Regrowth of atrophied skeletal muscle in adult rats after ending immobilization. The recovery time course of muscle atrophied by immobilization was followed after removal of hindlimb casts from adult female rats. Increases of only 9% in body weight, 4% in gastrocnemius weight, and 10% in soleus weight occurred in controls during the 78-day duration of the experiment. There were no increases in the amounts of total protein or of citrate synthase activities in gastrocnemius or soleus during the first 3 days after removal of hindlimb casts; thereafter, there were increases in these paramters. Citrate synthase activities per mg of gastrocnemius protein were significantly higher at the 16th and 50th day of recovery. No significant differences for citrate synthase activity per mg of soleus occurred during recovery. Until the 50th day of recovery, no significant differences for total protein in soleus and for total protein and wet weight of gastrocnemius were observed between control and recovery values. However, the wet weight of the soleus returned rapidly during recovery and was not significantly different from control during recovery.


Exercise ventilatory mechanics at increased ambient pressure. Transpulmonary pressure, lung volume, and flow rate were recorded in two healthy subjects performing graded exercise between 1 and 10 ATA. At simulated depths greater than 4 ATA, exercise was terminated by severe choking dyspnea at levels of work, oxygen consumption, heart rate, and ventilation significantly lower than during maximum exercise at 1 ATA. Comparison of exercise ventilatory mechanics with corresponding maximum expiratory flow-volume and expiratory isovolume pressure-flow (IVPV) curves demonstrated that the reduced aerobic capacity was associated with expiratory flow limitation. We conclude that dynamic airways compression limited aerobic capacity at these depths by causing a persistent cough making it seem difficult to continue exercise. Analysis of the IVPV curves suggested that maximum expiratory flow was reduced at depth below the rate allowing adequate exercise ventilation because increased gas density raised the resistance downstream from equal pressure points. At each level of sumbaximal exercise, end-expiratory position and alveolar CO2 tension increased with ambient pressure due primarily to the density dependence of airways resistance. In these respects, healthy subjects breathing dense gas resemble patients with obstructive lung disease.


Closing capacity in awake and anesthetized-paralyzed man. Functional residual capacity (FRC), closing capacity (CC), and (FRC--CC) were determined in 61 supine patients using the 133Xe bolus test. In 28 of the 61 patients measurements were made both while the patients were awake and during anesthesia-paralysis. Both FRC and CC decreased significantly after induction of anesthesia-paralysis. The magnitude of the reduction in CC, but not of FRC, was dependent on the relationship between FRC and CC in the awake state. Patients whose FRC was larger than their CC while awake (group I) showed less decrease in CC than FRC, i.e., (FRC--CC) decreased. By contrast, those patients whose CC was larger than their FRC while awake (group II) showed a greater decrease in CC than in FRC, i.e., (FRC--CC) became less negative. The reduction in CC after induction of anesthesia-paralysis may result from an increased elastic recoil of the lung. The larger reduction in CC in group II patients may have been due to a larger increase in elastic recoil, possibly due to the development of atelactasis.


Colloid osmotic pressure changes of dog's blood exposed to different mixtures of CO2 and air. Hansen's membrane manometer method for measuring plasma colloid osmotic pressure was used to obtain the osmolality changes of dogs breathing different levels of CO2. Osmotic pressure was converted to osmolality by calibration of the manometer with saline and plasma, using freezing point depression osmometry. The addition of 10 vol% of CO2 to tonometered blood caused about a 2.0 mosmol/kg H2O increase of osmolality, or 1.2% increase of red blood cell volume. The swelling of the red blood cells was probably due to osmosis caused by Cl- exchanged for the HCO3- which was produced rapidly by carbonic anhydrase present in the red blood cells. The change in colloid osmotic pressure accompanying a change in co2 tension was measured on blood obtained from dogs breathing different CO2 mixtures. It was approximately 0.14 mosmol/kg H2O per Torr Pco2. The corresponding change in red cell volume could not be calculated from this because water can exchange between the plasma and tissues.


Sweating response in man during transient rises of air temperature. Nude men were exposed to neutral environments (Ta = 28 degrees C, Pw = 20 mbar) changing to warm environments (Ta = 50 degrees C, Pw = 20 mbar). The transient period from neutral to warm environment lasted 4 min (dTA/DT = 5.50 degrees C/min) or 20 min (DTa/dt = 1.10 degrees C/min) or 40 min (dTa/dt = 0.55 degrees C/min) or 60 min (dTa/dt = 0.37 degrees C/min). Continuous measurements were made of rectal and mean skin temperatures and of body weigth loss. Sweating started before appreciable variation in rectal temperature. Onset of sweating could be explained by a peripheral proportional and rate control. Unsteady-state sweating can be predicted by summated stimulation of skin and rectal temperatures. This stimulation could be increased for some subjects by a multiplicative effect due to differences in local skin temperatures. This multiplicative effect occurred during the first transient period.


Functional residual capacity and body position in the dog. Resting lung volumes in the supine position (FRCs) were determined by N2 washout method in 67 dogs under pentobarbital anesthesia and computed in ml/kg body weight (BW). In 21 other dogs, FRCs and the change in FRC from the supine to upright positions (deltaFRC) were determined; these lung volumes were expressed in ml/kg BW and in percentage of TLC40 (lung volume at 40 cmH2O positive-pressure inflation). It was found that a) FRCs averaged 38.6 plus or minus 8.2 and 42 plus or minus 5.9 ml/kg BW in the two groups of dogs; b) deltaFRC averaged 23 plus or minus 4 ml/kg BW resulted in large data dispersion, a large coefficient of variation (CV) and a poor correlation (r) of lung volume to BW; D) on the contrary, marked uniformity of FRCs and FRCu (upright FRC) was obtained by expressing the resting lung volumes in %TLC40, allowing an accurate prediction of FRC from the inspiratory capacity (IC). Relationship of FRCu to TLC was comparable to human data reported in the literature. FRCs (%TLC40) was smaller than values previously reported for awake human subjects, probably due to the FRCs reduction in our dogs by anesthesia.


Maturation of respiratory control in unanesthetized newborn rabbits. The maturation of control of breathing was studied in unanesthetized rabbit pups breathing room air in a body plethysmograph during the first 8 postnatal days. Measurements include pulmonary ventilation (VE), tidal volume (VT), inspiratory (TI) and total breathing cycle (Ttot) durations, TI/Ttot, mean inspiratory flow (VT/TI), and tracheal pressure developed by the inspiratory muscles 0.1 and 0.2 s after the onset of inspirations with airways occluded at functional residual capacity (P0.1 and P0.2). All of the above variables increased progressively from the 1st to the 8th day, except for P0.1, P0.2, and TI/Ttot which remained constant. The constancy of TI/Ttot implies that the increase in VE with age was due entirely to increased VT/TI. The constancy of P0.1 and P0.2 implies that the increase in VT/TI with age was due to decreased "effective" impedance of the respiratory system. The latter probably mainly reflects increased compliance and decreased flow resistance with growth. The results also show that during the first 8 days of life there is a progressive shift to the right in the VT vs. TI relationship.


Control of ventilation during intravenous CO2 loading in the awake dog. The ventilatory response to venous CO2 loading and its effect on arterial CO2 tension was determined in five awake dogs. Blood, 200-500 ml/min, was diverted from a catheter in the right common carotid artery through a membrane gas exchanger and returned to the right jugular vein. CO2 loading was accomplished by changing the gas ventilating the gas exchanger from a mixture of 5% CO2 in air to 100% CO2. The ventilatory responses to this procedure were compared with those resulting from increased inspired CO2 concentrations (during which ventilation of the gas exchanger with the air and 5% CO2 mixture continued). The ventilatory response to each form of CO2 loading was computed as deltaVE/deltaPaco9. The mean ventilatory response to airway CO2 loading was 1.61 1/min per Torr PaCO2. The mean response for the venous CO2 loading was significantly higher and not significantly different from "infinite" CO2 sensitivity (i.e., isocapnic response). The results provide further evidence for a CO2-linked hyperpnea, not mediated by significant changes in mean arterial PCO2.


Determining P50 in the presence of carboxyhemoglobin. A method is proposed, using mathematical solutions to the Roughton-Darling analysis, that theoretically can correct the measurements used for P50 determinations, whenever HbCO is present in blood, either initially or after equilibration. The resulting P50 is calculated in a "CO-free" state, independent of the actual presence of HbCO. This can be converted to a term designated T50, which indicates the in vivo shift caused by the concentration of HbCO measured. The concept was tested by comparing the P50 measurements in subjects with normal hemoglobins who differed by smoking habits (smokers vs. nonsmokers). The results showed similar mean values for this definition of P50, despite an average 10-fold difference in initial concentrations of HbCO. The method is applicable both to the single point principle, wherein P50 is estimated directly from venous blood measurements, and to standard tonometry-mixing techniques, as long as instrumentation includes the measurement of CO saturation.


Selected brain amino acids and ammonium during chronic hypercapnia in conscious rats. Glutamic, aspartic, and gamma-aminobutyric acid (GABA), glutamine, and ammonium were measured in the brains of unanesthetized normocapnic and hypercapnic (10% CO2; 5 min to 3 wk) rats. Hypercapnia increased glutamine and GABA and decreased glutamic and aspartic acids. Changes occurred within 1 h and were maintained during the observation period of 3 wk. On return to normocapnia amino acid concentrations were almost normal after 1 h. Based on the time course it is concluded that intracerebral hypercapnia is more likely the stimulus for change than acidosis. Ammonium content was unchanged for at least 1 h after the onset of hypercapnia but increased thereafter. Experiments in which glutamine synthesis by brain was impeded by inhibiting the enzyme glutamine synthetase favor the hypothesis that the rise of ammonium content in hypercapnia is initially not seen because of increased glutamine synthesis. The changes observed may have a role in metabolic pH homeostasis of brain tissue and may also be relevant to the modified brain excitability in hypercapnia.


Adrenergic influences on histamine-mediated bronchoconstriction in the guinea pig. Intravenous infusion of histamine results in a fall in dynamic pulmonary compliance (Cdyn) in the unanesthetized guinea pig. During a prolonged infusion of histamine (30-200 s) a minimum value of Cdyn is reached and sustained after 30 s. Propranolol (10 mg/kg) enhanced this steady-state response in both normal and adrenally ablated guinea pigs to the same degree. After the infusion was terminated, normal guinea pigs recovered briskly, whereas guinea pigs whose adrenals had been ablated recovered slowly. Propranolol prolonged the recovery from the histamine-mediated fall in Cdyn in normal guinea pigs, but had no significant effect on the pattern of recovery in adrenally ablated animals. These experiments suggest that during a histamine infusion nonadrenal adrenergic factors modify the histamine response, but the brisk recovery from histamine is under the influence of adrenal factors.


Influence of limb length on a stepping exercise. The effect of limb length on responses to the step-test exercise was studied in 10 subjects of various heights; oxygen consumption values served as an indicator. Mean oxygen consumption was found to be significantly related to limb length in the step-test exercise. This was demonstrated when subjects with different limb lengths were tested as a standard work load (i.e., 10 m/min x body wt) but at seven different bench heights and correspondingly adjusted stepping rates. The minimum values for oxygen consumption were obtained when the bench height was near 50% (45-55%) of subject's limb length.


Lactate release in relation to tissue lactate in human skeletal muscle during exercise. In four healthy volunteers, muscle lactate concentration and the release of lactate from the leg were determined at rest and at 4 and 12 min of sitting bicycle exercise at four intensities (30, 50, 70, and 90% of maximal oxygen uptake). The muscle biopsies were obtained by needle biopsy technique from m. vastus lateralis. The rate of lactate release was calculated from the femoral venous-arterial differences of lactate and the leg blood flow was determined by constant rate dye infusion. Both leg blood flow and leg oxygen consumption increased linearly with work intensity. The release of lactate rose approximately linearly with the muscle lactate concentration up to about 4-5 mmol/min but then the relationship revealed a clear leveling off. These results indicate a maximal level for the lactate release from the exercising muscles with a translocation hindrance for lactate within the muscles.


Solute permeability of the alveolar epithelium in alloxan edema in dogs. The permeability of the alveolar epithelium following alloxan challenge was studied  in dogs by determining transfer of radiolabeled solutes between alveolus and blood. Two days after injection of 131-Ialbumin into the blood, anesthetized dogs had the air space of part of one lung isolated by a balloon catheter lodged in a bronchus. We infused the atelectatis-isolated area with normal saline containing trace amounts of Blue Dextran, 125Ialbumin, and 57Co-cyanocobalamin; challenged six animals with intravenous alloxan, and six animals with alloxan added to the alveolar saline. During the pulmonary edema, 57Co-cyanocabalamin and 125I-albumin appeared in the blood and 131I-albumin entered the alveolar saline. The animals challenged by alveolar instillation showed a greater permeability change (P less than 0.05). The bidirectional transfer of macromolecules indicates that alloxan produces a change in the permeability of the alveolar epithelium, allowing diffusional exchange of macromolecules. Since alveolar flooding in hemodynamic edema does not show a similar change in the permeability of the epithelial lining, alveolar flooding in alloxan edema is not due solely to an effect on the endothelial membrane, but also to a direct effect on the epithelial membrane.


2-Deoxy-D-glucose uptake by lung slices from fed and fasted rats. We studied the uptake and phosphorylation of 2-deoxy-D-[1-14C]glucose (2-[14C]DG) by lung slices from fed and fasted rats to obtain information on the effect of starvation on surgar transport by the lung. We found that 2-[14C]DG is taken up and phosphorylated by the lung, but that, as in other tissues it is not metabolized beyond the phosphorylation step. The accumulation of 2-[14C]DG as free 2-DG does not require energy, fails to show saturation in the range studied (5-100 mM), and is not inhibited by exogenous glucose. The phosphorylation of 2-DG by the lung is energy dependent, saturable, and competitively inhibited by exogenous glucose. Fasting does not interfere with the intracellular accumulation of unphosphorylated 2-DG but causes about a 40% decrease in the accumulation of phosphorylated 2-DG. We conclude that membrane transport does not limit uptake of 2-DG; fasting decreases the phosphorylation of 2-DG.


Effects of arachidonic acid and prostaglandins on lung function in the intact dog. The effects of the prostaglandin (PG) precursor, arachidonic acid (AA), and the primary PG's, PGF2alpha, and PGD2, on lung function were compared in 39 intact-chest, paralyzed, artificially ventilated dogs. Intravenous AA decreased dynamic compliance (Cdyn) and functional residual capacity and increased airway resistance (Rl) and transpulmonary pressure at end-passive expiration. The decrease in Cdyn correlated closely with a rise in pulmonary arterial pressure (Ppa). Indomethacin abolished airway and vascular responses to AA, but did not attenuate responses to the PG's. The effects of AA, PGD2, and PGF2alpha on lung function and Ppa were similar, whereas PGE2 had little effect. Vagotomy attentuated the RL increase in response to AA, PGE2alpha, and PGD2 and the Cdyn decrease in response to the PG's. The effects of the PG's on compliance were greater than those produced by mechanically increasing pulmonary venous pressure. The present studies suggest that the PG precursor is rapidly converted to agents that have marked effect on both pulmonary vessels and airways, particularly peripheral airways, in the dog.


Continuous distributions of specific ventilation recovered from inert gas washout. We describe a new technique for recovering continuous distributions of ventilation (V) as a function of tidal ventilation/volume ratio (V/V0) from the nitrogen washout. The analysis yields a continuous distribution of V as a function of V/V0 represented as fractional ventilations of 50 compartments plus dead space. The procedure was verified by recovering known distributions from data to which noise had been added. Using an apparatus to control the subject's tidal volume and FRC, mixed expired N2 data gave the following results: a) the distributions of young, normal subjects were narrow and unimodal with a mean ln standard deviation of 0.56 plus or minus 0.13; b) those of subjects over age 40 were broader (ln SD 0.86 plus or minus 0.19) with more poorly ventilated units; c) patients with pulmonary disease of all descriptions showed enlarged dead space; d) patients with cystic fibrosis showed multimodal distributions with the bulk of the ventilation going to overventilated units; and e) patients with obstructive lung diseases fell into several classes, three of which are illustrated. These results suggest that our approach is well suited for clinical investigation.


Emptying patterns of the lung studied by multiple-breath N2 washout. Changes in the nitrogen concentration seen during the single-breath nitrogen washout reflect changes in relative flow (V) from units with differing ventilation/volume ratios (V/V0). The multiple-breath washout provides sufficient data for the V for units with varying V/V0 to be plotted as a function of the volume expired. Flow from the dead space may also be determined. In young normals the emptying patterns are narrow and unimodal throughout the alveolar plateau with little or no flow from the dead space at the end of the breath. Older normals show more flow from the dead space, particularly toward the end of the breath, and some show a high V/V0 mode early in the breath. Patients with obstructive lung disease have a high flow from the dead space which is present throughout the breath. A well ventilated mode at the end of the breath is seen in some obstructed subjects. Patients with cystic fibrosis showed a poorly ventilated mode appearing at the end of the breath as well as a very high dead space.


Temperature and corticosterone relationships in microwave-exposed rats. Plasma corticosterone and colonic temperature were measured in unanesthetized male rats exposed to 2,450-MHz continuous wave (cw) radiation to characterize the response of the pituitary-adrenal axis to microwave exposure. The rats were exposed in the far field of a horn antenna for 30 or 60 min at power densities of 0, 13, 20, 30, 40, 50, or 60 mW/cm2, or for 120 min at 0, 13, 20, 30, or 40 mW/cm2. The average energy absorption rate of the rats was 0.16 W/kg absorbed per mW/cm2 incident. Plasma from individual rats decapitated immediately after exposure was collected for analysis. Colonic temperature was significantly elevataed after exposures to power densities of 13 mW/cm2 or greater, with progressively larger increases after high intensity exposures. Plasma corticosterone was significantly elevated above control levels only after exposures at 50 or 60 mW/cm2 for 30- or 60-min exposures, and at 20, 30, and 40 mW/cm2 for 120-min exposures. The relationship between the increased levels of circulating corticosterone and colonic temperature suggested that the increases in corticosterone levels may reflect a level of physiological response to the body temperature elevations caused by microwave exposure.


Quantitative lung morphology in Japanese waltzing mice. We have reinvestigated the question of whether exercise stimulates lung growth by determining body weight (BW), lung volume (LV), alveolar surface area (SA), and alveolar number (N) in Japanese waltzing mice, in their phenotypically normal littermates, and in normal albino mice. BW, LV, SA, and N were all less in waltzing mice than in their littermates; LV/BW, SA/BW, and N/BW were indistinguishable in the two groups. Age-matched albino mice had larger BW and smaller N/BW than the waltzers or their littermates. The results indicate that both waltzing mice and their nonwaltzing littermates have more highly compartmentalized lungs than albino mice of the same age. However, the data provide no support for the hypothesis that sustained exercise enhances lung growth.


Pulse rate-rectal temperature relationships during prolonged work. Forty-six industrial workers performed a total of 653 1-h work bouts over a wide range of work rates )142-273 W/m2) and environmental temperatures (11-35 degrees C corrected effective temperature) in the summer and winter seasons. Simultaneous measurements of heart rates and rectal temperatures were made at the end of each work period, and the relationship between heart rate and rectal temperature was computed with standard multiple linear regression techniques. The overall regression equation is: heart rate (beats/min) = 29.3 (beats/min. degrees C) x rectal temperature (degrees C) - 988.6 (beats/min). The relationship is significantly altered by work rate and job- and season-acquired acclimatization of the subjects. These data suggest that the average heart rates of industrial workers during prolonged work can be predicted from rectal temperatures with a reasonable degree of confidence if the degree of acclimatization and work rates are known. For a rectal temperature of 38.0 degrees C the expected heart rate during prolonged work ranges from 109 beats/min in acclimatized men working at low rates to 143 for unacclimatized men working at heavy rates.


Familial aspects of decreased hypoxic drive in endurance athletes. One difference between endurance athletes and nonathletes is decreased ventilatory responsiveness to hypoxia and hypercapnia. It has never been clear whether these decreased responses are a consequence of conditioning or precede participation in endurance athletics. Recent studies demonstrating clusters of decreased ventilatory responses to hypoxia in families of patients with unexplained respiratory failure suggest that decreased responses in endurance athletes might be familial. To investigate this possibility, ventilatory response to hypoxia and hypercapnia were measured in 16 nonathletic, healthy parents and siblings of five successful long-distance runners. Response were compared to 34 nonathletic controls. As measured by the shape parameter A, hypoxic response was decreased to a similar extent in runners 74 +/- 6.4 (mean +/- SE) (P less than 0.05) and their relatives 69 +/- 15.2 (P less than 0.01) compared to control 128 +/- 11.3. Hypercapnic responses were slightly, but not significantly, decreased in runners and their families. We conclude familial influences made a major contribution to the decreased hypoxic ventilatory response seen in long-distance runners.


Influence of abdominal gas on the Boyle's law determination of thoracic gas volume. In a body plethysmograph we have demonstrated differences in total lung capacity (TLC) derived from panting maneuvers performed at different levels in the vital capacity. In almost all cases, the discrepancies were due to the magnitude of the abdominal gas volume (AGV) and the relative magnitude of abdominal and thoracic pressure swings during the panting mandeuver. When panting was performed at functional residual capacity (FRC), the effect of AGV compression on the determination of thoracid gas volume (TGV) was small. Of 11 individuals studied 2 were known to have mild asthma. Compression and decompression of AGV appeared to be an insufficient explanation for discrepancies in derived TLC's in these two, suggesting that other as yet unidentified factors may influence the plethysmographic determination of TGV.


Respiratory mechanics in conscious sheep: response to methacholine. Most currently used animal models of allergic airway diseases differ from human asthma in that induced bronchospasm in the former is not accompanied by pulmonary hyperinflation. In the present investigation, we chose unsedated, restrained sheep to determine the effect of cholinergic bronchial provocation on respiratory mechanics, functional residual capacity (FRC), and arterial blood gases. Seven animals had been actively sensitized by intramuscular injections of Ascaris suum extract, and four untreated animals served as controls. After inhalation of nebulized 1% methacholine solution, mean pulmonary resistance increased significantly in the sensitized sheep from a base line of 2.4 +/- 0.7 (SD) cmH2O/(l/s) to a peak value after 5 min of 7.9 +/- 4.0 cmH2O/(l/s). This was accompanied by a significant increase of mean FRC from 0.99 +/- 0.14 liters to 1.31 +/- 0.24 liters. The observed changes were transient, and after 60 min, pulmonary resistance and FRC had returned to base-line values. No significant changes occurred in static lung compliance, PaO2, PaCO2, and pH. In the control animals, methacholine provocation did not produce changes in pulmonary function. These results indicate that, in sensitized conscious sheep, induced bronchospasm is associated with pulmonary hyperinflation.


Calibration of a new ear oximeter in humans during exposure to centrifugation. An optoelectronic ear oximeter (Hewlett-Packard, model 47201A) was evaluated as a noninvasive method for determining arterial oxygen saturation (SaO2) in human subjects during exposure to various levels of accelerative forces. This physiological calibration involved exposing five subjects, while breathing air and wearing the ear oximeter for 60 s to each of three levels of accelerative forces (3, 5, and 7 G); arterial blood samples were withdrawn concurrently. SaO2 was calculated indirectly from the oxygen tensions (PaO2) measured from the sampled arterial blood with a blood gas analyzer and corrected for pH and base excess. These data were compared, as were similar data taken from the same subjects breathing three different hypoxic gas mixtures while resitng at earth's gravity (1 G). Regression analyses of these data for both experimental groups (a, G exposure, or b, hypoxic exposure), comparing the ear-oximeter SaO2 with the calculated SaO2, showed the ear oximeter to be accurate with correlation coefficients of 0.95 and 0.98, respectively.


Epidemiologic findings on canine and feline intestinal nematode infections from records of the Iowa state University Veterinary Clinic. A wealth of data was available on intestinal nematode infections of dogs and cats in the Ames, Iowa, practice area served by the Iowa State University Veterinary Clinic. From July 1966 through June 1974, 8.5% of 33,594 dogs and 4.5% of 11,995 cats hospitalized for 1 or more days were recorded as infected with at least 1 type of intestinal nematode. For both dogs and cats, infection rates were generally higher in males than in females and in those that were sexually intact, compared with those that were neutered. The highest prevalence of hookworm infections in both dogs and cats was recorded in June and July, and the highest prevalence of ascarid infections in cats, but not in dogs, was in December. Seasonal patterns were not demonstrated for other nematode infections. Ascarid infections in dogs and cats and hookworms infections in dogs were most prevalent in 2-week-old to 2-month-old animals. Whipworm infections in dogs and hookworm infections in cats generally appeared at greater ages, reaching their highest prevalence in hosts 1 to 2 years old.


Heritability of multifocal retinal dysplasia in American Cocker Spaniels. Multifocal retinal dysplasia was detected in 96 American Cocker Spaniels. Affected dogs were part of approximately 500 American Cocker Spaniels examined from 1972 to 1976 in statewide survey clinics for inherited cataracts and progressive retinal atrophy. The dysplastic retina was observable ophthalmoscopically in dogs as young as 3 to 4 weeks and as old as 7 years. Ophthalmoscopic features varied, depending on whether the dysplastic foci were in the tapetal or non-tapetal portion of the fundus. Over the tapetum, dysplastic foci appeared as multiple small irregularities of diminished and altered tapetal reflectivity. In the non-tapetal fundus, dysplasia appeared as areas of decreased pigmentation. Blindness or other apparent visual deficits were not observed in affected dogs. Histologic examination of affected eyes revealed focal dysplasia limited to the retina and optic nerve. Abnormalities included localized areas of retinal folding, rosettes, and retinal pigment epithelium hypertrophy and hyperplasia. Pedigree analysis demonstrated that 71 of the 96 affected dogs were related and could be identified in an extended pedigree. A recessive mode of inheritance was suggested, because in 5 litters (born to 10 affected parents), all 23 progeny that lived were affected. A dominant or polygenic mode of inheritance could not be ruled out.


Hair chewing associated with suspected hyperadrenocorticism in a dog. Hair chewing in a 14-year-old female Toy Poodle was associated with signs of hyperadrenocorticism. Complete remission of both the psychosis and signs of hyperadrenocorticism followed mitotane therapy.


Scrotal and testical changes in canine brucellosis: a case report. An Irish Setter with scrotal enlargement had an agglutination titer to Brucella canis of greater than or equal to 1:200, but B canis was not cultured from the blood. Unusual findings included draining ulcers in the scrotum from which B canis was cultured, a necrotic testicle, and inflammation of the entire scotum. Other clinical and postmortem findings were as previously described in other cases of naturally occurring and experimentally induced canine brucellosis.


Bilateral torsion of uterine horns in a nongravid bitch. Hematometra was diagnosed in a 13-year-old, nulliparous Toy Poodle by fine needle aspiration and radiographic studies, including pneumoperitoneography. Attempted uterine evacuation with simultaneous use of diethylstilbestrol and ergonovine was unsuccessful. Exploratory celiotomy revealed 1,080-degree torsion of the right uterine horn around the left horn, which was itself twisted 270 degrees. Ovariohysterectomy was performed without complications.


Brain macrophages in rats following intravenous labelling of mononuclear leucocytes with colloidal carbon. Intravenous injections of colloidal carbon were used to label circulating mononuclear leucocytes. In the neonatal rats (3-5 days old), either 24 or 48 hours later, carbon-labelled macrophages were seen in the brain tissue. In the areas examined, notably the corpus callosum and the cerebral cortex, labelled macrophages were distributed randomly. They were either perivascular, perineuronal or lay between the nerve fibres. The labelled cells were mostly spindle-shaped with an eccentric nucleus and the cytoplasm at one pole of the cell was engorged with dark carbon particles. Abundant labelled cells were also seen over the brain surface in the layers of meninges. There was no evidence of leucocytic infiltration into the brain tissue of mature animals. It is concluded from the present work that a proportion (but not all) of the macrophages in the neonatal rat brain are derived from the blood stream.


Ultrastructure of fibrils in developing human and guinea-pig tympanic membrane. The development of connective tissue of the tympanic membrane has been examined in guinea-pig embryos and fetuses from 22 days of gestation (10 mm CR length) to birth (at about 67 days, 100 mm), in neonatal animals for the first 3 weeks of life, and in human embryos and fetuses aged 6, 11, 15 and 22 weeks. In the guinea-pig up to 34 days of gestation there was little evidence of fibril formation, but from 37 days onwards fibrils were an obvious feature of the tympanic membrane area. When first observed they resembled small collagen fibrils but there was no cross-banding, and within a week to 10 days they showed the foursubunit configuration typical of adult tympanic membrane fibrils in this species. There was no increase in fibril diameter and elastic fibres were not seen. In the human fetuses small groups of collagen fibrils (already of larger diameter than guinea-pig fibrils) were apparent at 11 weeks, together with small groups of elastic microfibrils. Later specimens showed increasing numbers of cross-banded collagen fibrils and the development of the amorphous elastin component in association with the microfibrils.


Electron microscopic and cytochemical observations on the membrane systems of the chicken thrombocyte. A combined electron microscopic and cytochemical study of the thrombocytes of the mature chicken has demonstrated the existence of two membrane systems, the surface connected system (SCS) and the dense tubular system (DTS). The SCS consists of light tubules and vacuoles which are in continuity with the plasmalemma. A ruthenium red-positive reaction product was observed on the inner surface of this membrane system. Ferritin particles were present in the tubules and the vacuoles of the SCS after the thrombocytes had been incubated in vitro with ferritin. The DTS consisted of the nuclear envelope, the rough-surfaced endoplasmic reticulum, dense tubules and concentric double membrane structures, all of which give a peroxidase-positive reaction. Although actual continuity between the DTS and the SCS of thrombocytes was not observed in this study, close approximation between the two systems was observed at many points. These results are discussed in relation to those from comparable studies of human platelets.


Succinic dehydrogenase activity during palate formation in the Mongolian gerbil. The palatal shelf epithelium of the Mongolian gerbil was examined for succinic dehydrogenase activity prior to, during and the after palatal fusion (days 18-20 post coitus). Enzyme activity was present during all stages examined, and was noted even in the epithelial pearls of fused palates. The presence of SDH activity in these epithelial pearls lends support to the theory 'epithelial stretching', and questions the theory of 'programmed cell death' in relation to the loss of the epithelium along the plane of fusion.


Cell specialization in the small intestinal epithelium of adult Xenopus laevis: structural aspects. The structure, cellular composition and organization of the small intestinal epithelium of adult Xenopus laevis are described. The mucosa is thrown into a system of longitudinal folds and is composed mainly of columnar cells and goblet cells. Leucocytes comprise about 28% of the mucosal cells. The degree of structural specialization of columnar cells varies according to their position in the folds. Cells at the crest of the folds display the greastest degree of specialization, evidenced by the degree of development of their inter- and intracellular membrane systems. Cells in the trough regions between the folds are much less specialized. These cell types are structurally analogous to mammalian villous and crypt cells respectively, although they are not segregated to the same degree.


The development of the epithelium and its innervation in the avian extra-pulmonary respiratory tract. The development of the epithelium and its innervation was investigated with the electron microscope in the larynx and trachea of embryo domestic fowls between the 10th and 21st days of incubation. From days 10 to 16 the airways were lined by undifferentiated cells which on day 15 or 16 became arranged in two distinct layers. Differentiation of the epithelial cells commenced on day 17, so that at hatching on day 21 fully differentiated ciliated cells, goblet cells, non-ciliated columnar cells, granular cells and basal cells were arranged into a simple or pseudostratified columnar epithelium. Comparisons are made with the differentiation of the respiratory epithelium in other vertebrates. Intra-epithelial axons were first seen on day 15 and increased rapidly in number. The varicose axon profiles contained both granular and agranular vesicles. The axons lay close to most types of epithelial cell, and some approached near to the surface of the epithelium. A synaptic complex, possibly efferent, involving a granular epithelial cell is described.


A fine structural study of coronal and root dentinogensis in the mouse: observations on the so-called 'von Korff fibres' and their contribution to mantle dentine. The fine structure of mantle dentine formation has been studied in the mouse molar. No evidence was found for the presence of collagenous von Korff fibres arising from the dental papilla, passing between odontoblasts and fanning out to form the collagenous matrix of mantle detine. Instead, large collagen fibrils were first demonstrable in the matrix peripheral to the dential aspect of an extensive junctional complex system occurring at the necks of the odontoblasts. The orientation of the fibres was at right angles to the future amelo-dentinal junction in coronal dentinogenesis, but parallel to the root surface in radicular dentinogenesis. These large collagen fibrils formed the mantle dentine. It is concluded that von Korff fibres, as strictly defined, are artefacts. Photographs in the literature purporting to show von Korff fibres are attributable to obliquity of section. Also, is suggested that the difference in fibril orientation in coronal and root mantle dentine is the reason for the conflicting opinions on the pattern of fibril orientation in this tissue.


Non-metric variation of the infracranial skeleton. 196 skeletons of known age, sex and rac from the Terry Collection were studied in order to document 30 non-metric infracranial traits. Each trait had the ability to be expressed bilaterally, although significant side dimorphism was not observed. Sex differences were statistically significant for some of the traits within a racial group, but these differences were not as pronounced as the differences generated by non-metric cranial traits in the same populations, and were not effective in all racial groups. In general, these infracranial traits show some age dependency when correlation statistics are used, but this dependency is lost when the more robust chi 2 statistic is used. These data suggest that infracranial non-metric traits may be superior to cranial non-metric traits for population comparisons. Infracranial traits may be more durable than cranial traits having regard to the nature of most archaeological material.


A study of the artrial arteries in man.

A study of the artrial arteries in man. The arterial blood supply to the atria of 40 human hearts has been studied using a clearing technique which leaves the anatomy of the heart intact. The general arrangement of the atrial arteries is described. The arterial supplies to the S.A. and A.V. nodes are considered in more detail as these ar probably the most important and most consistent vessels found. In over 50% of the hearts both the S.A. and A.V. nodes were supplied from branches of the right coronary artery. In only 7% were both nodes supplied by branches of the left coronary artery. In the remainder the S.A. node was supplied by one coronary artery and the A.V. node by the other, in either combination. The actual site of origin of the S.A. nodal artery varied considerably, but the single most common site (47%) was from the first part of the right coronary artery. The second most common site was from the circumflex branch of the left coronary artery (27%). The site of origin of the A.V. nodal artery was predominatly from the terminal part of the right coronary artery (80%), the remainder being supplied from the left. Some of the relatively large atrial vessels not primarily concerned with the supply of nodal tissue are described. A nomenclature has been suggested both for these and for the nodal arteries. There were many small and variable vessels that defied classification.


An electron microscopic study of the premineralizing zone of the condylar cartilage of the mouse mandible. The mandibular condylar cartilage of young ICR mice was used for ultrastructural studies with special emphasis on the cells and matrix in the premineralizing zone. It became evident that cytodifferentiation of chondroblasts was accompanied by deposition of intracellular lakes of glycogen, followed within the premineralizing hypertrophic chondrocytes by a gradual reduction in the glycogen content of the lakes and its replacement by lipid-like droplets. The premineralizing condylar cartilage, like that of other cartilage involved in mineralization, showed numerous matrix vesicles (Types I and II) unassociated with appreciable amounts of collagen.


Normal structure and dimensions of the pulmonary arteries in the rat. The normal structure and pattern of branching of the pulmonary arteries of the rat have been described and quantified, using precise morphometric techniques, after injection of the pulmonary arteries with a radio-opaque medium. Rats of the same strain from three different sources have been compared, and similarities between rat and man have been assessed. In rat, as in man, the pulmonary artery accompanies the airway and branches with it and, in addition, there are extra arterial branches. Most arteries within the rat lung are muscular and have no more than two central elastic laminae. The percentage wall thickness of the muscular arteries is between 1 and 3%, similar to that found in adult human lungs. At the periphery of the arterial tree there is a mixed population of muscular, partially muscular and non-muscular arteries. The distribution by size is similar in man and rat, but there are few wholly muscular arteries within the acinar region in the rat. The similarity of the features of the pulmonary arteries of rat and man makes the rat a suitable experimental animal for study of changes in pulmonary hypertension. However, any change must be interpreted in relation to arterial structure in normal rat lungs from the same source, since some significant differences have been found between rats from different sources in respect of wall thickness and number of vessels.


Postnatal development of the epidermis in a marsupial, Didelphis virginiana. At birth the epidermis of the opossum is 43 micron thick and consists of a basal layer of columnar cells, an intermediate layer of fusiform cells, a layer of incompletely cornified cells and a single surface layer of cells (the periderm). The latter shows central nuclei and distinct cell boundaries. Adjacent surface cells. are contiguous and show extensive interdigitations of the lateral cell membranes. The periderm is lost during the first week of postnatal development. The epidermis attains its greatest thickness (58 micron) at the 4.0 cm stage (18 days postnatum), and this is due primarily to an increase in thickness of the spinous layer. After this the epidermis thins to 14 micron in the adult. The epidermis of the adult consists of a thin Malphighian layer and a desquamating cornified layer. Hair follicles begin to differentiate at the 2.5 cm stage (7 days postnatum). They continue to differentiate and develop while the epidermis is increasing, and then decreasing, in thickness. The young are fully furred prior to the time they first venture from the protection of the pouch.


Ileumycin, a new antibiotic against Glomerella Cingulata. A new antifungal antibiotic, named ileumycin, was isolated from culture broth of streptomyces H 698-SY2, which was identified as S. lavendulae. The antibiotic was recovered from the culture filtrate by adsorption on Amberlite XAD-II and elution with aqueous methanol and was further purified by ion-exchange column chromatography on SE-cellulose and followed by partition chromatography on silica gel. The antibiotic was named ileumycin, because isoleucine was detected in the acid hydrolyzate of the antibiotic. Ileumycin exhibited antimicrobial activity against only a few species of fungi.


The structure of mycoheptin, a polyene macrolide antifungal antibiotic. The complete structure of mycoheptin, a heptaene macrolide antifungal antibiotic, has been established as I by chemical degradation methods and MS and 1H NMR analysis of the products.


Netilmicin synergy with carbenicillin or cefamandole against Serratia. Twenty clinical isolates of Serratia sp. were tested against netilmicin, gentamicin, carbenicillin and cefamandole alone (broth and agar dilution) and in combination (agar dilution). Broth and agar dilution minimal inhibitory concentrations agreed to within a two-fold dilution in 96% of the tests. Overall, 95% of the isolates were susceptible to netilmicin regardless of susceptibility to gentamicin or carbenicillin. Netilmicin-carbenicillin synergy was seen in 55% of the strains and netilmicin-cefamandole in 70%. These results indicate that combinations of netilmicin with carbenicillin or cefamandole may be clinically useful.


Some observations on the nature of reactive groups involved in reversal of mycobacillin inhibition by sterols and lipids. Antifungal action of di- and triacetyl derivatives of mycobacillin is not antagonised by cholesterol or lecithin which antagonise the action of its heptamethyl derivative. Considering the reactive groups of mycobacillin, the antagonism may involve the tyrosine hydroxyl groups of the antibiotic and the 3-hydroxyl group of cholesterol of the oleic acid component of lecithin having unsaturation at the 9:10 position with cis-configuration.


Butyrate-binding protein from rat and mouse liver. A novel component which specifically binds butyrate was found in rat and mouse liver. This component, termed butyrate binding protein (BBP), is localized in the cytosolic fraction and exhibits protein characteristics, such as heat- and protease-sensitivity. The size of BBP was found to be 7.6S, while it was converted to subunits of 45,000--48,000 dalton by treatment with sodium dodecyl sulfate. The dissociation constant of the binding of BBP with butyrate was 2.22 X 10(-6) M in the standard assay. 30-Fold purification of BBP was achieved by batch-wise adsorption and elution from CM-cellulose and hydroxylapatite column chromatography. BBP is clearly distinguishable from the fatty acid-binding protein found previously on the basis of its size and binding specificity.


Ferredoxin-Sepharose 4B as a tool for the purification of ferredoxin-NADP+ reductase. Ferredoxin immobilized on Sepharose 4B was prepared by reaction of CNBr-Sepharose 4B with spinach ferredoxin. The ferredoxin-Sepharose 4B conjugated ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, [EC 1.6.7.1]) in dilute buffer solution and released it in high salt concentrations. A novel method of preparation for the reductase was established by a combination of affinity adsorption on the ferredoxin-Sepharose 4B column with usual purification procedures. It was found using the new method, that there are two forms of ferredoxin-NADP+ reductase, FNR I and FNR II, in spinach. Comparative studies of the two components suggest that FNR I may be a dimer of FNR II.


Synthesis and metabolism of 5beta-[11,12-3H]cholestane-3alpha, 7alpha-diol. 5beta-[11,12-3H]Cholestane-3alpha,7alpha-diol was synthesized as follows. 5beta-Cholestane-3alpha,7alpha,12atriol 3,7-diacetate was treated with phosphorus oxychloride in pyridine solution and then the product, 5beta-cholest-11-ene-3alpha,7alpha-diol diacetate, was hydrogenated in acetic acid solution using platinum oxide as a catalyst under an atmosphere of tritium gas. 5beta-[11,12-3H]Cholestane-3alpha,7alpha-diol thus obtained was readily hydroxylated at C-26 by mitochondria in the presence of isocitric acid, magnesium chloride and potassium cyanide.


Interaction of subunits of polypeptide chain elongation factor I from pig liver. Formation of EF-1alpha.EF-1betagamma and EF-1beta complexes. In the preceding papers, we showed that one of the two complementar factors of polypeptide chain elongation factor 1 (EF-1) from pig liver, EF-1alpha, functionally corresponds to bacterial EF-Tu (Nagata, S., Iwasaki, K., and Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, 168), while the other, EF-1betagamma, as well as one of its subunits, EF-1beta, corresponds to bacterial EF-Ts (Motoyoshi, K. and Iwasaki, K. (1977) J. Biochem. 82, 703). Therefore, the interaction between EF-1alpha and EF-1 betagamma or EF-1beta was was examined and the following results were obtained. i) EF-1betagamma catalytically promoted the exchange of [14C]GDP bound to EF-1alpha with exogenous [3H]GDP. ii). In the absence of the exogenous guanine nucleotide, EF-1betagamma as well as EF-1beta could displace GDP bound to EF-1alpha to form an EF-1alpha.EF-1betagamma as well as an EF-1alpha.EF-1beta complex. iii) The occurrence of EF-1alpha.EF-1betagamma and EF-1alpha.EF-1beta complexes was demonstrated by gel filtration on Sephadex G-150. These results strongly indicate that the mechanism of the action of EF-1betagamma or EF-1beta in converting EF-1alpha.GDP into EF-1alpha.GTP is analogous to bacterial EF-Ts, and the reaction is accomplished by the following reactions; EF-1alpha.GDP + EF-1betagamma (or EF-1beta) in equilibrium EF-1alpha.EF-1betagamma (or EF-1beta) + GDP; EF-1alpha.EF-1beta (or EF-1beta) + GTP IN EQUILIBRIUM  EF-1alpha.GTP + EF-1betagamma (or EF-1beta).


Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation. Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.


Inhibition of protein synthesis in mouse L cells by poly-L-ornithine. A snythetic polypeptide, poly-L-ornithine (pLo; average molecular weight, 13,000), inhibited protein synthesis in mouse L cells in suspension culture. The inhibition was dependent upon both the concentration of pLo and the cell density, and the dose for half-inhibition was correlated with the cell density. Cell viability remained high in the concentration range of PLo causing inhibition of protein synthesis. This inhibition was prevented by addiction of calf serum with pLo, but not by washing the treated cells with serum or a high concentration of salt. PLo had no effect on protein synthesis in a cell-free system prepared from L cells or rabbit reticulocytes. The polysome profiles of cells treated with pLo were similar to those of control cells. Experiments on the effect of interchange of the ribosomes and supernatants of control and pLo-treated cells showed that the ribosomes from pLo-treated cells were inactive. The inactivation of these ribosomes was partially prevented by the presence of 120 mM K+ in the medium during pLo treatment. The inhibition of protein synthesis by pLo, therefore, may result from the binding of pLo to the cell membranes, causing leakage of intracellular K+ and thus inactivating ribosomes.


Hormonal effects on the biosynthesis of sulfated glycoprotein in a microsomal fraction of the endometrium of rabbit uterus. A crude microsomal fraction (M-Fr) was separated from the endometrial scrapings of uteri of ovariectomized rabbits with or without hormonal treatment. The effects of estrogen and progesterone on the incorporation into M-Fr of L-[U-14C]-fucose and N-acetyl-D-[6-3H]-glucosamine from their nucleotides were investigated. Estrogen increased the incorporation of these sugars, whereas progesterone suppressed this effect. The results of fractionation on a DEAE-Sephadex A-25 (Cl- form) column of the isotope-labelled complex saccharide mixtures, obtained by pronase digestion of the incubation mixtures, indicated that biosynthesis of sulfated glycoprotein was most sensitive to the hormones among the complex saccharides in M-Fr. Thus, a hormonal effects on the biosynthesis of sulfated glycoprotein in the endometrium of ovariectomized rabbit has been unambiguously confirmed at the microsomal level.


Urinary metabolites of polyamines in rats. Urinary metabolites of polyamines in rats were studied systematically by the intraperitoneal injection of radioactive polyamines. Urinary metabolites were fractionated into 4 fractions containing non-polar and acidic compounds, acidic and neutral ampholytes, basic ampholytes and polyamines. A large amount of radioactivity was detected in the fractions containing non-polar and acidic compounds and polyamines of urine of rats injected with radioactive putrescine, while in the case of the injection of radioactive spermidine or spermine a relatively large amount of radioactivity was found in the basic ampholyte fraction as well as in the polyamine fraction. Analysis of these fractions indicated that gamma-aminobutyric acid, N-monoacetylputrescine, 2(3)-hydroxyputrescine, putreanine, N-(3-aminopropyl)-4-aminobutyric acid (isoputreanine), spermic acid, N-(3-aminopropyl), N'-(2-carboxyethyl)-1,4-diaminobutane, and N-monoacetylspermidine A and B were excreted as urinary metabolites of the polyamines in addition to putrescine, spermidine and spermine.


Abnormal expression of a serine protease in human dystrophic muscle. The activities of serine protease in muscles from normal persons and from patients with progressive muscular and neuromuscular diseases have been determined. A significant increase in the level of serine protease was found in muscle of patients with Duchenne-type muscular dystrophy and with Becker-type muscular dystrophy, but the activity was not increased in muscle of a patient with amyotrophic lateral sclerosis.


Photoaffinity labeling of insulin receptor of rat adiopocyte plasma membrane. A photosensitive insulin derivative was synthesized by reacting radioactive iodinated bovine insulin with N-hydroxysuccinimide ester of 4-azidobenzolic acid. The photo-sensitivity and specificity of this insulin derivative were established by its covalent nonspecific cross-link to albumin and its covalent specific cross-link to the heavy and light chains of anti-insulin immunoglobulin. Plasma membrane preparations of rate adipocytes were incubated with the photosensitive insulin derivative and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization with sodium dodecyl sulfate and reduction with beta-mercaptoethanol showed that a protein having a molecular weight of 130,000 was specifically labeled by the radioactive photosensitive insulin, suggesting that this protein may be the insulin receptor.


Site-specific phosphorylation of beta-casein by proetin kinases from rabbit reticulocytes. The B variant of beta-casein was phosphorylated with [gamma-32P]ATP using four different protein kinases isolated from rabbit reticulocytes. Casein was maximally phosphorylated by the individual protein kinase activities and subjected to chymotrptic digestion. The peptides were separated by a two-dimensional peptide fingerprinting technique, and the phosphorylated peptides were identified by autoradiography, The two phosphorylated peptides obtained from the action of casein kinase I were shown to have different migration patterns from those obtained with casein kinase II. The cAMP-regulated protein kinases had the same substrate specificity with beta-casein B, and the two phosphorylated peptides obtained using these enzymes were distinct from those phosphorylated by the cAMP-independent enzymes. Thus, the different protein kinases can be identified by substrate specificity using beta-casein.


Complex formation between the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and valinomycin in the presence of potassium. Spectroscopic evidence is presented which indicates that the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the peptide antibiotic valinomycin form a complex in the presence of potassium. Complex formation has been observed both in aqueous and nonaqueous media. Several techniques have been used to indicate the existence of a complex in aqueous solution. In the presence of valinomycin and K+, the absorption spectrum of FCCP is significantly perturbed, and there is also a large induced circular dichroism signal. In addition, the previously characterized complex which forms between valinomycin, K+, and the fluorescent probe 8-anilino-1-naphthalene-sulfonate (ANS) in aqueous solution is apparently disrupted by the addition of FCCP. The result is an effective quenching of the fluorescence due to the bound probe as it is displaced from the valinomycin.K+ by the uncoupler. In a nonpolar solvent, the absorption spectrum of FCCP is also perturbed by valinomycin in the presence of K+, again indicating the formation of a complex. These data point to the importance of considering the role of valinomycin.K+.uncoupler complex in interpreting physiological or ion transport data in which these substances have been used together.


Characterization of azidobenzamidines as photoaffinity labels for trypsin. Meta- and para-azidobenzamidine have been prepared and evaluated as photoaffinity labels. The compounds inhibit trypsin reversible in the dark and are competitive with substrate binding. Upon photolysis, irreversible noncompetitive inhibition is observed and is dependent upon concentration, photolysis time, and pH. Specificity of the probes is indicated by experiments in which p-tosyl-l-arginine methyl ester, a trypsin substrate, is used to protect against photoinactivation. Maximum inactivation of trypsin is achieved at pH 6.2 using either azidobenzamidine derivative. Evaluation of the pH dependence of photoaffinity labeling may provide a sensitive tool for probing conformational changes in inhibitor binding sites. These studies provide a basis for the use of azidobenzamidines as photoaffinity analogs of lysine and arginine side chains.


Evidence that thyroglobulin contains nonidentical half molecule subunits. Bovine thyroglobulin was extracted from unfrozen glands, purified by sucrose gradient centrifugation, and fractionated into a narrow range in iodine content by RbCl isopycnic centrifugation. The subunit composition of these preparations was studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The extent of dissociation of 19 S into 12 S half-molecules followed the known relationship with iodine, i.e. decreased dissociability of 19 S with increased iodine content. The undissociated 19 S band always consisted of three closely spaced, equidistant bands. Reduction of the disulfide bonds of thyroglobulin by mercaptoethanol in SDS solution resulted in the formation of two major and one minor components (S, F, and A). The concentration of A was always less than 10% of the total. The ratio of S to F was, however, about equal in thyroglobulin preparations which ranged in iodine content from 0.2 to 1%. The final ratios were obtained before all the disulfides were reduced. The relative mobilitis of S, F, and A, decreased with increasing extent of reduction. Fully reduced S and F, but not A, migrated slower than unreduced 12 S. The three reduced alkylated polypeptides were purified by preparative SDS-polyacrylamide gel electrophoresis and their molecular weights were determined by sedimentation equilibrium in 8 M urea. Their Mw and Mz values agreed closely with that of the unreduced 12 S half-molecule subunit, thus indicating that reduction of the disulfide bonds changes the shape but not the molecular weights of the subunits.


Carnitine-acylcarnitine translocase. Inhibition by alpha-cyano-4-hydroxycinnamate and evidence for separate identity from the pyruvate transporting system of mitochondria. Some of the known inhibitors of pyruvate transport inhibited the activity of carnitine-acylcarnitine translocase. Their order of effectiveness with millimolar concentration required for 50% inhibition given in parentheses, was: Compound UK-5099 (alpha-cyano-beta-(1-phenylindol-3-yl)acrylate) (0.1); alpha-cyano-4-hydroxycinnamate (0.17); alpha-cyano-3-hydroxycinnamate (1); alpha-cyanocinnamate (1); alpha-fluorocinnamate (7); transcinnamate (10); p-hydroxycinnamate (10); phenylpyruvate (22); p-hydroxyphenylpyruvate (25). Kinetically, the alpha-cyano-4-hydroxycinnamate inhibition was mixed and the p-hydroxyphenylpyruvate inhibition was noncompetitive with respect to external (-)-carnitine. The alpha-cyano-4-hydroxycinnamate inhibition was reversible and resulted from its ability to act as a thiol reagent. In general, alpha-cyanocinnamate and its derivatives inhibit carnitine transport at concentrations 100 to 5000 times as high as those known to pyruvate transport. At millimolar concentrations, alpha-cyano-4-hydroxycinnamate inhibited the mitochondrial transport of molecules other than carnitine as well as the activity of carnitine acyltransferases. Pyruvate and carnitine did not complete for transport into and out of mitochondria. These results establish that transmitochondrial transport mechanisms for carnitine and pyruvate involve different carriers.


Pressure effects on folded proteins in solution. Hydrogen exchange at elevated pressures. The observed rate constants for base-catalyzed hydrogen exchange reactions between solvent water and peptide nitrogen in lysozyme, ribonculease A, oxidized ribonuclease A, and poly(DL-lysine) are all enhanced by an increase in pressure. Activation volumes have been calculated from the pressure effect on these rate constants. For the folded proteins lysozyme and ribonuclease A, deltaV for base-catalyzed exchange changes from about +9 ml/mol at atmospheric pressure -3 ml/mol at 2500 kg/cm2. The same quantity, determined for the random coil polypeptides oxidized ribonuclease A and poly(DL-lysine), does not show this dependence upon pressure. These effects can be understood either in terms of solvent penetration on the folded proteins or the onset of a small degree of pressure induced unfolding. Possible mechanisms by which such penetration could occur are discussed.


Reactions of tubulin-associated guanine nucleotides. Only exchangeably bound nucleotide (E-site) is involved in the reaction of the transplhosphorylase activity in microtubular protein. Contrary to earlier reports, we find that the nonexchangeable nucleotide (N-site) is not a substrate. This conclusion is based upon comparison of: (a) rates of hydrolysis of endogenous tubulin-associated GTP and added [32p]GTP: (b) hydrolysis rates for added [32p]GTP and [3h]GTP; (c) the 32P/3H ratio in bound and free GTP after reaction with [3h, 32p]gTP. During the course of the above studies we have made the unusual observation of a time dependent augmentation in the expected amount of GTP relative to GDP at the E-site; there is either a net conversion of E-site GDP to E-site GTP, or a means for providing additional E-site GTP from another source.


Interaction of bilirubin with lipids studied by fluorescence quenching method. The interaction of bilirubin and various lipids was studied by a static fluorescence quenching method. A quenching equation was developed to determine the binding constant. This method was tested first by a 5-dimethylaminoaphthalene-1-sulfonyl (dansyl).bovine albumin system and then applied to a fluorescent probe containing lipid. Both systems can yield binding parameters. For certain lipid systems, some dynamic quenching was observed at the part of the curve where bilirubin concentrations were at the extreme. Of the lipids tested, sphingomyelin showed the highest association constant; this may be related to the central nervous system toxicity of this bile pigment. The association constants determined with pure lipids were 2.4 x 10(5) M-1 for phosphatidylserine; 3.7 x 10(6) M-1 for sphingomyelin; 2.4 x 10(6) M-1 for diphosphatidylcholine with 5% cholesterol; 9.8 x 10(5) M-1 for diphosphatidycholine alone; 2.2 x 10(5) M-1 for phosphatidylethanolamine; and 1.6 x 10(5) M-1 for diphosphatidylglycerol (cardiolipin); and those with mixed lipids were 4.2 to 5 x 10(5) M-1 for total brain lipid if cow and 3 to 3.6 x 10(5) M-1 for total skin lipid of pig.


The mode of conversion of proparathormone to parathormone by a particulate converting enzymic activity of the parathyroid gland. The cleavage products from the conversion of proparathormone to parathormone by a bovine and porcine parathyroid microsomal converting activity have been analyzed. In the conversion reaction, the first 6 amino acid residues of the prohormone (Lys-Ser-Val-Lys-Lys-Arg-) are released as an intact hexapeptide. This is rapidly converted to a pentapeptide by removal of the NH2-terminal lysine and then to a tetrapeptide by removal of the COOH-terminal arginine. In order to test for the presence of a postulated COOH-terminal extension of the parathormone sequence in proparathormone, mixtures of 14C-proparathormone and 3H-parathormone were subjected to digestion by trypsin or Staphylococcus aureus protease. The resulting radioactive peptides from the hormone and its precursor were compared. There was no evidence that any fragments different from those from the hormone were released from the prohormone except those accounted for by the NH2-terminal hexapeptide adduct on proparathormone. Thus, the conversion of the prohormone to the hormone catalyzed by the microsomal membrane activity requires only the cleavage of this hexapeptide.


Electrophoretic studies on liver endoplasmic reticulum membrane polypeptides and on their phosphorylation in vivo and in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to examine the polypeptide patterns of rat liver rough and smooth endoplasmic reticulum (ER) membrane fractions stripped of ribosomes. Approximately 67 polypeptides were resolved from the rough ER membrane fraction. The polypeptide pattern of the smooth ER membrane fraction was similar to that of the rough ER membrane fraction, but exhibited substantially lower amounts of some seven polypeptides. Three of these polypeptides, of apparent molecular weights 63,000, 65,000, and 87,000, were of particular interest, as they could not be ascribed to contamination of stripped rough ER membrane fractions by residual ribosomal polypeptides. Conditions of treatment with low concentrations of trypsin were established that markedly diminished the capacity of the stripped rough ER membrane fraction to bind ribosomes in vitro and that also effected a partial detachment of ribosomes from nonstripped rough ER membranes; the results of electrophoretic analyses of rough ER membrane fractions treated in these manners are described. Comparison of the polypeptide patterns of guinea pig, mouse, and rabbit liver ER membrane fractions with rat liver ER membrane fractions revealed considerable variations in the distribution of the polypeptides of 63,000, 65,000, and 87,000 molecular weight among the ER membrane fractions of these species. The combined results of these studies indicate that the polypeptide of 87,000 molecular weight, although particularly sensitive to attack by trypsin, is not involved in the binding of ribosomes to the rough ER membrane fraction. Studies by others (cf. Kreibich, G., Grebenau, R., Mok, W., Pereyra, B., Rodriguez-Boulan, E., and Sabatini, D. D. (1977) Fed. Proc. 36, 656) have implicated the polypeptides of 63,000 and 65,000 molecular weight in this process. The patterns of phosphorylated polypeptides of rough and smooth ER membrane fractions of rat and mouse liver were also examined, using labeling in vivo with sodium [32p]phosphate or in vitro with [gamma-32P]ATP. Approximately 25 phosphorylated components were resolved by electrophoresis in the ER membrane fractions of both species. Evidence is presented that suggests that the great majority of these components are phosphopolypeptides. Differences were noted in the patterns of phosphorylation produced by in vivo and in vitro labeling; minor differences were also observed between the patterns of phosphorylation of the rough and smooth ER membrane fractions in either situation. The overall results afford an indirect approach toward evaluating the possible involvement of specific rough ER membrane polypeptides in ribosome-binding and reveal that liver ER membranes contain a substantially greater number of phosphorylated polypeptides thatn previously reported.


Physical map of the Bombyx mori DNA containing the gene for silk fibroin. A physical map of the DNA containing the gene for silk fibroin was developed from direct hybridization analysis of restriction endonuclease digests of total Bombyx mori DNA using fibroin 125I-mRNA. The orientation of mRNA transcription relative to this map was deduced from the sensitivity of the mRNA coding strand within certain DNA restriction segments to lambda-exonuclease and exonuclease III. The map includes the entire gene coding region (Mr approximately 11 x 10(6)) and large DNA elements which flank the gene at its 5' end (Mr approximately 3 x 10(6)) and 3' end (Mr approximately 6.5 x 10(6)). The coding region is remarkably uniform in its sensitivity to restriction endonucleases. It is completely devoid of sites for most of the enzymes tested, including Hae III, the recognition sequence (d-pG-G C-C) of which might be expected to occur frequently in this large DNA block of 60% G + C content. The fibroin coding region does contain an enormous number of sites for enzymes predicted to have activity from known fibroin mRNA sequences. These results suggeste that the fibroin gene core is a large homogeneously repetitive block of DNA with little evidence for sequence divergence, or the presence of qualitatively different sequences, which might creat other restriction sensitivities. The map also allowed a comparison to be made of the fibroin gene "context" in DNA from tissues either active or inactive in fibroin synthesis.


Crystalline ricin D, a toxic anti-tumor lectin from seeds of Ricinus communis. A toxic lectin, ricin D, present in the seeds of Ricinus communis has been purified and crystallized in a form suitable for high resolution crystallographic structure studies. This protein is different from a previously found form of ricin (also present in the same seeds), the only ricin for which a preliminary x-ray investigation has been reported so far. Ricin D crystallizes from an aqueous solution in an orthorhombic unit cell of symmetry P2(1)2(1)2(1) and a = 79.0, b = 114.7, and c = 72.8 A. The asymmetric unit contains one molecule with an average molecular weight of 62,400. The crystal is fairly stable to x-radiation and has a water content of approximately 54% by volume. It appears to comprise two closely related species of proteins, the major species corresponding to recin D and the other presumably corresponding to a deamidation product of ricin D. The two species have nearly identical molecular size and amino acid compositions, but different charges.


Effect of reductive lactosamination on the hepatic uptake of bovine pancreatic ribonuclease A dimer. Lactose has been coupled to the lysine residues of the cross-linked dimer of bovine pancreatic ribonuclease A by reductive amination with cyanoborohydride. Derivatives of ribonuclease dimer that contained up to 10 Nepsilon-1-(1-deoxylactitolyl)-lysine residues per molecule had greater than 75% of the enzymic activity of the unmodified enzyme toward yeast RNA. Upon intravenous injection of the 14C-labeled (enzymically inactivated by 14C-carboxymethylation) derivatives into rats, their uptake by the liver was a function of the number of lactose residues coupled. At 10 min, 69% of the injected derivative of ribonuclease dimer containing eight 1-deoxylactitolyl-lysine residues/molecule was found in the liver; with the non-glycosylated enzyme, the liver uptake at 10 min was only 4%, and 75% of the radioactivity was found in the kidneys.


Localization of the alpha-chain cross-link acceptor sites of human fibrin. The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain.


Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter. Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.


Translation and characterization of the fatty acid synthetase messenger RNA. Fatty acid synthetase messenger RNA was obtained from rat liver polysomal RNA and then injected into Xenopus laevis oocytes. The radioactive fatty acid synthetase protein synthesized in the oocytes was identified by immunoprecipitation with anti-fatty acid synthetase antibody and the immunoprecipitate was then characterized by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel. Co-migration of authentic fatty acid synthetase and the labeled product synthesized in oocytes was observed. Based on sucrose density gradient analysis, the rat liver fatty acid synthetase mRNA has a sedimentation coefficient of approximately 33 S, which agrees with the predicted minimum size necessary to code for the fatty acid synthetase protein. In addition, this mRNA was partially purified with oligo(dT)-cellulose, which indicates that it has a polyadenylate region. The relative in vivo rate of synthesis of fatty acid synthetase and the level of fatty acid synthetase mRNA in liver were also determined during the course of dietary induction of this enzyme. The results indicate that the dietary-induced increase in the level of fatty acid synthetase is probably due to an increased level of the fatty acid synthetase mRNA.


Effect of the beta6 Glu replaced by Val mutation on the optical activity of hemoglobin S and of its beta subunits. The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.


Glycosylated minor components of human adult hemoglobin. Purification, identification, and partial structural analysis. Human hemolysate contains several minor components designated Hb A1a, Hb A1b, Hb A1c, which are post-translational modifications of the major hemoglobin component A0. Individuals with diabetes mellitus have elevated levels of Hb A1c, a hemoglobin modified with a glucose moiety at the NH2 terminus of each beta chain. A new chromatographic technique using Bio-Rex 70 is described which not only allows complete separation of Hb A1a from Hb A1b but also resolution of Hb A1a into two components, designated Hb A1a1 and Hb A1a2. Carbohydrate determinations with the thiobarbituric acid procedure revealed that Hb A1a1, Hb A1a2, and Hb A1b as well as Hb A1c were glycosylated. Total phosphate analysis revealed 2.06 and 1.01 mol of phosphorus/alphabeta dimer for Hb A1a1 and Hb A1a2 respectively; Hb A1b and Hb A1c contained no detectable phosphate. Hemoglobin incubated with D-[14C]glucose-6-P co-chromatographs precisely with Hb A1a2, strongly suggesting that Hb A1a2 is glucose-6-P hemoglobin. Levels of Hb A1a1 and Hb A1a2 are normal in individuals with diabetes mellitus. Furthermore, diabetic red cells contain normal levels of glucose-6-P. Therefore, glucose-6-P hemoglobin does not serve as a significant precursor to Hb A1c. Instead Hb A1c is formed by the direct reaction of hemoglobin with glucose. This suggests that hemoglobin can serve as a model system for nonenzymatic glycosylation of protein.


Prolactin induction of casein mRNA in organ culture. A model system for studying peptide hormone regulation of gene expression. The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.


Isolation of vitamin K2(35) from Nocardia restrictus and Corynebacterium simplex. A natural electron acceptor in microbial steroid ring A dehydrogenations. Isooctane extraction of cells from Nocardia restrictus and Corynebacterium simplex followed by chromatographic separation gave yellow crystals which were identified as vitamin K2(35) by its ultraviolet absorption, nuclear magnetic resonance spectrum, and mass spectrometry. The purified vitamin K2(35) was found to stimulate steroid 1,2-dehydrogenase obtained from cell free extracts of N. restrictus, C. simplex, Cylindrocarpon radicicola, and Septomyxa affinis as well as 4,5alpha-dehydrogenase and 4,5beta-dehydrogenase obtained from N. restrictus. Evidence is presented to show that vitamin K2(35) can act as an efficient electron acceptor in steroid ring A dehydrogenations which may be coupled to other systems during microbial transformations of steroids.


Synthesis and characterization of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid, a new fluorescent analogue of diphosphoglyceric acid. The synthesis of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid (DIPANS) is described. It entails the synthesis of 2,4-diphosphobenzaldehyde from the action of POCl3 on 2,4-dihydroxybenzaldehyde. This is followed by coupling of the 2,4-diphosphobenzaldehyde to 1-amino-5-naphthalenesulfonic acid. Subsequent reduction with NaBH4 yields the desired product. The DIPANS exhibits an excitation maximum at 337 nm and a fluorescence emission maximum at 504 nm. This dye is quantitatively displaced by inositol hexaphosphate and is an effective analogus of diphosphoglyceric acid (DPG), possessing a KD at pH 7.0 in 0.05 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (bis-Tris) plus 0.1 M chloride of 6.88 microgram, with 1.0 molecule bound/hemoglobin tetramer. Like DPG its binding to deoxyhemoglobin decreases with increasing pH; in the presence of 0.1 M chloride it binds 0.031 times as tightly to CO hemoglobin and it yields a value for free energy coupling of 2.0 kcal/mol. The presence of 1 mM DIPANS decreases the affinity of hemoglobin for oxygen in the absence of salt from p1/2 of 0.8 mm Hg to 12.4 mm Hg. Using DPG as a competitor of DIPANS binding, a dissociation constant of 11.4 micrometer was calculated for DPG binding to deoxy-Hb at pH 7.0 in the presence of 0.05 M bis-Tris and 0.1 M chloride.


Structural changes in the T4 gene 32 protein induced by DNA polynucleotides. Alterations in the structure of the DNA-binding protein specified by gene 32 of bacteriophage T4 have been detected using partial trypsin digestion as a conformational probe. Limited tryptic hydrolysis of the gene 32 protein removes a fragment ("B" region), of 21 amino acids from the NH2 terminus and a 6,200-dalton fragment ("A" region) from the COOH terminus. Poly(dT), poly(dC), and single-stranded DNA increase the rate of tryptic hydrolysis of the "A" region but decrease the rate of tryptic hydrolysis of the "B" region. Oligonucleotides, which are too short to permit cooperative binding of the gene 32 protein, do not alter the rate of tryptic hydrolysis of either the "A" or "B" regions. A model which accounts for these findings requires that the "B" region be involved in gene 32 protein:gene 32 protein interactions when the gene 32 protein: DNA complex is formed. As a consequence of the gene 32 protein:DNA interaction, the "A" region should be able to participate more effectively in vivo and in vitro with other proteins involved in T4 DNA metabolism.


Synthesis of full length cDNAs from four partially purified oviduct mRNAs. Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.


Changes in gene expression accompanying neoplastic transformation of Syrian hamster cells. Proteins solubilized from the chemically transformed, highly tumorigenic Syrian hamster cell line, BP6T, and the untransformed parental embryo cells, have been analyzed by two-dimensional gel electrophoresis. Differences in seven major polypeptides have been identified in cytoplasmic and nuclear cell fractions from these two related cell types. The tumorigenic cells have lost the ability to synthesize detectable amounts of five major polypeptides which are found in untransformed cells; in addition, the tumorigenic cells synthesize two new major polypeptide species not found in the untransformed cells. Butyric acid, an agent which suppresses in vitro cellular properties frequently associated with neoplasia, induces in a reversible fashion synthesis of two of these missing polypeptide species in the tumorigenic cells. The results indicate that a change in the synthesis of less than 1% of the major polypeptide species is associated with a chemical mediated induction of the high tumorigenic state of Syrian hamster cells.


Light-driven ATP formation from 32Pi by chloroplast thylakoids without detectable labeling of ADP, as measured by rapid mixing and acid quench techniques. The labeling of ATP and ADP in the first few milliseconds of exposure of chloroplast thylakoids to light, 32Pi, and ADP has been measured. At least 4 mol of ATP/mol of coupling factor 1 (CF1)-ATPase can be formed without detectable labeling of membrane-bound or free ADP. Such results are consistent with ADP and not AMP as the primary acceptor of Pi in photophosphorylation. Evidence is presented demonstrating that quenching with perchloric acid, as used in these and earlier experiments, is satisfactory for the measurement of the amount and nature of membrane-bound nucleotides.


Identification of circulating apolipoproteins synthesized by rat small intestine in vivo. Apolipoproteins synthesized by intestine were identified in mesenteric lymph and plasma collected from an animal preparation in which exposure to [3H]leucine was restricted to the mucosal epithelial cells of a fat-transporting small intestinal segment in situ. The intestine released labeled chylomicrons and very low density lipoproteins only into lymph but labeled high density lipoproteins into both plasma and lymph. Of the 3H incorporated into individual apolipoproteins, separated by polyacrylamide gel electrophoresis, 25% was recovered in apolipoprotein B (apoB), 50% in apoA-I, 21% in apoA-IV, and 4% in C apolipoproteins, nearly all in apoC-II and apoC-III-0. Only traces of radioactivity were associated with the arginine-rich protein and none was detected in apoA-II. Labeled apoB was found in chylomicrons and very low density lipoproteins and labeled apoA-I and apoA-IV in all lipoproteins. Evidence suggested that substantial amounts of labeled apoA-IV were also present in the lipoprotein-free lymph fraction (d greater than 1.21 g/ml), further indicating that apoA-IV is a major apolipoprotein product in intestine. Thus, the pattern of apolipoprotein synthesis by intestine is notably different from that reported for liver.


Ketogenesis and malonyl coenzyme A content of isolated rat hepatocytes. We have measured rates of ketogenesis and malonyl-CoA contents of hepatocytes isolated from meal-fed rats under a variety of incubation conditions in order to determine the relationship between the intracellular malonyl-CoA level and the rate of ketogenesis. Evidence obtained from rat liver homogenates suggested that malonyl-CoA, which is a major determinant of fatty acid synthesis in vivo, also inhibits carnitine acyltransferase I (EC 2.3.1.21) and thereby decreases the rate of ketogenesis (McGarry, J.D., Mannaerts, G.P., and Foster, D.W. (1977) J. Clin. Invest. 60, 265-270). In hepatocytes from meal-fed rats, malonyl-CoA could be increased by glucose or lactate plus pyruvate and decreased by glucagon, oleic acid and the fatty acid synthesis inhibitor 5-(tetradecyloxy)-2-furoic acid. Malonyl-CoA varied from 14.8 +/- 1.2 to 1.4 +/- 0.1 nmol/g wet weight of cells. Rates of ketone body production varied from 0.10 +/- 0.01 to 0.96 +/- 0.06 mumol/min/g wet weight of cells and varied inversely with the malonyl-CoA content. Dixon plots and Cornish-Bowden plots of data suggest that malonyl-CoA is a competitive inhibitor of ketogenesis with a Ki of 2 nmol/g wet weight of cells. We conclude that in hepatocytes from meal-fed rats the cellular content of malonyl-CoA and the concentration of long chain fatty acid available to the cells are major determinants of the rate of ketogenesis.


Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. Procedures for the controlled addition of one or more deoxyribonucleotide residues to the 3' end of an oligodeoxyribonucleotide primer are described. Polynucleotide phosphorylase (EC 2.7.7.8), purified from Escherichia coli B, catalyzes the reaction using a deoxyribonucleoside 5'-diphosphate as substrate, with Mn2+ as cofactor. Reaction occurs rapidly in aqueous solution, and no protecting groups are required, simplifying recovery and purification of the products. The concentrations of sodium chloride and manganous chloride in the incubation mixture are critical to obtaining good yield of the required product. Primers of chain length from 3 to 12 have been extended by up to 9 deoxyribonucleotide residues to obtain oligodeoxyribonucleotides of chain length up to 13. Yields of single addition products varied from 8 to 59%. Factors which influence these yields are discussed. The effects of added polyamines and some organic solvents on the reaction are described. Spermidine or dimethylsulfoxide in the incubation medium tend to favor the addition of several residues of deoxyribonucleotide to the primer.


The effect of "cap" analogs on reovirus mRNA binding to wheat germ ribosomes. Evidence for enhancement of ribosomal binding via a preferred cap conformation. A variety of compounds related to the 5'-terminal "cap" (m7GpppN) of eukaryotic mRNA's were chemically synthesized and tested as inhibitors of reovirus mRNA binding to wheat germ ribosomes. Under our conditions of mRNA binding to ribosomes, 7-methyl-, 7-ethyl-, and 7-benzyl-GDP, but not GDP, decreased stable initiation complex formation by 70 to 80% at a concentration of 0.1 mM indicating that 7-substitution, but not a specific substituent, was required for the effect. Elimination of the positive charge on the imidazole of the 7-substituted compounds by treatment with alkali destroyed their inhibitory activity. Similarly, reduction to 8-hydro-m7GDP reversibly decreased the activity of m7GDP. The results were consistent with the hypothesis that the positive charge resulting from 7-alkylation provides an active cap conformer for binding via interaction of phosphate oxygens with the positively charged imidazole moiety. In accord with this suggestion, 7-carboxymethyl GDP and 7,8-dimethyl GDP were found to be less inhibitory than m7GDP. A 2-amino group was also important since m7IDP was less effective than m7GDP and 0.1 mM m7XDP did not inhibit ribosome binding. Other poor inhibitors were 6-Cl-m7GDP and 1,7-dimethyl GDP but N2,7-dimethyl GDP, 2'-deoxy-m7GDP, and m7GppI had essentially the same activity as m7GDP.


Evidence for a dual mechanism of lipolysis activation by epinephrine in rat adipose tissue. Whole homogenates prepared from tissue previously exposed to epinephrine displayed a 3-fold increased rate of lipolysis of endogenous substrate. When the aqueous infranatant phase of such homogenates was collected by centrifugation and assayed against exogenous triolein emulsions, no hormone effect could be demonstrated. Treatment of such infranatants with cAMP-dependent protein kinase prepared from muscle increased their lipase activity against exogenous triolein by 80%. Employing [3H]triolein emulsions as exogenous substrate, rates of lipolysis of both endogenous and exogenous glycerides were measured simultaneously in whole tissue homogenates. Prior treatment of the tissue with epinephrine increased the rate of lipolysis of endogenous glycerides an average of 3-fold but had no effect on the hydrolysis of exogenous triolein. By contrast, treatment of whole homogenates with protein kinase accelerated lipolysis of exogenous triolein without altering the rate of hydrolysis of endogenous glycerides. The data suggest that a second pathway of lipolysis activation occurs in response to epinephrine in addition to that involving a cAMP-mediated increase in the state of phosphorylation of the hormone-sensitive lipase.


Subunit structure of Mycobacterium smegmatis fatty acid synthetase. Evidence for identical multifunctional polypeptide chains. Fatty acid synthetase from Mycobacterium smegmatis has been purified to near homogeneity as judged by a variety of electrophoretic criteria under both native and dissociating conditions. A single protein band was obtained on gel electrophoresis in sodium dodecyl sulfate or 8 M urea at various pH values and on isoelectric focusing in 8 M urea. A subunit molecular weight of about 290,000 was found by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by sedimentation equilibrium ultracentrifugation in 6 M guanidine HCl. Quantitative Quantitative determination of pantetheine, of flavin, and of the number of fatty acids synthesized during a single enzyme turnover all yield values corresponding to a stoichiometry of about 1 mol per mol of subunit, providing strong evidence that M. smegmatis fatty acid synthetase is an oligomer of identical, multifunctional polypeptide chains.