability of PT axon collaterals was as high as that of PT cells. 2. These axon collaterals extended as far as 1.0 mm horizontally from the PT cells. 3. The low threshold area for activation of a given PT cell was as wide as 3--4 mm2 on the surface of the cortex. 4. Intracortical microstimulation (ICMS) delivered to the PT cell layer produced direct (D) and indirect (I) descending volleys in the pyramidal tract, but ICMS to the superficial layer (III) produced only I-waves. 5. These I-waves grew significantly larger after 15--20 msec from the start of the train of stimuli. 6. It is concluded that either surface stimulation, or short train of ICMS is inadequate for delineating fine localization of motor function within the cortex. Longer train (30--40 msec) with high frequency pulses (300--400 cy/sec) can produce muscle contraction with much smaller currents, increasing the accuracy of measuring the localization of motor function.


[Effect of trace elements on the hydroxylation of benzo(a)pyrene]. The trace elements are surrounding factors which are able to act on the yield of benzo(a)pyrene hydroxylation reaction of the hepatic tissue. This action was studied for each element and for various concentrations. The action of these elements may occurred to arylhydrocarbon hydroxylase by activating or inhibiting it, to epoxyde hydrase or to glutathione S epoxydase, favouring or inhibiting the way epoxyde dihydrodiol, and finally to arylhydrocarbon hydroxylase induction. Our results, obtained in vitro on hepatic tissue with many metallic salts, showed that some trace elements may have a cocarcinogenic action.