The effects of pregnancy and lactation on copper and zinc retention in the rat.

1. The accretion of copper and zinc in maternal tissues, conception products and postnatal offspring of the rat was determined at different stages of pregnancy and lactation. 2. Equations relating the weight of the bodies of the developing young and of the amounts of Cu and Zn in them to the time that has elapsed since conception showed that, early in pregnancy, the specific rates of accretion of Cu or Zn were greater than that of weight, but declined more rapidly as development continued. The instantaneous rates of accretion of both metals rose throughout pregnancy but only that of Cu continued to increase during lactation. 3. The amount of Cu in the maternal body rose significantly during pregnancy and declined thereafter, but relatively small changes in its Zn content occurred. 4. The results are discussed in relation to the relative demands for these metals during pregnancy and lactation.


The effect of pregnancy and lactation on the absorption of zinc and lysine by the rat duodenum in situ.

1. The absorption of zinc by the duodenum of the rat was greatly enhanced at late stages of pregnancy and during lactation. 2. During pregnancy no increase in lysine uptake could be demonstrated, but during lactation, when further increases in Zn absorption occurred, uptake of lysine was increased. 3. The increased absorption of Zn at different stages of pregnancy and lactation appeared to be related to the demand made by the developing foetuses and post-natal offspring.


Rates of rumen fermentation in relation to ammonia concentration.

1. Four sheep were fed from automatic continuous feeders on whole barley fortified with graded levels of a urea solution. This approach was to a large extent successful in maintaining relatively steady states of rumen ammonia concentration. 2. Rates of barley fermentation in the rumen at various rumen NH3 concentrations were assessed by measuring the disappearance of barley dry matter from polyester bags suspended in the rumen of these sheep. 3. The minimal NH3 concentration for maximal rate of fermentation was estimated as 235 mg/l rumen fluid.


The effects on glucose metabolism of feeding a high-urea diet to sheep.

1. Sheep were given either a basal diet of 107 g crude protein (nitrogen X 6.25)/kg or the same diet to which urea was added to increase the crude protein content to 221 g/kg. Isotope-dilution techniques with [U-14C]glucose and [2-3H]glucose were used to measure various criteria of glucose metabolism. The plasma concentrations of urea and potassium were determined. The sheep were then given the alternative diet and the experiment was repeated. 2. Plasma K concentrations were decreased on feeding urea (P less than 0.05). 3. Plasma glucose concentrations were reduced on the urea treatment (P less than 0.05), while glucose space and metabolic clearance rate were not significantly reduced (P greater than 0.05). 4. Some implications for the feeding of non-protein-N to ruminants are discussed.


The influence of protein source and basal dietary component on survival in acute experimental fowl typhoid in the chick (Gallus domesticus).

1. The influence of diet on the survival of chicks inoculated orally at 15 d of age with Salmonella gallinarum was determined, and in the first experiment the numbers of salmonellae were counted in blood 5 or 7 d after inoculation to assess their value in predicting survival. 2. Significantly more chicks survived among those given beef powder as the protein supplement in a starch-based diet than among those where fish flour replaced beef powder. The number of salmonellae in blood was significantly higher in chicks given the fish-flour diet than in those given the beef-powder diet, but the relationship between the number of viable organisms in blood and the final survival rate was not close. 3. Whale meat and soya-bean protein gave high survival rates, similar to that given by beef powder, and casein plus gelatin was slightly poorer in this respect. 4. For each high-protein food the level of survival from the acute disease was significantly greater with wheat than with starch as the basal component of the diet. 5. The addition of either condensed beef extract or fish solubles to a standard diet did not influence survival.


The protein-sparing effect of carbohydrate. 1. Nitrogen retention of growing pigs in relation to diet.

1. Measurements were made of the losses of nitrogen in the faeces and urine of sixty pigs of approximately 33 kg, given dietary regimens comprising twenty-nine combinations of fish flour (0-800 g/d) and maize starch (0-1200 g/d). 2. The results were used to develop a generalized equation describing N retention as a joint function of N intake and starch intake. 3. The protein-sparing effect of starch was exerted in all circumstances. It was greatest when protein intake exceeded 220 g/d but some effect persisted with protein-free diets. With a high protein supply, the increase in N retention per unit increase in dietary starch decreased from 36 mg/g with the first increment to 3 mg/g with the highest attainable starch intake. 4. From the generalized equation the relationships between net protein utilization and protein concentration and food intake could be described as continuous functions. The equations may be of use in comparing the protein values of diets measured under non-standardized conditions.


The protein-sparing effect of carbohydrate. 2. The role of insulin.

1. In five experiments with growing female pigs of 38-63 kg, insulin (2 mU/kg) per min) and glucose (9-17 mg/kg per min) were infused continuously for 3-7 d. In three further experiments, glucose (9 mg/kg per min) was infused alone for 5 d. 2. In response to the combined infusion, plasma insulin increased 2-7-fold, plasma glucose decreased, on average, by 50% and plasma urea concentration was reduced by 40%. Urinary excretion of urea and nitrogen decreased after the first day of infusion to values averaging 70% of control levels. 3. The infusion of glucose alone provoked only a small increase in plasma insulin. The reduction of urinary urea and of N excretion were approximately 25% of those observed with the combined infusions.


Polypeptides in the succinate-coenzyme Q reductase segment of the respiratory chain.

Complex II (succinate-coenzyme Q reductase) was resolved into ten different polypeptides by polyacrylamide gel electrophoresis. Four polypeptides, CII-1, CII-2, CII-3, and CII-4 with molecular weights of 70 000, 24 000, 13 500, and 7000, were present in large amounts in all preparations examined. CII-1 and CII-2 are the flavoprotein and iron-sulfur protein, respectively, of succinate dehydrogenase; CII-3 and CII-4 have not been functionally indentified. Six polypeptides were present in much smaller amoumts as judged by staining intensity, and each of these comigrated with components in complex III. The amino acid compositions of several of the minor components in complex II were identical with that of an equivalently migrating polypeptide in complex III. We conclude that succinate-coenzyme Q reductase contains four different polypeptides and is contaminated with variable amounts of complex III when isolated as complex II.


Characterization of heterogeneous solutions using laser light scattering: study of the tubulin system.

Laser light scattering has been applied to a systematic study of a heterogeneous solution of tubulin at low temperature--conditions under which tubulin assembly into microtubules does not take place. Methods of analyzing laser light scattering results obtained from solutions containing multiple components are discussed. Data analysis techniques are described and their application to the determination of diffusion constants from experimental data is extensively illustrated. Multiple components were found under the conditions that the tubulin was studied. We have identified one component having D20,w = 4.41 X 10(-7) cm2/s (sigma = 0.54 X 10(-7) cm2/s) which has the expected value for tubulin dimer. In addition, we have found two components which are significantly larger than tubulin. One large component has D 20,w approximately 0.55 X 10(-7) cm2/s and is present in all samples at 4 degrees C even after centrifugation to remove components greater than 10 S. Another large component having 3.2 X 10(-7) cm2/s greater than or equal to D20,w greater than or equal to 1.5 X 10(-7) cm2/s has been found to sediment with 10 S less than or equal to s less than 20 S.


Equilibrium components of tubulin preparations.

Laser light scattering techniques are used to examine the existence of a tubulin-ring or tubulin-aggregate equilibrium. Three principal components in addition to tubulin dimer have been observed. One large component having D20,w = 0.65 +/- 0.25 X 10(-7) cm2/s is in equilibrium with tubulin (dimer) at 4 degrees C. A second large component having D20,w = 0.74 +/- 0.05 X 10(-7) cm2/s is metastable at 4 degrees C and is formed when tubulin solutions are warmed to 36 degrees C and cooled back to 4 degrees C. The properties of the component reported in the previous paper (Gethner, J.S., Flynn, G.W., Berne, B.J., and Gaskin, F. (1977), Biochemistry 16 (preceding paper in this issue)) to sediment at 10 S to 20 S are shown to be consistent with a small, metastable aggregate of tubulin.


The shape of myosin subfragment-1. A equivalent oblate ellipsoid model based on hydrodynamic properties.

The molecular weights of the two heads of myosin subfragment-1, S-1(A1) and S-1(A2), based on sedimentation equilibrium are 120 000 and 110 000. Hydrodynamically, the two heads are indistinguishable, with intrinsic viscosity, [eta], of 0.064-0.065 dL/g and sedimentation coefficient, s(0)20,w, of 5.8 S. Together with the rotational correlation time taken from the literature (235 ns), all three hydrodynamic properties can be better fitted with an equivalent oblate ellipsoid of revolution than a prolate model. The width of the equatorial axis of the ellipsoid is about 135 A (the axial ratio is about 6). Probably, the S-1(A1) and S-1(A2) molecules have a half-doughnutlike or a flattened pearlike shape rather than an elongated one.


Purification and properties of the hemagglutinin from Maclura pomifera seeds.

Maclura promifera seeds contain a protein which agglutinates human erythrocytes at concentrations as low as 4 ng/mL. This property is related to its ability to bind with high specificity various alpha-D-galactopyranosides. The agglutinin, which was pruified by affinity adsorption, exhibits one band on immunoelectrophoresis and displays one peak during ultracentrifugation, isoelectric focusing, and gel permeation chromatography. The active protein has a molecular weight of 40 000-43 000 and contains two dissimilar polypeptide chains of 12 000 and 10 000, respectively.


Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts.

The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins.


Partial amino acid sequence of brain actin and its homology with muscle actin.

Actin was purified from calf brains by chromatography on DEAE-Sephadex and hydroxylapatite. The protein was then subjected to amino acid sequence analysis by isolating and sequencing its cyanogen bromide peptides. CB-1, 3, 4, 5, 6, 9, 10, and 12 correspond to equivalent segments of rabbit skeletal muscle actin, while subsitutions involving methionines give rise to some new peptides. The region that corresponds to CB-13 in muscle actin becomes two peptides in the brain protein because of a Leu leads to Met replacement at position 16, while Met leads to Leu substitutions at positions 176 and 298 give rise to two larger peptides, CB-15 + 7 and  CB-8 + 2, which correspond to muscle actin CB-15 fused with CB-7 and CB-8 fused with CB-2, respectively. The peptides that have been isolated from brain actin contain 267 of the 374 residues in actin, of which 157 have been unequivocally identified. When the data are compared with those for rabbit skeletal muscle actin, 11 replacements are seen; thus the two actins differ at about 7% of the positions examined.


Methylation of glucagon, characterization of the sulfonium derivative, and regeneration of the native covalent structure.

The methylation of the single methionine residue of glucagon is accomplished at a pH of 3.5 in 8 M urea with methyl iodide. The reaction product is a soluble sulfonium derivative, S-methylglucagon, which can be isolated in a highly purified form. This derivative is characterized by amino acid analysis and its effect on the adenylyl cyclase system of rat liver plasma membranes. S-Methylglucagon does stimulate the adenylyl cyclase system; however, its activity is approximately 500 times less than that observed with the native hormone. The solubility of this derivative is great enough to allow for further modifications of the molecule which can be followed at a later stage by demethylation. Demethylation of S-methylglucagon regenerates the original covalent structure and is accomplished by treatment with Cleland's reagents at a pH of 10.5. The regenerated hormone is indistinguishable from native glucagon by its amino acid composition and its ability to stimulate the adenylyl cyclase system. The entire methylation-demethylation reaction sequence has been carried out with yields that approach 75%. The technique is suitable for the isotopic enrichment of native glucagon and may well be applicable to selected other methionine-containing peptides.


Diazomethyl ketone substrate derivatives as active-site-directed inhibitors of thiol proteases. Papain.

The diazomethyl ketones of z-Phe-Phe inactivate papain by a stoichiometric reaction at the active-center thiol. Since the reagents are stable in mercaptoethanol, their reaction with papain is judged to be the result of complex formation characteristic of affinity-labeling reagents. The diazomethyl ketones react by a mechanism different from that of chloromethyl ketones, since the pH dependence of their inactivation of papain is different, the rate increasing with decreasing pH. This relationship has been observed in other cases, such as in the reaction of azaserine with glutamine amidotransferases [Buchanan, J. M. (1973), Adv. Enzmol. Relat. Areas Mol. Biol. 39, 91], and is interpreted as an indication of reaction with a thiol group in its protonated form.


Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue.

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.


Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature.

Light-induced absorbance changes were measured at temperatures between --30 and --55 degrees C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the "primary" to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870+ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870+. A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artifical donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.


Does the rate of cooling affect fluorescence properties of chloroplasts at -196 degrees C?

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to -196 degrees C rapidly, as very thin samples adsorbed into substrates whick were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.


Energy transfer from photosystem II to photosystem I in Porphyridium cruentum.

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of porphyridium cruentum which had been frozen to -196 degrees C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at -196 degrees C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.


Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants.

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.


Cooperative interactions between calcium-binding sites on glycerinated muscle fibers. The influence of cross-bridge attachment.

A double isotope technique and EGTA buffers were used to measure the binding of Ca2+ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca2+-binding sites in the intact filament lattice. In the presence of 5 mM MgCl2 (no ATP), reduction of filament overlap was associated with a reduced binding of Ca2+ over the entire range of free Ca2+ concentrations (5.10(-8)-2.10(-5) M). With maximum filament overlap (sarcomere length 2.1-2.2 micrometer) the maximum bound Ca2+ was equivalent to 4 mol Ca2+/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8-4.4 micrometer) the maximum bound Ca2+ was equivalent to 3 mumole Ca2+/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca2+ binding site appear, but cooperative properties are imposed upon the binding sites.


Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. I. Oxidation kinetics after a flash.

P-700, plastocyanin and cytochrome f redox kinetics were measured after one flash, using dark-adapted Chlorella in the presence of hydroxylamine and 3(3,4-dichlorophenyl)-1,1-dimethylurea. Plastocyanin becomes increasingly oxidized with a half-time of 70 microseconds, then undergoes reduction with a half-time of 7ms. Cytochrome f oxidation has a sigmoidal time-course and a half-time of 100 microseconds. Its redution exhibits a half-time of 4 ms. These results are interpreted in a linear scheme: (formula: see text). An equilibrium constant of 2 between cytochrome f and plastocyanin (PC), which contrasts with the large equilibrium constant between PC and P-700 is computed. The presence of cytochrome b6 in a cyclic path around Photosystem I is confirmed under these conditions.


Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. II. Reduction kinetics and electric field increase in the 10 ms range.

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC+ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern. The reduction of PC+ and cytochrome f+ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267-284). Both need the presence of a compound R' in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R' as terminal reducant, PC+ and cytochrome f+ as terminal oxidants, would account for this field generation. Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.


Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria.

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca2+ uptake and H+ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca2+ efflux and H+ uptake. Conversely, ruthenium red blocks Ca2+ uptake and H+ production but does not prevent dinitrophenol-induced Ca2+ efflux and H+ uptake by mitochondria. These results suggest that mitochondrial Ca2+ uptake and release exist as two independent pathways. The efflux of Ca2+ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca2+ uptake carrier.


Proton evolution from photosystem II. Stoichiometry and mechanistic considerations.

1. In a sequence of flashes given to dark-adapted chloroplasts, the flash yield of proton release oscillates with a period of 4, which is similar but not identical to the oscillation of the O2 flash yield. 2. Using the proton release associated with ferricyanide reduction as a calibration, we computed that two protons are released in the terminal O2-liberating reaction; the other two protons are released in precursor conversion steps. 3. Analysis of the effect of preflashes on the oscillation pattern showed that the S1 leads to S2 transition releases no proton, the S0 leads to S1 transition somewhat less than one (0.75), and the S2 leads to S3 transition somewhat more than one (1.25). 4. The precision of the data was sufficient to exclude the possibility that in the four-step water oxidation, proton release follows a simple 1, 0, 1, 2 pattern. A possible model to interpret the observed flash yield patterns is discussed.


Synthesis of chloroplast membrane lipids and chlorophyll in synchronous cultures of Chlamydomonas reinhardi.

Chloroplast membrane lipid synthesis has been studied in synchronously growing cultures of Chlamydomonas reinhardi. The synthesis of sulfolipid and phospholipid were measured by incorporation of 35SO4(2-) and 32PO4(3-) during a 1-h pulse. Galactolipid synthesis was measured by H14CO3- incorporation into lipid fractions separated by thin layer chromatography. Lipid synthesis occurs principally during the light portion of the synchronous cycle. Phosphatidylglycerol is synthesized between 3-4 h in the light and sulfolipid is labeled between 7-9 h in the light. Galactolipid synthesis appears to reach maximal rates shortly after the lights go on and again at 7 h. Chlorophyll reaches maximal rates of synthesis after 7 h. These lipids are made and inserted into the chloroplast membrane prior to major increases in photosynthetic capacity. Our results also show that chloroplast membrane lipids are synthesized in a sequential or multistep process.


Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts.

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.


Metabolism of eicosa-11,14-dienoic acid in rat testes. Evidence for delta8-desaturase activity.

The metabolism of [14C]eicosa-11,14-dienoic acid was investigated in rat testes in vivo and in vitro. Intratesticular injection of [1-14C]eicosa-11,14-dienoic acid resulted in the appearance of radioactivity (4-30% of 14C in total fatty acids) in 20-carbon trienoic fatty acids and a small amount (2-3.5%) in arachidonic acid. Analysis of the 20-carbon trienoic acid fraction by ozonolysis indicated that 15 to 34% of the 14C in this fraction was in an 8-carbon fragment originating from eicosa-8,11,14-trienoic acid. The rest (66 to 84%) was in a 5-carbon fragment, presumably originating from eicosa-5,11,14-trienoic acid. Incubation of testicular tissue minces or microsomes with [1-14C]eicosa-11,14-dienoic acid yielded labeled eicosa-8,11,14- and eicosa-5,11,14-trienoic acids in proportions similar to those obtained in vivo. Added unlabeled acetate had no effect on the formation of [14C]eicose-8,11,14-trienoic acid in vitro. Therefore, it is unlikely that the labeled eicosa-8,11,14-trienoic acid arose from elongation of octadeca-6,9,12-trienoic acid with labeled acetate derived from bio-oxidation of the labeled substrate. These results are compatible with a limited desaturation of eicosa-11,14-dienoic acid to eicosa-8,11,14-trienoic acid and provide evidence for delta8 desaturate activity in rat testis.


Elimination of low steady-state concentrations of [5,6-3H2]prostaglandin E1 in the pulmonary and the systemic circulations of anaesthetized rats.

The elimination of [3H]prostaglandin E1 in anaesthetized rats was studied by continuous intravenous or intraarterial infusions, producing steady-state concentrations at the level of endogenous prostaglandin E2 in mixed venous blood. Blood samples (0.5 ml) were collected from the carotid artery or the right atrium, respectively. The levels of [3H]prostaglandin E1 were measured at different infusion time intervals and the 3H-labeled hydrophobic metabolites characterized. Cardiac output was estimated by a modification of the dye injection method, using 125I-labelled albumin as the marker. From the cardiac output and the rate of infusion, the fractional clearance of the lung and the systemic beds in the steady-state situation were estimated to 88.3 +/- 3.2% and 54.1 +/- 15.2% (mean +/- S.D.), RESPECTIVELY. The hydrophobic metabolites were characterized chromatographically on Sephadez LH-20 columns, using synthetically prepared [14C]prostaglandin metabolites as internal standards and markers. The identities of some metabolites were further established by derivative formation to a constant [3H]/[14C] ratio. The major metabolite was 15-keto-13,14-dihydro-[3H]prostaglandin E1, while 15-keto-[3H]prostaglandin E1 and 13,14-dihydro-[3H]prostaglandin E1 could not be demonstrated.


The action of purified lysophospholipases on microsomal membrane-bound lysophosphatidylcholine.

Rat liver membranes were labelled by intraperitoneal injection of [Me-14C]choline chloride. Isolated microsomal membranes were briefly treated with pancreatic phospholipase A2 to produce different levels of membrane-bound lysophosphatidylcholine. The hydrolysis of this lysophosphatidylcholine by two purified lysophospholipases from beef liver was studied. The specific activity of enzyme I at saturating membrane concentrations appeared to increase linearly with the lysophosphatidylcholine level in the membranes until the lysoderivative represented 15% of the original phosphatidylcholine. In contrast, the specific activity of enzyme II was independent of the lysophosphatidylcholine level, at least in the range of 4.9-34.0% tested. These different kinetics are discussed in terms of the possible functions of both enzymes in liver.


Effect of polyol detergents on cholesterol and triglyceride absorption. Hypolipidemic action of chronic administration of hydrophobic detergent.

Nonionic detergents were found to affect absorption of neutral lipid and cholesterol. The effects of these detergents were related to their physicochemical properties. The most effective detergents for producing an inhibition of lipid absorption were detergents containing 90% hydrophobic components. Hydrophilic detergents did not interfere with lipid absorption and in some cases, it was facilitated. Studies done over a period of one month demonstrate that addition of a hydrophobic detergent to a high fat-high cholesterol diet results in lower serum levels of cholesterol and triglyceride and prevents cholesterol accumulation in the liver of the rat. This effect was also associated with significant loss of body weight.


Fluorescent study of tobacco mosaic virus protein.

Protein fluorescence properties of tobacco mosaic virus [3 Trp residues per monomer (positions 17, 52, 152)] and of two tobacco mosaic virus mutants [green tomato atypical mosaic virus, 2 Trp (52, 152) and cucumber virus4, 1 Trp (unknown position)] have been studied. Emission spectra, fluorescence quantum yields and lifetimes were determined. Results showed that protein fluorescence is due to buried Trp only, except for the cucumber virus4 strain, in which Tyr also contributed to the emission. Comparison of the three strains showed that Trp 17 and Trp 52 have high fluorescence yields (phi17 = 0.29; phi52 = 0.37) whereas Trp 152 (probably present in cucumber virus4) is strongly quenched (phi152 = 0.035). An unusually efficient Tyr leads to Trp energy transfer was observed in tobacco mosaic virus protein, indicating that most of four Tyr residues are located near the highly fluorescent Trp.


On the molecular conformation of human haemopexin. II. Analysis of circular dichroic spectra.

The circular dichroic (CD) spectra of haemopexin in the far ultraviolet region exhibit an atypical positive maximum at 231 nm, which prevents determination of the secondary structure by the usual methods. We have developed a modified analytical method by which it was possible to calculate the secondary structure of the protein (8% of alpha-helix, 10% of beta-conformation) and the actual wavelength (229 nm) and intensity of the above-mentioned positive band. Measurements and analysis of the CD spectra of haemopexin in an alkaline medium gave information ascribing a major part of intensity of the band at 229 nm to the B1u transition of Tyr; we believe that some other electronic transitions contribute to this band, too. An assignation of the individual extremes of the CD spectrum in the near ultraviolet region to the individual electronic transitions is propounded. The dichroic bands of Trp in this region and the temperature dependence of the CD spectra in the far ultraviolet region corroborate our idea that the haemopexin molecule contains a very compact hydrophobic peptide core. Similarities of the CD spectra of haemopexin to those of the haem. haemopexin complex throughout the ultraviolet region suggest a conformational likeness of these two molecules.


Fractionation of rhodopsin and other components in the rod outer segment membrane by ammonium sulfate salting-out.

Purified bovine rod outer segment membrane was solubilized in a mixture of 1.5% cholic acid/20% saturated ammonium sulfate and 0.05 M phosphate buffer (pH 7.5). The solubilized rod outer segment membrane was fractionated with ammonium sulfate and 70--90% of rhodopsin (A278/A498= 1.6--1.9) was recovered in the fraction of 50 to 60% saturation with ammonium sulfate, giving a highly concentrated solution of purified rhodopsin (A1CM 498 = 83). By the method of ammonium sulfate salting-out, the solubilized rod outer segment membrane was divided into several fractions without a loss of components. The components in each fraction were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rhodopsin and opsin amounted to 93% of the total protein in the membrane. Other proteins with molecular weights of 46 000, 52 000, 56 000, 70 000, 95 000, 105 000, 130 000 and 270 000 were also detected. Most of phospholipids in the rod outer segment membrane remained in the supernatant above 60% saturation with ammonium sulfate.


Affinity labelling of human transcortin.

The binding site of transcortin has been studied by using bromoacetyltestosterone and bromoacetylated derivatives of progesterone which were monohydroxylated at different positions of the steroid nucleus. Specificity of affinity labelling was demonstrated by the displad cortisol analog was added to a [3H]cortisol-transcortin complex solution. The binding site crevice was found to be very narrow in the vicinity of the A and B rings of steroid since 2alpha-hydroxyprogesterone, 6alpha- or 6beta-bromoacetoxyprogesterone and dexamethasone could not displace bound cortisol. A specific affinity labelling was obtained with 11alpha-bromoacetoxyprogesterone, 16alpha-bromoacetoxyprogesterone and 17beta-bromoacetyltestosterone. The results of the affinity labelling by these hormone analogs suggested that one methionine and one histidine residues were located within the active site:methionine might interact with the 11beta-hydroxyl group and histidine with the 20 keto group of cortisol.


Electrophoretic isolation and partial characterization of a glycoprotein of the submandibular glands of the mouse.

A protein has been isolated from the water-extract of the submandibular glands of the mouse, after Biogel P-300 column passage, followed by preparative polyacrylamide gel electrophoresis at pH 4.3 and subsequently at pH 8.9, designated the AM1 protein. The isolated protein was electrophoretically pure in 7.5, 10 and 15% polyacrylamide gels both at pH 4.3 and at pH 8.9. Likewise, by electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel only one protein band could be detected. Of the total amount of the water-extractable submandibular proteins the recovery of this protein component comprised 3 to 5 per cent. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 28 000, both in 7.5 and 15% gel. The isoelectric point was determined by isoelectric focusing in 4.8% polyacrylamide slabgel to be 4.85. The amino acid analysis showed that the ratio of acidic amino acids (Glx plus Asx) to basic amino acids (Lys plus Arg) is 2.3. The glycoprotein consists of protein for 77.4 per cent and of carbohydrate for 22.6 per cent. The molar ratio of the carbohydrates was GlcNH2:GalNH2:Man:Gal:Glc:Fuc:sialic acid = 22.0:1.3:3.0:1.7:10.0:2.6:0.3. The glycoprotein was not secreted from the submandibular glands by stimulation with cholinergic (acetylcholin) or adrenergic (noradrenalin) drugs both in vitro and in vivo. So, it appeared that this glycoprotein could be characterized as a cellular, non-secretory component of these salivary glands.


Isolation of vitamin B-12-binding proteins by combined immuno and affinity chromatography. Comparative studies on the isolated and unisolated proteins.

Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.


Purification of C-reactive protein on DEAE-cellulose by a simple two-step procedure utilizing the calcium-dependency of the protein.

A simple procedure for isolating C-reactive protein is presented. The method consists of two chromatographic separations on diethylaminoethyl (DEAE)-cellulose columns and utilized the difference between the binding of C-reactive protein to DEAE in the presence and absence of calcium. Electrophoretically pure C-reactive protein can be recovered from serum or ascitic fluid with a yield of approx. 50--60% within one day.


Structure of a peptide antifreeze and mechanism of adsorption to ice.

Sequence studies of an alpha-helical peptide antifreeze isolated from winter flounder have revealed the presence of clusters of polar amino acids separated by long sequences of alanine. Most of the polar residues are threonine and aspartate and are separated by 4.5 A, a repeat distance that also separates the oxygens in the ice lattice along the a-axis of an ice crystal. Such a lattice match suggests that the peptide binds to ice by means of hydrogen binding.


Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifolii which induce nitrogenase activity.

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growing mutants were selected which were unable to utilize NH+4 as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine. Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH+4 to a medium containing glutamate as the nitrogen-source resulted in a 50--70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH+4. Biochemical analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP+ -and NAD+ -dependent activities) and glutamate dehydrogenase (NAD+-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or beta-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.


Regulation of thyroid ornithine decarboxylase by the polyamines. Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction.

When spermidine, putrescine or 1,3-diaminopropane was injected (12.5 mumol/100 g body weight) into rats 1 h before thyrotropin, ornithine decarboxylase activity was increased by 75--150% over control levels. However, when greater than or equal to 75 mumol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70--95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx 35%. The polyamines also inhibited thyrotropin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2--5 . 10(-4)M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentrations of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity. A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats and in vitro by incubating bovine thyroid slices with 2--10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide. We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.


High affinity estrogen binding by rabbit liver.

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.


Factors involved in the uptake of corticosterone by rat liver cells.

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent Kt values of the saturable systems were 64 +/- 40 nM (n = 3) and 1085 +/- 313 nM (n = 12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluoro-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35--45%. In the presence of cortisone, cortisol, dexamethasone, aldosterone, testosterone, estradiol-17beta and estrone (2 muM each) the uptake decreased 30--50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.


Hormonal effect on glycosaminoglycans and glycoproteins in uteri of ovariectomized rats.

1. Glycosaminoglycans such as chondroitin sulfate A (or C), chondroitin sulfate B (dermatan sulfate), heparitin sulfate (heparan sulfate) and hyaluronic acid were identified as major glycosaminoglycan components in whole uteri as well as in uterine stroma of rats. Two types of sialoglycoproteins with different electrophoretic mobilities (fast- and slow-migrating) were detected in the glycosaminoglycan fraction from the luminal epithelia. 2. Treatment of ovariectomized rats with estradiol-17beta markedly increased the uterine contents of glycosaminoglycans. Chondroitin sulfate A (or C) was found to increase more than chondroitin sulfate B. Furthermore, it was found that the estrogen treatment specifically increases the fast-migrating sialoglycoprotein level in the luminal epithelia and results in the appearance of it in the uterine fluid. 3. Administration of progesterone to ovariectomized rats slightly increased the uterine glycosaminoglycan content without appreciable alteration of the uterine glycosaminoglycan pattern.


Proton NMR and spin lattice relaxation study of nucleoside di- and triphosphates in neutral aqueous solutions.

The average conformations of adenosine, inosine and guanosine di- and triphosphates in neutral aqueous solution have been investigated by 1H vicinal couplings, chemical shifts and T1 relaxation time measurements at 250 MHz. Comparison of chemical shifts with those of the corresponding nucleotide monophosphates suggests that the beta-phosphate group is in all cases oriented towards the base and close to H3'. The vicinal coupling constants indicate that the proportion of the S conformer of the ribose moiety is 55--60% and that the gauche-gauche rotamer of the CH2-OP exocyclic group is predominant. The preferential orientations of the base have been determined by minimization of the standard deviation about the mean of the molecular reorientation correlation times derived from the H8, H1', H2' and H3' relaxation times and computed interproton distances. The problem of the correlation between the syn-anti equilibrium and the N equilibrium S interconversion has been examined. Typical magnetization recovery curves after a 180 degree pulse have been simulated in the case of ATP, taking into account cross relaxation effects. It is shown that in most of the molecules under consideration the syn orientation of the base is predominant whereas for ATP the syn and anti are equivalent.


The isolation of a new compound from urine of humans with porphyria cutanea tarda.

A method is reported for the isolation of phyriaviolin, a new compound from human porphyria cutanea tarda urine. The substance has been crystallized and some of its properties are described.


Identification of acetylcholine and propionylcholine in bull spermatozoa by integrated pyrolysis, gas chromatography and mass spectrometry.

Acetylcholine was identified in fertile bull spermatozoa using combined pyrolysis gas-chromatography and mass spectrometry. Bull spermatozoa contain other minor choline esters in smaller quantities than acetylcholine. One of the minor choline esters is possibly propionylcholine. The spermatozoa from infertile bulls exhibited low motility and very low levels of acetylcholine.


[Solid-state enzymatic reactions. II. Chymotrypsin hydrolysis of N-succinyl-L-phenylalanine n-nitroanilide].

There was studied the solid-state enzyme reaction of specific substrate N-succinyl-L-phenylalanine-p-nitroanilide hydrolysis in contact with alpha-chymotrypsin (Cht). It is shown that product yield is dependent on hydration of the protein. The product is formed only when the relative pressure of water vapours (p/ps) was higher than certain magnitude (p/ps) crit; which depends on the amount of the salt in the sample: the higher the salt concentration the lesser the (p/ps) crit value. It is suggested that the counter-ions may interact with some of primary hydration sites of the Cht molecule. Because of that for the formation of the active Cht conformation is enough to bind lesser number of water molecules than in salt-free samples. But in the presence of salt excess in Cht sample it is necessary to bind of the protein surface at least yields to 80 mol H2O/mol Cht.


[Energy transformation in mechanoreceptors].

The values of relative threshold deformations and of the mechanosensitivity coefficent are evaluated for some mechanoreceptors. The thermodynamical model of energy transformation in a primary act of mechanoreception is presented, the values of mechanical elastic energy stored in the membrane are calculated. The model of the mechanosensitive center as the ion-selective channel, changing dimensions of its protein part due to closing and opening of the channel is shown to give satisfactory values of the free energy change of the system at the threshold stimulus.


[Equilibrium surface charge distribution in phospholipid vesicles. I. Method of calculation].

This paper presents a method of calculation of the surface charge equilibrium distribution between the two surfaces of a spherically closed phospholipid bilayer suspended in aqueous electrolyte solution. The net surface charge is supposed to be provided by the ionized polar groups of the phospholipid molecules. Its equilibrium distribution is found by minimization of the free electrostatic energy. The procedure of minimization  utilizes the solution of the Poisson-Boltzmann equation which describes the double electric layers of the membrane and an expression for the membrane potential derived under the assumption of absence of charges in the membrane phase. An analytical solution of the problem in the range of validity of the linearized Poisson-Boltzman equation is obtained. It is shown that in this case an equilibrium transmembrane potential exists, and the surface charge density is greater at the outer surface of the vesicle.


[Possible study of period turnover in the solutions of biological systems using the optical displacement method].

The spectrum of light scattered by particle solution with periodically changing properties is under discussion. Besides the central line the displaced ones are shown to appear. Both the central and displaced lines are broadened due to the diffusion movement of particles. Experimental conditions necessary for the resolution of these lines are found. Solution of functioning ribosomes is selected as a model. The results can be nevertheless also applied to other similar systems.


[Equilibrium surface charge distribution in phospholipid vesicles. II. Results of calculations].

The results of the calculations of the equilibrium potential and surface charge distribution in a vesicle with radii r1=55 A and r2=100 A are presented. The calculations were carried out for the electrolyte  concentrations 100 mM, 10mM, 1mM and 0.1 mM. The digitally obtained solutions of the Poisson-Boltzman equation for the case of spherical symmetry were utilized in the exact solution of the problem. The predictions of the exact solution differ significantly from these of the linear approximation in the range of low electrolyte concentrations. The influence of the membrane dielectric permeability on the charge distribution is negligible.


[Delayed chloroplast luminescence. Activation and suppression].

Effect of diethyl ether, detergent triton X-100, glycerine, sucrose and preliminary heating on delayed luminescence (DL) of chloroplasts has been studied. Ether and triton X-100 in concentrations activating electron transport inhibit DL acting similarly to photophosphorylation uncouplers. Preliminary heating to 42-42C, glycerine and sucrose activate both the electron transport and DL of chloroplasts. Activation of electron transport under these agents is suggested to result not from photophosphorylation uncoupling, but from the changes in the conformation of chloroplast memebranes.


[Phase transition studies of model and biological membranes. I. Use of hydrophobic fluorescent probes for studying phase transitions in liposomes].

Phase transition induced by temperature changes in liposomes prepared from synthetic dimyristoyl-(DML), dipalmytoyl-(DPL) and distearyl-(DSL) lecithins and also from equimolar mixture of DML and DPL and mixtures of DPL and cholesterol were studied with fluorescent probes such as 3-metoxybenzantron (MBA) and dimetylaminocholkone (DMC). The significant changes of fluorescence intensity and maximum position of MBA and DMC were found in the regions of phase transitions at 23, 42, 54 and 31c in liposomes from DML, DPL, DSL and mixture of DML and DPL, respectively. Cholesterol incorporation into liposomes from DPL led to a decrease of transition temperature and cooperativity for these liposomes. At cholesterol concentration of 20 mol. percent or more the transition disappeared completely. It is concluded that MBA and DMC can be used for the investigation of biological membranes phase transitions.


[Formals for histogram analysis of Purkinje cell interspike intervals].

A hypothesis has been advanced proposing that the resting discharge of Purkinje cells depends on two factors: 1) tonic excitatory inflow and 2) Puasson's inhibitory inflow. This is why variety of interspike intervals is supposed to be the result of non-regular arrival of inhibitory impulses. From these assumptions an analytical expression was derived allowing to calculate probabilities of interspike intervals of various durations. The histograms of interspike intervals obtained analitically were found to coincide satisfactory with those obtained in experiments (Fig. 3, a, b). The analysis of real histograms based on the presented theory allowed to compare excitatory and inhibitory inflows to individual Purkinje Cells. It was found that, on the average, those cells which had large excitatory inflow, had also large inhibitory one, and vice versa (Fig. 4). It was also found that small decrease (about 20 percent) of excitatory inflow, or corresponding increase of inhibitory inflow, resulted in complete inhibition of most Purkinje cells.


[Refinement of the A- and B-forms of DNA by the atom-atomic potential founctions method].

Structures of poly(dA). poly(dT), poly[d(A-T)].poly[d(T-A)], and poly (dG). poly (dC) were refined by an empirical method, taking into account flexible sugar moieties and simmetry conditions of DNA. The energetical optimal forms were found using Arnott's forms as starting points. These forms were shown to depend only slightly on the polynucleotide type and sequence, and characterized by C(2') endo-C(3') endo and the polynucleotide type and sequence, and characterized by C(2') endo-C(3') endo and C(3') endo and C(3') endo-C(2') endo sugar conformations. The resulting structures have the helix parameters corresponding to X-ray findings  and characterized by augment resemblance between structural parameters (dichedral angles and sugar conformation) of A- and B-basic DNA forms.


[Adaptation effects in the analyzer of movement direction].

A prolonged observation of a point-like stimulus moving in a given direction influences the perception of movement direction of subsequent stimuli. The prolonged observation of the same stimulus results in a subjective drift of the perceived movement towards horizontal or vertical direction depending on which of these directions is nearer to the stimulus trajectory. If, however, the stimulus moves in vertical, diagonal and horizontal directions its perception does not change under prolonged observation.


[New intermediates in the photochemical transformation of rhodopsin].

The conditions of preferential accumulation of intermediates of the photochemical reaction cycle of bacteriorhodopsin (BR) P550 and P419 at low temperature are found. Upon illumination P550 and P419 undergo photochemical conversions into the light-adapted form of BR (P570), forming during this conversions a number of new intermediates: P550 leads to P560-- -- -- leads to P570; P419 leads to P421-- -- -- leads to P565-- -- -- leads to P585-- -- -- leads to P570; P419 leads to P470-- -- -- leads to P570. All intermediates are photoactive. All light reactions are photoreversible and give formation to the products with absorption maximum shifted to the red as compared to the initial state. The absorption spectra of intermediates are complex and include several bands which are more pronounced in the spectrum of P419 (maxima at 442, 419, 398 nm, a shoulder at 375 nm) and P421, less in the spectrum of P570 (maximum at 578 nm, shoulders at 540 and 608 nm) and others.


[Oscillating mechanism of chromosome conjugation].

The macroscopic theory of selective interaction of large molecules suspended in liquid medium is developed. The structure of such molecules permits them to maintain their excited oscilation at the expense of the medium thermal fluctuations. Interaction is realized through the oscillatory energy transfer from one macromolecule to another in the type of beatings. The resonant attraction forces, appearing in the second order of the bond constant, are proportional to the third degree of inverce distance between the oscilators.


Local spectral analysis in the visual cortex.

On the basis of experimental evidence presented earlier a model of local spectral analysis of the image performed by the complex receptive fields of the visual cortex has been proposed. An essential feature of the model is that the generalized piece-wise Fourier transform is performed not over the image luminance function but over the logarithm contrast function resulted from analysis of the image by the round receptive fields of the preceding levels. Such an assumption removes a number of experimental objections offered against the hypothesis of two-dimensional Fourier transform in the visual system. The consequences from the piece-wise expansion in a series of basic functions have been considered and among them: the channel frequency characteristics which can have more than one maximum; the possibility of describing the image by a limited number of channels with overlapping frequency characteristics; the existence of mechanisms for estimation of phase shift between frequencies.


Time constants of vestibular nuclei neurons in the goldfish: a model with ocular propioception.

A simple model of the vestibular-ocular reflex with a proprioceptive eye velocity feedback loop is used to simulate recent data on the vestibular responses of neurons in the vestibular nuclei of spinal goldfish. The data support the hypothesis that a proprioceptive feedback loop elongates the vestibular nucleus time constant to equal that of the slow phase eye movements of vestibular nystagmus.


[Pentose phosphate pathway and nucleic acid metabolism in red and white muscles of fish].

The activities of the pentose phosphate cycle enzymes, the content of nucleic acids and the activities of acid and alkaline RNAses in the heart and red and white muscles of cartilaginous and teleost fish were determined. It was found that the rates of the dehydrogenase and transferase reactions of the pentose phosphate pathway and the nucleic acid metabolism in the red muscles and heart are much higher than those in the white muscles of the species under study.


[The substrate specificity of trypsin. The interrelationship between the structure and reactivity for quasi-substrates, derivatives of O-alkylmethylphosphonic acid and carboxylic acids].

The kinetics of trypsin phosphorylation by thioesters of O-n-alkylmethylphosphonic acids, and reactivation of corresponding phosphoryl enzymes as well as kinetics of trypsin-catalyzed hydrolysis of p-nitrophenylcarboxylates have been studied. The rate constants for phosphorylation and dephosphorylation of trypsin depend on hydrophobicity of non-polar fragments in both substrate series in the same degree. On the other hand, the deacylation rate constants for a series of acyl trypsins do not change significantly while the apparent Michaelis constants change consistently with variations of non-polar acyl substituent. The study of substrate specificity of trypsin in terms of the transition state theory has allowed to elucidate the basis for low reactivity of trypsin towards the quasisubstrates.


[Correlation of mitochondrial ribonucleotide reductase and thymidine kinase activities with the synthesis of mitochondrial DNA in rat liver during regeneration].

3H-thymidine incorporation into DNA of heavy mitochondria from regenerating rat liver and the change of mitochondrial thymidine kinase and ribonucleotide reductase activities are studied in vivo in regenerating rat liver within 6--48 hours after hepatectomy. Synthesis of mitochondrial DNA and changes in the activity of the enzymes studied are found to be undulate. Thymidine kinase activity maxima coincide with those of 3H-thymidine incorporation. Maximal activity of ribonucleotide reductase pre-exists maxima of mitochondrial DNA synthesis.


[O-demethylation kinetics of anisole and p-chloroanisole by cumene hydroperoxide and cytochrome P-450 of liver microsomes].

The kinetics of anisole oxidation by microsomes and tertiary butylhydroperoxide have been studied in phosphate buffer (pH 7,4) at 37 degrees C. About 20% of the substrate are shown to undergo demetylation and about 80% were hydroxylated in the p-position. The kinetics of the oxidative o-demethylation of anisole and p-chloroanisole by cumene hydroperoxide and hepatic microsomes has been studied within the temperature range of 20--37 degrees C. The catalytic rate constants for o-demethylation of anisole and chloroanisole have been determined in a general form to equal 2,04.10(7) exp (--11400/RT) and 5,5.10(7) exp (--11800/RT) sec-1, respectively. The reactions of o-demethylation and hydroperoxide have been proved to be independent. The data obtained are indicative of the electrophilic nature of the hydroxylating agent in the system "microsomes-hydroperoxide".


[Partial purification and characterization of protease A of germinating vetch seeds, hydrolyzing native reserve proteins].

Protease A is 870-fold purified by means of isoelectric precipitation, DEAE-cellulose chromatography and gel filtration through Sephadex G-50, the yield of the enzyme being 28%. The purified preparation is free of contaminant proteolytic activity and is almost homogenous chromatographically, but it produces a complex pattern under electrophoresis in 30% polyacrylamide gel, which is probably due to enzyme autolysis. As evidenced from the effect of protease A on A and B chains of insulin, the enzyme has a wide substrate specificity. It hydrolyses native vetch legumin and vicilin up to peptides having on average 9 and 16 amino acid residues respectively. No free amino acids were found in hydrolysates of both vetch proteins. Thus, protease A is an endopeptidase, which probably plays the main role in the process of reserve proteins degradation.


[Mechanisms of heart adenylate cyclase activation by epinephrine and fluoride ions].

The effects of epinephrine and NaF on the membrane preparations of adenylate cyclase from rabbit heart were studied. After preincubation with epinephrine or NaF at 37 degrees C and subsequent washing of the membranes at 4 degrees C from the effectors, adenylate cyclase passes into the activated state and loses its sensitivity to epinephrine and NaF. The effect may be "reversed" by preincubation of the membranes at 37 degrees C. The addition of ATP to the preincubation media does not affect the regulatory and catalytic properties of the enzyme. It is assumed that adenylate cyclase regulation by hormones and fluoride ions may occur without hypothetical processes of phosphorylation and dephosphorylation of the enzyme. The effect of preincubation is probably due to the temperature-dependent association and dissociation of the enzyme-receptor complex in the membrane. Epinephrine and NaF partially protect the cyclase against trypsin-induced inactivation, which is indicative of structural or conformational changes of the adenylate cyclase complex during its interaction with activators.


[Properties of alpha-chymotrypsin enclosed into polycarbonate microcapsules. Estimation of the diffusion effect].

The conditions for the microencapsulation of alpha-chymotrypsin into semi-permeable polycarbonate membranes are selected. The diameter of the microcapsules is 100-150 microns. The microencapsulation can be carried out at any value of pH. The effect of the diffusion in the hydrolysis of some esters by microcapsulated alpha-chymotrypsin was estimated. The thermostability of the microencapsulated enzyme was studied.


[Interaction of ascorbate oxidase with inorganic anions].

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.


[Incorporation of 32P into membrane phospholipids during chloroplast biogenesis].

The composition of membrane phospholipids during chloroplast biogenesis was studied. The maximal level of phosphatidic acid was observed in the membrane fraction of proplastids. Phosphatidylglycerol was found to be the most abundant phospholipid component of grana thylakoids. The evidence from the in vivo experiments indicates that phosphatidic acid and phosphatidylglycerol incorporate the 32P label at a high rate at all stages of the chloroplast biogenesis. It is concluded that plastids are the site of the phosphatidylglycerol biosynthesis in the plant cell.


[Effect of chloramphenicol and cycloheximide on synthesis of delta-aminolevulinate dehydratase in green and greening barley shoots].

The synthesis of sigma-aminolevulinate dehydratase (EC 4.2.1.24) in green or greening barley shoots was shown to increase, when the plants were grown on chloramphenicol solutions of varying concentrations for 48 hrs upon illumination. This was evidenced from the increase in the enzyme activity of the chloroplast preparations isolated from the shoots as compared to the controls grown in aqueous media. Similar treatment by cycloheximide resulted in inhibition of the enzyme synthesis as observed in the experiments with green and greening shoots. The activity of porphobilinogenase (the porphobilinogene deaminase and uroporphirinogene III cosynthetase complex) showed similar dependence on the effect of the antibiotics. The results obtained are discussed in terms of localization of the chloroplast enzyme syntheses inside the cell.


[9-(Adenylyl)alkylcobalamins as inhibitors of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes].

The behavior of two coenzyme analogs, [(5-aden-9-yl)methoxyethyl] cob (III) alamin and [(5-aden-9-yl)pentyl] cob (III) alamin modified at the nucleoside ligand sugar moiety was studied in the system of adenosyl-cobalamin-dependent glycerol dehydratase from Aerobacter aerogenes. It was shown that neither of the analogs possesses coenzyme properties and that both are strong competitive inhibitors for adenosylcobalamin (AdoCbl). The affinity of the two analogs for the apoenzyme is higher than that of AdoCbl. The data obtained are indicative of the essential role of the ribofuranoside fragment of AdoCbl in the manifestation of the coenzyme activity. The apoenzyme interaction with the analogs under study is discussed in terms of the Dreiding stereomodels for AdoCbl and its analogs.


Cerebrospinal fluid homovanillic acid in male alcoholics: effects of disulfiram.

It is well know that if an individual maintained on disulfiram (Antabuse) ingests alcohol, excess acetaldehyde is formed, resulting in a toxic reaction. In addition to this toxic interaction with alcohol (the basis of its use as a deterrent), there are both behavioral and biochemical observations to suggest that disulfiram alone has a direct effect on the CNS. The possibility that some of disulfiram's effects are related to alterations in biogenic amine metabolism led to the present study of cerebrospinal fluid amine metabolites in a group of male alcoholics. In this group, disulfiram treatment was associated with a significant reduction in homovanillic acid, the major metabolite of dopamine, while no change was noted in 5-hydroxyindoleacetic acid, the major metabolite of serotonin. Prior to disulfiram, patients with withdrawal symptoms had significantly lower homovanillic acid than those without such symptoms.


Tyramine-binding by synaptosomes from rat brain: effect of centrally active drugs.

Incubation of p-tyramine (TRM) with rat midbrain plus corpus striatum (MB+ CS) crude synaptosomal preparations, under conditions which reduce to a minimum amine uptake, results in appreciable binding of this amine by synaptosomes. This process is inhibited by preincubation with a number of drugs active in the CNS, e.g., chlorpromazine, desipramine, d-amphetamine, and diphenhydramine. Morphine, however, does not affect this binding. The Ki for each one of these compounds, as well as the K association constant and concentration of the binding sites of TRM, were determined. These results suggest a role for TRM in synaptic transmission mechanisms occurring in the nigrostriatal system, a function which could be regulated by a number of substances representing some of the major chemical classes of centrally active drugs (CD).


Pharmacological treatment following experimental cerebral infarction: implications for understanding psychological symptoms of human stroke.

Middle cerebral artery ligation in the rat produces decreases in catecholamine concentrations at brain sites uninjured by ischemic damage and a transient increase in spontaneous horizontal activity. Development of this hyperactivity can be blocked by postoperative treatment with the antidepressant, desmethylimipramine, or by preoperative destruction of norepinephrine terminals with 6-hydroxydopamine. These results suggest that ischemic damage to the cerebral cortex which injures some axonal branches of elaborately arborizing catecholamine-containing neurons may alter the biochemical and functional state of the entire system in its intact collateral axons. Thus the concept of stroke as a local injury producing symptoms by local structure-function relationships is conceptually inadequate, and poststroke symptoms must be evaluated with these "whole brain" concepts in mind. We suggest that the poststroke symptoms of apathy and depression may represent emotional changes which result from pathophysiological processes in catecholamine neurons far from the site of the stroke.


Hepatic and pancreatic dysfunction in anorexia nervosa: a report of two cases.

Two patients with anorexia nervosa with evidence of pancreas and liver injury are reported. Both showed evidence of organ dysfunction in the presence of cachexia. Serum amylase, ASAT (GOT), and ALAT (GPT) became normal parallel with body weight. On the basis of reports on the effects of malnutrition on pancreas and liver function, it is suggested that nutritional pancreas and liver dysfunction might represent hitherto unrecognized complications of anorexia nervosa.


Significance testing to establish equivalence between treatments, with special reference to data in the form of 2X2 tables.

In this paper, the role of significance testing in establishing equivalence between treatments is described. In place of the more customary null hypothesis of no difference between treatments, the hypothesis that the true difference is equal to a specified delta is tested. The particular case of comparing two binomial samples is described in detail. Results using a method due to Gart and approximations based on the chi2 and normal distributions are compared. It is found that the test based upon chi2 with continuity correction agrees best overall with Gart's (1971) test and is therefore recommended for use when computational facilities needed for carrying out the latter are not available.


Monitoring treatment differences in long-term clinical trials.

When the data from long-term clinical trials are reviewed continually over time for evidence of adverse or beneficial treatment effects, the classical significance tests are not appropriate. A stimulation procedure is described which provides, for different mortality rates and different patterns of patient enrollment, the correct critical regions corresponding to specified frequencies of looks at the data over the course of the study. The power of the test and the robustness of the critical regions for differences in pattern of enrollment, length of study, mortality model, and sample size are discussed. An application is made to a drug trial in coronary heart disease.


The effect of errors of diagnosis and frequency of examination on reported rates of disease.

Follow-up studies of certain diseases such as cervical dysplasia and carcinoma in situ may employ repeated screening to determine disease incidence in selected cohorts. Where errors of diagnosis occur, the true cohort experience will be distorted by an amount dependent upon the frequency of screening and the magnitude of the probabilities of incorrect diagnoses. These effects are investigated through a simple model of follow-up which employs three basic assumptions: that the probability of false positive and false negative diagnoses are constant; that individuals diagnosed as positive are treated and removed from the study, irrespective of whether or not they actually have the disease; and that fixed intervals occur between follow-up exams. The analysis permits one to assess the amount of bias introduced, and numerical examples are provided.


A randomization test for comparison of groups of growth curves with different polynomial design matrices.

Well defined methods for comparing mean polynomial growth curves exist when subjects are measured at identical times, modeled with polynomials of identical degree and when multivariate normality of the measurements can be assumed. Relaxing these conditions creates problems solved here for the special case of a completely randomized design by developing a randomization test which is both practical and interval specific. The procedure is applied to longitudinal data from a study of human growth.


Pair-matching-a reappraisal of a popular technique.

Pair-matching is undoubtedly one of the most popular techniques for controlling variation in both medical and other investigations involving human populations. Given the obvious advantages of ease of implementation and comprehension, apparent efficiency and simplicity of analysis, this popularity is understandable. Despite this appeal, however, pair-matching frequently involves high cost (particularly in the loss of unmatchable units), cannot claim efficiency when appropriate comparisons of precision are made, and suffers some important limitations in the analysis which directly affect inference. The persistence of the technique in the face of these limitations is discussed with reference to the disjunction between theoretical models and practical research constraints. It is concluded that this design may not be the optimal choice in many, if not most, research situations.


Predicting the stability of biological standards and products.

A high level of stability is essential for any biological standard and is desirable in most other biological products. It is in general impossible to observe directly the rate of degradation of a biological standard since no independent scale of measurement is available. An indirect method is therefore required. The most common approach is the accelerated degradation test in which samples are stored for a time at elevated temperatures and then compared with samples stored continuously at low temperature. The relative degradation rates are used to fit the Arrhenius equation (relating degradation rate to temperature) and hence to predict stability under normal storage conditions. Previous statistical work on this problem is reviewed and a maximum likelihood ML approach is suggested which overcomes some of the limitations of the existing methodology. The accelerated degradation test also finds wide application in the shelf-life prediction of biological products where the same statistical methods are appropriate.


Unchanged electrophoretic mobility in human cryopreserved lymphocytes.

Cell surface charge, assessed by the analytical electrophoresis of fresh and cryopreserved human peripheral blood lymphoctes, is changed in any perceivable manner by freezing and thawing. This was confirmed by different membrane markers (E, EA and EAC rosettes).


Creatine kinase activity in suicide by burning.

In the course of a systematic study of serum enzymes activities from muscular tissues, applied to fifty six burnt patients taken at random, the authors have observed a significant and isolated raised of serum CPK in six burnt people when they were admitted to hospital. There were no known causes of raised CPK essentially due to muscular isozyme. The only common point between those six persons is that they all attempted to commit suicide by burning.


In vivo effect of cyclic nucleotides on velocity and chemotaxis of neutrophil granulocytes.

Neutrophil granulocytes velocity and chemotaxis were obeserved by a direct method. Only chemotaxis could be inhibited by dibutyryl Cyclic AMP, this phenomenon could be reversed by cyclic GMP.


Changes in biochemical parameters associated with experimental arthritis in rabbits.

Peripheral venous blood samples were drawn before and after induction of adjuvant arthritis in rabbits at intervals of 15 days. Using these samples, biochemical parameters were measured, including total protein, albumin, globulin, cholesterol, alkaline phosphatase, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase. The various biochemical tests exhibited an abnormal pattern in adjuvant animals as compared to the normal rabbits. The significance of these findings has been discussed.


Fumarase activity in skeletal muscle of man.

Soluble extracts of human skeletal muscle have a fumarase activity of 31.2 +/- 7.27 (M. vastus lateralis quadricipitis) or 30.9 +/- 8.0 U/g wet weight at 37 degrees C (M. deltoideus). The distribution of activities in the 36 muscle samples studies is not gaussian. There is a significant correlation between the activities of citrate synthase and fumarase (r = + 0.881; p less than 0.001) in all investigated muscles, excepting M. vastus lateralis quadricipitis.


Smoking during pregnancy and placental pathology.

The pathological study of all placentas from women smoking at least five cigarettes daily during pregnancy (248 cases) and of those placentas from a corresponding control group randomised among all the non-smokers (196 cases) has been set up at the maternity hospital of Haguenau (France) since 1974. The systematic histological examination has shown among smokers a higher frequency of abonormal trophoblast and especially of nuclear clumps in the syncytiotrophoblast. On the other hand, according to a standard protocol, the grouping of various abnormalities has shown, among smokers, a higher frequency of "signs of hypoxia". The lack of relationship between these "signs of hypoxia" and the well-known decreased birthweight among smokers suggests that smoking during pregnancy could always go with a decreased birthweight without a corresponding decreased placental weight, and seldom with an intra-uterine hypoxia. These two effects are independent.


Study of carboxyl groups on the human blood platelet membrane.

The authors have studied the whole carboxyl groups of the platelet membrane using liquid phase electrophoresis and an initial step of activation of the acid group by a water soluble carbodiimide (ethyl-l-diméthyl-aminopropyl-3-carbodiimide), followed by the action of a nucleophilic molecule. The calculation based on the theory of Gouy-Chapman have shown the existence of 24, 7.10(5) groups per cell. That is a number about 2.5 fold superior to the carboxyl groups of sialic acid as determined by the action of neuraminidase.


Synthesis of a soluble catalase-dextran conjugate.

Results of a systematic study of the conditions for preparation of soluble catalase-dextran conjugates, using the cyanogen bromide activation procedure, are reported. A protocol for the synthesis of such a conjugate with satisfactory retention of enzymatic activity and high efficiency of coupling is described.


Effect of mass transfer resistance on the Lineweaver-Burk plots for flocculating microorganisms.

It is shown that the mass transfer resistance can significantly distort the linearity of the Lineweaver-Burk plot of the kinetic data for a microbial culture which forms aggregates. For small flocs, the linearity of the Lineweaver-Burk plot is largely retained, but a different slope and intercept will be obtained compared with flocs free from mass transfer resistance. For large flocs, the Lineweaver-Burk plot shows pronounced curvature at high limiting substrate concentrations. Hence, if the true intrinsic kinetic parameters are to be extracted from a highly flocculating microbial culture, sufficient agitation has to be provided to remove the effect of mass transfer resistance. If the behavior of the flocculating microbial culture is to be explored, additional values for some physical parameters, such as the effective diffusion coefficient of the substrate in floc, the floc density, and the mean floc radius, are needed.


Selection of a chemically defined medium for submerged cultivation of Streptomyces aureofaciens with high extracellular caseinolytic activity.

A chemically defined medium was developed for the submerged cultivation of Streptomyces aureofaciens with a high secretion of caseinolytic activity. The medium composition is: 40 g/liter maltose; 1.640 g/liter L-leucine (0.0125M); 1.765 g/liter L-lysine (0.0125M); 6.976 g/liter K2HPO4 (0.04M); 4 g/liter CaCO3; 0.2 g/liter MgSO4.7H2O; 0.01 g/liter ZnSO4.7H2O; 0.01 g/liter FeSO4.7H2O: 0.01 g/liter MnSO4H2O, and 0.005 g/liter CoSO4.7H2O. Quantitative correlations were established between the concentrations of nutrients in the medium and the secretion of proteolytic activity. In this medium the secretion of proteolytic activity parallels growth, reaching a maximum after 70 hr at 30 degrees C in shaker cultures. The secretion appears to be an active process and to require aerobic conditions.


Combined surgery and intermittent catheterization for neurogenic bladder dysfunction in children.

Intermittent catheterization is a valid treatment modality in the management of the neuropathic bladder. Numerous adjunctive surgical procedures have been tried to improve continence and to improve or preserve renal function. These procedures, performed on 38 of 82 patients on long-term intermittent catheterization, are preferable to urinary diversion and possibly to the implantation of prosthetic or artificial urinary sphincters. Uretero-ileocecocysto-urethroplasties have not been successful in our hands, but the other procedures certainly offer a preferable alternative to the treatment modalities in use up to only 4 years before this bicentennial year!


Pharmacologic modification of therapeutic measures used in managing the neurogenic bladder.

In the past 2 years, no supravesical urinary diversions have been done for myelodysplasia at St. Louis University Medical Center. Twenty-six patients with neuropathic bladders have been managed by intermittent catheterization and mechanical devices. The addition of anticholinergic and alpha-adrenergic agents to the therapeutic regimen has markedly increased the numbers of satisfactory results. Based on this group of patients, we are encouraged to believe that supravesical urinary diversion is rarely necessary for the management of both the medical and social problems of the urinary tract associated with myelodysplasia.


Cutaneous transureteroureterostomy in children with neurogenic bladders.

Our experience with 25 children in which cutaneous transureteroureterostomy had to be done because of neurogenic bladder is very gratifying. The procedure is very simple with few immediate and late complications. The cutaneous ureteral stoma works efficiently and so far has not offered important problems. We feel this method is superior to the bowel conduit methods.


Technical aspects of megaureter repair.

Megaureter is a correctable problem. Repair should be considered in appropriate cases; it is demanding surgery, however, which should not be attempted on an occasional basis. Results can be satisfactory in more than 90% of cases if certain technical points are followed, as enumerated in Table 2.


The surgical repair of membranous urethral strictures in children.

Eighteen patients have been treated for membranous urethral strictures following rupture of the prostatomembranous urethra. In 16, satisfactory results have been attained, 1 patient has been diverted permanently, and the remaining patient has not yet undergone complete repair.


Fused kidneys: morphologic study and theory of embryogenesis.

A study was made of 50 fused kidneys; 41 postmortem specimens and 9 living patients' records were reviewed. Three patterns of renal fusion were identified--midline fused horseshoe kidney [15], laterally fused horseshoe kidney [21], and crossed renal ectopia [14]. These morphologic renal patterns were correlated with coexisting abnormalities of the urinary tract and other organ systems. A high incidence of hydronephrosis of varied etiology, vertebral, and anorectal defects was found in the laterally fused horseshoe and crossed renal ectopic groups. A theory of embryogenesis was developed to explain the asymmetric patterns of renal fusion exemplified by laterally fused and crossed fused kidneys. It was based on the effects that the lateral flexion and rotation of the hind end in the 4- to 5-mm embryo exert on the relative positions of the nephrogenic cords and wolffian ducts.


Simultaneous bilateral pyeloplasties.

Eleven patients with bilateral pelviureteric junction obstruction are presented. The operative technique for simultaneous bilateral pyeloplasty is briefly described and the results discussed. The importance of adequate extraperitoneal loin drainage is stressed and the incidence of subsequent adhesion obstruction is recorded.


Fate of the kidney after antireflux surgery.

Representative case reports of children subjected to ureteroplasty in the management of reflux have been presented. Five- to 12-year follow-up studies were analyzed with reference to renal growth and pyelonephritic changes; the effect of the degree of preoperative reflux was recorded. The following conclusions appear warranted: 1. Severity of reflux is a major factor determining renal status; the more marked the reflux, the greater the frequency and extent of CPN. 2. Kidneys which appear normal initially usually remain so;however, CPN may develop, sometimes to a severe degree (Fig. 9). 3. The growth potential of pyelonephritic kidneys is quite variable and not readily predictable (Fig. 9). 4. Prolonged exposure to reflux appears to be an unfavourable situation possibly accelerating renal deterioration.


Overactivity of the integral storage and voiding reflexes in the juvenile urinary incontinence syndrome.

The physiologic importance of 12 integral storage and voiding reflexes has not been generally recognized and appreciated by clinicians. Pathologic overactivity of reflexes No. 9 and No. 10, the urethrodetrusor facilitatory reflexes, appears to be a very common factor in the etiology of the juvenile incontinence syndrome. Mild but urodynamically significant obstructive lesions of the distal urethra are a common predisposing factor to chronic urethritis and are potentially remediable by simple surgical procedures. Cystometric as well as symptomatic improvement is commonly observed postoperatively. The micturition reflex is extremely complex. Its excitability is dependent upon the balance of multiple vectors of both facilitatory and inhibitory influence from many sources, peripheral as well as central. The popular clinical concept of "uninhibited neurogenic bladder," based on the cystometrogram, is naive and simplistic. It deserves to be replaced by the term "unstable bladder" which acknowledges the neurologic and urologic complexities of micturition reflex instability and introduces no erroneous connotations as to the cause and potential treatability of an incontinence condition.


Posterior urethral obstruction: valvular or diaphragmatic? Endoscopic diagnosis and treatment.

Young's classification of posterior urethral valves has been criticized because clinically only type 1 valves have been observed. However, with a certain technique of postmortem examination the obstruction has been found to be caused by a fenestrated diaphragm. When reevaluating the photographs and motion pictures taken during endoscopies it was concluded that the obstructions were diaphragmatic. The earlier controversial conclusions can be attributed to questionable methods of examination and misinterpretation of the findings. On the basis of the anatomic structure of diaphragmatic obstruction, the endoscopic anterior resection used by the author can be considered a logical and effective method of treatment.


[Gating current in the membrane of nodes of Ranvier under conditions of linear alteration of the membrane potential].

Asymmetrical displacement currents (Id) in the frog Ranvier node (R. ridibunda) treated with tetrodotoxin and tetraethylammonium were studied by the use of ramp-voltage pulses. In some experiments both ramp- and step-voltage pulses were used. The net Id consists of two components, one of which Id (I) can be blocked by local anesthetic trimecaine, or inactivated with the 10 ms depolarizing prepulse sufficiently large to inactivate the sodium current in the same node (before Na+ removal and TTX application). Parameters of the steady state charge distribution are very close (though not identical) to that of the peak sodium permeability vs. potential relation. The charge carrying Id (I) is estimated as 0.3--0.5 of the net displaced charge. The results suggest that trimecaine- and inactivation-sensitive component of Id may be the true gating current of Na+ channels.


[Analysis of the inhibitory influence of whole human erythrocytes on plasma heparin activity].

The accelerating effect of intact human erythrocytes upon the thrombin time of plasma (deprothrombinized, thrombocyte depleted) was independent of the presence of calcium ions, preincubation, erythrocytes and plasma, and was not accompanied by a decrease of the free heparin level in the plasma. Acceleration of the process of plasma fibrinogen transformation into fibrin caused by the whole erythrocytes was not conditioned by the endogenous heparin inhibition, but reflected the cell fibrinoplastic activity. The results obtained are against the idea advanced in literature on the regulating influence of the whole erythrocytes upon the endogenous heparin level in the blood.


[Oxygen supply to isolated rabbit heart during its perfusion with eryhem and blood].

Coronary vessels of the isolated rabbit heart were perfused with eryhem--a solution containing 2.5--3.0 g% of hemoglobin and with the rabbit own blood diluted to the Hb content corresponding to such in eryhem. The oxygen capacity of eryhem as per 1 g of Hb constituted 72% only of the oxygen capacity of erythrocyte Hb. It was shown that eryhem was capable of uptake, transportation and of giving up oxygen. At the same time the myocardium perfused with eryhem was in worse conditions of oxygen supply than the muscle perfused with blood. The required level of oxygen uptake was reached with the increase of coronary blood supply and more complete utilization of blood oxygen reserves.


[Experimental study of the immunotherapy of burns].

Experiments were conducted on rats. The influence of the serum of a burn convalescent on the toxic properties, the level of proteolytic enzymes activity, and morphological changes following burn were studied. After the burn the blood serum and the extracts of the organs acquired toxic properties; there was an increase in the activity of proteolytic enzymes, and marked morphological changes developed Administration of the serum of aburn convalescent promoted detoxication, diminished the activity of proteolytic enzymes distinctly, and decreased the morphological disturbances.


[Alteration of systemic hemodynamics in dogs with adrenal hypertension].

Arterial hypertension was reproduced in 20 dogs by suturing the adrenal glands with ligature. Arterial pressure showed a significant fall in 2 weeks; cardiac output diminished, and the general peripheral resistance displayed a sharp elevation. The phasic syndrome of hypodynamia, a reduction of the contractility index, of the volumetric rate of cardiac output, of the cardiac index, and of the rate of increase of the intraventricular pressure pointed to reduction  of the myocardial contractility. Three months after the suturing there was an even greater elevation of arterial pressure, and hemodynamic shifts were analogous to the two-week hypertension period.