Perception of time-compressed consonant-nucleus-consonant monosyllables by non-native speaker/listeners of English. Recent investigations of time-altered speech have dealt with the effect of time compression and sensation level on intelligibility scores of native speaker/listeners of English. In the present investigation, the intelligibility of time-compressed consonant-nucleus-consonant monosyllables was studied using English speaker/listeners whose native languages are Spanish or Indo-Dravidian. Results supported earlier findings in that intelligibility decreased as a function of increasing percentage of time compression and decreasing sensation level. This effect was more prominent for the Indo-Dravidian than for Spanish speaker/listeners. The Spanish group of subjects showed generally lower difference scores than did the Indo-Dravidian group when compared to native English speaker/listeners.


Modified rhyme test and synthetic sentence identification test scores of normal and hearing-impaired subjects listening in multitalker noise. Articulation functions were established for 35 normal hearing, 6 flat sensorineural, and 6 high frequency sloping sensorineural hearing-impaired subjects using the modified rhyme test (MRT) and synthetic sentence identification test (SSIT) in three message competition ratios (MCRs) of multitalker noise. An analysis of variance revealed significant differences between tests, subject groups, presentation conditions, and interactions between variables. Post hoc analyses revealed that SSIT functions were not significantly different among groups, and that MRT functions were different for each group. The MRT and SSIT were found to be two significantly different tests, each taxing different areas of discrimination. Results suggest that both tests can be used clinically with various MCRs of multitalker noise, but that the poorest MCR for each test does not aid in defining discrimination abilities.


Surgical repair of atrial septal defect in a dog. Atrial septal defect, a relatively rare canine congenital cardiovascular defect, caused syncope during excitement in a 5-year-old male Boxer dog. The defect was successfully repaired by open heart surgery.


Traumatically induced bullous lung lesions in the dog: a radiographic report of three cases. Three dogs with automobile-induced trauma had radiographic evidence of multiple bullous pulmonary lesions. Two of these dogs were subsequently reexamined, and resolution of their lesions was demonstrated radiographically.


Study of the classical and modified alternate binaural loudness balance tests in normal and pathological ears. This study was carried out to examine recruitment of loudness measured by the classical and the modified, self-recording alternate binaural loudness balance tests in 20 normal subjects and in 14 patients with various kinds of ear pathology. In spite of individual variability, the modified method, when used as described in this study, does provide an indication of the presence or absence of loudness recruitment.


Evaluation of monensin toxicity in the horse. Gelded horses of mixed breeding, ranging in weight between 360 and 455 kg, were fed pellets that contained monensin at concentrations of 279, 124, 31, and 0 ppm. Of 2 horses fed 279 ppm (253 g/T), both died. Of 3 horses fed 125 ppm (113 g/T), 1 died; feed intake was reduced in the 2 survivors. Of 3 horses fed 31 ppm (28 g/T), 1 had a transitory reduction in feed intake and the other 2 remained clinically normal. The 2 horses fed 0 ppm remained clinically normal. Monensin was also given to horses by gavage, at dosages of 1, 2, 3, 4, or 20 mg/kg of body weight. The LD50 was estimated to be between 2 and 3 mg/kg of body weight. The signs of toxicosis were partial to complete anorexia, ataxia, and intermittent profuse sweating.


Naturally occurring and experimentally induced eastern encephalomyelitis in calves. Eastern encephalomyelitis virus was isolated from the brain of 2 calves with encephalomyelitis. Using the isolant from 1 of these calves, the disease was reproduced in a clinically normal calf. Histopathologic features conformed with those described for eastern encephalomyelitis in the horse.


Coagulopathy and encephalopathy in a dog with acute hepatic necrosis. Disseminated intravascular coagulation developed secondary to hepatic necrosis in a 5-year-old Saint Bernard. Although the coagulopathy responded to treatment with heparin, the dog died from the combined effects of gastric hemorrhage and encephalopathy, both of which are complications of hepatic necrosis.


A muscle disorder of Labrador retrievers characterized by deficiency of type II muscle fibers. A muscle disorder was detected in 5 Labrador Retrievers. The age of onset was less than 6 months. The clinical signs, which were exaggerated with exercise, cold, or excitement, included abnormal head and neck posture, and a stiff, forced, hopping gait. A marked deficiency of skeletal muscle mass resulted in poor conformation. Both black and yellow Labrador Retrievers were affected, and pedigree studies indicated the disorder may have been inherited as an autosomal recessive trait. Some relief of the clinical signs was afforded by treatment with diazepam. Marked creatinuria and low serum creatine phosphokinase activity were the only consistent, abnormal laboratory findings. Histologic and cytochemical examinations of muscle biopsy specimens revealed a relative predominance of type I and a deficiency of type II skeletal muscle fibers. Electromyographic studies of the affected dogs indicated a myotonic defect.


Intravenously administered ketamine HCl and diazepam for anesthesia of raptors. A combination of ketamine HCl and diazepam given intravenously was successfully utilized to induce anesthesia for various surgical procedures in 40 raptors. A dosage of 30 to 40 mg of ketamine HCl/kg of body weight and 1.0 to 1.5 mg of diazepam/kg of body weight was satisfactory for diurnal raptors. Owls were more sensitive to the anesthetic combination, necessitating greater care in anesthetizing them. Abundant body fat was found to be an important factor in calculation of the proper dose of this combination.


Intestinal nematodes of white-tailed deer in southeastern United States. The small intestine, large intestine, and cecum of 975 white-tailed deer (Odocoileus virginianus) from 83 localities in 13 southeastern states were examined for nematodes. In order of prevalence, the following parasites were found: Capillaria bovis, Eucyathostomum webbi, Monodontus louisianensis, Nematodirus odocoilei, Oesophagostomum venulosum, Trichuris sp, Cooperia punctata, Trichostrongylus longispicularis, Strongyloides sp, Cooperia oncophora, Cooperia spatulata, Cooperia sp, and Trichostrongylus calcaratus. Over one-half of the deer examined were free of intestinal nematodes, and most infected deer harbored few parasites. Of the most prevalent species, C bovis, N odocoilei, O venulosum, and Trichuris sp were widely distributed, whereas M louisianensis and E webbi were restricted in distribution. Severe pathogenicity was associated with only heavy Strongyloides sp infections in 2 penned deer. The findings of this study suggested that white-tailed deer of the Southeast are insignificant in the epizootiology of important intestinal nematodes of domestic livestock.


Prevalence of Toxoplasma gondii antibodies in wild and domestic animals in northern California. Wild and domestic animals from 3 geographic-climatologic areas in northern California were tested for antibodies against Toxoplasma gondii. A total of 2,796 serum samples representing 37 species of wild mammals, 35 species of wild birds, and 5 species of domestic animals were tested by the indirect hemagglutination test. Of 1,174 wild mammal serums tested, 10.8% were positive, which compared with 14.7% of the 1,221 domestic mammal serums. Of 229 wild carnivores tested, 45% were seropositive, including 69% of 86 bobcats, 28% of 58 coyotes, 48% of 25 raccoons, 27% of 26 gray foxes, 22% of 32 striped skunks, a civet cat, and a mink. Serologic evidence of infection was found in 38% of 47 rural domestic cats, but none of the 7 dogs tested was seropositive. Of 160 murid rodents (rats and house mice) in rural habitats, 4% were seropositive, which compared with 2% of 399 cricetine rodents (mostly deer mice) collected from wilderness habitats. Seven percent of 56 wild Artiodactyla (deer and feral pigs) were seropositive, which compared with 15% of 1,048 domestic sheep tested. Of 401 birds tested, 3.5% had antibodies against T gondii. The highest prevalence of antibodies among birds was in crows (14%). Toxoplasma was isolated from 1 raven, by mouse inoculation. In general, the highest prevalence of seropositive carnivores, rodents, and sheep was in the coastal region below 100 ft elevation, where the weather is cool and damp for much of the year. In the central valley the highest prevalence among sheep was in areas under irrigation. The prevalence of antibodies was lowest in the mountain areas, where climatologic extremes prevail at various seasons of the year.


Mycobacterium tuberculosis infection in two East African oryxes. Tuberculous lesions were observed at necropsy of 2 East African oryxes (Oryx gazella beisa) at a municipal zoological park in Jackson, Ms. Microscopic examination revealed granulomas containing acid-fast bacilli in the lungs and liver of both animals, as well as in the uterus and mediastinal lymph nodes of 1 animal. Mycobacterium tuberculosis was isolated from the tissues of both oryxes and from fluid aspirated from the mammary gland of 1 oryx; the organism was pathogenic for guinea pigs but not for rabbits.


Pulmonary tuberculosis in a sulfur-crested cockatoo. An adult, female sulfur-crested cockatoo (Kakatoe sp) was examined because of dyspnea associated with hemoptysis. On radiographic examination there was a large cystic pulmonary mass that, on aspiration, was found to contain thick sanguineous fluid. Ziehl-Neelsen staining of aspirate smears revealed numerous acid-fast organisms. Gross necropsy and microscopic findings suggested a diagnosis of tuberculosis, with primary focus of infection in the pulmonary tissues. Bacteriologic isolation and typing confirmed a diagnosis of tuberculosis and established Mycobacterium avium as the etiologic agent.


Tuberculosis in captive exotic birds.

Tuberculosis in captive exotic birds. Avian tuberculosis was studied clinically and pathologically in 137 affected birds from the National Zoological Park during a 7-year period (1969-1975). Twelve of 22 orders exhibited were affected by the disease, and the highest annual mortality was 4% (in 1975). Antemortem diagnosis of early cases of the disease, based on tuberculin testing, and serologic, hematologic, and radiographic studies, was inconsistent and often not conclusive. Pathologically, the diseases primarily involved digestive organs and spleen. There was a spectrum of lesions consisting of nodules of large foamy histiocytes packed with acid-fast bacilli to giant cell-containing granulomas that were often caseous but not cavitated or calcified. Amyloidosis was seen in approximately 20% of the cases. Mycobacterium avium serotype 1 was isolated from 30 tuberculous birds cultured. There was no sex predilection, and most of the affected birds were adults ranging from 1 to 10 years of age.


Systemic mycoses in marine mammals.

Systemic mycoses in marine mammals. Thirty-four cases of systemic mycosis in marine mammals were represented by 9 genera of fungi. All cases were characterized by pulmonary involvement, with miliary spread to abdominal organs. Cases were geographically distributed among Hawaii, northern United States, Europe, New Zealand, and southern Asia.


Dermatophilosis among wild raccoons in New York State. Dermatophilus congolensis was established as the cause of dermatitis in 8 raccoons, as determined from examination of carcasses and untreated hides. Encrusted lesions were restricted to or more pronounced in the areas around the eyes, along the snout, and on the skin in the tarsal and carpal regions. The causative agent was demonstrated through direct examination of stained paraffin sections, isolation in pure culture, or examination of stained smears from lesions induced experimentally in rabbits with material from the affected raccoons.


Myopathy and myoglobinuria in a wild white-tailed deer. Fatal myopathy similar to "capture myopathy" described for African game was diagnosed in a wild white-tailed deer. Clinical signs included depression, inability to rise or stand, and myoglobinuria. Values for serum creatine phosphokinase, glutamic oxaloacetic transaminase, and blood urea nitrogen were high. The deer died 42 hours after capture. At necropsy the muscles of the limbs had a waxy, "cooked" appearance and the kidneys were brown. Microscopic findings included severe degeneration and fragmentation of skeletal muscle fibers, nephrosis, centrilobular hepatic necrosis, myocardial degeneration, and anoxic neuronal degeneration.


Fluorescein angiography of the normal and diseased ocular fundi of the laboratory dog. A method was determined for fluorescein angiography of the ocular fundus of the laboratory Beagle, using a portable fluorescein fundus camera. A 10% solution of sodium fluorescein was injected by a syringe pump in the right cephalic vein of each dog. Acepromazine maleate (0.5 mg/kg, IV) with ketamine hydrochloride (10 mg/kg, IM) provided chemical restraint. Of the 10-, 25-, and 50-mg/kg dosages of fluorescein, 25 mg/kg permitted satisfactory fluorescence of the choroidal and retinal vasculature and the recirculation phase. High-speed color film permitted good quality photography of the tapetal and nontapetal retinal vessels. Reduced retinal blood vessel and pigment epithelium permeability was demonstrated by fluorescein angiography in dogs with chorioretinitis associated with systemic blastomycosis, retinitis associated with distemper, serous retinal detachment, and preretinal hemorrhage.


Immune response of channel catfish under different environmental conditions. Channel catfish were maintained under conditions of low (15.4 C) and fluctuating (15.4 to 26.9 C/24 hours) temperatures, low (126 mg/L NO3-N) and high (289 mg/L NO3-N) nitrate in recirculating systems, crowding (171 g body mass/L), and fasting. They were vaccinated with formalin-killed enteric red-mouth bacterium, and antibody titers were monitored weekly for 10 weeks. Only those fish maintained in low or fluctuating temperature environments had significant (P less than 0.01) immunosuppression. The other environmental conditions studies, which are commonly encountered in intensive fish culture operations, did not compromise the humoral immune response of channel catfish.


An ultrastructural survey of cat dentinal tubules. The extent of the odontoblast process, the distribution of intratubular nerve-like structures, and tubule dimensions, have been measured in cat dentine. The odontoblast process varies in extent throughout the tooth. At most levels it is limited to inner dentine, extending further out only in the cervical region. This variation supports the view that the limited extent of the process occurs in vivo and is not an artefact of fixation. No nerve-like processes were found within dentinal tubules in this study, indicating the rarity of such structures. The tubules in the dentine of the cat are more variable in number but narrower than those of man and occupy a relatively smaller proportion of the volume of the tissue. Some factor other than tubule numbers and size must be responsible for the greater access which chemical stimuli seem to have to nerve terminals beneath cat, as opposed to human, dentine.


Compensatory muscular hypertrophy in the extensor digitorum longus muscle of the mouse. Hypertrophy of the extensor digitorum longus muscle after surgical removal of its companion tibialis anterior muscle has been studied in mice. Early, intermediate and late stages in the process of hypertrophy have been defined. The proportional increase in the man cross sectional area of fibres in a hypertrophic muscle consistently exceeded the proportional increase in the weight of the same muscle in all animals. This suggests that hypoplasis (loss of fibres) was occurring. This work was carried out with the aid of grants from the Medical Research Council and Science Research Council of Great Britain.


Musculature of facial scent glands in the muntjac. The muscles associated with the very large preorbital glands of muntjacs (Muntiacus) are described. Although the two muntjac fawns examined were only 10 days old, their muscles were proportionately larger than those in adult specimens of North American cervids. A muscle, not found in North American cervids, but well developed in muntjacs, is probably responsible for the eversion of muntjacs' preorbital glands.


The development of the dorsal funiculus in the human spinal cord. 1. The development of the dorsal funiculus of the human cord is described from the second month to the end of the fourth. 2. The fasciculus cuneatus arises as a distinct bundle of fibres near the mid-line in the ninth week. This gradually spreads laterally, and to it is joined the more medial fasciculus gracilis. 3. The original embryonic dorsal marginal layer is displaced laterally, and may possibly become the dorsolateral tract adjacent to the dorsal horn. 4. The appearance of the future 'epicritic' pathway is accompanied by a marked wave of differentiation in cervical and thoracic ganglia. 5. The implications of these findings are discussed in relation to the problems of peripheral and central factors in nervous development.


The ultrastructure of implanted trophoblast cells of the yellow agouti mouse. An electron microscope study of ultrathin sections of trophoblast cells of implanting blastocysts resulting from the crossing of heterozygous yellow agouti mice has been conducted. Several conditions for successful preparation of the blastocysts have been described. These include avoidance of the use of sucrose in the buffers, reduction of specimen washing times and exclusion of propylene oxide from the dehydration procedure. Electron micrographs of cells from a blastocyst considered, on the basis of light microscopy, to be 'abnormal' revealed a cytoplasm with many 'empty' areas and numerous 'vacuolated' mitochondria. Electron micrographs of cells from blastocysts considered to be 'normal' revealed, in one instance, a preponderance of cells with very few of these 'abnormal' features, and, in the other instance, a preponderance of cells with many abnormalities. Both 'normal' blastocysts revealed cellular features usually regarded as either primitive or pathological, namely tight junctions, vacuolated mitochondria and mitochondria with cristae oriented parallel to their long axis. It was generally concluded that the appearance of the blastocysts, in the light microscope, at 100-105 hours of development, could not be used as a valid criterion of normality or abnormality, for, although a blastocyst classified as 'abnormal' under the light microscope appeared 'abnormal' also in the electron microscope, those classified as 'normal' under the light microscope appeared either 'normal' or 'abnormal' in the electron microscope. This disturbing situation may indicate that the electron microscope is diagnosing 'abnormality' before the light microscope. The presence of immature forms of organelles (e.g., vacuolated mitochondria) indicates either a failure to mature or regression. Failure to mature could be benign or of sinister import.


An ultrastructural study of keratinized epithelia in the rat soft palate. The three kinds of keratinized epithelium evident on gross examination of the soft palate of Wistar albino rats were studied by scanning and transmission electron microscopy. Scanning indicated a central area where the epithelium showed numerous fine folds and papillae, more lateral areas which were smooth, and most lateral areas with fine folds only. At high magnifications the cell surfaces were seen to be pitted with numerous small craters. However, all three epithelia had a similar appearance under the transmission electron microscope, namely that of keratinized oral epithelia. The gross surface differences probably reflect functional modifications.


Evaluation of intrahepatic arterial branching patterns in corrosion casts. A study of the branching pattern of the hepatic artery in 85 human livers has been undertaken in corrosion casts. The various branching patterns observed are described in detail and compared with observations of other workers.


Changes in articular cartilage following intraarticular injection of tritiated glyceryl trioleate. The effects of intra-articular injections of non-radioactive and tritium-labelled glyceryl trioleate into the mandibular and knee joints of adult rabbits have been investigated using autoradiographic and histochemical techniques and electron microscopy. As observed at the fourth day after operation, fat droplets accumulate in cells of the fibrous, intermediate and cartilaginous layers of mandibular condylar, and in the superficial and upper middle (rather than the deeper) zones of femoral condylar cartilage. Autoradiography of frozen sections shows that numerous silver grains are located over these fat-laden cells following injection of trioleate which has been labelled in the fatty acid moiety of the molecule. In the knee joint the number of grains is directly related to the amount of lipid in the cell. Following injection of glyceryl-labelled trioleate no such result is obtained; it seems doubtful whether or not there is any uptake of this label. However, synovial membrane from the knee joint appears to take up both kinds of trioleate. Results of histochemical methods of NADH diaphorase, lactic dehydrogenase, acid phosphatase and beta-glucosaminidase are consistent with ultrastructural evidence of degeneration in some chondrocytes and of loss of ground substance from the matrix. A raised level of alpha-glycerophosphate dehydrogenase activity is probably associated with synthesis of endogenous glycerol for re-esterification of absorbed fatty acids, and enhanced activity of UDPglucose dehydrogenase with the chondrocytic reaction to matrix depletion. Apart from the increase in fat content, ultrastructural features in injected knee joints include flattening of cell processes against the chondrocyte surface and more abundant intracytoplasmic filaments. Injected mandibular joints show little evidence of these changes although the number of cells in the cartilage appears to be greatly reduced. No extracellular fat droplets occur in femoral cartilage, but material similar in electron density to intracellular fat is observed at the external aspect of some mandibular chondrocytes. The findings indicate that the fatty acid portion of triglyceride injected intra-articularly is taken up by the chondrocytes and retained until at least the fourth day after injection. It is suggested that prior lipolysis takes place either in the synovial cavity (or membrane) or at the chondrocyte surface, but it is uncertain how or in what form fat traverses the matrix. Lipoarthrosis appears to produce changes in the chondrocytes which are thought to be pathological; a number of cell deaths occur. The possibility that gross degeneration of the articular cartilage may ensue is subject to further investigation.


A multiple-baseline analysis of social-skills training in chronic schizophrenics. Social-skills training was applied to two male chronic schizophrenics. Component behaviors of social skill requiring modification were identified for each patient by rating videotapes of role-played interactions. Training involved instructions and feedback for one subject and instructions, feedback, and modelling for the second. Target behaviors were treated sequentially and cumulatively in a multiple-baseline format. Training was applied for both positive and negative assertion and for situation involving males and females. The results were positive for all behaviors for both patients. Follow-ups at 2, 4, 6, and 8 weeks after training indicated that most effects were maintained at near-treatment levels.


Reducing time limits: a means to increase behavior of retardates. A common assumption in special education is that temporal limits for a task should be expanded so that ample time is provided for completing the work. This study describes the opposite strategy of restricting temporal limits to augment academic performance. Three educable retarded children received token reinforcement contingent on the number of correct math problems answered during daily sessions. A reversal design was used to assess the effects of an abrupt reduction in time limits (20-5-20 min) and a graduated sequence of reductions (20-15-10-5-20 min). The graduated sequence resulted in rate increases of correct responding ranging from 125% to 266% and these gains endured when temporal limits were again expanded. In contrast, the abrupt shift produced interfering emotional behaviors and rate decreases in academic performance of 25% to 80%. The findings indicate that systematically restricting temporal limits for an academic task can further enhance the performance of slow learners already maintained by a token system.


The cellblock token economy: token reinforcement procedures in a maximum security correctional institution for adult male felons. Two experiments were conducted (1) to explore the application of token reinforcement procedures in a maximum security correctional institution for adult male felons and (2) to determine to what extent the reinforcement procedures disrupted the day-to-day lives of inmate participants. In Experiment 1, an expanded reversal design revealed that the combination of praise and token reinforcement was more effective than the combinations of praise and noncontingent token award or direct commands on four common institutional activities. The latter two combinations were not found to be any more effective than praise alone. Experiment 2, which also employed a reversal design, indicated that the high levels of performance observed during the token reinforcement phases of Experiment 1 could be attained without subjecting participants to undue hardship in the form of increased deprivation of either social intercourse or the opportunity to engage in recreational and entertainment activities. Client safeguards are discussed in detail.


Employing task arrangements and verbal contingencies to promote verbalizations between retarded children. This study investigated the effects of arranging task events for interdependence, to increase the probability of social responding. During task interdependence, the subjects, participating in dyads and a four-person group, obtained task materials (a puzzle piece) from their partner before completing their task (appropriately placing the puzzle piece). The verbal contingency required a verbal request to precede a subject's receiving a task material from his partner. The verbal contingency yoked with task interdependence made task completion contingent on the appropriate verbalization. The findings from two experiments suggested that task interdependence was sufficient to increase partner-directed verbalizations for three of the four subjects. When the verbal contingency was added, all subjects increased their requests and other verbalizations to partner. Applied to a four-person group, the verbal contingency yoked with varying levels of task interdependence correspondingly affected the pattern and level of group communications. The greater the task interdependence, i.e., the more members each subject depended on to complete his task, the more complex the social network of verbal contacts, and the higher the level of both requests and other verbalizations for the group.


The development and functional control of reading-comprehension behavior. Reading comprehension, indicated by motor behavior and multiple-choice picture selection called for in written instructions, was taught to an autistic child using verbal prompts, modelling, and physical guidance. The child was rewarded for correct behaviors to training items; nonrewarded probes were used to assess generalization. Probable maintaining events were assessed through their sequential removal in a reversal design. Results showed: (a) following acquisition, performance was maintained at a near-100% level when candy, praise, attention, and training were removed, (b) absence of other persons was correlated with a marked decrease in performance, whereas their presence was associated with performance at near 100%, and (c) performance generalized to probes and across experimenters. Rewards, which may have been reinforcing during acquisition, did not appear necessary to maintain later performance. Instead, presence of others (a setting event) was demonstrated to have control over maintained performance.


The generalization effects of parent training across stimulus settings. Five parents of nonverbal children were trained in two home settings to modify antecedents and consequences to their children's vocalizations. Generalization effects of the parent training on both the parent's and children's behaviors under different stimulus conditions were investigated using multiple-baseline designs. Increases in parent prompting and reinforcing their children's vocalizations generalized only minimally to a new setting in the home where parent training had not occurred. Child increases in vocalizations produced by the parents in the training settings did generalize to this new setting in the home. There was minimal generalization of child vocalizations to a free-play setting at school. In a formal speech session conducted by a behavior specialist at school, only one child showed definite increases in acquisition rate as a function of the parents starting to train the sound at home.


Comparison of an electronic spirometer with a pediatric Wright Peak Flow Meter and their relationship to clinical symptomatology in asthmatic children. To evaluate the correlations between the Wright Peak Flow Meter and an Electronic Spirometer with clinical symptomatology in chronic asthmatic chidlren, objective measurements of Peak Flow (PF) on both electronic (Vanguard Electric Spirometer) and mechanical (Wright Peak Flow Meter) Spirometers, Forced Vital Capacity (FVC), Forced, Expiratory Volune 1/2 second (FEV 0.5), Forced Expiratory Volume 1/2 per cent (FEV 0.5%), AND Maximal Midexpiratory Flow Rate (MMEFR) were obtained. Subjective observations of clinical wheezing were recorded immediately before each flow rate by trained nurses. PF determinations of both the mechanical and electronic spirometers were in close agreement. PF on the electronic spirometer correlated best with clinical wheezing in this study. The MMEFR calculated from the electronically produced graphys had the nex best coefficient of corelation. These were followed by the PF measured mechanically on the WPFM.


Release of enzymes by normal and wall-free cells of Chlamydomonas. The phosphatase produced by the wild-type strain of Chlamydomonas reinhardi in media deprived of inorganic phosphate are found partly inside and partly outside the cells. The same enzymes are almost completely released by a mutant strain defective in cell wall formation. It is proposed that the failure of cell wall mutants to survive in certain conditions is related to their inability to retain certain essential compounds that are normally associated to the cell wall.


Selective inactivation of nitrogenase in Azotobacter vinelandii batch cultures. When the exhaustion of sucrose or sulfate or the induction of encystment (by incubation in 0.2% beta-hydroxybutyrate) leads to termination of growth in Azotobacter vinelandii batch cultures, the nitrogenase levels in the organisms decreased rapidly, whereas glutamate synthase and glutamine synthetase levels remained unaltered. Glutamate dehydrogenase activities were low during the whole culture cycle, indicating that ammonia assimilation proceeds via glutamine. Toward depletion of sucrose or during induction of encystment, slight secretion of ammonia with subsequent reabsorption was occasionally observed, whereas massive ammonia excretion occurred when the sulfate became exhausted. The extracellular ammonia levels were paralleled by changes in the glutamine synthetase activity. The inactivation of the nitrogenase is explained as a result of rising oxygen tension, a consequence of a metabolic shift-down (reduced respiration) that occurs in organisms entering the stationary phase.


Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain. Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids.


Biosynthesis of saturated and unsaturated fatty acids by a T-strain mycoplasma (Ureaplasma). A human T mycoplasma (Ureaplasma urealyticum) incorporated radioactivity into its lipids from [1-14C]acetate in the growth medium. Methanolysis of the lipids showed the label to be confined almost entirely to the methyl esters of the fatty acids. About 80% of the label was associated with the methyl esters of the saturated fatty acids, and the rest was found in the unsaturated methyl ester fraction. Gas-liquid chromatography of the saturated methyl esters showed the label to be present in the peaks of palmitate, myristate, and stearate, whereas in the unsaturated methyl ester fraction most of the radioactivity emerged in the peak of palmitoleate. The addition of either oleic or palmitic acid to the growth medium markedly decreased the organisms' incorporation of radioactivity from acetate. It is concluded that the T mycoplasma strain is capable of de novo synthesis of both saturated and unsaturated fatty acids, in this respect differing from all of the Mycoplasma and Acholeplasma strains investigated to date.


Isolation and characterization of a glycerol auxotroph of Rhodopseudomonas capsulata: effect of lipid synthesis on the synthesis of photosynthetic pigments. A glycerol auxotroph was isolated from Rhodopseudomonas capsulata for use as a system for studying membrane synthesis and function. When the mutant was deprived of glycerol, net phospholipid synthesis ceased immediately and a small amount of free fatty acids accumulated. A turnover of lipid occurred in both deprived and supplemented cultures. Deoxyribonucleic acid and protein synthesis continued for one doubling of cell massand then slowed down in deprived cells. Net ribonucleic acid synthesis slowed down more dramatically. Oxidative phosphorylation activity of membrane preparations from aerobically and semi-anaerobically grown cells appeared unaffected by glycerol deprivation, indicating that simultaneous lipid synthesis is not a requirement for new oxidative phosphorylating activity. In the absence of net phospholipid synthesis, bacteriochlorophyll and carotenoid syntheses were reduced to 30% of the activity of supplemented cultures. Delta-Aminolevulinic acid synthase, the first enzyme on the bacteriochlorophyll pathway that is subject to regulatory control, increased in activity in deprived cultures. Lascelles and Szilagyi (1965) showed an association between phospholipid synthesis and pigment production. They found an increased lipid content associated with pigmented cells. The present results indicate that not only is there an association between lipid and pigment synthesis, but also there is actually a dependence of bacteriochlorophyll synthesis on phospholipid synthesis.


Kinetics and catalytic properties of coenzyme A transferase from Peptostreptococcus elsdenii. Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii was purified to homogeneity, and some of its physical and catalytic properties were determined. The native enzyme has a molecular weight of 181,000 and is composed of two alpha subunits (molecular weight, 65,000) and one beta subunit (molecular weight 50,000). Heat treatment of the crude cell extract to 58 degrees C causes proteolysis of the native enzyme and yields a catalytically active enzyme with an approximate molecular weight of 120,000. The native CoA transferase is specific for CoA esters of short-chain alkyl monocarboxylic acids. With acetate as CoA acceptor the enzyme is active with propionyl-, butyryl-, isobutyryl-, valeryl-, isovaleryl,- and hexanoyl-CoA but not with heptanoyl or longer-chain CoA esters. There is no activity with acetoacetyl-CoA or the CoA esters of dicarboxylic acids. Steady-state kinetics indicated that the reaction proceeds via a classical bi-, bi-ping-pong mechanism. Maximal activity is obtained with propionyl- or butyryl-CoA, and both the Vmax and Km decrease as the alkyl chain length of the CoA ester increases. All CoA esters apompetitive inhibitor although it is not active as a substrate. Evidence for an enzyme CoA intermediate was provided by demonstration of an exchange between 14C-free acids (acetate and butyrate) and their corresponding CoA esters and by isolation of a 3H-labeled CoA enzyme after incubation of the enzyme with 3H-labeled acetyl-CoA. Approximately 2 mol of CoA was bound per mol of enzyme.


Requirement of a cell division step for stalk formation in Caulobacter crescentus. Penicillin G at low concentrations blocked cell division in Caulobacter crescentus without inhibiting cell growth. The long filamentous cells formed after two to three generations under these conditions had a stalk at one pole and usually one or more flagella at the opposite pole. The failure of the filaments to form a second stalk at the flagellated pole indicates that stalk formation was dependent upon completion of a step that was also required for cell division. Two observations support this conclusion. (i) Penicillin did not stop the normal development of synchronous swarmer cells into stalked initiation and stalk elongation. (ii) When the action of penicillin was reversed by the addition of penicillinase to cultures of filaments, stalks were not formed at the nonstalked pole until after cell division had occurred; thus the normal order of development events was maintained: cell division leads to stalk formation. These results are consistent with a model in which the organization of the developmental program for stalk formation occurs before cell division as a consequence of steps that branch from the cell division pathway.


Plasmid deoxyribonucleic acid in Rhizobium trifolii. In deoxyribonucleic acid of Rhizobium trifolii centrifuged in cesium chloride-ethidium bromide equilibrium was found a sattelite peak containing covalently closed circular deoxyribonucleic acid. The plasmid had a molecular weight of about 64 x 10(6) shown by sedimentation in sucrose gradients and electron microscopy.


Occurrence of 3-O-methylmannose in the polysaccharide of Leptospira biflexa Urawa. 3-O-methylmannose was identified by gas-liquid chromatography-mass spectrometry in the acid hydrolysate of the polysaccharide of Leptospira biflexa Urawa.


Execution times of macromolecular synthetic processes involved in the induction of allophanate hydrolase at 15 degrees C. We have observed that transcription, involved in production of allophanate hydrolase, is completed 2.5 min after the addition of inducer at 15 degrees C. The rna1 gene product must be functional up unti 10 min; protein synthesis is initiated at 20 min and is terminated by 24 min. Two minutes later, active enzyme appears. The results confirm our earlier observations and eliminate any uncertainty that might have clouded identification of the time within the lag period that is occupied by ribonucleic acid synthesis.


Light-induced inhibition of sporulation in Bacillus licheniformis. Sporulation of Bacillus licheniformis is inhibited by broad-spectrum light. This phenomenon is intensity dependent and is a near-ultraviolet and blue light effect.


Temperature-sensitive mutant of Schizosaccharomyces pombe exhibiting enhanced radiation sensitivity. A conditional lethal and radiation-sensitive mutant of Schizosaccharomyces pombe is described in which both characteristics result from a single gene mutation. Confirmation of the pleiotropic nature of this mutant was obtained by tetrad analysis and by testing the radiation sensitivity of a large number of revertants that grew normally at the restrictive temperature. The colony-forming ability of the mutant after ultraviolet radiation, gamma radiation, and ethyl methane sulfonate treatment is considerably altered by the post-treatment incubation temperature, showing higher survival at 25 than at 30degreesC. The radiosensitivity of the mutant is also influenced by the stage of growth. The difference in radiation sensitivity between the wild type and mutant is greater when log-phase cultures are compared. The characteristics of this mutant suggest that it is defective in a step common to both deoxyribonucleic acid replication and repair.


Pentose metabolism in Mycobacterium smegmatis: specificity of induction of pentose isomerases. The induction of D-xylose, D-ribose, L-arabinose, and D-lyxose isomerases by various sugars was studied to determine the configuration necessary for induction. D-Xylose isomerase was only induced by D-xylose, whereas D-ribose isomerase was induced by D-ribose, L-rhamnose, and L-lyxose. L-arabinose isomerase was induced by L-arabinose, D-galactose, L-arabitol, D-fucose, and dulcitol, whereas D-lyxose isomerase was induced by D-lyxose, D-mannose, D-ribose, dulcitol, and myoinositol. Some compounds such as dulcitol, D-galactose, and D- or L-fucose which do not support growth are still able to serve as inducers for various pentose isomerases.


Arginine catabolism by Treponema denticola. Treponema denticola, an anaerobe commonly present in the human mouth, ferments various amino acids and glucose. Amino acid analyses indicated that substrate amounts of arginine were utilized by T. denticola growing in a complex, serum-containing medium. Cell suspensions metabolized L-arginine to citrulline, NH3, CO2, proline, and small amounts of ornithine. CO2, NH3, ornithine, and proline were produced from L-citrulline by cell suspensions. Determinations of radioactivity in products formed from L-[U-14C]ornithine indicated that cell suspensions converted this amino acid to proline. Furthermore, proline was excreted by cells growing in a complex, arginine-containing medium. Arginine iminohydrolase (deiminase) and ornithine carbamoyltransferase activities were detected in T. denticola cell extracts. Carbamoylphosphate dissimilation by extracts yielded adenosine triphosphate. The data indicate that T. denticola derives energy by dissimilating L-argine via the arginine iminohydrolase pathway. However, unlike some of the other bacteria that utilize this pathway, T. denticola converts to proline much of the ornithine derived from L-arginine.


Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI. The bacteriophage Pa16, isolated from soil on Streptomyces albus G, was restricted when transferred from an alternative host back to S. albus G. Extracted unmodified Pa16 deoxyribonucleic acid was cleaved at a single site by a cell-free extract of S. albus G. Fractions cleaving Pal6 deoxyribonucleic acid contained the endonuclease SalI first described by J. Arrand, P. Myers, and R. J. Roberts (unpublished data). A mutant of S. albus G was isolated which was defective in both restriction and modification of Pal6. This mutant lacked SalI activity. It is concluded that SalI is the agent of restriction of Pal6 by S. albus G.


Cytochemical evidence of extramembranous carbohydrates on Ureaplasma urealyticum (T-strain Mycoplasma). Cells of Ureaplasma urealyticum that were prepared by a ruthenium red technique demonstrated an extramembranous layer of polyanions of about 15 to 30 nm in width. Application of the concanavalin A-iron dextran strain indicated that the outer surface of this layer contained glucosyl-like residues.


Dermatan sulfate-chondroitin sulfate copolymers from ambilical cord. Isolation and characterization. Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactosaminoglycan fraction was obtained from this tissue by papain [EC 3.4.22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or D-glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgalactosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetyl-galactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in electrophoresis on cellulose acetate and gel chromatography on Sephadex G-200.


Further studies on the possible compartmentation of the precursor pool of cholesterol for the biosynthesis of cholic acid in the rat. In vivo and in vitro experiments with rats were carried out to investie precursor for the biosynthesis of cholic acid. When rats with a bile-fistula were given a mixture of [2-14C]mevalonate and [1,2-3H]cholesterol intravenously, the 14C:3H ratio in cholic acid in both whole homogenate and cytosol prepared from their lives was higher than that in free cholesterol in any subcellular fraction of the livers. When [2-14C] mevalonate was administered intravenously to bile-fistula rats, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other fraction, and the specific radioactivity of biliary cholic acid was remarkably high, exceeding that of microsomal free cholesterol. In similar experiments with [4-14C] cholesterol, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other subcellular fraction and the specific radioactivity of biliary cholic acid was lower than that of free cholesterol in any hepatic subcellular fraction. Tissue suspensions of rat livers in Krebs-Ringer bicarbonate (pH 7.4)-5.5 mM glucose were incubated with [2-14C]mevalonate in O2-CO2 (95:5, v/v) at 37 degrees. The specific radioactivity of free cholesterol in the microsomal fraction prepared from the incubated tissue exceeded the specific radioactivities of free cholesterol in the other subcellular fractions. The estimated specific radioactivity of taurocholate formed during the incubation was far higher than that of microsomal free cholesterol. These data indicate that hepatic microsomal free cholesterol which was newly synthesized in situ was preferentially incorporated into cholic acid.


Determination of the best-fit values of kinetic parameters of the Michaelis-Menten equation by the method of least squares with the Taylor expansion. The best-fit values of the Michaelis constant (Km) and the maximum velocity (V) in the Michaelis-Menten equation can be obtained by the method of least squares with the Taylor expansion for the sum of squares of the absolute residual, i.e., the difference between the observed velocity and the corresponding velocity by calculation. This method makes it possible to determine the values of Km and V not in a trial-and-error manner but in a deductive and unique manner after some iterative procedures starting from arbitrary approximate values of Km and V. These values can be said to be uniquely determined for a set of data as the finally converged values are no longer dependent upon the initial approximate values of Km and V. It is also very important to obtain initial approximate values of parameters for the application of the method described above. A simple method is proposed to estimate the approximate values of parameters involved in fractional functions. The method of rearrangement after canceling of denominator of a fractional function can be utilized to obtain approximate values, not only for cases of two unknown parameters such as the Michaelis-Menten equation, but also for cases with more than two unknowns.


Circadian rhythms of digestive enzymes in the small intestine of the rat. II. Effects of fasting and refeeding. The effects of fasting were examined on the rhythmic changes in the activities of maltase [EC 3.2.1.20] and leucine aminopeptidase [EC 3.4.11.1] in the small intestine of rats which has been kept under scheduled feeding conditions. Irrespective of whether the rats had been kept on a daytime or nighttime feeding schedule, the rhythms of maltase and leucine aminopeptidase persisted when the animals were starved. However, the amplitude of the leucine aminopeptidase rhythm began to decrease from the first day of fasting, while that of maltase did not. Conspicuous rhythms persisted for at least 2 days during fasting, but they gradually became vague and disappeared after 5 days. When rats were refed after fasting, the leucine aminopeptidase activity increased within a few hours, but the maltose activity did not. It is suggested that the rhythms of the digestive enzymes in the small intestine of rats are not a direct consequence of food intake, but are triggered off by the anticipatory mechanism which operates when rats expect to be fed. The rhythmic change of leucine aminopeptidase seemed to be intensified by food intake.


Isolation, properties, and crystallization of an iron-chlorin protein from Aspergillus niger. A green iron-chlorin protein was purified 160-fold from a lyophilized extract of Aspergillus niger by ion-exchange chromatography and gel filtration with a yield of 25%. The purified preparation appeared nearly homogeneous on sedimentation analysis and the sedimentation coefficient of the protein at infinite dilution was 13.4S. Its molecular weight was calculated to be 2.8--3.2 X 10(5) from sedimentation and gel filtration data. The ferric form of the protein had absorption maxima at 587.5 and 708 nm in the visible region and a Soret band at 404 nm. High-spin ESR signals of a rhombically distorted ferric iron-chlorin complex were observed at g = 6.5 and 5.3 together with unidentified, weaker signals at g = 4.3 and 2.0. The ferric form reacted readily with cyanide to give a complex showing absorption peaks at 422 and 632 nm and a shoulder at about 595 nm. When the protein combined with cyanide its high-spin ESR signals disappeared and low-spin signals at g = 1.88, 2.29, anous form having absorption maxima at 622 and about 410 nm. The rate of reduction by dithionite was slightly reduced by the presence of either nitrite or sulfite, and greatly accelerated by the presence of hydroxylamine. The reduced spectrum obtained in the presence of hydroxylamine had maxima at 620 and about 420 nm. The ferric cyanide complex did not show any spectral change on addition of dithionite. The green prosthetic group could be extracted with acidified acetone and the absorption maxima of the pyridine ferrihemochrome were at 413 and 599 nm. On removal of metal from the prosthetic group the characteristic spectrum of a chlorin was obtained. The absorption maxima of a solution of the chlorin in benzene were at 403, 501, 537, 576, 595, and 655 nm, the 655 nm band being strongest of those in the visible region. No significant amount of flavin was detected in the purified preparation. The iron-chlorin protein catalyzed methyl viologen-linked reduction of hydroxylamine and also that of nitrite at a slower rate under the same conditions, but not evidence that it reduced sulfite was obtained in the present study. The purified preparation also had high catalase [ec 1.11.1.6] activity. Crystalline material was obtained by gradual concentration of the purified preparation at about pH 6.


New types of gangliosides in starfish with sialic acid residues in the inner part of their carbohydrate chains. Three gangliosides were isolated from the starfish by Folch's partition, dialysis, silicic acid column chromatography, Iatrobead column chromatography and preparative thin-layer chromatography. Partial acid hydrolysis of the three lipids in 0.1 N HCl at 80 degrees for 90 min liberated all the lactosyl ceramide and water-soluble degradation products, but no sialic acid. Gas-liquid chromatography of the trimethylsilylated methyl glycosides of the water-soluble products demonstrated the presence of arabinose in addition to the usual sugar units of gangliosides.


Quantitative analysis of the action of Taka-amylase A on maltotriose. The action of Taka-amylase A from Asp. oryzae was studied quantitatively by the product analysis method using unlabeled maltotriose and maltotriose labeled at the reducing end as substrates. It was found that the ratio of the unlabeled products, maltose (G2) and glucose (G1) exceeded unity, and that the ratio of the labeled products, G2/G1 was strongly dependent on the initial substrate concentrations. The results can only be explained by a transglycosylation or condensation mechanism or both. Analysis of maltose labeled at the nonreducing or reducing end reveal that the ratio of the transglycosylation to the condensation mechanism was about 20:1 with about 80 mM maltotriose. A computer simulation was made on a reaction scheme involving the termolecular-shifted complex, transglycosylation and condensation besides hydrolysis, by using reported subsite affinities as modified by the authors. The simulation reproduced the experimental results satisfactorily.


A clottable protein (coagulogen) of horseshoe crab hemocytes. Structural change of its polypeptide chain during gel formation. A clottable protein (coagulogen) isolated from a hemocyte lysate of the Japanese horseshoe crab (Tachypleus tridentatus) was incubated with an endotoxin-activated clotting enzyme(s) partially purified from the same lysate, and its structural change during gel formation was examined. The results indicated that the enzymatic formation of gel involved limited proteolysis of the Arg-Gly and Arg-Thr linkages located in the N-terminal portion of the coagulogen, liberating peptide C.


Photoelectrochemical effects in the electrolyte-pigment-metal system. I. Metal-free phthalocyanine film description of short-circuit photocurrents for thin films of pigment. Using a semitransparent metallic electrode covered with a thin film of metal-free phthalocyanine, photoelectrochemical effects have been studied in the electrolyte-pigment-metal system. Measurement of the short-circuit photocurrent, produced by continuous illumination, allows description of a photodependent kinetic phase corresponding to the establishment of a stationary state in the charge separation region of the system after the creation of the current carriers.


Photoelectrochemical effects in the electrolyte-pigment-metal system. II. Metal-free phthalocyanine film action spectra of short-circuit photocurrents with increase of the film thickness. The electrolyte-pigment-metal system can be described as analogous to a photosensitive junction region. When the thickness of the pigment film is increased, the action spectra of the maximum short-circuit photo-current under continuous illumination differ from the absorption spectra both in direct illumination (pigment-electrolyte) and in back illumination (metal-pigment). One is led to believe that there exist two photoactive regions in the system for the production of the short-circuit photocurrent; these two active regions are associated respectively with each interface. When the metallic semitransparent electrode is made of aluminum the two interfaces have opposite sign contributions to the photocurrent; this allows the determination of conditions in which one can observe specifically the contribution of the pigment-electrolyte interface, that is the interactions between excited pigment molecules and the redox system in the electrolyte.


Photoelectrochemical effects in the electrolyte-pigment-metal system. III. Chlorophyll films short-circuit photocurrent transients light energy conversion efficiency. Short-circuit photocurrents produced by a semitransparent metallic electrode covered with multilayers of chlorophyll and immersed in an electrolyte have been studied. The action spectrum of maximum photocurrent is identical to the absorption spectrum of the film of pigment. The kinetics of rise of the photocurrent are photoindependent for thin multilayers but dependent on thickness. Comparison of efficiency of light energy conversion on the basis of short-circuit photocurrent seems to show that the stack of Chl a monolayers is the more efficient chlorophyll solid system. Introduction for the same number of Chl a molecules of vitamin K1 and Phytol shows that vitamin K1 acts probably like an excitation quencher as in monolayers at the water-air interface.


Chemical models of oxidative phosphorylation. Chemical models for coupling oxidation to phosphorylation are summarized and examined both from the standpoint of organic reaction mechanisms and with respect to their relevance to mitochondria and chloroplasts. In order to accelerate the progress of our research in bioenergetics, it is suggested to focus at least as much attention on structural biochemistry as on phenomenological observations of energy-transducing membranes.


Ecto-enzymes of mammary gland and its tumors. Lectin inhibition of 5'-nucleotidase of the 13762 rat mammary ascites carcinoma. Lectin inactivation of the cell surface enzyme 5'-nucleotidase has been studied in intact 13762 mammary ascites cells, cell membrane envelopes, membrane vesicles, and solubilized membranes as a means of understanding the effects of lectins on biochemical processes and the behavior of the enzyme in the membranes. The properties of the enzyme are essentially the same in the intact cells and in membrane envelopes prepared after zinc treatment of the cells. The enzyme has a Km of 25 muM and is inhibited by concanavalin A (Con A) by an apparent noncompetitive process, which is reversed by alpha-methylmannoside. Half-maximal inhibition requires about 50 mug of Con A per mg of membrane protein. The inhibition for the intact cells and membrane envelopes does not exhibit significant cooperativity. Membrane vesicles, prepared by EDTA extraction, and solubilized membranes show differences in the behavior of the nucleotidase toward Con A even though the Km of the enzyme (25 muM) and inactivation (noncompetitive) are essentially unchanged. The Hill coefficient for the inactivation process in solubilized membranes and membrane vesicles is shifted to near 2, indicating significant cooperativity in the Con A-enzyme interaction. It is suggested that this change in cooperativity may reflect a different mode of interaction with the membrane and increased enzyme mobility, although an alteration in enzyme subunit interactions cannot be ruled out at present.


Mechanism for the several activities of the glutathione S-transferases. The catalyzed reactions of GSH with organic nitrate and thiocyanate esters and with a series of chloronitrobenzene substrates have been investigated and the results used to formulate a mechanism for glutathione S-transferase catalysis. All the homogeneous preparations of the glutathione transferases that have been tested catalyze the reaction of GSH with organic nitrates and thiocyanates. The nature of the reaction with nitrate esters, resulting in the formation of GSSG rather than a thioether, has been investigated further. The presence of an additional nonsubstrate thiol decreased the formation of GSSG to an extent that cannot be explained by disulfide interchange. These results are interpreted to reflect the enzymatic formation of an unstable glutathione sulfenyl nitrite that undergoes subsequent non-enzymatic decomposition. Hammett plots of the catalytic constants of rat liver transferases B and C obtained with a series of 4-substituted 1-chloro-2-nitrobenzene substrates demonstrate a linear relationship with sigma- substituent constants, reflecting the nucleophilic nature of the enzymatic reactions and their strong dependence on the electrophilicity of the nonthiol substrate. These data suggest that the many diverse reactions catalyzed by the glutathione transferases may be formulated as a nucleophilic attack of enzyme-bound GSH on the electrophilic center of the second substrate. The final products observed reflect this primary event and the existence of subsequent nonenzymatic reactions.


Purification of a glycoprotein from mouse ascites fluid by immunoaffinity chromatography which is related to the major glycoprotein of murine leukemia viruses. Immunologic and structural comparison with purified viral glycoproteins. Proteins immunologically related to the major glycoprotein from murine leukemia viruses, gp70, are expressed in the tissue and body fluids of unmanipulated mice in the absence of virus particle production. Cross-reactive gp70 was isolated from ascites fluid of NZB and (NZB X NZW)F1 mice utilizing immunoaffinity chromatography. An enriched antibody preparation bound to Sepharose was used as an immunosorbent to purify gp70 over 600-fold in one step with a 30% recovery. The gp70s from ascites fluid were compared with gp70s purified from laboratory virus strains Friend, Rauscher, Moloney, and Scripps leukemia viruses. Direct antibody binding studies showed that the viral gp70s were more closely related to each other than to the gp70s from ascites fluid. Furthermore, the gp70s from ascites fluid were more reactive with antibody to xenotropic virus than were the gp70s from the laboratory viruses tested. Tryptic peptide map analysis of radioiodinated proteins showed that gp70 from Scripps virus is closely related to gp70 from Moloney virus in primary structure and less closely related structurally to gp70 from Rauscher virus and gp70 from NZB ascites fluid.


Methylisocyanate as an antisickling agent and its reaction with hemoglobin S. Reviewing the reaction of potassium cyanate, an antisickling agent, with alpha-amino groups of hemoglobin, it was found that the reaction was a slow process and requires a large excess of the reagent. The reason for the slow reaction rate of carbamylation of hemoglobin by cyanate is that cyanate itself does not react with hemoglobin. It is rather isocyanic acid, the reactive species, that reacts with hemoglobin. Since the pK of isocyanic acid is 3.8, only one out of 4,000 cyanate ions is present as isocyanic acid at physiological pH. Therefore, it appears that a large excess of cyanate ions is required to achieve the carbamylation of hemoglobin S, both in vivo and in vitro. Furthermore, the pH optimum for carbamylation of carboxyhemoglobin and deoxyhemoglobin is 5.5 and, at pH 7.4, the reaction velocity drops to one-third for carboxyhemoglobin and one-half for deoxyhemoglobin. To seek an approach to reduce the dosage of cyanate and to increase the reaction velocity, an isocyanate derivative, methylisocyanate which is already in the reactive form, was tested for its antisickling activity and its reaction with hemoglobin S. It was found that methylisocyanate had antisickling activity and that only a stoichiometric amount to 2-fold excess of the reagent over hemoglobin S alpha-amino groups was required to prevent the sickling of erythrocytes. Methylisocyanate-treated sickle erythrocytes showed an increased oxygen affinity compared to untreated methylisocyanate reacted with alpha-amino groups of hemoglobin S and the reaction was complete in less than 1 min. Methylcarbamylated hemoglobin S had a higher minimum gelling concentration than the untreated hemoglobin S. There was no detectable reaction of free sulfhydryl and epsilon-amino groups of hemoglobin S with methylisocyanate. These results indicate that methylisocyanate, and probably other isocyanate derivatives, possesses powerful antisickling activity.


Isolation and characterization of rat olfactory marker protein. The olfactory marker protein was isolated and characterized from rat olfactory bulbs. Its properties and those of the olfactory marker protein isolated from the mouse are described. The rat protein was less acidic (pI = 5.0) than the mouse protein (pI = 4.7). However, the amino acid compositions were very similar: in both proteins arginine plus lysine accounted for 13 mol% and glutamate plus aspartate for 30 mol% of the total residues. Molecular weights of both proteins estimated by sodium dodecyl sulfate gel electrophoresis were indistinguishable and estimated to be 16,500. The molecular weight of the native rat olfactory marker protein estimated by gel filtration techniques was 30,000, which is identical to the molecular weight of the native mouse and garfish olfactory marker proteins. This suggested a dimeric structure. The purified rat and mouse proteins behaved like species of 35,000 molecular weight on gel filtration.


Vitamin K-dependent carboxylase. Solubilization and properties. Vitamin K is required for an enzymatic carboxylation of glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid. The enzyme system (carboxylase) which catalyzes this reaction has now been solubilized by extraction of the microsomes with Triton X-100 and has been shown to fix H14CO3- as gamma-carboxyglutamic acid residues in biologically active prothrombin. Enzyme activity requires O2 and vitamin K hydroquinone or vitamin K + NADH. Unlike the microsomal-bound carboxylase, soluble carboxylase activity is independent of either ATP or Mg2+ addition and is unaffected by either the ATP analog, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P, or EDTA. These observations suggest that the energy required to drive the carboxylation reaction is derived from the oxidation of the reduced form of vitamin K. Although the membrane-bound carboxylase is inhibited by Warfarin, this anticoagulant is ineffective as an inhibitor of the soluble enzyme. A second anticoagulant, 2-chloro-3-phytyl-1,4-natpthoquinone (chloro-K), differs from Warfarin in that it effectively inhibits both the membrane-bound and soluble carboxylases.


Interaction of manganese with bovine prothrombin and its thrombin-mediated cleavage products. The binding of the paramagnetic metal, Mn(II), to bovine prothrombin and the thrombin-mediated cleavage products of prothrombin, i.e. fragment 1 and the prethrombin 1 has been investigated. Analysis of the Scatchard plots of the binding data reveals that prothrombin has two high affinity Mn(II) binding sites with a Kd of 1.2 +/- 1.0 X 10(-5) M and approximately two to three lower affinity Mn(II) sites with a Kd of 1.3 +/- 1.0 X 10(-4) M. Positive cooperativity in Mn(II) binding to prothrombin was observed for the strong sites. Fragment 1, the phospholipid-binding region of prothrombin, possesses two high affinity Mn(II) sites with a Kd of 2.2 +/- 1.0 X 10(-5) M and at least two lower affinity sites with a Kd of approximately 2.5 +/- 1.0 X 10(-4) M. Positive cooperativity was not observed for the binding of Mn(II) to fragment 1. Prethrombin 1 binds one Mn(II) with a Kd of 3.2 +/- 1.0 X 10(-4) M. Using the values of free Mn(II) concentration, as determined by EPR measurements and the observed enhancements of the water proton relaxation rates at various concentrations of Mn(II) and protein, the binary enhancement values (epsilon b) of the metal-protein complexes were obtained. The extrapolated values are 11 +/- 0.4 for the initial prothrombin-binding sites, and 10 +/- 0.3 for the tight binding sites of fragment 1. The unique epsilon b value obtained for prethrombin 1 was 5.3 +/- 0.7. When Mn(II) was used in a Factor Xa-metal ion-phospholipid system for activation of prothrombin, the rate of generation of thrombin was less than or equal to 5% of that obtained when Ca(II) was employed in this activation system. Addition of Mn(II) to the same activation system containing Ca(II) resulted in a marked decrease in the rate of thrombin generation, suggesting that Mn(II) probably competes for the same sites on prothrombin as Ca(II). In agreement with this is the observation that the Mn(II) sites on prothrombin could be displaced by Ca(II) at high concentrations of Ca(II).


Crystallization of proteins from polyethylene glycol. Twenty-two proteins available to this laboratory were examined for their ability to crystallize from polyethylene glycol (PEG). Of the 22, 8 had been crystallized previously by conventional means and the remainder had yielded no crystals by other methods. Using a single broad screen with four different concentrations of five different molecular weight sizes of polyethylene glycol, 13 of these proteins were crystallized. Of the 13, 6 proteins were crystallized for the first time, one yielded crystals readily where only infrequent and irreproducible results were obtained before, and one other produced excellent quality crystals where only very disordered or twinned crystals were previously grown. In only one case did polyethylene glycol fail to yield crystals where other agents had succeeded. It is concluded that polyethylene glycol may be the best initial trial reagent for crystallization of proteins for x-ray diffraction analyses.


Bovine adrenal medulla troponin-C. Demonstration of a calcium-dependent conformational change. A high affinity calcium-binding protein has recently been purified from the adrenal medulla (Kuo, I.C.Y., and Coffee, C.J. (1976) J. Biol. Chem. 251, 1603-1609). This protein is closely related in its chemical and physical properties to troponin-C (TN-C) of muscle tissue. Further examination of the adrenal medulla protein indicates that the removal of calcium is accompanied by a marked change in the conformation. This change in structure is similar, if not identical, to the calcium-dependent conformational change which has been described for skeletal muscle TN-C (Murray, A.C., and Kay, C.M. (1972) Biochemistry 11, 2622). The far ultraviolet circular dichroism spectrum of native adrenal medulla calcium-binding protein (AM-CBP) shows characteristic helical ellipticity bands at 222 and 207 nm. The helical content, as estimated from these data, is between 40 and 45%. Removal of calcium is accompanied by a change in ellipticity which corresponds to a decrease from 40 to 20% in the helical content. The near-ultraviolet circular dichroism spectrum shows negative dichroic bands at 262 and 268 nm which are characteristic of phenylalanine. These bands are relatively insensitive to changes in the calcium ion concentration. Sedimentation velocity studies likewise are indicative of a calcium-dependent structural alteration. The sedimentation coefficient of the native protein was observed to be 1.89 S. Similar measurements performed in the presence of 3 mM ethylene glycol bis(beta-aminoethyl ether) N,N, N', N'-tetraacetic acid (EGTA) gave a sedimentation coefficient of 1.50 S. The molecular weight, as determined by sedimentation equilibrium studies, was 16,000 regardless of whether the measurements were made in the presence of CaCl2 or EGTA. From the elution properties of AM-CBP on Sephadex G-100, the Stokes radius was observed to be 19.8 A in the presence of calcium and 21.9 A in the presence of EGTA. All of these changes which were induced by the addition of EGTA were completely reversible by the readdition of excess CaCl2. These data suggest that the removal of calcium from AM-CBP is accompanied by a pronounced conformational change which occurs without a molecular weight change. The decreased sedimentation coefficient, the increased Stokes radius, and the reduced helical content, which are observed for the apoprotein, indicate that removal of calcium results in a transformation from a compact symmetrical structure to one that is less ordered and more asymmetrical.


Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization. Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.


Studies on nicotinic acetylcholine receptors in mammalian brain. Preliminary characterization of membrane-bound alpha-bungarotoxin receptors in rat cerebral cortex. Binding of alpha-bungarotoxin, labeled with 125I, has been studied in crude membrane preparations of rat cerebral cortex. Membrane-bound receptors appear to consist of at least two saturable subsets: one accounts for the binding of about 50 fmol of toxin/mg of protein, the other binds 120 fmol/mg. For the high affinity sites the dissociation constant for interaction of toxin and receptor extrapolated to zero protein concentration is 9.2 X 10(-10) M. Competition with a variety of agonists and antagonists indicates that toxin binding exhibits the pattern expected for nicotinic acetylcholine receptors. Toxin binding is inhibited by low concentrations of Ca2+ ions (50% inhibition at 3 mM) while higher concentrations are required for Mg2+, Na+, K+, and Li+ (50% inhibition at 0.5 M).


Binding of chick skin collagen alpha 1 chain by isolated membranes from human platelets. Binding of a denaturated polypeptide chain derived from chick skin collagen, the alpha 1(I) chain, by isolated membranes of human platelets has been demonstrated. The process is reversible, and time- and protein concentration-dependent. The binding is specific, with an association constant of 1.88 X 10(-6) M. Prior treatment of the isolated membranes with trypsin, chymotrypsin, and pronase, resulted in significant inhibition of the 14C-labeled alpha 1 chain binding, but neuraminidase or collagenase treatment had no effect. Dissociation of the bound radioactivity and subsequent chromatographic analyses on carboxymethylcellulose and agarose A-1.5m revealed that the alpha 1 chain was unaltered. Scatchard plot analysis suggested that there are approximately 20,000 binding sites per platelet. The binding of the alpha 1 chain was inhibited by a glycopeptide derived from alpha 1, alpha 1-CB5 and by purified glucosylgalactosyl hydroxylysine, but was not affected by other cyanogen bromide peptides of alpha 1, namely alpha 1-CB3, -CB4, -CB7, and -CB8. Kinetic studies demonstrated that inhibition by the hydroxylysine glycoside is competitive. Dose-response curves of platelet aggregation induced by alpha 1 and the binding of alpha 1 by platelet membranes correlate closely. These results indicate that there are specific binding sites for collagen alpha 1 chain on platelet membranes, and that the carbohydrate moiety of the alpha 1 chain plays a role in the binding. The findings also support the hypothesis that the chick skin alpha 1 chain mediates platelet aggregation and the release reaction by acting on platelet membranes.


Nucleotide sequence of 5 S ribosomal ribonucleic acid of Iguana iguana. Cell cultures of the reptile Iguana iguana have been labeled with 32PO4(3-) and the resulting radioactive 5 S rRNA isolated. Enzymatic digests of the radioactive RNA were fractionated by standard procedures, and the products of the digestions sequenced. From the sequences of the oligonucleotides produced by enzymatic digestion of the Iguana 5 S rRNA it is possible to propose a primary sequence for this RNA which differs in only two positions (2 and 25) from the sequence known for mammalian 5 S rRNAs. This sequence is closer to that of mammals than is that of any other nonmammalian species yet examined.


Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver. Perfused rat hearts catalyze the hydrolysis of AMP added to the perfusion fluid at a rate of 35 mumol/g dry weight/min. The activity is specific for 5'-nucleoside monophosphates, little activity being observed with 2' and 3'-AMP. The enzyme exhibits Michaelis-Menten kinetics in situ and is inhibited competitively by adenosine-5'-alpha, beta-methylene diphosphonate (Ki = 13 muM). This, as well as the nucleotide specificity, confirms that the hydrolysis is catalyzed by 5'-nucleotidase. The maximum activity of 5'-nucleotidase in perfused hearts is equal to or greater than that found in heart homogenates; thus, all of the enzyme is accessible to AMP added externally. Hydrolysis of endogenous AMP was studied in the perfused heart. Under aerobic conditions hearts contain very low amounts of purine nucleosides, and little or no nucleoside is found in the effluent perfusate. Under anaerobic conditions hearts accumulate adenosine, inosine, and hypoxanthine and release all three substances into the perfusate. Hydrolysis of externally added AMP was also observed in perfused skeletal muscle and liver, at rates of 10 and 17 mumol/g dry weight/min, respectively.


Protein synthesis in rabbit reticulocytes. Characteristics of mRNA (AUG codon)-dependent binding of Met-tRNAfMet to 40 S and 80 S ribosomes. The characteristics of Met-tRNAfMet binding to ribosomes (40 S and 80 S) were studied using a two-stage assay method (Gupta, N.K., Chatterjee, B. and Majumdar, A. (1975) Biochem. Biophys. Res. Commun. 65, 797) and the complexes formed were analyzed either by Millipore filtration or by sucrose density gradient centrifugation. The results are summarized as follows: (a) with both assay methods, Met-tRNAfMet binding to 40 S ribosomes was entirely dependent upon addition of a partially purified mixture of initiation factors and AUG codon; (b) this binding occurred over a wide Mg2+ concentration range; significant binding was observed even at 20 mM Mg2+; (c) upon addition of 60 S ribosomes, a significant part of Met-tRNAfMet bound to 40 S ribosomes was transferred to 80 S complex. This transfer reaction had a sharp Mg2+ optimum around 2 mM. Met-tRNAfMet-80 S-AUG complex thus formed was active in Met-puromycin synthesis; (d) Met-tRNAfMet deacylase present in crude 0.5 M KCl ribosomal wash is a potent inhibitor of the binding reaction as it deacylates Met-tRNAfMet in the Met-tRNAfMet-40 S-AUG complex; (e) glutaraldehyde (0.5%) degrades Met-tRNAfMet-40 S-AUG complex but increases the background binding of Met-tRNAfMet to 40 S ribosomes in the absence of AUG codon; (f) polynucleotides containing uracil and adenosine are strong inhibitors of Met-tRNAfMet binding to 40 S ribosomes. The order of inhibitory activities of the polynucleotides tested was as follows: poly(rU)-poly(rA) (2:1) greater than poly(rU)-poly(rA) (1:1) greater than poly(rU) greater than poly(rA). Other RNAs tested such as poly(rC), poly(rI)-poly(rC) and phi6 bacteriophage RNA (double-stranded) were without significant effects on the Met-tRNAfMet-binding reaction.


Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation. Thyroid slices were found to incorporate radioactivity from 14C-labeled sugars into the carbohydrate moiety of a polar lipid soluble in chloroform/methanol/water, 10/10/3. This radiolabeled glycolipid was purified by chromatography on DEAE-cellulose and was shown to have as its monosaccharide constituents mannose, glucose, and glucosamine. This compound cloud also be labeled by incubation of thyroid slices with [3H]mevalonic acid or [32P]phosphate as demonstrated by the coincidence of elution profiles upon DEAE-cellulose chromatography. This suggested that the lipid portion of the molecule is a polyprenol derivative and the lipid-saccharide linkage region involves a phosphate bridge. Mild acid hydrolysis of the glycolipid labeled with 14C in its carbohydrate released a neutral oligosaccharide which on the basis of Bio-Gel filtration studies was shown to have a molecular weight of approximately 2,400. This oligosaccharide contained [14C]mannose and [14C]glucose in about the same ratio as that occurring in the intact glycolipid. The oligosaccharide-lipid appeared to be distributed rather widely in thyroid particulate fractions obtained after differential and density gradient centrifugation. Its highest specific activity occurred in fractions rich in endoplasmic reticulum. By means of pulse-chase experiments in slices a relationship was demonstrated between the disappearance of radioactivity from the lipid-bound oligosaccharide and its appearance in protein-bound form. When protein synthesis was inhibited by the addition of puromycin during the chase period of the experiment transfer of oligosaccharide from the lipid to protein appeared to be blocked and the level of radiolabeled oligosaccharide-lipid increased. The observation that mannose and glucose were similarly affected during the pulse-chase studies suggests that transfer of the intact oligosaccharide unit was involved in the addition of carbohydrate to protein.


Lipid-saccharide intermediates in glycoprotein biosynthesis. II. Studies on the structure of an oligosaccharide-lipid from thyroid. Structural studies have been performed on an oligosaccharide-lipid from thyroid believed to be an intermediate in glycoprotein synthesis. For these investigations the compound was isolated from the gland in unlabeled form as well as differentially radiolabeled in its saccharide, lipid, and phosphate portions by incubation of slices with [14C]- or [3H]glucose, [3H]mevalonic acid and [32P]phosphate, respectively. The unlabeled oligosaccharide-lipid was obtained in a chloroform/methanol/water (10/10/3) extract in a yield of about 1 nmol/g of thyroid and was purified therefrom by DEAE-cellulose chromatography. The saccharide moiety released from the glycolipid by mild acid hydrolysis was isolated by gel filtration and contained 11 mannose, 1 to 2 glucose, and 2 N-acetylglucosamine residues. The reducing terminal position of the oligosaccharide was occupied by 1 of the glucosamine residues and from these analyses a molecular weight of 2,415 was calculated. That glucose is an integral part of the molecule was further demonstrated by the finding that during Dowex 50 chromatography it remained as a constituent of the positively charged oligosaccharide produced by deacetylation with alkaline borohydride at 80 degrees. The phosphorus content of the purified unlabeled oligosaccharide-lipid was determined to be 2 residues per molecule, suggesting the presence of a pyrophosphate bridge between its carbohydrate and lipid portions. Further evidence for such a linkage region was provided by characterization of the products from mild acid and alkaline hydrolysis of the differentially radiolabeled glycolipid. These included dolichyl mono- and pyrophosphate, oligosaccharide phosphate, and free oligosaccharide. Digestion with alpha-mannosidase of the radiolabeled glycolipid led to the release of 39% of its mannose while from the free oligosaccharide 53% of this sugar was removed. Acetolysis of the [14C]oligosaccharide yielded a mannobiose and mannotriose as well as larger fragments consisting of mannose, glucose, and glucosamine. Smith periodate degradation gave rise to a small core segment (6 glycose residues) made up only of mannose and glucosamine from which half of the mannose residues could be released by alpha-mannosidase digestion. From these studies a tentative structure for the carbohydrate moiety of the oligosaccharide-lipid has been proposed. In this formulation an inner core (periodate-resistant) made up of 4 mannose and 2 N-acetylglucosamine residues is attached to the pyrophosphate group by the most internal glucosamine. This core, as well as an additional mannose and 1 to 2 glucose residues, constitutes the alpha-mannosidase-resistant fragment. More peripherally are found other mannose residues, all in alpha-linkage. In this structural scheme the glucose is located so as to prevent the enzymatic release of more internally situated alpha-linked mannose residues.


Lipid-saccharide intermediates in glycoprotein biosynthesis. III. Comparison of oligosaccharide-lipids formed by slices from several tissues. The synthesis of oligosaccharide-lipids thought to play a role in the attachment of carbohydrate to protein has been studied in incubations of slices from calf kidney, pancreas, thymus, and liver, as well as from hen oviduct. These compounds were characterized after radiolabeling of their saccharide moiety by incubation with [14C]glucose or [14C]mannose and a comparison was made with the oligosaccharide-lipid produced by thyroid slices. Furthermore, the unlabeled glycolipid was prepared from hen oviduct for the purpose of quantitating its sugar constituents. Purification of the oligosaccharide-lipids extracted with chloroform/methanol/water (10/10/3) was achieved by DEAE-cellulose chromatography and their carbohydrate moieties were released by mild acid hydrolysis. On the basis of gel filtration it was determined that the lipid-bound oligosaccharides formed by oviduct, thymus, kidney, and liver had molecular weights comparable to that from thyroid (about 2400). The saccharide moiety of the glycolipid from pancreas was however distinctly smaller in size with a molecular weight of approximately 1800. Analyses of the radiolabeled oligosaccharide-lipids from oviduct, kidney, and thymus indicated that they, like the compound from thyroid slices, but unlike those believed to be formed by cell-free systems from various tissues, contained glucose in addition to mannose and N-acetylglucosamine as their monosaccharide constituents. This compositional data was supported by the finding that the unlabeled oligosaccharide from oviduct consists of 10 mannose, 1 glucose, and 2 N-acetylglucosamine residues. Sodium borohydride reduction of this oviduct saccharide moiety indicated that 1 of the 2 glucosamines was situated in a reducing terminal position. The radiolabeled oligosaccharide from the glycolipid produced by pancreas differed from the others analyzed in that it contained only trace amounts of glucose. Upon treatment with alpha-mannosidase this glucose-deficient pancreatic oligosaccharide was extensively digested (85% of the mannose released). In contrast, the carbohydrate moieties of oviduct, kidney, and thymus, like that of thyroid, underwent a more limited digestion with the alpha-mannosidase (55% or less of the mannose released) suggesting that the presence of glucose may serve to block a more complete degradation of these oligosaccharides by this enzyme.


The NH2-terminal region of the beta chain of sickle hemoglobin. I. Synthesis and purification of oligopeptides. Five peptides from the NH2-terminal region of the beta chain of hemoglobin S, betaS (1-13), betaS (3-13), betaS (1-8), betaS (4-10), And betaS (4-8), have been synthesized by a rapid solid phase method based on the Merrifield procedure. In addition, one peptide, betaS (3-13), has also been synthesized by the original Merrifield method. We have shown that the products of the two methods are comparable, that gel filtration is a useful method for removing truncated fragments of the desired oligopeptide, and that measurement of the efficiency of coupling at each step is an important adjunct to amino acid analysis in determining purity. Peptides of the purity achieved by these methods may be used to fractionate antibodies to the native hemoglobin S, in the characterization of antigen-binding properties of specific antibodies, and in other studies of peptide-protein interactions.


The NH2-terminal region of the sickle hemoglobin beta chain. II. Characterization of monospecific antibodies. We have previously shown that antibodies specific for hemoglobin S could be fractionated by absorption of an antiserum to hemoglobin S to Sepharose containing a synthetic oligopeptide. betaS (1-13), corresponding to the first 13 amino acid residues of the beta chain of hemoglobin S. We report here that this antibody population, anti-betaS (1-13), shows considerable restriction of heterogeneity in isoelectric focusing studies and monospecificity on velocity ultracentrifugation in the presence of hemoglobin S. The binding of various hemoglobin species to anti-betaS (1-13) was studied using a double antibody radioimmunoassay with [14C]carbamoylated hemoglobin S. Carbonmonoxy-, oxy-, met-, and cyanmethemoglobin S reacted equally with the antibody, but deoxyhemoglobin (with or without organic phosphates) reacted differently. Hemoglobin A and several hemoglobin mutants with alterations in the NH2-terminal region of the beta chain did not displace labeled hemoglobin S from anti-betaS (1-13). BETAS chains reacted with the antibody, but less well than hemoglobin S, while betaA and alpha chains, and globins did not react with the antibody. The synthetic peptide, betaS (1-13), used for fractionation, reacted with the antibody about 300-fold less efficiently than hemoglobin S. BetaS (3-13) was even less reactive, while smaller peptides which included the valine residue at position 6 displaced little of the tracer [14C]carbamoylated hemoglobin S at concentrations as high as 10(-2) M. We interpret these results to indicate that this method of immunoabsorption has produced a monospecific subfraction of antibodies which is specific for the NH2-terminal region of the beta chain of hemoglobin S in its native conformation.


Proteoglycans from bovine proximal humeral articular cartilage. Structural basis for the polydispersity of proteoglycan subunit. Polydisperse proteoglycan subunit from bovine proximal humeral articular cartilage has been separated into a series of relatively monodisperse fractions which have been chemically and physically characterized. The proteoglycan subunit species of the lowest molecular weight contains the least chondroitin sulfate and had an amino acid composition relatively low in serine and glycine and relatively high in cysteine, methionine, and aspartic acid, almost identical to that of the hyaluronic acid-binding region of proteoglycan subunit isolated by Heinegard and Hascall (Heinegard, D., and Hascall, V.C. (1974) J. Biol. Chem. 249, 4250-4256). The molecular weight of proteoglycan subunit increases in proportion to its chondroitin sulfate content. As the molecular weight and chondroitin sulfate content of proteoglycan subunit increase, there is a parallel increase in the serine and glycine contents, and a decrease in the cysteine, methionine, and aspartic acid contents of proteoglycan subunit core protein. The pattern of polydispersity observed strongly supports the concept that proteoglycan subunit core protein contains a hyaluronic acid-binding region of constant size and composition and a polysaccharide attachment region of variable length and composition, composed of repeating peptide sequences containing serine and glycine in equimolar amounts.


The anomeric configuration of the D-mannosyl retinyl phosphate formed in rat liver microsomes. Alkaline hydrolysis at 65 degrees converted D-mannosyl retinyl phosphate primarily into D-mannose 2-phosphate which was identified by its behavior on paper chromatography in two solvent systems, as well as by its resistance to acid hydrolysis under conditions that converted authentic alpha-D-mannosyl phosphate to mannose. When the D-mannosyl retinyl phosphate was hydrolyzed in alkali at 100 degrees, the main product was beta-D-mannosyl phosphate and not the alpha anomer (as shown by chromatography with authentic samples on thin layers of silica gel). No evidence for enzymatic hydrolysis of D-mannosyl retinyl phosphate by alpha-mannosidase was obtainable. It is concluded that the compound under investigation is beta-D-mannosyl retinyl phosphate.


Effects of estrogen on gene expression in the chick oviduct. Control of ovalbumin gene expression by non-histone proteins. Chromatin was isolated from the oviducts of chicks which had either received diethylstilbestrol stimulation for 14 days (stimulated chromatin) or been treated and then withdrawn from hormone administration (withdrawn chromatin). These chromatin preparations were dissociated and then reconstituted in the presence of various non-histone protein fractions. cDNAov was used as a probe for measuring the level of mRNAov sequences synthesized in vitro from various reconstituted chromatins. Our results indicate that extractable non-histone proteins from stimulated chromatin are capable of activating the in vitro transcription of the ovalbumin gene when included in the reconstitution of withdrawn chromatin. On the other hand, addition of extractable non-histone proteins from withdrawn chromatin to stimulated chromatin does not affect the synthesis of mRNAov sequences. Therefore, the extractable non-histone proteins from stimulated chromatin contain a positive regulator(s) which controls the in vitro expression of the ovalbumin gene.


Purification and properties of an endo-beta-N-acetylglucosaminidase from hen oviduct. An endo-beta-N-acetylglucosaminidase has been purified extensively from hen oviduct extracts and found to have a somewhat broader substrate specificity than the previously reported endoglycosidases from Dipolcoccus pneumoniae and Streptomyces plicatus (Arakawa, M., and Muramatsu, T. (1974) J. Biochem. (Tokyo) 76, 307-317; Tarentino, A. L., and Maley, F. (1975) Biochem. Biophys. Res. Commun. 67, 455-462. The enzyme was shown to hydrolyze and di-N-acetylchitobiosyl bond in such compounds as Asn(GlcNAc)2(Man)3, Asn(GlcNAc)2(Man)5, Asn(GlcNAc)2(Man)6 and a glycopeptide from immunoglobulin M, (aa)x-Asn(GlcNAc)2(Man)3(Fuc)1. The capacity to hydrolyze the latter compound is a characteristic of the endoglycosidase from D. pneumoniae, but not that from S. plicatus. Competitive inhibition studies indicate that a single enzyme in the hen oviduct endoglycosidase preparation is probably responsible for hydrolyzing both fucose-containing the fucose-depleted substrates. The existence of an endo-beta-N-acetylglucosaminidase in animal tissue may explain why oligosaccharides with N-acetylglucosamine on the reducing terminus accumulate in certain lysosomal storage diseases.


Formation of glutathione conjugates of prostaglandin A1 in human red blood cells. When prostaglandin A1 was incubated with a Tris/saline suspension of washed human red blood cells, a substantial amount was converted to polar metabolites. These were purified by solvent extraction, XAD-2 column, and cellulose thin layer chromatography and characterized by chromatography, amino acid analysis, and mass spectrometry. The polar metabolites were a mixture of two glutathione conjugates of prostaglandin A1. The first (approximately 40%) was identical with the product of the nonenzymic reaction of glutathione with prostaglandin A1. The second (approximately 60%) was formed from the first by reduction of the 9-keto group of the prostaglandin moiety. The latter compound was also prepared synthetically by treating the glutathione conjugate of prostaglandin A1 with sodium borohydride.


Purification and properties of filamin, and actin binding protein from chicken gizzard. Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.