Effect of work-induced hypertrophy on skeletal muscle of tumor- and nontumor-bearing rats. Synergistic muscles (soleus and plantaris) from a gastrocnemius-tenotomized hindlimb were compared to the same muscles in a sham-operated hindlimb in both tumor-bearing and non tumor-bearing rats. In nontumor-bearing animals muscle from the tenotomized hindlimb had a significant increase in wet weight (26%), percent-water (5%), and total nitrogen/muscle (10%) consistent with muscular hypertrophy. In tumor-bearing animals, muscle from the tenotomized hindlimb had a significant increase in wet weight and percent water, but there was no significant difference in total nitrogen/muscle. As the tumor burden increased, the final muscle weight, in both the tenotomized and sham-operated hindlimb, was found to decrease proportionately. Only in animals with large (23% body wt) tumor burdens did hypertrophied muscle weigh significantly less than sham-operated muscle from nontumor-bearing animals. This indicated that, in rats with large tumor burdens, work-induced hypertrophy was unable to preserve muscle mass despite the fact that hypertrophied muscle was 24% heavier than contralateral sham-operated muscle.


Demonstration of interlobar collateral ventilation. In an ongoing series of experiments on mongrel dogs, three dogs were observed to have incomplete lobar fissures (interlobar connection present) between the left upper and left lower lobes. With the interlobar connection unclamped, selective hypoxia of the left lower lobe caused the electromagnetically measured blood flow to the left lower lobe to decrease 41 +/- 4%, whereas with the interlobar connection clamped selective hypoxia of the left lower lobe caused a 52 +/- 4% decrease in left lower lobe flow (P less than 0.05). Following 100% O2 ventilation and with the interlobar connection unclamped, complete occlusion of the left lower lobe bronchus caused no atelectasis or decrease in left lower lobe blood flow, whereas with the interlobar connection clamped complete occlusion of the left lower lobe bronchus caused complete left lower lobe atelectasis and a 58 +/- 3% decrease in left lower lobe blood flow. Histological confirmation of interlobar pulmonary parenchymal connection was obtained in each dog. We conclude that a sufficient amount of interlobar collateral ventilation can occur in dogs with incomplete lobar fissures to prevent absorption atelectasis and alter lobar blood flow reduction usually caused by selective lobar hypoxia.


Catecholamines in dogs during cold adaptation by repeated immersions. Dogs were immersed in cold water 1 h/day for 30 consecutive days. During the first immersion, oxygen uptake increased approximately sevenfold and colonic temperature decreased; a large increase in plasma epinephrine and norepinephrine concentrations was found; plasma glucose, lactic acid, and free fatty acid (FFA) concentrations were also increased. An adaptative response to cold was evidenced during the 10th immersion by a further increase in oxygen uptake and a reduction in the fall of colonic temperature; plasma epinephrine, glucose, and lactic acid were diminished whereas FFA were higher. Consequently 10 h spent in cold are sufficient to induce an improved capacity for heat production. An enhanced ability to produce nonshivering thermogenesis mediated by epinephrine or norepinephrine is not obvious because after the 30th immersion no enhanced calorigenic response to epinephrine or norepinephrine infusion was found. In dogs mechanisms other than norepinephrine-enhanced sensitivity might be involved in this kind of cold adaptation.


Graded inspiratory inhibition: specific effects of flow rate. The effect of inspiratory flow rate on graded inspiratory inhibition was examined in barbiturate-anesthetized paralyzed cats normally ventilated by a phrenic-driven servorespirator. Periodically, the animals were inflated, for a single breath, by a constant-flow volume ramp begun at one of several delays following phrenic onset. The phrenic activity during these volume-ramp test inflations was compared with phrenic activity in the absence of volume feedback. The relation between volume and phrenic inhibition was determined for two different flow rates. Inhibition of phrenic activity began when volume above functional residual capacity (FRC) exceeded a time-dependent threshold and, beyond this threshold, continued in a graded fashion. The threshold for the first detectable inhibition declined as inspiratory time progressed both in intact animals and in pneumotaxic-lesioned animals when phrenic activity reached an apneustic plateau. Inspiratory inhibition was associated with similar lung volume above FRC regardless of whether volume resulted from high or low flow rates, suggesting that the processing of the receptor feedback information offsets flow effects on receptor firing.


Intermittent flow in tendon capillary bundles. The capillary bed has been observed in the Achilles tendon of 40 bullfrogs and 10 guinea pigs for periods of up to 2 h. The opening and closing of adjacent capillaries in a perfused area follows the pattern originally described by Krogh for skeletal muscle but the frequency is slower, as anticipated from the lower metabolic rate of tendon. However, superimposed on this "flickering is a much slower process whereby whole bundles of 20-147 capillaries open and close with little overlap in the tissue areas perfused by each. Periods of no flow averaged 39 min in 70 bundles followed in bullfrogs and 43 min in 34 bundles followed in guinea pigs, although a few failed to open in 100 min. This bundle phenomenon is discussed in relation to the serious implications in mathematically modeling the exchange of gases and nutrients between blood and tissue and the possible errors in assuming time averaging when determining blood perfusion rates. Also mentioned are the likely effects of decompression on closed bundles and the notion is introduced that the bundle phenomenon may be a factor determining which tissues can be injured by decompression.


Fluid shifts during initial phase of immersion diuresis in man. The object was to study fluid shifts in man during the 1st h of immersion diuresis. Control experiments were done on subjects lying down in air for 4 h with and without vasopressin. During immersion up to the neck, seven of nine subjects had significant diuresis and natriuresis. In the first 20 min of sitting in 33 degress C water, a hemodilution of 2% of blood volume was observed. As diuresis progressed, hemoconcentration began. When vasopressin was given just before immersion to prevent the diuresis, the hemodilution observed was greater and lasted longer. Thus the hematocrit fell by 1.7 U, plasma osmolality by 6.0 mosmol/kg, plasma proteins by 0.33 g/100 ml, and plasma sodium by 5.0 meq/l. We conclude that a hemodilution of about 4% of blood volume occurs during the early plasma of immersion and the degree of hyposmolality observed suggests that the fluid shifted was more hyposmotic than the interstitial fluid alone, possibly because some intracellular water may have shifted into the bloodstream during immersion.


Respiratory mechanics in recumbent dogs anesthetized with thiopental sodium. Pressure-volume (PV) and conductance-volume (GV) curves were obtained in trained dogs awake and then anesthetized with thiopental sodium, in the prone left lateral decubitus, and supine positions. By paired analyses, induction of anesthesia had no significant effect on lung volumes, static PV curves of the lung or chest wall, or static compliance of the lung or chest wall. In addition, change of posture had little effect on these variables in either the awake or the anesthetized state. Frequently, however, individual dogs showed changes in lung PV curves after induction of anesthesia or change of posture. These PV curve shifts were not accompanied by predictable changes in GV relations. Therefore, we concluded that such changes in the lung PV curve reflect either local artifacts or local deformation of the respiratory system.


Training-induced changes in hormonal and metabolic responses to submaximal exercise. Plasma glucagon and catecholamines increase during prolonged submaximal exercise, but the magnitude of the increase is less in endurance-trained individuals than in untrained subjects. We have studied the rapidity at which this adaptation occurs. Six initially untrained healthy subjects exercised vigorously (on bicycle ergometers and by running) 30-50 min/day, 6 days/wk, for 9 wk. Prior to the beginning of training and at 3-wk intervals thereafter, participants were subjected to 90-min bicycle ergometer test work loads that elicited 58 +/- 2% of the subjects' initial maximal oxygen consumption. The major proportion of the training-induced decrement in plasma glucagon and catecholamine responses to exercise was seen after 3 wk of training. We conclude that the hormonal component of the training adaptation occurs very early in the course of a vigorous endurance training program.


A neuromuscular mechanism maintaining extrathoracic airway patency. The ability of the extrathoracic airway (ETA) to remain open when exposed to negative pressure was investigated in rabbits. Postmortem, the ETA collapsed at -6.3 +/- 0.6 cmH2O whereas, during airway occlusion maneuvers in lightly anesthetized animals, it remained patent at pressures as low as -80 cmH2O. This discrepancy suggested that a neuromuscular mechanism maintains ETA patency. Four findings indicated that the genioglossus and geniohyoid muscles, which pull the tongue and hyoid bone anteriorly, help maintain ETA patency: 1) anterior movement of the hyoid bone increased the negative pressure at which the ETA collapsed postmortem, 2) ETA closure during occluded inspirations occurred after 12th nerve section abolished electromyographic activity in these muscles and 3) after deep anesthesia depressed such activity, and 4) closing pressure was linearly related to peak integrated electromyograms of the two muscles. After 12th nerve section, ETA closing pressure became more negative with progressive asphyxia greatly exceeding postmortem closing pressure, which suggests that other muscles also help maintain ETA patency. Blood gas tensions, respiratory system mechanoreceptors, and depth of anesthesia appear to influence genioglossus and geniohyoid activity.


Body carbon dioxide storage capacity in exercise. Body CO2 storage capacity was measured in nine subjects at two levels of exercise, approximating 30 and 60% VO2max, by measuring the excess CO2 output associated with hyperventilation at constant end-tidal PCO2, and the change in mixed venous PCO2 (PVCO2) measured by rebreathing. CO2 output was measured during 20-s periods and monitored throughout the procedure; hyperventilation was continued until CO2 output had returned to control values. Washout of CO2 was more rapid than previously found at rest, 90% of the change following an increase in ventilation occurring within 4 min. CO2 storage capacity was 1.83 +/- 0.552 (SD) ml-kg--1.Torr--1 at the lower power output and 1.19 +/- 0.490 ml-kg--1.Torr--1 at the higher power output. The CO2 storage capacity was inversely related to PVCO2. It was concluded that the body's capacity to store CO2 decreases with increasing PVCO2; this may be one factor leading to the progressive increase in pulmonary CO2 output at high levels of exercise.


Effects of chronic changes in hemoglobin-O2 affinity in rats. We have assessed the characteristics of oxygen transport following chronic intraperitoneal administration of sodium cyanate (NaCNO, 90 mg/kg; P50 decreased), o-iodosodium benzoate (OISB, 300 mg/kg; P50 increased), or sodium chloride (NaCl; P50 unchanged) to rats at a rate of 5 times/wk for 16 wk. At the end of this period, the animals were exposed to a low inspired O2 concentration and were subjected to exercise stress. Hill's n50 at pH 6.90-7.60, hematocrit, and the saturation dependency of the Bohr effect (PCO2 constant) were altered by NaCNO only. Cyanate-treated rats gained less weight, probably due to a toxic side effect of the drug. Hypoxemia-induced lactatemia was greatest with a high P50 (OISB). Physical effort (swimming) was tolerated poorly at normal arterial PO2 when P50 was low (NaCNO). Although a left-shifted curve appears beneficial when FIO2 is reduced, reasons for the physiological advantage may be the result of other characteristics of the O2 dissociation curve, not P50 alone.


Acute hypertrophic response of skeletal muscle to removal of synergists. The purpose of this project was to study the initial rapid enlargement phase of skeletal muscle in response to ablation of synergistic muscles. The first experiment tested the hypothesis that this initial phase is due to inflammation in the enlarging muscle. The wet weight of the plantaris muscle increased significantly (14%) within 1 h after removal of the ipsilateral gastrocnemius muscle. This increase was due to inflammation, as evidenced initially by edema, and within the next few hours, by leukocyte invasion of the interstitium and enhanced hexose monophosphate shunt-reducing capacity. In the second experiment we followed the time course of this inflammatory reaction. The response peaked at 1-5 days following surgery, after which it subsided to control levels by 16 days postsurgery. The final experiments were designed to uncover the factor(s) causing the inflammatory response. Two possible mechanisms were hypothesized: 1) trauma resulting from surgical manipulation of the tissue, and 2) trauma due to elevated stretch and/or tension on the muscle following removal of the synergists. The data indicate that surgical trauma is the causative factor of the inflammatory response in this hypertrophy model.


Fecal fat and trypsin in dogs fed a meat-base or cereal-base diet. Fat balance studies and quantitative analysis of fecal trypsin were done on 2 groups of Beagles fed either a meat-base (8.8% fat) or a cereal-base (10.5% fat) diet. In the amount fed, the meat-base diet provided 2.5 to 3 times more fat than the cereal-base diet, yet the mean daily fecal fat output of the 2 groups was not significantly different. Thus the percentage assimilation of fat in cereal-base diet (90.0%) was significantly (P less than 0.01) lower than that in the meat-base diet (96.9%). Dietary composition made no significant difference in fecal trypsin output, although the mean output from dogs fed the meat-base diet 4 times higher than that from dogs fed the cereal-base diet.


Determination of fecal fat and trypsin output in the evaluation of chronic canine diarrhea. Quantitative fat and trypsin analysis was done on the feces of dogs with chronic diarrhea. The results of clinical examination, quantitative fecal analysis, and other laboratory tests permitted assignment of the dogs into one of 4 groups: (1)pancreatic exocrine insufficiency,(2)small intestinal malabsorption,(3)colitis, and(4)other nonspecific or incompletely diagnosed diarrhea. The mean 24-hour fat output was significantly higher (p less than 0.01) in dogs with malabsorption or pancreatic insufficiency than in clinically normal dogs, dogs with colitis, or dogs with nonsteatorrheic diarrheas. The mean 24-hour trypsin output with pancreatic insufficiency was significantly (P less than 0.01) lower, and in dogs with malabsorption, significantly (P less than 0.05) higher than in clinically normal dogs. Normalization of the output data for body weight enhanced the value of fat and trypsin analyses in the differentiation of pancreatic insufficiency and intestinal malabsorption from other causes of chronic canine diarrhea.


Primary hyperparathyroidism in a dog.

Primary hyperparathyroidism in a dog. A 6-year-old male Standard Poodle had hypercalcemia, hypophosphatemia, and a urinary concentrating defect. A parathyroid adenoma involving the left caudal parathyroid gland was surgically removed. Transient hypocalcemia followed, which resolved without treatment. A renal biopsy did not demonstrate renal parenchymal mineral deposition. Urinary concentrating ability returned after surgery.


Colorectal polyps of the dog: a clinicopathologic study of 17 cases. Seventeen cases of colorectal polyps in the dog were seen at the Ontario Veterinary College over a 12-year period. The mean age of occurrence was 6.9 years, and males and females were almost equally affected. The Collie was the most frequently affected breed (4/17). The clinical signs were typically dyschezia, periodic intermittent diarrhea and melena, and rectal prolapse of the polyp. The histopatholic classifcation were hyperplastic polyp (1), papillary adenoma (1), tubular adenoma (4), papillotubular adenoma (10), and unclassified (1). Severe epithelial atypia, likely carcinoma in situ, was apparent in 5 of the papillotubular adenomas. Retrospective analysis of survival data after polypectomy suggested a good prognosis, but the results were inconclusive and it is possible that some of the polyps represented premalignant lesions. Large polyps (greater than 1 cm in diameter) frequently had marked epithelial atypia and were considered more likely to recur or to become malignant.


A new broad-spectrum aminoglycoside antibiotic complex, sporaricin. II. Taxonomic studies on the sporaricin producing strain Saccharopolyspora hirsuta subsp. Kobensis nov. subsp. Morphological, cultural and physiological characteristics of a new nocardioform actinomycete are reported. The microorganism which produces the antibiotic complex sporaricin has been selectively isolated from a sample of soil obtained from Kobe City, Hyogo Prefecture, Japan. By whole-cell analysis of the actinomycete, meso-diaminopimelic acid, arabinose and galactose were identified. But lipid LCN-A (lipid characteristic of Nocardia) and nocardomycolic acid were not detected. The taxonomic characteristics of this strain is closely related to the genus of Saccharopolyspora, described by LACEY and GOODFELLOW. Based on the taxonomic comparison with Saccharopolyspora hirsuta ATCC 27875, the strain was considered to be a subspecies of Saccharopolyspora hirsuta. Therefore, the proposed subspecies is named Saccharopolyspora hirsuta subsp. kobensis.


A new broad-spectrum aminoglycoside antibiotic complex, sporaricin. III. The structures of sporaricins A and B. The structures of sporaricins A and B have been determined to be 2-amino-1-O-(2,6-diamino-2,3,4,6,7-pentadeoxy-beta-L-lyxo-heptopyranosyl)-2,3,5-trideoxy-5-(N-glycyl-N-methyl-amino)-4-O-methyl-D-chiro-inositol and 2-amino-1-O-(2,6-diamino-2,3,4,6,7-pentadeoxy-beta-L-lyxo-heptopyranosyl)-2,3,5-trideoxy-4-O-methyl-5-methylamino-D-chiro-inositol, respectively.


Capsimycin, a new antibiotic. I. Production, isolation and properties. Capsimycin is a new antifungal antibiotic produced by a strain of Streptomyces sp. C 49--87. The active substance in the fermented broth was isolated by solvent extraction followed by silica gel column chromatography. The antibiotic melts at 186 degrees C (decomp.) and has a molecular formula C30H40N2O6. It exhibits marked inhibitory activity against Phytophthora capsici (Leaf blight disease of cucumber) and Pythium debaryanum (Damping-off disease of cucumber).


New daunorubicin analogs. 3-Amino-2,3,6-trideoxy-alpha- and beta-D-arabino- and 3,6-diamino-2,3,6-trideoxy- alpha-D-ribo-hexopyranosides of daunomycinone. Glycosidation of 2,3,6-trideoxy-3-trifluoroacetamido-4-O-trifluoroacetyl-alpha-D-arabino-hexopyranosyl chloride (19) (or the corresponding 4-p-nitrobenzoate, 20) with daunomycinone under Koenigs-Knorr conditions afforded, after separation of the anomers and removal of the protecting groups, the individual target glycosides 8 (alpha anomer; major product) and 9 (beta; minor) in acceptable yields. In contrast, the title diamino sugar, suitably protected with N-trifluoroacetyl and O-acetyl (or O-p-nitrobenzoyl) groups, underwent stereospecific coupling to the anthracycline aglycon by the glycal procedure to give, after deprotection, the alpha glycoside 12. All three analogs were assayed in vivo against P388 lymphocytic leukemia. They showed little (T/C 125 for 8; T/C 115 for 9) or no (compound 12) activity, but were essentially devoid of toxicity at the dose-levels tested.


The synthesis of epsilon-rhodomycinone- and carminomycin-11-methyl ethers. The conversion of epsilon-rhodomycinone to its 11-methyl ether via selective hydrolysis of the 4,6,7,11-tetraacetate is described. This series of reactions was used as a model for the conversion of carminomycin to its 11-methyl ether. The anti-tumor activity of the latter compound was less than that of both carminomycin and its 4-methyl ether (daunomycin).


Allergenic significance of some fungi prevailing in the respiratory tract of allergy patients--A preliminary study. The allergenic significance of some fungi prevailing in the respiratory tract of allergy patients was studied. The percentage of markedly positive intradermal reactions observed was higher in patients to the extracts of homologous fungi found in their respiratory passages as compared to the patients who did not show the same fungi in theri respiratory tact.


Specific interaction of the tetragonally arrayed protein layer of Bacillus sphaericus with its peptidoglycan sacculus. Tetragonal layer protein (T-layer) isolated from Bacillus sphaericus NTCC 9602 (wild type) or 9602 Lmw (variant) bonded specifically to the sacculi (peptidoglycan) of either cell type. Only uncleaved T-layer subunits were capable of specific recognition of the B. sphaericus sacculi; other Bacillus strains and gram-positive bacterial sacculi would not adsorb B. sphaericus strain 9602 T-layer. The peptidogylcan did not function as a template since isolated T-layer subunits self-assembled into characteristic pattern. Upon reassociation with sacculi, T-layer assemblies were randomly oriented patches compared with more continuous strictly oriented pattern on cells or fresh cell walls. T-layer associated with the sacculus was less susceptible to conditions that dissociated in vitro-assembled T-layer. Mild proteolysis of both wild-type and variant T-layer subunits by a variety of enzymes reduced the molecular weight by 18,000 in all cases, indicating that one region of the molecule was particularly susceptible to cleavage. Subunits from which the minor fragment had been cleaved upon aging retained the capacity to assemble in vitro, but would no longer adsorb to sacculi. Thus, the ability of T-layer to form networks was separate from its ability to bind cell walls, and the 18,000-dalton piece of the T-layer polypeptide was necessary for attachment to the cell wall.


Aflatoxin biosynthesis: detection of transient, acetate-dependent intermediates in Aspergillus by kinetic pulse-labeling. A simple technique was developed for the detection of intermediary metabolites of Aspergillus versicolor that are putative precursors of aflatoxin. Minicolony populations were allowed to metabolize [1,2-14C]acetate over various time intervals. The biosynthetic reactions were quenched by quick-freezing the minicolonies, the cells were disrupted, and the metabolites were extracted into acetone. Small silica thin-layer chromatographic plates were then used to separate any radioactive metabolites present. Elution in two or three different directions was often necessary. Radioautography of the thin-layer chromatography plates provided a sensitive assay for the appearance of the various intermediates in a timing pattern which implicated the sequence of formation. Transient intermediates were distinguished from dead-end metabolites by the rapid formation and disappearance of the former. At least five unknown precursors of versicolorin A, a dead-end metabolite, were recognized. The kinetic pulse-labeling technique should be generally applicable to other fungal species whenever the entrapment of intermediary metabolites in the mycelium poses and technical problem.


Excretion of glutamic acid in Citrobacter intermedius C3 associated with plasmid deoxyribonucleic acid. Several mutants of Citrobacter intermedius C3 lacking both the ability to synthesize proline and the ability to excrete glutamic acid were isolated by treatment with nitrosoguanidine. No revertants for either characteristic were obtained from these mutants. The ability to excrete glutamic acid was transferred to those mutants with very high frequencies in mating experience by using auxotropic excreting strains as donors. Moreover, the ability to synthesize proline was transferred together with the ability to excrete glutamic acid when an excreting strain was used as donor. The transconjugants showed a rapid spontaneous curing of both genetic markers. It was shown by two different methods that a band of covalently closed circular deoxyribonucleic acid is present in the cesium chloride gradients corresponding to the wild type and excretor mutants. Nonexcretor mutants described herein lacked such a band. Pro + transformants that were also excretors were obtained with plasmid deoxyribonucleic acid isolated either from wild type or from an excretor mutant. These data strongly indicate that glutamic acid excretion in C. intermedius C3 is related to the presence of extrachromosomal deoxyribonucleic acid.


Obligatory biosynthesis of L-tyrosine via the pretyrosine branchlet in coryneform bacteria. Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) utilize pretyrosine [beta-(1-carboxy-4-hydroxy-2,5-cyclohexadien-1-yl) alanine] as an intermediate in L-tyrosine biosynthesis. Pretyrosine is formed from prephenate via the activity of at least one species of aromatic aminotransferase which is significantly greater with prephenate as substrate than with either phenylpyruvate or 4-hydroxyphenylpyruvate. Pretyrosine dehydrogenase, capable of converting pretyrosine to L-tyrosine, has been partially purified from all three species. Each of the three pretyrosine dehydrogenases is catalytically active with either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as cofactors. The Km values for nicotinamide adenine dinucleotide phosphate in C. glutamicum and B. flavum are 55 microM and 14.2 microM, respectively, and corresponding Km values for nicotinamide adenine dinucleotide are 350 microM and 625 microM, respectively. The molecular weights of pretyrosine dehydrogenase in C. glutamicum and in B. flavum are both about 158,000, compared with 68,000 moleculr weitht in B. ammoniagenes. In all three species the enzyme is not feedback inhibited by L-tyrosine. Results obtained with various auxotropic mutants, which were used to manipulate internal concentrations of L-tyrosine, suggest that pretyrosine dehydrogenase is expressed constitutively. Pretyrosine dehydrogenase is quite sensitive to p-hydroxymercuribenzoic acid, complete inhibition being achieved at 10 to 25 microM concentrations. This inhibition is readily reversed by thiol reagents such as 2-mercaptoethanol. Coryneform organisms, like species of blue-green bacteria, appear to lack the 4-hydroxyphenylpyruvate pa thway of L-tyrosine synthesis altogether. The loss of pretyrosine dehydrogenase in extracts prepared from a tyrosine auxotroph affirms the exclusive role of pretyrosine dehydrogenase in L-tyrosine biosynthesis. Other reports in the literature, in which the presence in these organisms of prephenate dehydrogenase is described, appear to be erroneous.


Effect of sugars on D-arabitol production and glucose metabolism in Saccharomyces rouxii. The effect of sugars on the production of d-arabitol and on the glucose catabolic pathways was investigated in the osmotrophic yeast Saccharomyces rouxii. The activity of d-arabitol dehydrogenase, which served as a measure of total d-arabitol production, increased when cells were grown in the presence of increasing glucose concentrations. Growth in sucrose had no effect on the enzyme activity. A high intracellular concentration of d-arabitol could be demonstrated when the cells were grown in a 60% glucose medium and could be eliminated by anaerobic growth or growth in the presence of 4 mg of chloramphenicol per ml. A mutant was isolated that would not grow in 60% glucose; although the regulation of d-arabitol dehydrogenase was altered in this strain, the production of d-arabitol was not eliminated. The activity of d-arabitol dehydrogenase followed the growth phases of the parent strain when the cells were preadapted to 30% glucose. If the cells were adapting from 1 to 30% glucose, a large increase in enzyme activity was detected before growth occurred. Protein synthesis was found to be involved in this increase in activity. There was an increased participation of the pentose phosphate pathway when the cells were grown in the presence of increasing glucose concentrations. The mutant strain had only an 11% pentose phosphate pathway participation compared with 20% for the parent strain in glucose. The results suggest that the active pentose phosphate pathway is involved in glucose tolerance by providing a plentiful supply of reduced nicotinamide adenine dinucleotide phosphate which is necessary for cell survival.


Flagellar hook and basal complex of Caulobacter crescentus. Intact bacterial flagella possessing a membrane-free hook and basal complex were purified from Caulobacter crescentus CB15, as well as from mutants which synthesize incomplete flagella. The basal body consisted of five rings mounted on a rod. Two rings were in the hook-proximal upper set, and three rings (two narrow and one wide) were in the lower set. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. The lower rings were all approximately 21 nm in diameter, although they varied significantly in width. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. The hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component.


Isolation, characterization, and in vitro assembly of the tetragonally arrayed layer of Bacillus sphaericus. The tetragonally arranged cell wall layer (T-layer) of Bacillus sphaericus NTCC 9602 was isolated and characterized. Parallel studies were made on a spontaneous variant of the wild-type strain which had a T-layer subunit of altered molecular weight. A purification method for the T-layers was devised which involved separation of the cell walls from the cytoplasmic contents, urea dissociation of the T-layer from the cell walls, removal of soluble contaminants by differential centrifugation, and finally selective adsorption of uncleaved subunits to sacculi. The purified subunits retained the capacity to form an assembly in vitro with the same lattice parameters as that observed on whole cells or cell walls and could readsorb to the cell walls from which they had been extracted. Both the wild-type and the variant subunits behaved as single, homogeneous polypeptide chains. Carbohydrate assay and isoelectric point determinations revealed that both subunit types were acidic glycoproteins. Values obtained for thebuoyant density, isoelectric point, and extinction coefficient differed minimally; major differences were observed in the molecular weight and the characterisitc width of cylinders formed by in vitro-assembled T-layer of the wild-type and variant. Assembled T-layer was subject to alkaline or acid dissociation and in acid titration dissociated at its isoelectric point.


Partial purification and some properties of the Staphylococcus aureus cytoplasmic nitrate reductase. The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.


Effect of alanine-containing dipeptides on germination of Bacillus thiaminolyticus spores. Stereoisomeric alanylalanine (Ala-Ala) derivatives were examined for their effects on germination of Bacillus thiaminolyticus spores. L-Ala-L-Ala and L-Ala-glycine were effective in inducing germination, and their activities were completely inhibited by D-Ala. L-Ala-D-Ala and glycine-D-Ala competitively prevented L-Ala-induced germination. Sarcosine- or beta-Ala-containing L-alanyldipeptides and eight kinds of alanyltripeptides did not show any detectable effect on germinability or any inhibitory effect. No detectable amounts of Ala were found in germination exudates when alanylpeptides were incubated with spores. The ability of these peptides to induce or inhibit germination depends on their steric conformation and a certain distance between the primary amino group and the free carboxyl groups. Involvement of L-Ala dehydrogenase in the initiation of germination is unlikely because L-Ala-L-Ala was not a substrate and L-Ala-D-Ala was not an effective inhibitor of enzyme activity.


Distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species. The distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species was examined. Enzyme activity was demonstrated in cell-free growth media from all members of the family Vibrionaceae which were tested except Plesiomonas shigelloides. In each case, enzyme was produced in exponential to early stationary phase and was excluded from Sepharose 6B, indicating a complex of high molecular weight. In a limited survey of other families, Stahylococcus aureus was the only organism outside the Vibrionaceae which was shown to produce the enzyme. In this case, however, the enzyme exhibited much less activity against erythrocyte membranes and appeared to have a lower molecular weight. The reasons for these differences and the importance of the acyltransferase as a biochemical identification tool are discussed.


R-plasmid-mediated chromosomal gene transfer in Agrobacterium tumefaciens. Although several techniques are available for transferring the Ti plasmids from one strain of agrobacterium tumefaciens to another, there are no reproducible methods for analysis of chromosomal markers in this phytopathogen. The R plasmid, R68.45, is known to show chromosomal mobilizing ability in several bacterial genera including the closely related Rhizobia. R68.45 was transferred into the prototrophic A. tumefaciens strain 15955. Ten kanamycin-resistant transconjugant clones were tested for chromosomal mobilizing ability by mating with strain SA10, rifampin- and streptomycin-resistant histidine auxotroph of strain 15955. Of the 10 donor clones, 2 showed high chromosomal mobilizing ability. Between 1,000 and 2,000 His+ colony-forming units per ml were obtained, a value 10 to 20 times greater than can be accounted for by spontaneous reversion. Sequential recloning and matings resulted in the isolation of relatively stable donor cultures. Chromosome gene transfer is dependent upon the presence in the donor of R68.45. Donors lacking an R plasmid or harboring the closely related plasmid RP4 failed to yield His+ transconjugants. With strain SA11, a methionine auxotroph of strain SA10, coinheritance of histidine and methionine independence could be demonstrated. Approximately half of the transconjugants also inherited R68.45. These results indicate that A. tumefaciens 15955 is capable of undergoing host chromosomal genetic exchange.


Ribulose 1,5-bisphosphate carboxylase/oxygenase from Pseudomonas oxalacticus. Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified by a rapid, facile procedure from formate-grown Pseudomonas oxalaticus. The electrophoretically homogeneous enzyme had specific activities of 1.9 mumol of CO2 fixed per min per mg of protein and 0.15 mumol of O2 consumed per min per mg of protein. The amino acid composition was similar to that of other bacterial sources of the enzyme. The molecular weights determined by sedimentation equilibrium and by gel filtration were 421,000 and 450,000, respectively. Upon sodium dodecyl sulfate electrophoresis of enzyme purified under conditions which would limit proteolysis, two types of large (L) subunits and two types of small (S) subunits were observed with apparent molecular weights of 57,000, 55,000, 17,000 and 15,000. By densitometric scans at two different protein concentrations the stoichiometry of the total large to total small subunits was 1:1, implying an L6S6 structure. Electron micrographs of the enzyme revealed an unusual structure that was inconsistent with a cubical structure. The enzyme had an unusually high Km for ribulose 1,5-bisphosphate (220 microM) and was strongly inhibited by 6-phosphogluconate in the ribulose 1,5-bisphosphate carboxylase assay (Ki = 270 microM). One, 5, and 12 days after purification the enzyme was half-maximally activated at 0.13 microM, 0.23 mM, and 0.70 mM CO2, respectively, at saturating Mg2+. At saturating CO2, enzyme 1 day afer purification responded sigmoidally to Mg2+ and was half-maximally activated by 0.85 mM Mg2+ in the absence of 6-phosphogluconate (Hill coefficient, h = 2.0) and by 0.19 mM Mg2+ in the presence of mM 6-phosphogluconate (h = 1.7).


Amylooligosaccharides from Mycobacterium smegmatis. In early stationary phase of growth, Mycobacterium smegmatis cultures accumulate amylooligosaccharides (alpha 1 leads to 4-glucooligosaccharides) up to the undecasaccharide. Although M. smegmatis also makes an acylated polymethylpolysaccharide that is predominantly and alpha 1 leads to 4-glucan, we conclude that these oligosaccharides are precursors of glycogen rather than lopopolysaccharide.


General transduction in Rhizobium meliloti by a thermosensitive mutant of bacteriophage DF2. A simiplified version of bacteriophage DF2 transduction, using the thermosensitive mutant DF2tsC, is described. The transduction frequencies of Rhizobium meliloti markers by DF1tsC are of about 10(-6) or higher. The development of transductants at 34 degrees C does not require the addition of antiserum for phage neutralization.


Pseudomonas cepacia mutants blocked in the direct oxidative pathway of glucose degradation. Glucose dehydrogenase-deficient strains of Pseudomonas cepacia grew normally with glucose as carbon source, indicating that the direct pathway of glucose oxidation does not play an essential role in this bacterium.


Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria. A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.


Lipid and lipopolysaccharide composition of Acholeplasma oculi. The total lipid content of Acholeplasma oculi comprises 13.3% of the dry weight of the organism and is about equally distributed between the neutral lipids plus glycolipids and the phospholipids. The phospholipids were identified as phosphatidyl glycerol and diphosphatidyl glycerol. The glycolipid fraction contained O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol and O-alpha-D-glucopyranosyl-(1 leads to 2)-O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol. The neutral lipid contained pigmented carotenoids. Hot aqueous phenol extraction of lipid-extracted whole cells yielded a polymeric carbohydrate comprising 2.3% of the dry weight of the organism. The A. oculi lipopolysaccharide was found to contain only neutral sugars and no amino sugar, in contrast to other acholeplasmas. The neutral sugars consisted of fucose, galactose, and glucose in a ratio of 2:19:3.


Plasmids and transposable elements in Salmonella wien. The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.


Some effects of uncouplers and inhibitors on growth and electron transport in rumen bacteria. Uncouplers and inhibitors of electron transport affected growth and electron transport of rumen bacteria in various ways. Selenomonas ruminantium was not affected by inhibitor and uncoupler concentrations which affected growth and electron transport of Bacteroides ruminicola, B. succinogenes, and Butyrivibrio fibrisolvens. Inhibitors, when active, led to accumulation of reduced electron carriers before the site of action, but differences were found among organisms in the site of action of these inhibitors. Uncouplers reduced the glucose molar growth yields (Ygluc) of B. ruminicola, B. succinogenes, and B. fibrisolvens compared with those obtained without uncouplers. The extent of Ygluc reduction accompanying inhibitor exposure reflected electron transport chain structure. S. ruminantium appeared to obtain its adenosine 5'-triphosphate from substrate-level processes only. The other organisms studied appeared to obtain adenosine 5'-triphosphate both from substrate-level processes and from electron transport but differed in the amount of adenosine 5'-triphosphate obtained from glucose catabolism and in the proportions of adenosine 5'-triphosphate obtained from substrate-level reactions and electron transport.


Negative control of octopine degradation and transfer genes of octopine Ti plasmids in Agrobacterium tumefaciens. The regulatory system that controls the expression of the Ti plasmid-borne octopine degradation (uad) and transfer (tra) genes in Agrobacterium tumefaciens was studied. A deletion mutant derived from the cointegrate plasmid R702::Ti-B6S3 was isolated, which was compatible with a wild-type Ti plasmid and which had retained the uad genes. By means of this mutant plasmid pAL116, it was possible to make cells diploid for the uad genes. pAL116 was introduced into Rec- strains that contained different types of regulation mutants for the uad and tra genes. The repression pattern that was found in this complementation analysis indicated that the uad and tra operons are controlled by a common repressor system. Several results indicated that there may be additional transcriptional relations between both operons. The corresponding genes of the non-tumorigenic octopine plasmid pAt-AG60 appeared to be controlled by a repressor related to that of the octopine Ti plasmid.


5-n-Alkylresorcinols from encysting Azotobacter vinelandii: isolation and characterization. Azotobacter vinelandii was found to form novel lipid compounds when encystment was initiated by 0.2% beta-hydroxybutyrate. An examination of these compounds led to the isolation and characterization of 5-n-heneicosylresorcinol, 5-n-tricosylresorcinol, and their galactoside derivatives.


Response of Rhodopseudomonas capsulata to illumination and growth rate in a light-limited continuous culture. Rhodopseudomonas capsulata was grown under anaerobic, photosynthetic conditions in a continuous culture device. Under light limitation, at a constant dilution rate, it was shown that cell composition, including photopigment (bacteriochlorophyll and carotenoids) and ribonucleic acid content, was not affected by incident light intensity; however, steady state culture density varied directly and linearly with light intensity. On the other hand, photopigment and ribonucleic acid levels were affected by growth rate regardless of light intensity. Additional experiments indicated a high apparent Ks for growth of R. capsulata with respect to light. These results were interpreted to mean that near the maximum growth rate (D = 0.45 h-1) some internal metabolic process became the limiting factor for growth, rather than the imposed energy limitation. A mathematical expression for the relation between steady-state culture density and dilution rate was derived and was found to adequately describe the data. A strong correlation was found between continuous cultures limited either by light or by a chemical energy source.


Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens. The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a.


Phosphate-limited culture of Azotobacter vinelandii. Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly-beta-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.


Developmental regulation of invertase isozymes in Fusarium oxysporum. Fusarium invertase has two isozymes; one is developmentally regulated, whereas the other exists throughout the developmental stages of Fusarium.


Adenine inhibition of the synthesis of photosynthetic membranes in the chloroplast of Scenedesmus obliquus grown in the dark. In the presence of adenine, heterotrophically grown Scenedesmus obliquus lost most of its photosynthetic membranes. The rudimentary membranes appeared to be intact both functionally and morphologically.


Psychosis and water intoxication.

Psychosis and water intoxication. Three cases of psychosis, polydipsia, and water intoxication are presented as examples of a syndrome that is potentially unrecognized in psychiatric settings. Diagnostic and etiologic considerations, with particular attention to the syndrome of inappropriate antidiuretic hormone secretion (SIADH), are discussed through a review of relevant literature. A schema for a routine psychiatric evaluation is described that will minimize overlooking this association of psychosis and disturbed water balance.


Marriage and midlife: the impact of social change. This paper considers the impact of social change on marriage in the midlife period. Issues which ordinarily require adaptation in midlife e.g., awareness of the finiteness of time, the limits of options and the necessity to reassess goals and deal with losses are affected by societal changes. These changes include increased numbers of dual career families and a decreased birth rate. Marital partners must integrate these changes into their lives. The process of adaptation may be stressful and result in symptoms such as depression, anxiety, somatic complaints of sexual problems. Illustrative clinical examples and treatment approaches are provided.


Physical restraint and the nonpsychotic patient: clinical and legal perspectives. Recent legal challenges to the use of therapeutic seclusion in the treatment of the mentally ill have forced psychiatrists to defend this practice in court. The clinical characteristics of 10 nonpsychotic inpatients who required restraint are reviewed. These patients are characterized by intense labile affect, impulsive, manipulative behavior and transient micropsychotic episodes in the context of immature personality patterns suggestive of the borderline syndrome. Restraint was precipitated by transient micropsychotic episodes or impulsive behavior during sustained regression. Physical control in these cases is required to defend the social milieu more often than to prevent injury to self or others. The legal implications of these clinical findings are discussed.


Manic depressive illness and EEG abnormalities. In evaluating 40 manic-depressive (bipolar) patients who had clinical electroencephalograms, there was a significant relationship between abnormal EEG findings and a negative family history for affective disorder when a comparison was made between patients with various classes of family history.


Marital therapy with borderline patients: is it beneficial? Marital therapy has developed techniques which do not, for the most part, rely on the individual psychiatric diagnosis of the 2 partners. When both spouses suffer from Borderline personality disorders, the standard techniques do not seem to apply, are difficult to put into effect and the results of therapy are poor. The authors illustrate their experience with the description of a 3 month trial of marital therapy involving a borderline couple. The outcome suggests the need for careful individual and interactional diagnosis prior to marital therapy. Specific psychotherapeutic techniques should be tested for use with Borderline marriage partners.


Prosopagnosia and the Capgras syndrome.

Prosopagnosia and the Capgras syndrome. The Capgras syndrome is an unusual psychiatric disorder which has attracted much attention in recent years because of its striking nature and controversial etiology and pathogenesis. The question of whether some cases of Capgras syndrome are an expression of a perceptual disorder of facial recognition, i.e., prosopagnosia, is addressed in this report. Careful testing for a possible prosopagnosia revealed that in 2 newly identified cases of Capgras syndrome, the deficit was present. A deficit of facial recognition could not be appreciably detected in a group of normal female and schizophrenic controls.


Process of iodination of thyroglobulin and its maturation. I. Properties and distribution of thyroglobulin labeled with radioiodine in pig thyroid slices. Pig thyroid slices were incubated with Na131I and the 17--19S 131I-labeled thyroglobulin isolated was subjected to dissociation with 0.3 mM sodium dodecyl sulphate SDS) on sucrose density gradient centrifugation and to iodoamino acid analysis. During the incubation, initially dissociable thyroglobulin was gradually altered to 0.3 mM SDS-resistant species with increasing incorporation of iodine. Microsome-bound, poorly iodinated thyroglobulin and preformed thyroglobulin were chemically iodinated and then subjected to analysis of dissociability and iodoamino acid contents with newly incorporated iodine. The results indicated that the behavior of the former thyroglobulin resembled that of 131I-thyroglobulin obtained from the slices. Then, thyroid slices were incubated for 3 min with Na131I and 3H-leucine with or without 10-min chase incubation. The sucrose density gradient centrifugation patterns of 131I and 3H-radioactivity of cytoplasmic extracts indicated that 131I-thyroglobulin is contained in particulates, especially in vesicles with low density(d=1.12) and that some of them are released into the soluble fraction within 10 min. The vesicles contained peroxidase and NADH-cytochrome c reductase, and are probably exocytotic vesicles in the apical area of cytoplasm of follicular cells. No positive evidence was obtained that plasma membranes participate in the iodination of thyroglobulin under the present experimental conditions. These results suggest that, in the incubation of thyroid slices, iodine atoms are preferentially incorporated into newly synthesized, less iodinated thyroglobulin, rather than preformed thyroglobulin, and that the iodination occurs, at least to a certain degree, in apical vesicles before the thyroglobulin is secreted into the colloid lumen.


Purification and some properties of rabbit C1r. C1r, an activated subcomponent of the first component of the complement system, was highly purified from rabbit serum by affinity chromatography on IgG-Sepharose 6B followed by column chromatography on CM-Sephadex C-50. The C1r thus purified had a molecular weight of 105,000, consisting of two polypeptide chains connected by disulfide bonds; the molecular weights of the chains were 60,000 and 45,000. The C1r was found to reconstitute C1 complex when it reacted with rabbit C1q and C1s in the presence of Ca2+, since C1s was able to bind to C1q bound on sensitized sheep erythrocytes only in the presence of C1r. On the other hand, and active C1s fragment derived by hydrolysis of the H chain without any loss of C1s activity [J. Biochem. 80, 1423--1427 (1976)] could not bind to C1q even in the presence of C1r. This result indicates that a part of the H chain of C1s not contributing to the structural integrity of an active site may be involved in the binding of C1s to C1r.


Limited proteolysis of p-chloromercuribenzoate-dissociated hemoglobin by trypsin. p-Chloromercuribenzoate-treated hemoglobin was digested by trypsin. The hydrolysate was subjected to gel-filtration on Bio-Gel P-4 and Sephadex G-50 columns, ion-exchange chromatography on CM-Sephadex and DE 52 columns, and paper electrophoresis. Peptides obtained by this procedure were analyzed for amino acid compositions and amino-terminal amino acid sequences. The results showed that p-chloromercuribenzoate-treated hemoglobin was hydrolyzed to a limited extent by trypsin at the bonds involving the carboxyl group of a lysine or arginine residue in planes A--E in the parent hemoglobin, which represent the external region of the parent tetramer. It is concluded therefore that the slight modification of hemoglobin enhances the susceptibility of the protein to proteases and that the hydrolysis of the modified protein is limited.


Partial purification and properties of citrate synthase from sea urchin eggs. Citrate synthase [EC 4.1.3.7] was purified from sea urchin eggs about 14-fold with a 23% yield, based on the activity of the crude extract. The molecular weight of the enzyme was about 100,000 as determined by gel filtration. The optimum pH was about 7.8 in 100 mM Tris-HCl. The apparent Km values for acetyl-CoA and for oxaloacetate were 33 and 3.2 muM, respectively. Monovalent and divalent cations inhibited the enzyme. Iodoacetamide, pCMB, EDTA, NaF, and dithiothreitol did not affect the enzyme activity. Oxaloacetate protected the enzyme against heat denaturation. Among nucleotides, ATP was the most potent inhibitor of the enzyme. The inhibition by ATP was competitive with respect to acetyl-CoA and mixed with respect to oxaloacetate.


Comparison of carbohydrate-containing substances from non-calcified and calcified portions of bovine costal cartilage. Carbohydrate-containing substances were extracted from non-calcified (NCC) and calcified (CC) portions of bovine costal cartilage with 0.5 M LaCl3 by the method of Mason and his co-workers, followed by dilution of the extract with 9 volumes of water. The precipitate formed on dilution yielded Fr. P, while Fr. S was obtained from the supernatant. Fr. P was separated into two subfractions by gel filtration on Sepharose 2B. The experimental results showed that Fr. P contained proteoglycans with different molecular sizes and compositions, while Fr. S contained proteoglycan, hyaluronic acid, glycoproteins, and glycogen. The present data suggest that in the proteoglycan of Fr. P, the relative content of chondroitin sulfate decreases with a concomitant increment in that of keratan sulfate on calcification. In addition, elevation of the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate, together with a small increment of non-sulfated disaccharide units in the chondroitin sulfate chains appear to occur on calcification. The glycogen content in Fr. S diminished on calcification. The present observations suggest therefore that the remodeling of proteoglycan consumption of glycogen in bovine costal cartilage occur on calcification.


Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent. The optimal conditions for the activation of Sepharose by epichlorohydrin and subsequent immobilization of ligands were investigated. Under the optimal conditions for activation, namely, 30% Sepharose-5% epichlorohydrin-0.4 M NaOH, 40 degrees C, 2 h, the maximum amount of epoxy group was introduced into Sepharose with low cross-linking. The absorbents obtained by using N-acetyl-D-glucosamine, tri-N-acetylchitotriose, and glycoprotein as a ligand exhibited no nonspecific adsorption and good permeability for the high molecular substance to be purified, and were stable in an alkaline solution. Solanum tuberosum agglutinin was specifically adsorbed on a tri-N-acetylchitotriose-Sepharose column and was quantitatively recovered by elution with 0.2 M ammonia solution. Furthermore, the column could be repeatedly used under these conditions without reduction of its capacity.


Isolation and properties of a glycohydrolase specific for nicotinamide mononucleotide from Azotobacter vinelandii. A glycohydrolase that catalyzes the irreversible conversion of NMN to nicotinamide and ribose 5-phosphate has been partially purified from a sonic extract of Azotobacter vinelandii. The enzyme is highly specific for NMN. NAD, NADP, nicotinic acid-adenine dinucleotide, nicotinamide riboside and alpha-NMN are not significantly hydrolyzed by this enzyme, nor do they compete with NMN. The enzyme also exhibits an absolute dependence on guanylic acid derivatives with following order of relative effectiveness: GTP, guanosine 5'-tetraphosphate greater than dGTP, GDP, 2'-GMP, 3'-GMP greater than GMP, dGMP. A heat-resistant, nondialyzable factor which could replace the GTP requirement was found in the sonic extract. The Ka for GTP and the Km for NMN in the presence of GTP at 1mm were calculated to be 0.025 mM and 4.5 mM respectively. GMP, dGMP, and dCMP were found to be effective inhibitors of the enzyme when 1 mM GTP was also present. The kinetic data suggest that the binding site for these mononucleotides is distinct from the active site or the GTP binding site. The ability of this enzyme to cleave NMN is suggestive of a metabolic role of the enzyme in selective conversion of NMN to nicotinamide, which, in turn, would be re-utilized by the cell as a precursor of NAD via nicotinic acid.


Purification and properties of pig liver kynureninase. Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.


Correlation between circadian rhythms of polyamine synthesis and cell proliferation in rat liver. In rats fed ad libitum, a marked circadian rhythm with a peak at night was observed in the hepatic level of ornithine decarboxylase (ODC) [EC 4.1.1.17], the enzyme for the first step of polyamine synthesis. A similar rhythm was found in the hepatic content of putrescine, but not of spermidine or spermine. The mitotic activity of the liver also exhibited a clear rhythm with a peak in the daytime. The rhythms of both ODC and mitosis were generated by cyclic ingestion of proteinous food, since the peaks shifted when rats were meal-fed and both activities disappeared on starvation or protein deprivation. The close parallel between the rhythms suggested that synthesis of polyamine, especially that of putrescine, was a prerequisite for the rhythmic growth of liver. The dietary induction of hepatic ODC depended on the nutritive value of dietary protein; zein or gelatin was effective only when supplemented with limiting amino acids and there was a good correlation between the hepatic ODC level and the relative growth rate.


Fluorescence studies on the interaction of dansyl-L-arginine with trypsin and trypsinogen. The enhancement of fluorescence intensity of the dansyl group due to the formation of trypsin- or trypsinogen-dansyl-L-arginine complex was measured. Dansyl-L-arginine (L-DA) is a product in the trypsin-catalyzed hydrolysis of dansyl-L-arginine methylester. Trypsinogen was found to have only one binding site for L-DA with the dissociation constant of 6.9 x 10(-3)M, which is identical with the Michaelis constant for the trypsin-catalyzed hydrolysis of dansyl-L-arginine amide (Goto, S. and Hess, G.P., unpublished results). This finding and the results of X-ray diffraction studies (1,2) suggest that this binding site is located in the active site of the enzyme. On the other hand, the active enzyme, trypsin, was found to have at least two binding sites for L-DA. One is located in the active site. The dissociation constant for L-DA bound to this site is 6.7 x 10(-3)M. The other site is probably located in the allosteric site of trypsin. The dissociation constant for L-DA bound to this site is 4.8 x 10(-4)M.


Novel properties of DNA polymerase beta with poly(rA).oligo(dT) template-primer. Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.


Steroids as substrates for cholesterol: oxygen oxidoreductase, with special reference to 3beta-hydroxy bile acids. Cholesterol: oxygen oxidoreductase [EC 1.1.3.6] from Brevibacterium sterolicum (ATCC 21387) was found to catalyze the oxidation of steroids such as sterols, steroid hormones, and bile acids having a free C-3beta hydroxyl group. However, the enzyme was inactive towards estradiol and estriol and had a weak activity towards steroids with functional groups adjacent to the 3beta-hydroxyl group on the steroid nucleus. Variation in the length of the side chain of 3beta-hydroxy steroids had no marked effect on the activity. 3beta-Hydroxy bile acids with delta4 or delta5 were oxidized to almost the same extent as cholesterol. In contrast, 3beta-hydroxy bile acids without delta4 or delta5 were oxidized only to the extent of 1.4--2.1%. 3 beta-Hydroxychol-4 or 5-enoic acid was oxidized in the same way as cholesterol. This enzyme is useful as a simple tool for identification of 3 beta-hydroxy groups of bile acids.


NADH: quinone oxidoreductase as a site of Na+-dependent activation in the respiratory chain of marine Vibrio alginolyticus. The site of Na+-dependent activation in the respiratory chain of the marine bacterium, Vibrio alginolyticus, was investigated. The respiratory chain system contained ubiquinones (Q), menaquinones (MK), cytochromes b(560), c(553), d(630), and o(560). The membrane-bound and partially purified NADH dehydrogenase was stimulated 2- to 3-fold by the addition of 0.2 M Na+ or K+ and no specific requirement for Na+ was observed in this reaction step. The cytochrome oxidase showed no requirement for monovalent cations. The respiratory activity (NADH oxidase) of the membrane was lost on removal of the quinones, and the reincorporation of authentic Q-10 or MK-4 restored the activity. The rate of MK-4 reduction by NADH (menaquinone reductase) as measured using MK-4 incorporated membrane was activated by Na+, but only slightly by K+. The apparent Ka for Na+ was 78 mM for both menaguinone reductase and NADH oxidase. The requirement for Na+ of menaquinone reductase was greatly reduced in the presence of 0.2 M K+. Ubiquinone reductase as measured by using Q-10 incorporated membrane was also activated more effectively by Na+ than by K+. These results strongly suggested that the site of Na+-dependent activation in the respiratory chain of marine V. alginolyticus was at the step of NADH; quinone oxidoreductase.


Studies on glycosphingolipids of fresh-water bivalves. V. The structure of a novel ceramide octasaccharide containing mannose-6-phosphate found in the bivalve, Corbicula sandai. A ceramide octasaccharide containing mannose-6-phosphate was isolated from the fresh-water bivalve Corbicula sandai by solvent fractionation, followed by two types of silicic acid column chromatography, and finally QAE-Sephadex column chromatography. The structural analysis involved the following steps. (a) Gas-liquid chromatography of the component sugars, fatty acids, and long-chain bases. (b) Degradation with HCl and HF to elucidate the sugar sequence. (c) Permethylation analysis coupled with GC-MS to identify the positions of the glycosidic linkages between the sugar units. (d) Chromium trioxide oxidation to determine the anomeric configuration. (e) Smith degradation to determine the site of linkage of the ethanolamine residue. The structure of this novel glycolipid was determined to be: 4-O-MeGalp(bets1 yields 3)-GalNAcp(beta1 yields3)Fucp(alpha1 yields 4)GlcNAcp(beta1 yields 2)Manp(alpha1 yields 3)[Xylp(alpha1 yields 2)][2'-aminoethylphosphoryl(yields 6)]Manp(beta1yields 4)Glcp(beta1 yields 1)ceramide. It is very interesting that fucose was found to be internally linked in this sugar chain. To our knowledge, this is the first example of internal fucose in a glycolipid. The ceramide moiety consisted of normal saturated fatty acids, among which stearic acid was pr


Androgenic regulation of N-acetyl beta-glucosaminidase activity in the submandibular glands of mice. N-Acetyl beta-glucosaminidase [beta-2-acetamido-2-deoxy-D-glucoside acetylamido-deoxyglucohydrolase; EC 3.2.1.30] in the submandibular gland of mice was found to be androgen-dependent; the specific activities in males, females, and castrated males were 0.25, 0.11, and 0.11 unit/mg protein, respectively. The activities in females and castrated males were increased to the level of normal male mice by testosterone injection. Injections of progesterone and 17 beta-estradiol hardly affected the activity in males. In both males and females, the enzyme activity was detected in the convoluted tubular cells, not in acinous cells. The results of isoelectric focusing have shown that one enzyme having an isoelectric point of 9.0 is present in the glands of both sexes, indicating that the enzyme remains after castration and that the increases caused by testosterone represent the same molecular species. In addition, it was shown that the saliva from both sexes contained significant activity of N-acetyl beta-glucosaminidase, which also changed depending on the androgenic state of the animals. Most of the salivary activity was shown to originate from the submandibular gland, since the extirpation of this gland resulted in a significant decrease of the salivary activity.


Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8. (1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.


Water-soluble lipoproteins from yolk granules in sea urchin eggs. II. Chemical composition. The chemical composition of yolk lipoproteins (YLP-1, 2, and 3) was determined. YLP-1, 2, and  3 were quite similar as regards the chemical composition of lipids, proteins, and carbohydrate moieties. Each lipoprotein has an average dry weight composition of lipids (55--72%) and apo-lipoproteins (28--45%) containing protein, hexose, hexosamine, and sialic acid. In each lipoprotein, triacylglycerol is a major lipid component (70--83%), followed by phospholipid (8--16%), cholesterol (free and esterified, 8--10%), and free fatty acid (3--4%). Phosphatidylcholine and phosphatidylserine account for 68--74% and 16--24% of the phospholipids, respectively. The fatty acid compositions of total lipids from each lipoprotein are quite similar, with a high degree of unsaturation (63--65%). The carbohydrate content of apolipoprotiens from each lipoprotein is remarkably high (27--31% of apo-lipoproteins) and their composition is very simple: mannose and glucosamine are major constituents in the polysaccharide moiety of each lipoprotein and sialic acid is all in the N-glycolyl form. The amino acid compositions of apo-lipoproteins are quite similar in YLP-1, 2, and 3, with high contents of aspartic acid, glutamic acid, threonine, serine, and leucine. Furthermore, a small amount of glycolipids is present in the yolk lipoproteins. They were separated into six components on TLC. All of them are resorcinol-positive, indicating the presence of sialoglycolipids.


Post-mortem changes in skeletal muscle connectin. Changes in connectin and elasticity of skeletal muscle were determined during post-mortem ageing. The amount of connectin decreased with increasing time of post-mortem storage whereas the rate of the decrease depended on the source of muscles. The loss in elasticity of muscle coincided well with the decrease in connectin contents. Electron microscopically, a network structure between the Z discs vanished when the amount of connectin fell to zero. We have concluded that the continuous net structure of connectin is responsible for about 30% of the total elasticity of living skeletal muscle and its degradtaion is responsible for post-mortem tenderization of meat.


Calcium sensitivy of foot muscle myosin from clam (Meretrix lusoria). 1. It was found that Mg-ATPase of clam foot myosin is strongly activated by calcium or strontium ions and is as sensitive to those divalent cations as the Mg-ATPase and superprecipitation of rabbit skeletal acto-clam foot myosin are. 2. It was also found that desensitization and resensitization of clam foot myosin result in the loss of superprecipitation activity with acto-desensitized myosin and in its recovery with acto-resensitized myosin, respectively. However, the ATP-ASE activity in the absence of calcium ions rises with acto-desensitized myosin and falls again with acto-resensitized myosin. 3. It is thus proposed that the primary role of the EDTA-light chain component in calcium regulation is to inhibit myosin-ATPase rather than to inhibit the actin-myosin interaction.


Difference in the mechanisms of poly(dT) synthesis by DNA polymerases beta and gamma. Poly(dT) products which were synthesized depending on (rA)n . (dT)12-18 as a template . primer by mammalian DNA polymerases beta and gamma were analyzed by alkaline sucrose gradient centrifugation. The size of the population of poly(dT) chains synthesized by DNA polymerase beta increased slowly and consistently during incubation up to at least 30 min. On the other hand, the product size with DNA polymerase gamma reached the final size (7 s) within 5 min and the number of products increased during further incubation. Comparison of product number per enzyme molecule suggests that DNA polymerase beta acts on multiple primers in a distributive fashion while DNA polymerase gamma completes poly(dT) chains of large size in a one-by-one fashion.


The Association of Biomedical Communications Directors. The Association of Biomedical Communications Directors (ABCD) has recently completed the fifth in its series of surveys of biomedical communications units. The fourth survey, published in 1975, reported staffing patterns, salary data, degrees held, and a variety of other information from units directed by members of the ABCD in the United States and Canada. The current report covers similar data from the 1977-78 academic year and in addition includes information from units whose directors were not members of ABCD but who wished to cooperate in the survey.


Sequence-specific endonuclease Bgl I. Modification of lysine and arginine residues of the homogeneous enzyme. The sequence-specific endonuclease Bgl I from Bacillus globigii (RUB561) has been purified to homogeneity as determined by denaturing polyacrylamide gel analysis. The active form of the enzyme is a single polypeptide with a molecular weight of 32,000. The enzyme requires Mg2+ in the reaction mixture and displays a broad pH and monovalent cation requirement. Bgl I is not sensitive to sulfhydryl reagents but was affected by reagents that modify lysine and arginine residues. When lysine residues were modified by pyridoxal 5'-phosphate, both binding and catalysis were diminished while modification of arginine residues by 2,3-butanedione inhibited the enzyme activity but had no effect on its binding properties.


Purified eukaryotic initiation factor 2 from calf liver consists of two polypeptide chains of 48,000 and 38,000 daltons. Eukaryotic initiation factor 2 (eIF-2), which specifically binds Met-tRNAMetf and forms stable ternary complexes with GTP, has been purified from ribosomal salt wash proteins from calf liver. The purified factor exhibits only two polypeptide bands of Mr = 48,000 and 38,000 following electrophoresis in 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. Densitometric tracings show the two polypeptides are present in a molar ratio of 1:1. This suggests a Mr = 86,000 for the native enzyme, a value which agrees with the apparent molecular weight determined by physical methods. Less pure preparations of eIF-2 show additional polypeptide bands, including 50,000- and 46,000-dalton bands, all of which can be removed by further purification without affecting the activity of eIF-2.


Differentiation of ob 17 preadipocytes to adipocytes. Effect of insulin on the levels of insulin receptors and on the transport of alpha-aminoisobutyrate. Cells of a clonal cell line (ob 17) isolated from the epididymal fat pad of ob/ob mouse possess insulin receptors. Their number was increased 1.5-fold after growth arrest, with no significant change in the Kd values of the "high affinity" sites determined by extrapolation of the high affinity portion of the curvilinear Scatchard plots. With chronic insulin exposure for 3 to 11 days after confluence, ob 17 cells showed a decrease in insulin receptor concentrations from 8,000 to 1,600 high affinity sites/cell (Kd from 0.45 to 1.10(-9) M) while similar levels of "low affinity" sites were found (80,000 to 100,000 sites/cell; Kd from 10(-8) to 3 x 10(-8) M). The loss of the high affinity binding sites is accompanied by the disappearance of the stimulatory effect by insulin of alpha-aminoisobutyrate uptake. Therefore, in contrast to 3T3-L1 fibroblasts, the ob 17 cells present, in culture, a self-modulation of insulin receptors and a loss of insulin sensitivity after chronic exposure to insulin.


Activation of adenylate cyclase by molybdate. The effect of molybdate on adenylate cyclase (EC 4.6.1.1) in rat liver plasma membranes has been examined. The apparent K alpha for molybdate activation of the enzyme is 4.5 mM, and maximal, 7-fold stimulation is achieved at 50 mM. The observed increase in cAMP formation in the adenylate cyclase assay is not due to: (a) an inhibition of ATP hydrolysis; (b) a molybdate-catalyzed conversion of ATP to cAMP; (c) an inhibition of cAMP hydrolysis; or (d) an artifact in the isolation of cAMP formed in the reaction. Molybdate activation of adenylate cyclase is a general phenomenon exhibited by the enzyme in brain, cardiac, and renal tissue homogenates and in erythrocyte ghosts. However, like fluoride and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), molybdate does not activate the soluble rat testicular adenylate cyclase. Molybdate is a reversible activator of adenylate cyclase. Activation is not due to an increase in ionic strength and is independent of the salt used to introduce molybdate. Molybdate does not activate adenylate cyclase previously stimulated with Gpp(NH)p or fluoride. At concentration greater than 20 mM, molybdate inhibits fluoride-stimulated adenylate cyclase, and at concentrations greater than 100 mM, molybdate stimulation of basal adenylate cyclase activity is diminished.


Quantitation and localization of globin messenger RNA in rabbit reticulocyte lysate. A sensitive and quantitative method is described for the determination of globin mRNA distribution in rabbit reticulocyte lysate. The method uses high resolution sucrose density gradient centrifugation followed by [5'-3H]polyuridylate hybridization to poly(A)-mRNA in gradient fractions. Polyadenylate, purified globin mRNA, and ribonuclease-treated lysate are used to standardize the hybridization assay. It is demonstrated that changes of mRNA and ribosomal distribution do not affect quantitation of the total mRNA localization and Met-tRNAf which suggest that the monitoring of Met-tRNAf binding alone may not be sufficient to assess the mechanisms of control which affect the initiation of protein synthesis.


Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping. Adenine phosphoribosyltransferase (EC 2.4.2.7) has been purified 55,000-fold from normal human erythrocytes. The native molecular weight of the enzyme is 38,200 as determined by sedimentation equilibrium centrifugation. The subunit molecular weight is 18,000 as determined by sodium dodecyl sulfate gel electrophoresis and 17,000 as determined by gel filtration in guanidine hydrochloride, suggesting that the enzyme is a dimer in its native state. Cross-linking the enzyme with dimethylsuberimidate confirms the dimeric structure and peptide mapping data suggested that the subunits are quite similar if not identical. The amino acid composition reveals that 33% of the residues are hydrophobic.


Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures. NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.


Developmental regulation of creatine kinase isoenzymes in myogenic cell cultures from chicken. Levels of mRNA for creatine kinase subunits M and B. During the first 2 days of culture of myogenic chicken cells only minute amounts of mRNA for a subunit of creatine kinase, M-CK, could be found in cultures from the 3rd and 4th day; however, a dramatic increase of mRNA for the muscle-specific M-CK was observed. The mRNA for B-CK, another subunit of creatine kinase, was readily detectable in young cultures increased up to the 3rd day in culture but decreased significantly 1 day later. The comparison of the ratios of mRNA concentrations for M-CK and B-CK in polysomal and total cellular RNA did not reveal significant differences in polysomal and total cellular RNA from 24-, 48-, and 68-h cultures, indicating that specific accumulation of a nonpolysomal form containing translatable mRNA for M-CK prior to its appearance in the polysomes does not seem to represent a major mechanism of regulation. In RNA from cells grown in 5-bromo-2'-deoxyuridine (BrdUrd) for 3 days and subcultured into the same medium for another 3 days only mRNA for B-CK was found. If cells, however, were subcultured into standard medium both mRNAs, the one for B-CK and the one for M-CK could be detected. BrdUrd inhibition seems to affect only the mRNA for M-CK and not the one for B-CK. Removal of BrdUrd from the cultures probably allows new transcription or processing of mRNA for M-CK, or both.


The binding of substrates and a product of the enzymatic reaction to glutathione S-transferase A. The binding of substrates and a product to glutathione S-transferase A from rat liver was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrates glutathione and bromosulfophthalein as well as a product of glutathione and 3,4-dichloro-1-nitrobenzene, S-(2-chloro-4-nitrophenyl)glutathione, gave hyperbolic binding isotherms with a stoichiometry of 2 mol per mol of enzyme (i.e. 1 molecule per subunit). Glutathione (and glutathione disulfide) had an equilibrium (dissociation) constant for the binding of about 10 microM, whereas bromosulfophthalein and the product had equilibrium constants of about 0.5 microM. All ligands showed the same binding stoichiometry, and competition experiments involving unlabeled ligands indicated that glutathione and the glutathione derivatives were binding to the same site. Low affinity sites appeared to exist in addition to the specific high affinity sites (one per subunit) for all ligands tested. The binding studies are fully consistent with a steady state random kinetic mechanism for the enzyme.


The nucleotide sequence adjacent to poly(A) in silk fibroin messenger RNA. The 3'-terminal nucleotide sequence of fibroin mRNA has been determined. A cDNA transcript primed by oligo(dT) hybridized to the poly(A) segment of fibroin mRNA was used to determine the sequence immediately adjacent to poly(A). A specific primer complementary to this sequence then allowed synthesis of 5'-terminally homogeneous transcripts amenable to analysis by rapid sequencing methods. The 3' terminus of fibroin mRNA contains an unusually long oligo(U) stretch, as well as the sequence A2UA3 recently found in 3' non-coding regions of other eukaryotic mRNAs. It lacks the oligonucleotides characteristic of the repetitive coding region of fibroin mRNA.


Biological activity of 24,24-difluoro-25-hydroxyvitamin D3. Effect of blocking of 24-hydroxylation on the functions of vitamin D. To examine the question of whether 24-hydroxylation plays and importance role in the physiological functions of vitamin D, the biological activity of 24,24-difluoro-25-hydroxyvitamin D was compared with that of 25-hydroxyvitamin D in vitamin D-deficient rats. These two compounds were found almost identically active in the stimulation of intestinal calcium transport, the mobilization of calcium from bone, the healing of rachitic epiphyseal plate cartilage, the elevation of serum inorganic phosphorus, the mineralization of rachitic bone, and in the prevention of rachitogenesis in rats. Little or no difference was detected in the time course of response of intestinal calcium transport or bone calcium mobilization to the two forms of vitamin D. Therefore, in the rat no support could be obtained for the idea that 24,25-dihydroxyvitamin D3 plays an important role in the known physiological responses to the vitamin.


Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins. Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventional techniques and recoveries of both small (4-residue) hydrophilic and large (30-residue) hydrophobic peptides are excellent. The total amino acid composition of the bovine intestinal calcium-binding protein has been accounted for on the basis of two tryptic maps of 20 microgram of protein each.


Cholecystokinin/pancreozymin induces the parallel discharge of digestive enzymes from the in vitro rabbit pancreas. Considerable controversy has surrounded the question of whether the exocrine pancreas discharges digestive enzymes in a parallel or nonparallel fashion. A recent report (Rothman, S.S., and Wilking, H. (1978) J. Biol. Chem. 253, 3543-3549) claimed that the in vitro rabbit pancreas demonstrated nonparallel enzyme discharge after stimulation with cholecystokinin/pancreozymin, but that parallel discharge followed stimulation with the COOH-terminal octapeptide of cholecystokinin/pancreozymin. It was suggested that the full hormone acted to inhibit chymotrypsinogen secretion while stimulating trypsinogen secretion. Because of the fundamental importance of this question to our understanding of the exocrine secretion of exportable proteins, we have repeated these experiments using the same preparation and stimulant but have observed only parallel enzyme discharge. We conclude that it is unlikely that cholecystokinin/pancreozymin causes the selective inhibition of chymotrypsinogen secretion.


Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization. Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and certain short straight and branched chain fatty acyl-CoA esters as substrates while the other enzyme specifically used either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase inhibited the other. Glycine was the preferred acyl acceptor for both enzymes but either L-asparagine or L-glutamine also served. Peptide products for each transferase were identified by mass spectrometry. Enzymatic cleavage of acyl-CoA was stoichiometric with release of thiol and formation of peptide product. Apparent Km values were low for the preferred acyl-CoA substrates relative to the amino acid acceptors (10(-5) M range compared to greater than 10(-3) M). Both enzymes were inhibited by high nonphysiological concentrations of certain divalent cations (Mg2+, Ni2+, and Zn2+). In contrast to benzoyltransferase, phenylacetyltransferase was sensitive to inhibition by either 10(-4) M 5,5'-dithiobis(2-nitrobenzoate) or 10(-5) M p-chloromercuribenzoate; 10(-4) M phenylacetyl-CoA partially protected phenylacetyltransferase against 5,5'-dithiobis(2-nitrobenzoate) inactivation but 10(-1) M glycine did not. For activity, phenylacetyltransferase required addition of certain monovalent cations (K+, Rb+, Na+, Li+, Cs+, or (NH4)+) to the assay system but benzoyltransferase did not. Preliminary kinetic studies of both transferases were consistent with a sequential reaction mechanism in which the acyl-CoA substrate adds to the enzyme first, glycine adds before CoA leaves, and the peptide product dissociates last.


Transport and metabolism of thiamin in isolated rat hepatocytes. This study examines thiamin transport in isolated rat hepatocytes and its relationship to thiamin phosphorylation. In an Na+ medium, [35S]thiamin, 3 microM, was accumulated rapidly by the cells, and a near study state intra-/extracellular distribution ratio of 3 was attained in 1 min. However, the uptake of radioactivity continued to increase with time owing principally to the accumulation of [35S]thiamin pyrophosphate (TPP). In a choline, Li+ or K+ medium, the steady state intra-/extracellular distribution ratio of [35S]thiamin was decreased to less than or equal to 1.1. Accordingly, the rate of formation of [35S]TPP also decreased. Ouabain and uncouplers of oxidative phosphorylation significantly lowered the distribution ratio of intra-/extracellular [35S]thiamin. These data indicate that thiamin transport in liver is concentrative, Na+-dependent, and dependent on biological energy. Additionally, they suggest that thiamin transport plays a significant role in governing the rate of synthesis of TPP. Neither pyrithiamin, an inhibitor of thiamin pyrophosphokinase nor o-benzoylthiamin disulfide, a permeable thiamin analog, affected the distribution ratio of intra-/extracellular [35S]thiamin, but preferentially inhibited the phosphorylation of [35S]thiamin. By contrast, amprolium primarily inhibited uptake. These data suggest that thiamin transport and phosphorylation can be differentiated by the action of appropriate inhibitors.


Synthesis of high mobility group proteins in regenerating rat liver. Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.


Conformational changes associated with complex formation between a mycobacterial polymethylpolysaccharide and palmitic acid. The anomeric proton magnetic resonances of Mycobacterium smegmatis 3-O-methylmannose polysaccharide have chemical shifts intermediate betwen those of nonaglucoamylose and alpha-cyclodextrin, and on addition of palmitic acid most of these resonances are shifted upfield toward that of the cyclodextrin. This suggests that the methylated polysaccharide could have a conformation with some secondary structure intermediate between those of the two reference compounds, and that it forms a tightened coil upon addition of the lipid which yields an inclusion complex with the polysaccharide. The change in chemical shift is linear with lipid concentration, which indicates that the complex undergoes rapid exchange with free polysaccharide. The changes in proton chemical shifts of the polysaccharide and of the palmitic acid are consistent with the fatty acid being inserted in the coiled polysaccharide with its carboxyl group near the methyl aglycon.


Preliminary crystallographic data on monomeric and dimeric hemoglobins from the sea cucumber, Molpadia arenicola. Large single crystals of two distinct globin chains from coelomic cells of the sea cucumber Molpadia arenicola have been prepared and examined by x-ray crystallography. These hemoglobins exhibit a variety of ligand-dependent association states with the met-hemoglobins existing as monomers and liganded hemoglobins as dimers at physiological concentrations. Monomeric methemoglobin C chain crystallizes in space group P21, with a = 46.0 A, b = 45.3 A, c = 40.9 A, beta = 104.5 degrees, and one monomer per asymmetric unit. These crystals exhibit unusual spectroscopic behavior when examined with a polarizer, turning colorless in certain orientations. This implies that all the heme rings are nearly parallel within the crystals. Dimeric cyanmethemoglobin D chain crystallizes in space group P41212 (P43212), with a = b = 77.0 A, c = 61.5 A, and one-half a dimer per asymmetric unit. These homodimers thus possess a molecular 2-fold which is aligned with the crystallographic dyad.


Preliminary crystallographic data for glycolate oxidase from spinach. Glycolate oxidase, an enzyme that plays an important role in photorespiration in plants, has been purificant from spinach and crystallized in two different crystal forms. Form A which was obtained with tertiary butanol as precipitating agent belongs to space group I 422 with unit cell dimensions a = b = 148.1 A and c = 134.9 A. This form diffracts to high resolution and will be used for further crystallographic studies. Form B is also tetragonal, space group P42212, with cell dimensions a = b = 145.4 A and c = 104.2 A. This form was obtained from ammonium sulfate precipitations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows that the enzyme is built up from subunits of molecular weight 37,000. The asymmetric units of both crystal forms contain at least two such subunits.