Architecture and density of the connective tissue papillae of the human oral mucosa. The papillary body of the human oral mucosa was studied at six different sites. Biopsy and autopsy material from 57 individuals, 11-81 years of age, was split chemically along the basal lamina and the epithelium-connective tissue interface examined by light and scanning electron microscopy. Morphometric techniques were employed in order to determine: epithelial thickness, height and density of connective tissue papillae and the percentage of basal epithelial surfaces occupied by them. In the majority of sites, connective tissue plateaux or ridges carrying a variable number of single or grouped papillae were found to be the basic structural units of the papillary body. Three regions with diferent characteristics of the epithelium-connective tissue interface could be identified: (1) floor of the mouth, (2) lip and cheek, (3) gingiva and hard palate. The floor of the mouth showed the lowest connective tissue papillae density, the smallest papillae, and connective tissue plateaux separated by narrow grooves. Lip and cheek mucosae revealed an intermediate density, the papillae were frequently bifurcated and angulated. Gingiva and hard palate were characterized by the highest papillary density and by papillae which were cylindrical, slender and erect. The alveolar mucosa exhibited intermediate features between those of the floor of the mouth and those of the cheek mucosa.


Biosynthesis of streptolidine moiety of streptothricins by Streptomyces noursei JA 3890b. The incorporation of uniformly 14C-labeled compounds into the streptothricin-type antibiotic nourseothricin was studied with a strain of Streptomyces noursei JA 3890b. 6.5% of radioactivity from U-14C-L-arginine was incorporated into the antibiotic, while glutamic acid, aspartic acid, alanine, proline, glycine and leucine displayed much lower incorporations. Furhtermore, 95% of the activity incorporated from arginine was located in the streptolidine moiety supporting the suggestion that this subunit of streptothricin antibiotics is formed via the dehydroarginine pathway.


Isolation and characterization of a new nucleoside antibiotic, amipurimycin. A new antibiotic amipurimycin, active against Pyricularia oryzae in vitro and in vivo, was isolated from the culture filtrate of Streptomyces novoguineensis nov. sp. The antibiotic was purified by a combination of ion-exchange and adsorption chromatography based on its amphoteric water-soluble characteristics. Its molecular formula was estimated to be C20H27approximately31N7O8-H2O. Characteristic maxima in the UV spectrum and signals in the PMR and CMR spectra were similar to those of 2-aminopurine 9-(beta-D)-riboside. These findings indicated that amipurimycin is a new nucleoside antibiotic and the first example of a natural product containing 2-aminopurine.


A new aminoglycoside antibiotic complex--the seldomycins. I. Taxonomy, fermentation and antibacterial properties. A soil isolate named Streptomyces hofunensis sp. nov. was found to produce seldomycin factors 1, 2, 3 and 5, new aminoglycoside antibiotics. Taxonomy of the producing organism, a study of cultural conditions for seldomycin production, and antibacterial activity of seldomycins are reported. Seldomycin factor 5 was the most active both in vitro and in vivo against gram-positive and negative bacteria.


A new aminoglycoside antibiotic complex--the seldomycins. II. Isolation, physicochemical and chromatographic properties. An antibiotic complex consisting of four components, seldomycin factors 1, 2, 3 and 5 was isolated from the fermentation broth of Streptomyces hofunensis sp. nov. by use of a cationic exchange resin. After silica gel column chromatography, the purified components were characterized as new aminoglycoside antibiotics by their physicochemical, chromatographic and antimicrobial properties.


A new aminoglycoside antibiotic complex--the seldomycins. III. The structures of seldomycin factors 1 and 2+. The structures of seldomycin factors 1 and 2 have been determined by consideration of chemical degradation and spectral properties. Factor 1, also known as XK-88-1, is shown to be 6-O-(2-amino-2-deoxy-alpha-D-xylopyranosyl) paromamine (1) and factor 2, also known as XK-88-2, is shown to be 4'-deoxy-neamine (2). Mass spectral evidence has been obtained that suggests the most probable structure for seldomycin factor 3, also known as XK-88-3, is 6'-amino-6'-deoxyseldomycin factor 1 (12).


Microbial reduction of the side-chain carbonyl of daunorubicin and N-acetyldaunorubicin. Microorganisms reduced the side-chain carbonyl of daunorubicin to yield 13-dihydrodaunorubicin (daunorubicinol; daunomycinol). This microbial transformation occurred under aerobic conditions in agitated baffled shake flasks incubated at 37 degrees C. The microorganisms were first grown in a medium which supported dense growth. Daunorubicin-HCl was then added. Following a period of incubation, broths were adjusted to pH 10.0 and extracted with chloroform. Daunorubicinol was recovered and purified from the chloroform extracts by preparative TLC. Identity of the daunorubicinol was established by TLC and spectroscopy (UV-vis, IR, NMR, MS, CD and ORD). N-Acetyldaunorubicin was likewise reduced microbially to N-acetyldaunorubicinol. N-Acetyldaunorubicinol appears to be a new compound which is yet to be tested for antitumor activity.


Syntheses of 6'-C-aminomethyl-3'-deoxyparomamines. The first synthesis of 6'-C-aminomethyl derivatives (6'-epimers) of 3'-deoxyparomamine is reported starting from 3'-deoxyparomamine by way of 6'-O-tritylation, O-acetylation, hydrolysis of the trityl group, conversion of the 6'-hydroxyl group into an aldehyde group, nitromethane condensation of the aldehyde group and catalytic reduction of the nitro group into an amino group.


Distribution of chloramphenicol acetyltransferase and chloramphenicol-3-acetate esterase among Streptomyces and Corynebacterium. Chloramphenicol-3-acetate esterase activity was detected in cell-free extracts of strains of Streptomyces venezuela, Streptomyces sp. and Streptosporangium viridogriseum var. kofuense which produced chloramphenicol and also Corynebacterium hydrocarboclastus which produced chloramphenicol analogs (corynecins). None of the cell-free extracts of chloramphenicol- or corynecin-producing strains possessed chloramphenicol acetyltransferase activity under conditions which avoided the influenced of the esterase activity. Among 20 strains examined that did not produce chloramphenicol, chloramphenicol acetyltransferase was detected in cell-free extracts of one strain of Streptomyces coelicolor Müller and one strain of S. fradiae ISP5063.


Mutational biosynthesis by idiotrophs of Micromonospora purpurea. II. Conversion of non-amino containing cyclitols to aminoglycoside antibiotics. A mutant of Micromonospora purpurea, which produces the gentamicin complex only when 2-deoxystreptamine is added to the fermentation medium, produces a new antibiotic complex, 2-hydroxygentamicin, when streptamine or 2,4,6/3,5-pentahydroxycyclohexanone is added to the fermentation medium. This mutant also produces the gentamicin complex when 2,4/3,5-tetrahydroxycyclohexanone is added to the fermentation medium. The C1 and C2 components of 2-hydroxygentamicin have broad spectrum in vitro antibacterial activity similar to the gentamicin C1 and C2 components, but with greater activity against some gentamicin-resistant strains.


Muscle glycogen repletion after high-intensity intermittent exercise. Six subjects exercised to exhaustion on a cycle ergometer at intensities corresponding to approximately 140% of their maximal aerobic power. Subjects attempted to pedal for 1-min intervals with 3-min rest periods between, and continued until 30 s of exercise could no longer be maintained. Venous blood was sampled for lactate and glucose analysis. Muscle biopsies were extracted from the quadriceps before and immediately after exercise and at 2-, 5-, 12-, and 24-h intervals thereafter for total glycogen analysis. Three subjects consumed a mixed controlled diet (approx. 3,100 kcal) during the 24 h after exercise, and three consumed the same diet plus an additional 2,500/kcal carbohydrate. Following exercise, glycogen concentration had dropped to a mean value of approximately 28% of its preexercise value. After 2 h, it had recovered to 39%, at 5 h to 53%, at 12 h to 67%, and at 24 h to 102% of its preexercise value, with no difference in resynthesis rate between the two groups. It was concluded that, following glycogen depletion through intense intermittent exercise, complete recovery to preexercise values may be accomplished within 24 h; and that within this time period, the rate of resynthesis cannot be accelerated by a higher than normal carbohydrate intake.


Hemodynamic response to graded exercise after chronic beta-adrenergic blockade. The effect of sustained beta-adrenergic blockade (BB) on the hemodynamic response to graded exercise has been studied in 31 patients with high blood pressure. Hemodynamic investigations were conducted during a control period and were repeated after 1 mo of BB. Similar readjustments were observed at rest and during submaximal and maximal exercise. No significant change occurred in maximal physical working capacity during beta blockade. This resulted from hemodynamic readjustments. Maximal exercise heart rate was reduced by 34%, and this was compensated for by a 31% enhancement in stroke index. Consequently cardiac index decreased by only 14%. In the Fick equation the decrease in cardiac index was further compensated by an increase of the total arteriovenous O2 difference of 8%, thereby maintaining O2 delivery to the tissues. At maximal exercise mean brachial artery pressure dropped 14.5%, while mean pulmonary artery pressure increased by 20%. It is concluded that the compensatory action of the stroke volume, resulting from the interaction of an increased preload and a decreased impedance, played a major role in the hemodynamic readjustments following chronic BB to maintain maximal working capacity.


Role of bulk fluid flow in protein permeability of the dog lung alveolar membrane. In five anesthetized, closed-thorax dogs, we measured net tracer albumin (RISA) uptake rate from an isosmotic buffer-filled lung lobe for 6 h; 3 h at each of two different alveolar RISA concentrations. We calculated the permeability coefficient assuming a two-compartment (alveolar fluid and plasma) diffusional model. In every dog the permeability coefficient decreased after the alveolar RISA concentration was increased. After freezing the lungs terminally, we found the fluid-filled lobes had extensive free interstitial fluid perivascular cuffs, indicating a third compartment filled by bulk flow. In separate experiments, we filled isolated lung lobes with buffer containing RISA and microsampled free interstitial fluid. The free interstitial fluid RISA concentration averaged 90% of airway concentration. The interstitium appears to fill by bulk flow through low-resistance channels. Tracer protein uptake from a fluid-filled lung lobe involves three fluid compartments. We postulate fluid and protein enter the interstitium by bulk flow along a hydrostatic pressure gradient, and protein then diffuses into plasma from the interstitium.


Ouabain-sensitive liver and diaphragm respiration in cold-acclimated hamster. The in vitro respiratory rates of liver and diaphragm from hamsters were compared before and after prolonged cold exposure (5 degrees C, 3-4 wk). In the presence or absence of glucose, respiratory rates were elevated in both tissues from the cold-acclimated hamsters, and these cold-induced increases were significantly reduced by ouabain. This ouabain inhibition is consistent with the hypothesis that cold exposure of these rodents stimulates the energy demands of the Na+/K+ transport system in liver and diaphragm, with these demands providing a driving force, at least in part, for respiration and accompanying cellular thermogenesis.


Spinal cord thermosensitivity and sorting of neural signals in cold-exposed rats. In the present study, data relevant to the presence or absence of sorting of neural signals were obtained by evaluating the thermal responses to spinal warming in the chronically prepared rat. Specifically, shivering activity and the rate of oxygen consumption (VO2) were measured in unanesthetized rats during cold exposure (10-16 degrees C). Warming the spinal cord at the level of T2 resulted in a significant decrease in shivering (P less than 0.001), without a significant change in VO2. The shivering response was reversed upon cessation of heating. These results are interpreted as indicating a direct influence of spinal cord temperature on shivering but not nonshivering thermogenesis in the rat. Similarly, in previous work with the rat, we have obtained data supporting hypothalamic receptor control of nonshivering but not shivering heat production. These findings are thus consistent with the suggestion that in the rat there occurs a sorting of neural signals. That is, impulses from the three thermoreceptor locations are not integrated in an identical manner for the control of shivering and nonshivering thermogenesis.


Role of ketone bodies in nonshivering thermogenesis in cold-acclimated rats. The role of ketone bodies (KB) in nonshivering thermogenesis was investigated in warm- and cold-acclimated rats with and without norepinephrine (NE) loads. NE-induced calorigenesis, as evidenced by changes in rectal temperature, was highly developed in cold-acclimated rats, but the levels of blood KB and free fatty acids (FFA) remained almost unaltered. In contrast, FFA turnover rate in cold-acclimated rats under NE load was much greater than in warm-acclimated rats. Similarly, turnover rate of beta-hydroxybutyrate estimated from decay curves of the endogenous substrate in functionally eviscerated rats was significantly higher in cold- than warm-acclimated rats. Perfused livers from cold-acclimated rats produced more KB than warm-acclimated ones. No significant effect of NE load was observed in either group. Quantitative analysis shows that the turnover rate of KB in vivo essentially equals the production rate in the perfused liver when no exogenous NE is added. In contrast, under constant NE infusion the turnover rate in vivo was almost double that of the perfused liver. These results indicate that KB are an energy source as important as FFA in nonshivering thermogenesis. It may be further surmised that increased KB production in vivo, particularly in the cold-acclimated state, is affected by factor(s) other than NE.


Metabolic and cardiovascular responses to norepinephrine in trained and nontrained human subjects. Forty young male adults, aged 18-30 yr, with maximal oxygen intake (VO2 max) varying between 35 and 76 ml-kg-1-min-1 were studied. The fasting subjects were perfused for 15 min with saline and thereafter for 30 min with norepinephrine (0.1 mug/kg per min). Blood variables were determined at the end of slaine perfusion, at 15 and 30 min during norepinephrine perfusion and 15 min after the end of NE perfusion. Trained subjects are characterized by lower basal plasma glucose and insulin concentration. NE perfusion produced a larger increase in plasma glucose in the trained subjects. Levels of insulin in the postperfusion period were smaller in the trained group. Serum free fatty acids (FFA) and glycerol increased less in the trained subjects during NE perfusion and this difference persisted in the post-perfusion period. During NE perfusion, blood lactate increased only in the nontrained subjects. Initial heart rate and blood pressure were lower in trained subjects but, during NE perfusion, elevated levels of blood pressure and decreased levels of heart rate were comparable in both groups. Because of similarities in responses to exercise and to NE in trained subjects, the results of the present study suggest a possible role for NE in exercise training.


Fetal O2 changes in response to hypoxic stress: a mathematical model. We developed a mathematical model to compute the time course of PO2 changes in fetal blood vessels during hypoxia. The model represents the circulation and major organs as a system of paths and nodes. We calculated outflow O2 for a path from its inflow O2 content and its distribution of transit times. The O2 content at a given node equals the flow-weighted sum of O2 delivered by different paths. Placental O2 transfer and organ O2 consumption are related to their arterial PO2 levels. We simulated the effects of uterine contractions with Gaussian-shaped decreases in placental O2 transfer. Increasing the intensity and increasing the duration of hypoxic episodes have comparable effects. Liver O2 consumption decreases more than that of other organs during hypoxic episodes. At the peak of a contraction, fetal systemic PO2 values decrease only about one-fourth as much as those in end-capillary placental blood. This indicates that despite rapid circulation times, fetal O2 reserves protect it against severe, short term hypoxia.


Regulation of arousal from hibernation by temperature in three species of Citellus. The frequency of arousal from periods of hibernation of Citellus lateralis, C. tridecemlineatus, and C. columbianus was demonstrated to be regulated by environmental (and therefore body) temperature over the range of 2-12 degrees C in a log-linear relationship. The slopes of the lines of regression for all three species were not significantly different from one another. Durations of the periods of hibernation for the considerably larger C. columbianus, however, were significantly longer at all levels of temperature than those for the similar-sized C. lateralis and C. tridecemlineatus. The mechanism(s) regulating the duration of the period of hibernation was considered to be concerned with some aspect of metabolism and probably the same for all three species.


Mechanism of change in pulmonary shunt flow with hemorrhage. In the presence of regional atelectasis, hemorrhage increases the pulmonary shunt fraction (Qs/Qt). We wanted to determine if the increase in Qs/Qt occurs due to inhibition of hypoxic vasoconstriction or due to hydrostatic effects associated with a collapsed lung and decrease in cardiac output. We compared the effects of hemorrhage on flow to a hypoxic lung (Qh) produced in two ways in anesthetized dogs. In six dogs atelectasis of one lung was produced by bronchial occlusion and in a second group of six dogs one lung was ventilated with 100% nitrogen. The other lung in each group was ventilated with 100% nitrogen. The other lung in each group was ventilated with 100% O2. Hemorrhage (20 ml/kg) had no effect on Qh/Qt in dogs with a nitrogen-ventilated lung suggesting that hemorrhage did not inhibit hypoxic vasoconstriction. In contrast, hemorrhage caused a marked increase in Qh/Qt in dogs with a collapsed lung because the flow to the inflated ventilated lung fell, while flow to the collapsed lung did not change. The increase in Qs/Qt in the dogs with atelectasis can be explained solely on the basis of mechanical effects produced by the differences in lung heights and alveolar pressures between ventilated and atelectatic lung.


Lung lipid metabolism after 7 days of hydrocortisone administration to adult rats. Pathogen-free rats were given either hydrocortisone (4 mg) or saline by intraperitoneal injection twice daily for 7 consecutive days. Lung weight, body weight, DNA, and total phosphatidylcholine content in lungs were equal in the saline and hydrocortisone groups. Lungs of rats receiving hydrocortisone had 23% more saturated phosphatidylcholine (P less than 0.001) and 7% less unsaturated phosphatidylcholine (P greater than 0.05). When tissue slices of these lungs were incubated with radioactive glycerol and palmitate, the incorporation of radioactivity into saturated phosphatidylcholine from animals given hydrocortisone was significantly higher (P less than 0.001 and P less than 0.05, respectively). Incorporation of radioactivity from lysophosphatidylcholine into phosphatidylcholine was 10 times greater than from glycerol or palmitate, but hydrocortisone had no effect. These results suggest that lysophosphatidylcholine may be an important precursor for phosphatidylcholine synthesis, especially saturated phosphatidylcholine, and hydrocortisone may lead to increased de novo synthesis of phosphatidylcholine.


Influence of insulin on amino acid uptake by lung slices. We examined the transport of amino acids by rat lung slices using mainly 14C-labeled alpha-aminoisobutyric acid ([14C]AIB) as a nonmetabolized amino acid. We found that [14C]AIB is accumulated by the lung in an energy-dependent fashion against a concentration gradient. The uptake is saturable, stereospecific, and follows Michaelis-Menten kinetics suggesting enzyme or carrier mediation across the plasma membrane. Insulin increases the uptake of [14C]AIB and insulin plus glucose increases its uptake even more. The diffusion constant (KD) in the presence of glucose, insulin, or glucose plus insulin is the same, 0.29 h-1; the Vmax is also the same, 83.0 mmol-1-1-h-1, under these three conditions. The apparent Km is 14.0 mM with glucose, 9.0 mM with insulin, and 4.0 mM in the presence of glucose and insulin. We conclude that the uptake of [14C]AIB is increased by insulin, and insulin plus glucose, and, based on this kinetic analysis, this is due to an increased affinity of the transport sites for AIB (decreased Km, unchanged Vmax, and KD).


Effects of intravenous histamine on pulmonary mechanics in nonasthmatic and asthmatic subjects. Histamine is thought to be one of the primary mediators of the airways response in allergic asthma. We have studied in detail in nonasthmatic and asthmatic volunteers the effects of its intravenous infusion on pulmonary mechanics. The doses used were similar in both groups and limited by vascular rather than pulmonary effects. Measurements were made of lung resistance, the subdivisions of lung volume, maximal flow, density dependence, lung elastic recoil and dynamic compliance. The only mean group changes were a decrease in dynamic compliance and increase in frequency dependence of compliance in the nonasthmatics. Although in nonasthmatic individuals, statistically significant changes occasionally occurred in some parameters, the response was variable on repeated studies and sufficiently small to be of questionable physiological significance. Some of the asthmatics developed larger changes, but these only occurred in those individuals whose preinfusion pulmonary function was abnormal. Our results suggest the possibility that altered prechallenge pulmonary function rather than an unusual sensitivity to histamine may determine the greater responses observed in asthmatics.


Lung elasticity and airway dynamics in Peruvian natives to high altitude. To evaluate the role of genetic and environmental factors in the genesis of large lungs in high-altitude natives, we measured forced vital capacity (FVC), static lung pressure-volume characteristics and maximum expiratory flow-volume loops in 17- to 20-yr-old Peruvian natives to 3,850 m (highlanders) and 800 m (lowlanders). Forced vital capacity was 5.11 +/- 0.64 liters in highlanders, 116 +/- 11% of predicted; and 3.73 +/- 0.32 liters in lowlanders, 84 +/- 7% of predicted. Lung elastic recoil at functional residual capacity and at total lung capacity, and size-corrected pressure volume curves were similar in the two groups. Despite the larger volumes in highlanders, density-corrected maximum flow rates were similar in highlanders and lowlanders, and flow expressed in FVC'S-S-1 was less in highlanders. Upstream conductance at 50% FVC expressed in fvc's-s-1-cmH2O was 0.094 +/- 0.023 in highlanders vs. 0.147 +/- 0.050 in lowlanders. Flow rates did not change in sojourners to altitude, suggesting that the lower values of highlanders were due to anatomic factors. These findings suggest that airways, which form in fetal life, do not participate in adaptation to altitude, and that the large lungs of highlanders result from postnatal environmental hypoxic stimulation of lung growth. Our results illustrate the importance of "dysynaptic" lung growth in determining patterns of adult lung function.


Fluid shifts during thermal stress with and without fluid replacement. Six unacclimatized men rested for 4 h in a hot, dry environment (Ta 50 degrees C, Tw 26 degrees C; wind speed 0.5 m/s) without fluid replacement (DH). Another group of six men were exposed to the same thermal stress, replacing evaporative fluid loss with warm 0.1% NaCl solution (FRP). Total grams of circulating hemoglobin, determined by CO immediately prior to and again during the last minutes of heat exposure, increased an insignificant 1.6 and 1.3% during DH and FRP, respectively. With DH, body weight loss of 2.6% was accompanied by a 7.8% reduction in calculated plasma volume (PV). Even when body weight was maintained (FRP), PV decreased 2.9% during the heat exposure. Total circulating serum protein did not change as a result of the heat stress with either DH or FRP. In a test-retest series of experiments on four men, DH was not detrimental to sweat rate which averaged 251 g/m2-h compared with 239 g/m2-h with FRP. It was concluded that hemodilution is not a general response to acute heat exposure, and the disproportionately large reduction in PV during thermal dehydration was confirmed.


Cardiac responses to moderate training in rats. Left ventricular (LV) performance was studied in 30 rats conditioned by swimming and in 27 sedentary controls. Hearts were studied in the open chest both during sinus rhythm and during atrial pacing. Afterload was increased by occlusion of the aorta. Sinus rates were 328 beats/min in sedentary and 300 in conditioned animals (P less than 0.01). During ejection (E), peak left ventricular systolic pressure (PLVSP) and maximum LV dP/dt were significantly higher in conditioned than in sedentary animals. When data from hearts matched for similar heart rates were compared, increased LV performance was still seen during aortic obstruction in conditioned animals. Performance at pacing rates up to 400 beats/min was similar in hearts from conditioned and sedentary animals. At pacing rates above 400 beats/min cardiac performance was less in sedentary than in the conditioned animals. These data illustrate that although base-line cardiac performance may be the same in rats conditioned by a moderate swimming program and in sedentary animals, cardiac reserve is improved by the swimming program.


Interaction of lung volume and chemical drive on respiratory muscle EMG and respiratory timing. The effect of increased FRC on the change in respiratory muscle electrical activity (EMG) and the duration of inspiration (Ti) and expiration (Te) produced by increases in chemical drive (i.e., progressive hypercapnia and isocapnic hypoxia) was assessed in 15 anesthetized, spontaneously breathing dogs. FRC was raised by applying continuous positive pressure (4 and 8 cmH2O) to the airway. Progressive hypercapnia and hypoxia were produced by rebreathing techniques. At any PCO2 or PO2, increases in FRC decreased diaphragm EMG (D); increased abdominal muscle EMG (AB); and prolonged Te without affecting Ti. The effect of increased FRC on D, AB, and Te diminished as PCO2 increased or PO2 decreased. The effect of sustained increases in lung volume in the absence of phasic changes was assessed by performing airway occlusion for a single inspiration during rebreathing at both control and increased FRC. The effects of increases in FRC were present during airway occlusion but were eliminated by vagotomy. We conclude, therefore, that tonic vagal stimulation produced by increases in FRC modified the change in respiratory muscle electrical activity and timing produced by increasing chemical drive.


Effects of cold exposure and dehydration on renal function in black-tailed prairie dogs. Black-tailed prairie dogs (Cynomys ludovicianus) were deprived of food and water for several weeks during the fall and winter in a cold-room hibernaculum (Ta 5-8 degrees C), and for several days at room temperature during the summer. Body temperatures (Tb) were determined periodically in nine animals by radiotransmitters implanted in the abdomen. Animals deprived of food and water in the summer were killed when maximum urine concentration was achieved. Eight animals in the winter were active when killed after 7-35 days in the hibernaculum with Tb between 18 and 36 degrees C. Five animals that became torpid periodically in the winter were killed after 19-42 days in the hibernaculum when their Tb indicated torpor (Tb less than 13 degrees C). Active animals in the summer and winter possessed pronounced renal corticomedullary urea and sodium concentration gradients. Torpid animals lacked these gradients and had lower urine and plasma osmotic concentrations than active animals. Plasma urea values and terminal osmolal U/P ratios were lowest in torpid prairie dogs.


Ventilatory and gas exchange dynamics in response to sinusoidal work. The dynamic relationships between ventilation and gas exchange variables during exercise were determined utilizing frequency analysis techniques. Five subjects exercised on a cycle ergometer for 30 min at work rates which fluctuated sinusoidally between 25 W and 80% of the anaerobic threshold at sinusoidal periods of 0.7, 1,2,4,6, and 10 min. VE, VCO2, VO2, and HR were computed and displayed breath-by-breath. From these and steady-state response data, digital computer routines extracted amplitude and phase relations between each variable and the perturbing work load. These response characteristics were well described by first-order linear dynamics with time constants for VE, VCO2, VO2, and HR averaging 1.4, 1.2, 0.8, and 0.8 min, respectively. The time constants of VE and VCO2 were strongly correlated among subjects (r = 0.97). Further, there was no evidence that neural afferents from the exercising limbs induced fast components in the ventilatory response to these forcings. These results are consistent with the hypothesis that exercise hyperpnea is linked to metabolism via carbon dioxide production.


Temperature and per se hydrostatic pressure reversal of pentobarbital anesthesia in fish. Investigations have been made into the effects of temperature (between 10 and 19 degrees C) on the one hand, and hydrostatic pressure per se (101 ATA) on the other, on recovery time from anesthesia (pentobarbital) in a fish (Salmo gairdneri R). At each of the two pressures studied (1 and 101 ATA) the rise in temperature shortens the recovery period, after a treatment by a constant dose of pentobarbital. Under hyperbaric pressure conditions (101 ATA) the duration of anesthesia is lower than the control value obtained at barometric pressure, whatever the temperature. The analysis of the results shows a similarity between the effects of temperature and pressure on the recovery from pentobarbital anesthesia on the one hand and on chemical kinetics on the other. The results support the hypothesis of the direct action of hydrostatic pressure per se at molecular level.


Carotid and aortic chemoreceptor function in the rat. Carotid and aortic chemoreceptor function was studied in normal Wistar rats. Sodium cyanide, lobeline HCl, and doxapram HCl in the doses of 2-400 mug/kg injected into the external carotid artery stimulated respiration significantly. Injections of the drugs into the ascending aorta produced less effects which were abolished after section of the carotid sinus nerves. The drugs produced a significant increase in the carotid sinus nerve activity but failed to do so in the aortic depressor or recurrent laryngeal nerves. These results indicate that the carotid chemoreceptor function in Wistar rats in normal while functional aortic chemoreceptors are absent in them.


Effect of chronic exposure to cold on some responses to catecholamines. An objective of these studies was to test the responsiveness of cold-adapted (8 wk, 5 degrees C) rats to a specific beta-adrenergic agonist. Twenty-four hours after removal from cold, increases in tail skin temperature (Tsk) and colonic temperature (Tco) were measured for 2 h in air at 25 degrees C following subcutaneous (sc) administration of 28, 70 or 136 mug d,l-isoproterenol sulfate dihydrate/kg body wt to restrained male rats. Cold-adapted rats responded to each dose of isoproterenol with greater increases in Tsk than controls. Tco of both groups increased at the two highest doses, but cold-adapted rats showed a greater rise. Thus, cold-adapted rats showed greater metabolic (Tco) and vascular (Tsk) responsiveness to the beta-adrenergic agonist, isoproterenol, than nonadapted controls. No effect of the alpha-adrenergic agonist, phenylephrine (50 or 100 mug/kg body weight, sc), was observed on Tco or Tsk. A second objective was to study the tension developed by aortic smooth muscle rings of cold-adapted and control rats both during stimulation of alpha-adrenergic receptors by norepinephrine and membrane depolarization by KCl. Adaptation to cold air appeared to suppress alpha-adrenergic responsiveness in aortic segments but did not alter responsiveness to KCl. This suggests an unchanged contractile mechanism in aortic rings of cold-adapted rats and a reduced responsiveness either at the level of the alpha-receptor or at a site immediately beyond.


Linear increase in aerobic power induced by a strenuous program of endurance exercise. Eight subjects exercised for 40 min/day, 6 days/wk for 10 wk. For 3 days/wk they performed six 5-min intervals of bicycling on an ergometer against a resistance that elicited VO2 max, separated by 2-min intervals of exercise requiring 50-60% of Vo2 max. On the alternate 3 days, they ran as far as they could in 40 min. Our purpose was to obtain information regarding the time course and magnitude of the increase in Vo2 max and endurance that occur in response to strenuous exercise when the training stimulus is kept approximately constant relative to maximum aerobic capacity. Average Vo2 max increased 5% (P less than 0.05) during the 1st wk. Endurance, Vo2 max, and time to attainment of peak heart rate all increased linearly during the 10 wk. The average weekly increase in Vo2 max was 0.12 l/min. The total increase in Vo2 max averaged 16.8 ml/kg per min (44%). Four of the eight subjects attained Vo2 max levels approaching or exceeding 60 ml/kg per min. It appears from these results that aerobic work capacity can increase more rapidly and to a greater extent in response to training than has generally been thought.


Thermoregulatory heat production in man: prediction equation based on skin and core temperatures. The relationship of metabolic rate to skin and core temperatures was measured in eight subjects cooled in 10 degrees C water. Reference core temperatures, based on the tympanic and rectal sites, were 41.0 and 41.4 degrees C, respectively. Reference mean skin temperatures were 41.8 and 42.2 degrees C when determined in relation to tympanic and rectal temperatures, respectively. These results enabled construction of equations for predicting the steady-state metabolic rate (MR, W-kg-1) of nonexercising man according to thermal inputs from the skin (Tsk) and core (Tty or Tre) MR = 0.0356(Tsk - 41.8)(Tty - 41.0) or, MR = 0.0314(Tsk - 42.2)(Tre - 41.4). Each equation was limited by the low level of basal metabolic rate (1.1 W-kg-1) and by the high level of peak metabolic rate (6.4 W-kg-1). Evaluation of these equations showed satisfactory agreement with previous observations and concepts on the regulation of heat production in man.


Effects of hyperoxia on leg blood flow and metabolism during exercise. These experiments were designed to investigate the effects of O2 breathing on limb blood flow and metabolism during exercise. Six subjects took part in the study. Four subjects breathed air or 100% O2 while pedaling a Krogh bicycle at 150 W (55-70% of maximal aerobic capacity). Two subjects breathed either 60% or 100% O2 while working at a power output at or slightly in excess of their maximal aerobic capacities. The major findings of the study were 1) leg blood flow is reduced during exercise when comparing hyperoxia with normoxia; 2) VO2 of the exercising limb is not different during hyperoxia; 3) O2 delivery to the leg (the product of blood flow and arteriovenous O2 difference) is not significantly different in the two conditions; and 4) blood pressure is not markedly affected in the experiments at 150 W. Since BP was not different during hyperoxia, at a time when flow was reduced by 11%, this suggests an increased resistance to flow in the exercising limb. In general, these findings are consistent with those reported for the in situ dog muscle but are at variance with results of experiments with humans, especially the reports indicating substantial increases in O2 uptake during hypertoxic conditions.


Mechanisms responsible for decreased glomerular filtration in hibernation and hypothermia. Mechanisms underlying the elimination or marked depression of renal function in hibernation and hypothermia were investigated through measurements of blood pressure, heart rate, red blood cell and plasma volumes, and relative distribution of cardiac output. Hamsters (Mesocricetus auratus) were made hypothermic (rectal temperature (Tre), 7 degrees C) by exposure to helox and cold, or permitted to hibernate with several weeks of cold exposure (Ta approximately 5 degrees C). Mean arterial pressure, 120 Torr in normothermic control animals, demonstrated a 55% and 60% decrease during hibernation and hypothermia, respectively. As the animals rewarmed from hypothermia or aroused from hibernation, blood pressure increased rapidly at 8-12 degrees C, more gradually at 12-17 degrees C, and plateaued thereafter. Blood pressure rapidly returned to near control levels whereas heart rate remained at less than one-half control value at the highest temperature examined. Red blood cell volume, 26.2 +/- 0.6 ml/kg body wt in the control animals appeared unaffected by hypothermia. Plasma volume, by contrast, decreased from control values of 33.0 +/- 0.8 to 21.3 +/- 0.6 ml/kg body wt in hypothermia, a decrease of approximately 35%. Distribution of cardiac output to various organs in hibernation and hypothermia followed a similar pattern. Relative flow to the heart, lung, diaphragm, and brown fat increased while the fraction distributed to the visceral organs appeared to decrease. The normothermic control kidney received approximately 16% of the cardiac output while the hibernating and hypothermic kidneys received approximately 10% and 6%, respectively. The data are discussed in terms of the determinants of glomarular filtration rate and explain, in part, the elimination or marked reduction in renal function observed in depressed metabolic states.


Functions of selected biochemical systems from the exercised-trained dog heart. Mitochondria and sarcoplasmic reticulum (SR) fractions were isolated from exercised-trained (E-T) and sedentary control dog hearts. Measurements of mitochondrial respiratory functions indicated no changes in energy-producing (ATP synthesis) capacity in mitochondria from E-T compared to control dog hearts. However, the ability of isolated mitochondria from E-T hearts to retain accumulated calcium was markedly decreased compared to controls. Inhibition of mitochondrial rates of calcium uptake with the inhibitor, ruthenium red, revealed fewer binding and/or transport sites in mitochondrial membranes from exercised-trained heart preparations. ATP-dependent binding (- oxalate) and uptake (+ oxalate) of calcium by SR preparations from E-T hearts were unchanged compared to controls. In contrast, significant differences in the rates of release of bound calcium were found in SR isolated from E-T hearts. Total myocardial protein, nucleic acids, and connective tissue levels were unchanged in E-T hearts compared to controls. The results suggest subtle changes are occurring in the energy-utilizing mechanism(s) involving calcium transport of the myocardial cell during exercise training. These changes may be related to alterations in the performance of the exercised-trained heart.


Phagocytosis: quantification of rates and intercellular heterogeneity. We present a method which permits analysis of phagocytic behavior in small samples of macrophages. Both overall phagocytic kinetics and intercellular variability in particle uptake were measured. Macrophages lavaged from Syrian golden hamster lungs were incubated (5 min at 37 degrees C) with 2- to 4-mum plastic particles at 4.6, 6.9, 9.1, and 16.0 X 10(6) part./ml. Harvested macrophages ranged from 0.6 to 4.0 X 10(6) cells per animal. To concentrate the cells and separate them from cell-free substrate after termination of phagocytosis, 30-60% of each flask's contents were centrifuged (400 X g, 20 min) atop a Metrizamide subphase (34% wt/vol, rho = 1.18). Cells were collected from the interface, fixed, and visually scored for number of particles phagocytized. Phagocytic rates followed Michaelis-Menten kinetics with Vmax = 0.63 +/- 0.18 (SEM) part./viable cell per min and Km = 8.5 +/- 2.7 (SEM) X 10(6) part./ml. In every case (23/23 flasks from 6 animals) particle uptake exhibited greater (P less than 0.01) cell-to-cell variability in avidity for substrate than the Poisson distribution would predict from mean number of particles phagocytized per viable cell.


Mechanical response of pulmonary artery under aerobic and hypoxic conditions. The present study demonstrates the reactivity of isolated main pulmonary artery (MPA) from guinea pigs and rats to two vasoactive drugs, norepinephrine (NE) and histamine (H), in substrate-rich and substrate-free medium, under both aerobic (PO2 = 95 +/- 0.5 Torr) and hypoxic conditions (PO2 = 30 +/- 1 Torr). The sensitivity of MPA from guinea pigs to NE and H during aerobic conditions is not significantly affected by the absence of substrate in the experimental medium. Furthermore, it is demonstrated that in the substrate-rich experimental medium (5.5 mM glucose), the reactivity of MPA from guinea pigs to NE and H is not significantly affected by acute hypoxia as compared with the response of MPA during aerobic conditions. These experiments contrast with data obtained when substrate is absent from the experimental medium. The dose-response curves of MPA from guinea pigs to NE and H under this condition were significantly blunted during hypoxia. Following the completion of the dose-response curves during aerobic conditions, with both NE and H, spontaneous mechanical activities were seen in the guinea pig MPA. On the other hand, it was demonstrated that during aerobic and hypoxic conditions MPA's isolated from rats exhibit no physiological response to histamine even when administered in the dose required to produce the maximal response in MPA isolated from guinea pigs. The sensitivity of MPA from rats to NE during aerobic conditions is not significantly affected by the absence of substrate in the experimental medium. However, when the preparation was exposed to hypoxia, the presence of substrate failed to maintain the reactivity of MPA to norepinephrine. In addition, MPA isolated from rats demonstrated a smaller contractile response to NE than those from guinea pigs. Furthermore, no spontaneous mechanical activities were observed after norepinephrine or histamine administration. The present study, in addition to pointing out species differences, shows the important role of exogenous substrate in maintaining the reactivity of pulmonary vascular smooth muscle during hypoxia.


Combined effects of breathing resistance and hyperoxia on aerobic work tolerance. Effects of three curvilinear inspiratory resistances (R1, R2, R3) on the cardiorespiratory responses of seven well-trained men during incremental cycling tests to exhaustion were studied by comparison to the low resistance R0 (at 1 l/s, R0 = 0.2; R3 = 6.5 cmH2O-s/l). Submaximal VO2 and the gas exchange anaerobic threshold (AT) were not affected by increasing resistance. Although maximal work rates were not significantly changed, highly significant reductions were observed for VE (R0 = 166.3; R3 = 99.7 l/min BTPS), VO2 max (R0 = 4,26; R3 = 3.74 l/min), HR (R0 = 185; R3 = 176 beats/min), and endurance (R0 = 17.3; R3 = 15.5 min) suggesting that aerobic work tolerance was dependent on ventilatory capacity. In additional tests removal of R3 at exhaustion abruptly increased VE and VO2, and permitted work to continue. Ventilation and work tolerance were therefore limited by R3 before the legs fatigued. Breathing 35% O2 against R3 produced significant, although small, increases in AT, VO2 max, peak HR, and endurance while decreasing the hyperventilatory response to work above AT. Thus, aerobic work tolerance reduced with high inspiratory resistance was partly restored by moderate hyperoxia, apparently because the ventilatory limit was delayed.


Antirotatory device permitting free movement in animals with several catheters. A very simple antirotatory device permitting free movement of small and middle-sized animals is described. It allows the performance of chronic experiments in animals bearing several catheters and wires for perfusion or recording purposes. A set of eight metal plates, articulated end to end by small brackets, sets the animal's head at a fixed point on the ceiling of the cage. This device has been perfectly tolerated by rats during 2 wk to 3 mo.


Measurement of mucociliary clearance from the trachea of conscious and anesthetized rats. A technique is described whereby particles are injected as a suspension into the trachea of conscious rats. Tracheostomy and cannulation are performed under halothane, but the particles are injected 10 min after recovery. Clearance of BaSO4 particles labeled with 133Ba was determined with a collimated slit gamma-counter, either aligned with the region of particle deposition or used to scan along the animal. Compared with clearance up the trachea in conscous rats, the effect of halothane, thiopental and pentobarbital was to inhibit clearance. With halothane, clearance was resumed at the control rate 0-3 min after anesthesia lasting 7-9 min.


Anodal block of medullated cardiopulmonary vagal afferents in cats. In anesthetized cats examination was made of the conditions under which the application of DC current to a mixed nerve permits determination of whether a reflex response is mediated by medullated or nonmedullated afferents. The following precautions should be observed. The nerve must be stripped of its sheath and bleeding avoided. The anode should be 6 mm distant from the cathode. The temperature of the mineral oil surrounding the nerve should be 30-32 degrees C to avoid activation of nonmedullated fibers during the block. The major limitation is a time- and frequency-dependent block of nonmedullated fibers which makes the technique suitable only for differentiating between medullated fibers and nonmedullated fibers with low frequency traffic. Observing these criteria, anodal block of the cervical vagus in sino-aortic denervated cats resulted in a mean rise in aortic pressure of 8 Torr; subsequent cold block caused a further mean rise of 30 Torr. Thus 80% of the total increase in aortic pressure could be ascribed to interruption of vagal C-fiber activity.


Evaluation of rapid partitioning cleanup method for pesticides. A partitioning cleanup method performed in test tubes with small solvent volumes, and using syringes instead of separatory funnels for phase separations, has been tested for potential as a general cleanup method for a variety of pesticidal compounds. More than 50% recoveries were obtained for most of the 60 compounds tested, even though minimal manipulation rather than maximum recovery was the aim. Since the recoveries usually are constant, procedural losses can be compensated  for by correction factors.


Trace analysis of zearalenone and/or zearalanol in animal chow by high pressure liquid chromatography and gas-liquid chromatography. An analytical method is described for determining residues of the estrogens zearalenone and/or zearalanol in animal chow at levels as low as 10 ppb. The chow is extracted with methanol and cleaned up by a 3-step procedure employing a Sephadex LH-20 column, liquid-liquid partitioning at pH 13 and 8.3, and a silica gel column. Residues of the 2 compounds, separated on silica gel, are assayed by using high pressure liquid chromatography with ultraviolet detection. Additional data are also included concerning p-values of the compounds in several solvent systems, Rf values from thin layer chromatography with 9 solvent systems, solubilities in 3 solvents, and a procedure for preparing their pentafluoropropionyl derivatives for analysis by electron capture gas-liquid chromatography.


Butyl ester preparation for gas-liquid chromatographic determination of fatty acids in butter. Butter oil is extracted from butter with hexane, using a rolling boil technique. The resulting 10% butter oil in hexane is converted to butyl esters, using butanol in place of methanol as specified in the official final action method, 28.063-28.067. A water wash and centrifuge technique is used to remove butanol from the n-hexane-butyl ester solution. Conversions to butyl esters are quantitative by the boron trifluoride, sulfuric acid, and sodium butoxide procedures. When methyl ester methods are used, there are serious losses of the short chain volatile and water-soluble acids, making the methyl ester procedures questionable for determining the authenticity of milk fat.


Improved procedure for quantitative determination of aflatoxins in corn and wet milled corn products. Aflatoxins are extracted from the sample by high-speed blending with aqueous acetone, purified with ammonium sulfate, and partitioned into benzene. The benzene extract is purified on a silica gel-alumina column by washing with benzene-acetic acid and ether-hexane, and eluted with methylene chloride-acetone. The eluate is evaporated, redissolved in benzene-acetonitrile, and spotted on a thin layer chromatographic plate for resolution and quantitative determination against standards.


Modified Sutter's arginine dihydrolase medium for Pseudomonas speciation. Sutter's arginine dihydrolase medium has been modified to obtain maximum yields of arginine dihydrolase. Bromothymol blue is the indicator for alkalinity in Sutter's medium. By adding glucose and lowering the pH of the medium, more positive reactions were obtained in 24 hr as well as a sharper color contrast to the base medium which facilitated reading the reactions. The modified Sutter's medium was included in the routine biochemical schema for speciation of pseudomonads isolated from cosmetic products. Of the 706 strains belonging to the 5 species isolated form cosmetic products, only one false negative reaction was obtained with a strain of Pseudomonas putida. The medium was also used for the biochemical characterization of 122 Pseudomonas sp. isolates.


Biochemical localization of the alkaline phosphatase of Bacillus licheniformis as a function of culture age. Biochemical localization of the enzyme as a function of age of cell culture showed the alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity of Bacillus licheniformis MC14 predominantly in the particulate cell fraction in early- and mid-log cells. However, in late-log and stationary cells, increasing amounts of activity were found in the soluble fraction of lysed cells. Upon protoplast formation of these cells, the activity was released into the soluble fraction. No alkaline phosphatase activity was found in either the cytoplasmic fraction or in the cell medium during any phase of cell growth. The soluble fraction released on protoplast formation that contained alkaline phosphatase activity showed immunological cross-reactivity with antibody to the purified heat--salt-solubilized membrane alkaline phosphatase (F. M. Hulett-Cowling and L. L. Campbell, 1971). Theparticulate membrane fraction containing a firmly associated alkaline phosphatase also showed similar cross-reactivity. Further, the effectiveness of nonionic detergents, ionic detergents, bile salts, and various concentrations of magnesium and sodium as solubilizing agents for this membrane-bound alkaline phosphatase was investigated. Hexadecyl pyridinium chloride (0.03 M) and magnesium and sodium salts (above 0.2 M) were effective solubilizing agents. The substrate specificities of the various fractions were determined and compared to the substrate specificities of the purified membrane alkaline phosphatase.


Morphological changes associated with novobiocin resistance in Bacillus licheniformis. Spontaneously occurring novobiocin-resistant (Nov) mutants of Bacillus licheniformis ATCC 9945, resistant to low levels of novobiocin (15 mug/ml), were isolated with a frequency of 3 in 106 organisms. Such isolates grew well, but nearly all exhibited consistent plleiotropic alterations in colonial and cell morphologies. One mutant, nov-12, grew as chains of unseparated but clearly distinct daughter cells in the absence of novobiocin in liquid culture. When novobiocin was present, nov-12 grew as very long "filaments" which were, however, septate. Septa formed in the presence of the antibiotic were normal, except that no annular clevage of the septal wall was observed. Septa were also irregularly positioned along the filament. These observations were compared with previous findings on the effects of novobiocin and novobiocin resistance described for other organisms. It was concluded that the primary action of novobiocin might differ in gram-positive and gram-negative organisms. However, when the low-level novobiocin sensitivity, normally associated with gram-positive organisms, was genetically abolished in Nov strains of B. licheniformis they became susceptible to an action of novobiocin more analogous to that found for gram-negative organisms. The morphological alterations associated with the Nov phenotype in this organism, together with observations in other organisms, indicate that novobiocin resistance might be generally useful in the search for mutants of gram-positive organisms with altered cell walls.


Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant. nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.


Distribution of lipopolysaccharide and the detection of a new subfraction in the cell envelope of a marine pseudomonad. The three outer layers of the cell envelope of marine pseudomonad B-16, the loosely bound outer layer, the outer membrane, and the periplasmic space layer, are the only ones containing appreciable amounts of both lipid and carbohydrate. These layers and a fraction released into the medium during growth of the cells were examined for the presence of common antigens by double immunodiffusion using anti-whole serum. Each of the layers, the medium fraction, and lipopolysaccharide (LPS) isolated from the organism were shown to contain two or more diffusible components showing reactions of identity. Thus LPS is found in each of the three outer layers of the cell envelope of this gram-negative bacterium. The periplasmic space layer was found to contain a fraction accounting for 20% of the dry weight of the layer, which was sedimentable at 30,000 x g and contained lipid, protein, and carbohydrate. Double-immunodiffusion tests indicated that the fraction contained at least one of the two antigens present in isolated LPS. A  particulate material was released by the cells during growth which gave a positive test for 2-keto-3-deoxyoctulosonic acid and cross-reacted serologically with LPS.


Bacterial parasite of a plant nematode: morphology and ultrastructure. The life cycle of a bacterial endoparasite of the plant-parasitic nematode Meloidogyne incognita was examined by scanning and transmission electron microscopy. The infective stage begins with the attachment of an endospore to the surface of the nematode. A germ tube then penetrates the cuticle, and mycelil colonies form in the pseudocoelom. Sporulation is initiated when terminal cells of the mycelium enlarge to form sporangia. A septum within each sporangium divides the forespore from the basal or parasporal portion of the cell. The forespore becomes enclosed by several laminar coats. The parasporal cell remains attached to the forespore and forms the parasporal microfibers. After the newly formed spores are released into the soil, these microfibers apparently enable a mature spore to attach to the nematode. These results indicate that the endoparasite is a procaryotic organism having structural features that are more common to members of Actinomycetales and to the bacterium Pasteuria ramosa than to the sporozoans or to the family Bacillaceae, as previous investigatios have concluded.


Effects of incident light levels on photosynthetic membrane polypeptide composition and assembly in Rhodopseudomonas sphaeroides. Cells of Rhodopseudomonas sphaeroides were grown anaerobically with incident light levels ranging between 4,500 and 400 footcandles (ca. 48,420 and 4,304 lux). Cells grown with the higher light levels had lower contents of total bacteriochlorophyll and incorporated L-[U-14C]leucine into membrane protein at higher rates than cells grown with lower light levels. The former cells also contained relatively lower amounts of light-harvesting membrane polypeptides as compared with the latter cells. In contrast, the relative amounts of reaction center membrane polypeptides were approximately the same with varying incident light levels. The relative amounts of these membrane polypeptides were correlated with differences in rates of synthesis and assembly of the polypeptides into membrane by measuring the rates of incorporation of L-[U-14C]leucine into the membrane-bound polypeptides. No significant differences in rates of turnover of these polypeptides were detected under the varying incident light levels as measured in pulse-chase radioactive labeling experiments.


Purification of flagellar cores of Vibrio cholerae. A procedure is described for the purification of the cores of flagella sheared from Vibrio cholerae. V. cholerae is a monotrichous organism whose flagellar core (FC) is enclosed within a sheath. The purification procedure consists of several cycles of differential centrifugation and cesium chloride density-gradient ultracentrifugation in the presence of a neutral detergent, Triton X-100. Purity of the FC preparations is assessed by electron microscopy, polyacrylamide gel electrophoresis, and chemical analysis. The purified FC preparations are devoid of flagellar sheaths and free from detectable cell wall and cytoplasmic contamination. Antibody prepared in rabbits against purified FC reacts with the flagellum of intact V. cholerae or purified FC as seen by ferritin-labeled antibody studies. Purified FC is composed of a single protein subunit with an estimated molecular weight of 45,000 g/mol and a density of about 1.3 g/cm3.


Identical nutritional requirements of Streptococcus faecium f24 and a derived stable L-phase variant. The nutritional requirements of Streptococcus faecium F24 and a lysozyme-produced stable L-phase variant of this strain appear to be identical. Thus, conversion of bacteria to the stable L-phase can occur without changes in nutritional requirements.


Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry. Populations of symbiotic Rhizobium meliloti extracted from alfalfa nodules were shown by flow microfluorometry to contain a significant number of bacteroids with higher nucleic acid content than the free-living rhizobia. Bacteroids with lower nucleic acid content than the free-living bacteria were not detected in significant quantities in these populations. These results indicate that the incapability of bacteroids to reestablish growth in nutrient media may not be caused by a decrease in nucleic acid content of the symbiotic rhizobia.


Purification and properties of homoprotocatechuate 2,3-dioxygenase from Bacillus stearothermophilus. The enzyme 3,4-dihydroxyphenylacetate:oxygen 2,3-oxidoreductase (decyclizing) (homoprotocatechuate 2,3-dioxygenase) was purified from the thermophilic organism Bacillus stearothermophilus, grown with j-hydroxyphenylacetic acid as a source of carbon. The enzyme appeared to be homogeneous as judged by disc-gel electrophoresis and sedimentation equilibrium measurements. The average molecular weight determined by three independent procedures was 106,000; the protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 33,000 to 35,000. The enzyme was fairly stable on heating and showed maximal activity at about 57 degrees C. An Arrhenius plot of Km for homoprotocatechuate was concave upward, with a break at 32 degrees C; an increase in delta H above this temperature was compensated by lower values of --delta S. Several properties of this enzyme are contrasted with those reported for homoprotocatechuate 2,3-dioxygenase purified by other workers from Pseudomonas ovalis.


Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia. Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.


H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. Purple photosynthetic bacteria produce H2 from organic compounds by an anaerobic light-dependent electron transfer process in which nitrogenase functions as the terminal catalyst. It has been established that the H2-evolving function of nitrogenase is inhibited by N2 and ammonium salts, and is maximally expressed in cells growing photoheterotrophically with certain amino acids as sources of nitrogen. In the present studies with Rhodopseudomonas capsulata, nutritional factors affecting the rate and magnitude of H2 photoproduction in cultures growing with amino acid nitrogen sources were examined. The highest H2 yields and rates of formation were observed with the organic acids: lactate, pyruvate, malate, and succinate in media containing glutamate as the N source; under optimal conditions with excess lactate, H2 was produced at rates of ca. 130 ml/h per g(dry weight) of cells. Hydrogen production is significantly influenced by the N/C ratio in the growth substrates; when this ratio exceeds a critical value, free ammonia appears in the medium and H2 is not evolved. In the "standard" lactate + glutamate system, both H2 production and growth are "saturated" at a light intesity of ca. 600 ft-c (6,500 lux). Evolution of H2, however, occurs during growth at lithe intensities as low as 50 to 100 ft-c (540 to 1,080 lux), i.e., under conditions of energy limitation. In circumstances in which energy conversion rate and supplies of reducing power exceed the capacity of the biosynthetic machinery, energy-dependent H2 production presumably represents a regulatory device that facilitates "energy-idling." It appears that even when light intensity (energy) is limiting, a significant fraction of the available reducing power and adenosine 5'-triphosphate is diverted to nitrogenase, resulting in H2 formation and a bioenergetic burden to the cell.


H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of H2 by resting cells. Photoproduction of H2 and activation of H2 for CO2 reduction (photoreduction) by Rhodopseudomonas capsulata are catalyzed by different enzyme systems. Formation of H2 from organic compounds is mediated by nitrogenase and is nto inhibited by an atmosphere of 99% H2. Cells grown photoheterotrophically on C4 dicarboxylic acids (with glutamate as N source) evolve H2 from the C4 acids and also from lactate and pyruvate; cells grown on C3 carbon sources, however, are inactive with the C4 acids, presumably because they lack inducible transport systems. Ammonia is known to inhibit N2 fixation by photosynthetic bacteria, and it also effectively prevents photoproduction of H2; these effects are due to inhibition and, in part, inactivation of nitrogenase. Biosynthesis of the latter, as measured by both H2 production and acetylene reduction assays, is markedly increased when cells are grown at high light intensity; synthesis of the photoreduction system, on the other hand, is not appreciably influenced by light intensity during photoheterotrophic growth. The photoreduction activity of cells grown on lactate + glutamate (which contain active nitrogenase) is greatly activated by NH4+, but this effect is not observed in cells grown with NH4+ as N source (nitrogenase repressed) or in a Nif- mutant that is unable to produce H2. Lactate, malate, and succinate, which are readily used as growth substrates by R. capsulata and are excellent H donors for photoproduction of H2, abolish photoreduction activity. The physiological significances of this phenomenon and of the reciprocal regulatory effects of NH4+ on H2 production and photoreduction are discussed.


Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles. Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell.


Phototaxis mutants of Chlamydomonas reinhardtii. A group of 31 mutants of Chlamydomonas reinhardtii with altered phototactic responses was isolated. Growth of all the mutants was normal and, with one exception, they had no gross morphological aberrations. One mutant produced supernumerary flagella. The mutants can be subdivided into five phenotypic classes on the basis of their abnormal responses: slow positive, no reponse, slow negative, fast negative, and variable. Random motility of the mutants was also characterized. About half were indistinguishable from wild type; the others exhibited "jerky" or "twirly" movements instead of relatively straight paths. These mutants can now be used for genetic and biochemical studies of the cellular basis of phototaxis.


Induction of D-aldohexoside:cytochrome c oxidoreductase in Agrobacterium tumefaciens. D-Aldohexopyranoside:cytochrome c oxidoreductase (ACO) was strongly induced by cellobiose, alpha-methylglucoside, beta-methylglucoside, kojibiose, and sophorose. Induction was rapid, and ACO was readily detectable within 10 min after addition of cellobiose as inducer. Although not measurable for 30 to 40 min after addition of inducer, once started, the rate of induction with alpha-methylglucoside equaled or even exceeded that obtained with cellobiose. Induction by sucrose, maltose, alpha-alpha-trehalose, melibiose, and lactose was weak. In general, the active ACO inducers were poor glycosidase inducers; the converse also appeared to be true. Although ACO induction was not repressed by D-glucose, it was repressed by succinate, malate, and fumarate.


Control of arginine utilization in Neurospora. The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora.


Chromosomal and extrachromosomal deoxyribonucleic acid from four bacterial endosymbionts derived from stock 51 of Paramecium tetraurelia. Four variant lines of stock 51 kappa (Paramecium tetraurelia) were screened for the presence of covalently closed circular (CCC) deoxyribonucleic acid (DNA). Stock 51m43 kappa, a nonkiller resistant to 51 killing, contained four classes of CCC DNA: 2.9 X 10(7), 9.7 X 10(7), and 11.8 X 10(7) daltons. The buoyant densities of 51m43 kappa chromosomal and CCC DNA were 1.700 and 1.698 g/cm3, respectively. Stock 51m43 pi, a sensitive nonkiller, contained two CCC species: 0.3 X 10(7) and 4.4 X 10(7) daltons. The buoyant densities of both the chromosomal and CCC DNA were 1.694 to 1.695 g/cm3. Three sizes of CCC DNA were found in 51m1 pi: 0.3 X 10(7), 2.3 X 10(7), and 4.5 X 10(7) daltons. The buoyant densities of both the chromosoaml DNA and the CC DNA were 1.694 to 1.695 g/cm3. It is not known whether 51m1 kappa, a sensitive spinner killer, contains CCC DNA. The buoyant density of its chromosomal DNA was 1.703 g/cm3. Of the four variant lines, only 51m43 kappa appears to be a mutant of 51 kappa. The chromosomal and CCC DNAs of 51m43 kappa have the same buoyant densities as those of 51 kappa; in addition 51m43 kappa contain a CCC molecule the same size as that found in 51 kappa (2.8 x 10(7) daltons). The three other lines are probably bacterial species that are distinct from 51 kappa and which, at one time, were co-inhabitants with 51 kappa in stock 51 paramecia.


Phylogenetic relationships of bacterial endosymbionts of Paramecium aurelia: polynucleotide sequence relationships of 51 kappa and its mutants. Hydroxyapatite chromatographic procedures were used to investigate the deoxyribonucleic acid (DNA) sequence relationships of kappa of Paramecium tetraurelia stock 51 and the organisms that have been designated as mutants of 51 kappa. Of the "mutants" studied, only 51m43 kappa possessed a high percentage (89%) of DNA sequences homologous to those of 51 kappa. All other "mutant" strains possessed less than 25% polynucleotide sequence homology to 51 kappa DNA. The three strains of pi endosymbionts (51m1 pi, 51m43 pi, and 139 pi) share greater than 75% DNA sequence homology with each other and approximately 50% DNA sequence homology with 138 mu, the mate-killer endosymbiont found in P. octaurelia. Only 23% of the 51 kappa DNA sequences were found to be homologous with those of 51m1 kappa. The data indicate that of the "mutants" studied, only 51m43 kappa could be a mutant of 51 kappa. The pi endosymbionts comprise a closely related group of organisms that are also related to 138 mu but not to any of the kappas tested. The group of organisms designated as kappa appears to be comprised of at least two distinct phylogenetic groups.


Evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin. To obtain information on the mechanism of glycosylation of ovalbumin, three types of experiments were performed with either hen oviduct membrane preparations or tissue slices and the antibiotic tunicamycin. First, experiments involving the addition of tunicamycin to oviduct membranes demonstrated that this antibiotic inhibited the synthesis of a N-acetylglucosaminyl-lipid with the properties of N-acetyl-glucosaminylpyrophosphorylpolyisoprenol. No inhibitory effects on the other steps in the synthesis of oligosaccharide-lipid were observed. Thus, tunicamycin inhibits the lipid-linked pathway for membrane protein glycosylation by blocking the first step in the synthesis of oligosaccharide-lipid, namely, the synthesis of N-acetylglucosaminylpyrophosphorylpolyisoprenol. Second, in experiments using membranes prepared from oviduct tissue slices preincubated with tunicamycin, it was found that mannosylphosphoryldolichol was the only saccharide-lipid synthesized. This result indicates that tunicamycin administered in vivo depletes endogenous pools of N-acetylglucosamine-lipid precursors to oligosaccharide-lipid in a manner consistent with its activity in vitro. Finally, it was found that tissue slices incubated in the presence of tunicamycin synthesized the polypeptide chain of ovalbumin at almost normal rates. However, the protein newly synthesized in vivo did not contain labeled N-acetylglucosamine or mannose and had the properties of unglycosylated ovalbumin. These results indicate that saccharide-lipids participate in the assembly of the core oligosaccharide of the secretory glycoprotein, ovalbumin.


Structural analysis of nine oligosaccharides isolated from the urine of a blood group O, nonsecretor, woman during pregnancy and lactation. Nine oligosaccharides have been isolated from urine collected from an O(H), Le(a+ b-), nonsecretor, woman during her pregnancy and subsequent lactation. One of them is a new hexasaccharide denoted lacto-N-neo-difucohexaose II: beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-beta-D-GlcNAc-(1 leads to 3)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-D-Glc. Seven of the remainder were identified as the milk oligosaccharides lacto-N-difucohexaose II, lacto-N-fucopentaose II and III, lacto-N-tetraose, 6'-galactosyllactose, 3-fucosyllactose, and lactose. The ninth component is a glucose-containing tetrasaccharide, alpha-D-Glc-(1 leads to 6)-alpha-D-Glc-(1 leads to 4)-alpha-D-Glc-(1 leads to 4)-D-Glc, previously found in normal human urine. The oligosaccharides were isolated by gel chromatography, preparative zone electrophoresis, and paper chromatography. The structures were determined by sugar and methylation analyses. Sequence analysis of tri- and tetra-saccharides was carried out by gas-liquid chromatography-mass spectrometry of reduced and permethylated oligosaccharide derivatives. The monosaccharide sequence in penta- and hexasaccharides was deduced from gas chromatographic-mass spectrometric studies of di- and trisaccharides obtained from partial acid hydrolysis of the parent compound. Anomeric configurations were deduced from the optical rotation.


Structural analysis of oligosaccharides isolated from the urine of a blood group A, secretor, woman during pregnancy and lactation. Twenty different oligosaccharides have been isolated from urine collected from an A, Le(a- b+), secretor woman during her pregnancy and subsequent lactation. Nine of these have been found previously in the milk of Le (a- b+) individuals. Three new fucose-containing oligosaccharides denoted lacto-N-neotrifucoheptaose II (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-beta-D-GlcNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 4)-D-Glc), lacto-N-neodfucohexaose I (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]beta-D-GlcNAc-(1 leads to 3)-beta-D-Gal-(1 leads to 4)-D-Glc), and lacto-N-difucohexaose IV (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 3)-beta-D-GlcNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 4)-D-Glc) are described. Three new myo-inositol-containing oligosaccharides which are characteristic for pregnancy have also been isolated. Their structures are: alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 0)-[alpha-L-Fuc-(1 leads to 0)]-myo-inositol; alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 0)-[alpha-D-Fuc-(1 leads to 0)]myo-inositol; and alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 0)-myo-inositol, respectively. Three oligosaccharides containing only glucose were found and partially characterized. In addition, two oligosaccharides described previously (a glucose tetrasaccharide (Hallgren, P., Hansson, G., Henriksson, K.-G., Häger, A., Lundblad, A., and Svensson, S. (1974) Eur. j. clin. Invest. 4, 429-433) and a urine A pentasaccharide (Lundblad, A., and Svensson, S. (1973) Carbohyd. Res. 30, 187-189) were also isolated and characterized. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, and preparative paper chromatography, Structural determination involved sugar and methylation analyses. Tri- and tetrasaccharides were investigated by combined gas-liquid chromatography-mass spectrometry. Sequence analyses of larger oligosaccharides were performed by combined gas-liquid chromatography-mass spectrometry of di- and trisaccharides obtained after partial acid hydrolysis of the parent compound. Anomeric configurations were deduced from optical rotations and by comparison with authentic samples.


Quantitation of some urinary oligosaccharides during pregnancy and lactation. The excretion rates of different oligosaccharides in the urine of two individuals, a blood group O, nonsecretor woman, and a blood group A, secretor woman, were studied at different times during pregnancy and lactation. Quantitative determinations of di-, tri-, and tetrasaccharides were performed by combined gas-liquid chromatography-mass spectrometry. Larger oligosaccharides were quantitated by their content of total hexose or 6-deoxyhexose (fucose). The urinary oligosaccharide excretion during pregnancy is detectable by the 13th week. All oligosaccharides studied increased and reached the highest levels during the last trimester. During lactation more complex excretion patterns were observed. Lactose and oligosaccharides with a lactose backbone were the most predominant compounds. Oligosaccharides with a lacto-K-tetraose or a lacto-N-neotetraose backbone were present both during pregnancy and lactation, but the former type showed a much more pronounced increase of excretion during lactation. Four oligosaccharides containing only D-glucose were found in largest amounts during pregnancy. One of these oligosaccharides, a glucose tetrasaccharide, was quantitatively determined in the urine of 13 individuals and the excretion was found to decrease 2- to 5-fold in the first week post partum relative to the last week of pregnancy. Urine from a woman whose fetus died unexpectedly in the 24th week of pregnancy was also studied. No oligosaccharides characteristic of a normal pregnancy could be detected, suggesting that at least some oligosaccharides appearing specifically during pregnancy might reflect the function of the placenta or the fetus, or both.


Hormonal control of transcription in the rat uterus. Stimulation of deoxyribonucleic acid-dependent RNA polymerase III by estradiol. DNA-dependent RNA polymerases were extracted from rat uterine tissue, partially purified and resolved by DEAE-Sephadex chromatography. RNA polymerases I, II, IIIA, and IIIB eluted at the characteristic ammonium sulfate concentrations of 0.15, 0.28, 0.34, and 0.42 M, respectively. The sensitivity of each peak of polymerase to alpha-amanitin was examined and was shown to be essentially identical to the three classes of RNA polymerases in other mammalian systems. RNA polymerase I was insensitive to high levels of alpha-amanitin, RNA polymerase II was sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.006 mug/ml) and RNA polymerases IIIA and IIIB were sensitive to high concentrations of alpha-amanitin (50% inhibition at 18 mug/ml). The alpha-amanitin sensitivity curve of total RNA synthesis measured in isolated nucleo demonstrated that the activity of each class of RNA polymerase could be quantitated in uterine nuclei. Thus the initial decrease in activity at low concentrations of alpha-amanitin (50% inhibition at 0.005 mug/ml) was attributed to the inhibition of RNA polymerase II activity, the second decrease in activity at higher concentrations of alpha-amanitin (50% inhibition at 15 mug/ml) was attributed to the inhibition of RNA polymerase III activity, and the activity which was resistant to the highest alpha-amanitin concentration tested was attributed to RNA polymerase I activity. When estradiol was given to immature rats 6 h before killing both RNA polymerases I and III levels in nuclei were increased significantly over the control values. The time course of these changes demonstrated that the increases in RNA polymerases I and III were first evident between 1.5 and 3 h following hormone treatment. Significantly, these increases in polymerase I and III in nuclei parallel the published increases for rRNA and tRNA synthesis following hormone treatment. However, the amount of RNA polymerase I and III was not altered upon extraction, suggesting that these changes are due to the alteration in chromatin template activity. Both estradiol and estriol produced identical increases in uterine RNA polymerase I and III 6 h after treatment.


Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. A multifunctional protein from porcine liver. Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase from porcine liver have been co-purified more than 500-fold to apparent homogeneity. The inability of three sequential chromatographic procedures followed by affinity chromatography using NADP+- or ATP-substituted Sepharose to resolve the three activities demonstrates that they are physically associated. Molecular weight estimates of the native protein by gel filtration (Mr = 150,000) and by dodecyl sulfate gel electrophoresis (Mr = 100,000) indicate that the native structure is probably a single subunit. Since only one protein band is seen on dodecyl sulfate gels, it is concluded that the three activities are properties of a single polypeptide chain. The kinetic properties of the three activities are described, the most unusual feature being the susceptibility of the cyclohydrolase to competitive inhibition by NADP+, NAD+, ATP, and folate.


Characterization of a novel metabolic pathway of arachidonate in coronary arteries which generates a potent endogenous coronary vasodilator. Bovine coronary artery strips were incubated with [1-14C]arachidonic acid and the chemical properties of the various prostaglandins (PG) formed were studied. Arachidonate was converted to two major prostaglandin products, PGE2 and a novel prostaglandin having chemical (i.e. base hydrolysis and borohydride reduction) and chromatographic properties identical with 6-keto-PGF1alpha. This final compound was inactive on coronary artery strips. The endoperoxide intermediates, PGG2 or PGH2, previously shown to induce coronary relaxation, were not released into the medium from isolated bovine coronaries. The arachidonic acid-induced dilation may have been due to an intracellular action of PGH2 (or PGG2) or to the action of another, yet unidentified, labile intermediate formed in the enzymatic conversion of endoperoxides to 6-keto PGF1alpha. When PGH2 was incubated with bovine coronary microsomes, the PGH2 was completely metabolized (i.e. loss of rabbit aorta contraction) but a compound was generated which was a much more potent coronary relaxant. We suggest that this major novel metabolic pathway of arachidonate generates a substance, intermediate between PGH2 and the final 6-keto PGF1alpha-like product, which is a potent coronary vasodilator.


Differentiation of metal ion-induced transitions of prothrombin fragment 1. The intrinsic fluorescence of human and bovine prothrombin fragment 1 is quenched (approximately -35%) when calcium ions are bound. The intrinsic fluorescence of prothrombin is also quenched by the binding of Ca2+ but to a lesser extent (total change is approximately -6%). The prothrombin fragment 1 fluorescence transition is also effected by the binding of Mg2+, Mn2+, Gd3+, and Tb3+. With all ions the fluorescence quenching is readily reversed by titration with EDTA. Titration with any of the above ions results in a sigmoidal titration curve. The fluorescence transition midpoints (expressed as Tm) occur at the following concentrations of ions: Ca2+, 0.22 (0.35) mM; Mg2+, 0.22 (0.45) mM; Mn2+, 12.6 (12.6) muM; and Gd3+, 3.6 (5.3) muM (the values in parentheses indicate the concentration of the respective ions bound to bovine prothrombin fragment 1). The presence of phospholipid steepens the Ca2+ titration curve, significantly more so for the bovine system than for the human. Tm values for Ca2+ binding in the presence of phospholipid are 0.21 mM and 0.22 mM for binding to human and bovine prothrombin fragment 1, respectively. Sedimentation equilibrium and velocity studies indicate that prothrombin fragment 1 which is monomeric in the presence of EDTA undergoes concentration-dependent association in the presence of Ca2+. A plot of fraction monomer versus Ca2+ concentration is sigmoidal, with a Tm of approximately 1 mM Ca2+. Mg2+ is only marginally effective in promoting the dimerization, and Mn2+ and Gd3+ are even less effective. However, sedimentation studies performed in the presence of 1 mM Mg2+ at varying Ca2+ concentrations shifted the apparent dimerization transition markedly to the left, the midpoint of the transition occurring at 0.25 mM. Kinetic studies reveal that while Mg2+ by itself does not promote prothrombin activation to thrombin, Mg2+ will partially substitute for Ca2+ in prothrombin activation. In the presence of 1 mM Mg2+ maximal activation rates are attained with 0.3 mM Ca2+; higher Mg2+ concentrations (at this Ca2+ concentration) are inhibitory. Only Ca2+ and Gd3+ permit significant prothrombin-phospholipid binding. Scatchard plots derived from equilibrium dialysis experiments performed with bovine prothrombin fragment 1 suggest marked cooperativity in calcium binding and the existence of approximately six Ca2+ binding sites. Similar studies, but with 1 mM Mg2+ also present in the dialysate, suggested that Mg2+ promotes a greater degree of cooperativity in Ca2+ binding to prothrombin fragment 1 with an apparent decrease in the number of Ca2+ sites occupied. It therefore appears that prothrombin fragment 1 has two classes of metal ion binding sites. One class, probably comprising two of six sites, is apparently fairly nonselective with respect to which metal ion is bound; binding of metal ion to these sites is responsible for the fluorescence change and apparently triggers a conformational transition...


In vitro synthesis of a high molecular weight virion-associated RNA by vaccinia. Although the bulk of RNA synthesized in vitro by vaccinia virus is 8 to 12 S, a small amount of high molecular weight RNA can be detected. This RNA is virion-associated and is not extruded from the virus as high molecular weight RNA. It is sensitive to pancreatic RNase digestion in high salt, has a density in neutral CS2SO4 of 1.68 g ml-1 and remains large after digestion with DNase or denaturation in dimethyl sulfoxide. In the presence of high concentrations of virus in the in vitro RNA polymerase reaction, pulse-labeling experiments indicate an RNA sedimenting heterogeneously between 20 and 30 S. Pulse-chase experiments indicate that a fraction of this high molecular weight RNA can be chased into RNA sedimenting at 8 to 12 S. Cleavage into smaller fragments is not dependent on continued RNA synthesis but does require ribonucleoside triphosphates. In the presence of ethidium bromide, the RNA is not cleaved.


High molecular weight virion-associated RNA of vaccinia. A possible precursor to 8 to 12 S mRNA. The high molecular weight virion-associated RNA synthesized by vaccinia in vitro can be cleaved into smaller components, some of which are extruded from the virus as 8 to 12 S RNA. The high molecular weight virion-associated RNA fails to bind appreciably (5%) to poly(U) filters indicating that it is not polyadenylated. Its cleavage products will, however, bind to poly(U) (40 to 50%) after processing in the presence of ATP. The high molecular weight virion-associated RNA is methylated by the virus, and purified unmethylated RNA can be methylated by detergent-solubilized extracts of vaccinia virus cores. In the presence of GTP, methylation is stimulated 3-fold. The level of methylation of purified unmethylated high molecular weight RNA achieved by soluble core extracts is approximately 80% of the level of methylation achieved with purified unmethylated 8 to 12 S viral RNA, suggesting that more residues than the primary 5' termini became methylated. Approximately 85% of the methylated RNA binds to poly(U) when purified high molecular weight RNA is processed with soluble core extracts in the presence of S-adenosyl[methyl-3H]methionine, GTP, and ATP, conditions which also cleave the RNA. Nucleic acid hybridization-competition studies indicate that virion-extruded 8 to 12 S mRNA contains sequences found in the high molecular weight virion-associated RNA.


Prostaglandin endoperoxide E isomerase from bovine vesicular gland microsomes, a glutathione-requiring enzyme. Prostaglandin endoperoxide E isomerase catalyzes the isomerization of the endoperoxy group of prostaglandin H and produces prostaglandin E. The enzyme was solubilized with Tween 20 from bovine vesicular gland microsomes and purified 26-fold by successive column chromatography on DEAE-cellulose and omega-aminobutyl Sepharose 4B. The previously known requirement for glutathione was further investigated. The activity of freshly prepared enzyme was destroyed rapidly with a half-life of about 30 min at 24 degrees, pH 8.0. Various thiol compounds including glutathione protected the enzyme from such an inactivation. However, several findings indicated that glutathione was possibly specifically involved as a coenzyme in the isomerase reaction, but was not oxidized in a stoichiometric quantity. The enzyme also isomerized prostaglandin G at a rate approximately half of that of prostaglandin H.


Spinach nitrite reductase. Purification and properties of a siroheme-containing iron-sulfur enzyme. Ferredoxin-nitrite reductase (EC 1.7.7.1.) from spinach has been purified to homogeneity with a specific activity of 110 units/mg of protein. The enzyme, Mr = 61,000 has 3 iron atoms (of which one is in siroheme) and 2 labile sulfides, i.e. 1 (Fe2-S2) per molecule, with absorption maxima at 276, 386 (Soret), 573 (alpha), and 690 nm, with an E386 of 3.97 X 10(4) M-1-cm-1, and A276/A386 absorptivity ratio of 1.8. Anaerobic addition of dithionite results in the loss of the 690 nm peak and the splitting of the 573 nm absorption band into two broad peaks at 545 and 585 nm. Reduction by dithionite is enhanced by cyanide (Fig. 7) and requires about 3 electron eq per mol of enzyme. With nitrite or hydroxylamine (substrates of the enzyme), cyanide (a competitive inhibitor with respect to nitrite), or sulfite, the 690 nm absorption band of substrate-free enzyme disappears and the absorbance in the Soret and alpha region are altered. The high spin EPR signals disappear (J. M. Vega, H. Kamin, N. R. Orme-Johnson, and W. H. Orme-Johnson, unpublished observations). Titration permits calculation of 1 mol of nitrite bound/mol of enzyme with a Kdiss of 3.2 X 10(-6) M. Dithionite-reduced enzyme also forms complexes with added nitrite, hydroxylamine, or cyanide, characterized by marked alterations in the 573 (alpha) absorption band. THus, substrates or competitive inhibitors can be bound to the oxidized or reduced enzyme forms. CO inhibits nitrite reductase and forms a complex with reduced enzyme (epsilonmax at 395, 543, and 585 nm). Formation or dissociation of the spectrophotometrically detectable CO complex correlates with inhibition or inhibition-reversal of nitrite reduction catalysis. During steady state turnover with dithionite and nitrite, the enzyme forms a complex with added nitrite with absorption difference maxima at 445, 538, and 580 nm with respect to reduced enzyme. When nearly all substrate is depleted the spectrum of a new species appears, indicating that nitrite reductase may form complexes with nitrogen compounds of more than one oxidation state. Nitrite is stoichiometrically reduced to ammonia without detectable free nitrogen compounds of intermediate reduction state. p-Chloromercuribenzoate (pCMB) inhibits nitrite reductase activity and nitrite partially protects against this inhibition. Titration of native enzyme with the mercurial shows that 6 mol of pCMB can be bound/mol or nitrite reductase. The Soret absorption band of the native nitrite reductase is altered and partially bleached in the pCMB-treated enzyme, and the 573 (alpha) band disappears.


Identification of folate binding proteins in rat liver. Intraperitoneal injection of tritiated folic acid (PteGlu) into rats has revealed the presence of three separate protein fractions in the cytosol fraction of the liver and one in the mitochondria which bind folate derivatives. The proteins in the cytosol (cytosol I, II and III) have approximate molecular weights of 350,000, 150,000, and 25,000 and the protein in the mitochondria has an approximate molecular weight of 90,000 as estimated by gel filtration. The bound folate derivatives are primarily polyglutamate forms while cytosol II contains primarily bound 5-methyltetrahydrofolate polyglutamate derivatives. Little binding of radioactively labeled folic acid or 5-methyltetrahydrofolate to these fractions was observed when binding was carried out in vitro. Significant binding in vitro was observed, however, when a mixture of biosynthetically labeled natural folate derivatives was used. These proteins have not been purified, but cytosol III partially consists of the enzyme, tetrahydrofolate dehydrogenase (EC 1.5.1.3). Studies on the time course of folic acid incorporation into the liver showed that soon after injection nonmetabolized folic acid was bound to the plasma membrane fraction of the liver cell. It is suggested that at least one of the binding proteins in the cytosol may be involved in storage of the vitamin while the binding of nonmetabolized folic acid to the plasma membrane may reflect the existence of a carrier for folic acid transport into the cell.