Torque-velocity relationships and muscle fiber composition in elite female athletes.

The relationship between the predominance of fast and slow muscle fibers of the vastus lateralis and "in vivo" torque velocity properties in 22 female athletes was studied. Fiber types were classified according to the histochemical myofibrillar adenosine triphosphatase technique at a basic pH. Maximal extensor troques were recorded at 30 degrees from full extension at four selected velocities. While results confirm earlier reports on muscle fiber type and performance, an additional finding was that as knee extension velocities increased from 0 to 95 degrees/s angle specific extensor torque production did not decline as seen in in vitro muscle preparations. The difference in extensor torque between 0 and 96 degrees/s appeared far more critical than the differences observed between 96 and 288 degrees/s. Significant differences in torque were seen at 96, 192, and 288 degrees/s in thos with greater than 50% and less than 50% slow-twitch fibers. When expressed per kilogram of body weight the subjects with greater than 50% fast-twitch fiber produced the greatest torque at 192 degrees/s. These results suggest that the velocity at which torque begins to decline in vivo is related to the proportion of slow-twitch fibers in the vastus lateralismuscle.


Cage size and exercise affects infarct size in rat after coronary artery cauterization.

Left coronary occlusion in the rat was performed by cornary artery cauterization. A small amount of myocardial damage at the site of occlusion was noted, and myocardial infarction occurred in the distal distribution of the obstructed coronary. The effects of cage size and level of physical activity on estimated infarct size (as measured by creatine kinase depletion) 48 h after occlusion were determined. Isolation in small cages and moderate treadmill exercise resulted in an approximate doubling of the amount of infarction when compared to grouping of rats in large cages. Mild exercise did not increase infarct size. Total urinary catecholamines in normal rats placed in small cages for 48 h were elevated when compared to unconfined rats in larger cages. Cornary artery occlusion by cauterization is an easily performed technique. Cage size and level of activity (and their effects on sympathoadrenal function) are important independent determinations of infarct extent after coronary occlusion in the rat.


Effect of catecholamines on ion transport in dog tracheal epithelium.

The action of adrenergic agonists on ion fluxes across the epithelium of the canine trachea was determined. Isolated sheets of tracheal mucosal membranes were mounted in Ussing-type chambers, bathed with Krebs-Henseleit solution at 37 degrees C, pH 7.4, and gassed with 5% CO2 Iin oxygen. Various adrenergic agents, when added to the bathing medium elevated short-circuit current (SCC) (isoproterenol greater than epinephrine greater than norepinephrine greater than phenylephrine). Propranolol (10(-6) M) decreased SCC response to epinephrine. Epinephrine (1mM) increased both unidirectional 36Cl fluxes; net 36Cl secretion toward the lumen increased from 2.01 +/- 0.52 to 3.20 +/- 0.46 mueg/cm2.h (P less than 0.05); 22Na flux did not change. In epinephrine-stimulated tissues, propranolol (1mM) abolished net 36Cl secretion. The SCC response to epinephrine was blunted in the absence of Na from the submucosal reservoir; this blunting suggests that Cl entry across the basal membrane is coupled with Na. Both the relative sensitivities of SCC to various adrenergic agonists and the effect of propranolol suggest that Cl-secretory process is highly sensitive to beta-adrenergic receptor stimulation. Epinephrine increased cell membrane permeability to Cl and probably stimulated a specific Cl pump.


Cross-adaptive effects of cold, hypoxia, or physical training on decompression sickness in mice.

The effects of adaptation to cold, hypoxia, or exercise on hyperbaric decompression tolerance were investigated in two factorial experiments. For either 14 or 28 days, groups of mice were handled (control); exposed discontinuously for 4 h to cold (4 degrees C) or hypoxia (P approximately 379 or 320 Torr); or exercised by swimming (15 min at 31 degrees C) or treadmill excursion (8.1 m/min for 1 or 1.5 h). The animals were divided into subgroups, exposed to one of three hydrostatic pressures (7.6--11.1 ATA) for 30 min, decompressed, and observed to determine survival rate or bends incidence (type II decompression sickness). Decompression sickness was significantly reduced (P less than 0.05) in the treadmill-trained animals, was unchanged in cold-exposed and swim-exercised mice, and tended to increase in animals adapted to hypoxia. Enhanced tolerance by treadmill training is presumably due to lean body conformation, which could reduce nitrogen saturation of tissues, and greater muscle capillarization and cardiovascular fitness, which may improve nitrogen elimination. Reduced tolerance with adaptation to hypoxia may be attributed to rheological changes associated with polycythemia, which facilitate bubble production.


Prolonged methacholine-induced bronchoconstriction in dogs.

We studied the effect of prolonged airways obstruction induced by extended cholinergic stimulation in five anesthetized, mechanically ventilated dogs. A continuous intravenous metacholine infusion was utilized to maintain pulmonary resistance (RL) at 200--1500% preinfusion levels for 13--23 h. At maximum RL (18.86 +/- 7.74 vs. 2.09 +/- 0.18 (mean +/- SD) cmH2O/ (L/S) PREINfusion; P less than 0.01), dynamic lung compliance (Cdyn) fell from 67.5 +/- 14.6 to 32.7 +/- 11.6 ml/cmH2O (P less than 0.005) and arterial partial pressure of oxygen (PaO2) fell modestly from 95.8 +/- 6.1 Torr preinfusion to 83.2 +/- 12.7 Torr (P less than 0.05). Tachyphylaxis to methacholine developed, requiring increases in infusion rates to maintain elevated RL. Abnormalities in lung function resolved promptly upon termination of the infusion. Two similarly instrumented control animals ventilated for 19 and 25 h without metacholine infusion had no change in RL, Cdyn, or PaO2. Histological examination of the lungs revealed no differences between infused and control animals. In spite of marked increases in RL, prolonged cholinergic stimulation produced only mild changes in gas exchange and no sustained changes in lung function or structure.


Cardiac function in goats exposed to carbon monoxide.

To investigate the possibility that prolonged carbon monoxide (CO) exposure would depress myocardial function, six chronically instrumented, unsedated goats were exposed to 160--200 ppm CO for 2 wk, resulting in a mean carboxyhemoglobin saturation of 20%. Cardiac index and stroke volume remained unchanged during and after exposure. Hematocrit and hemoglobin concentration started increasing on the 10th day of exposure, this increase reached statistical significance (P less than 0.05) on the 6th postexposure day. Contractility (Vmax) of the left ventricular myocardium and heart rate were unchanged during exposure to CO, but both were significantly (P less than 0.05) decreased at some time during the 1st wk after removal from CO. If there was a decrease in intrinsic myocardial function during CO exposure, it may have been masked by increased sympathetic activity. The mechanism(s) that might produce the decrease in heart rate and contractility after removal from CO are not obvious. Possible explanations are discussed.


Recovery of skeletal muscle after 3 mo of hindlimb immobilization in rats.

During immobilization, skeletal muscle undergoes decreases in size and strength with concomitant atrophic and degenerative changes in slow-twitch muscle fibers. Currently there are no objective data in slow-twitch muscle demonstrating recovery of biochemical or physiological indices following termination of immobilization. The purpose of this study was to determine whether the soleus, a slow-twitch muscle, could recover normal biochemical or physiological levels following termination of immobliization. Adenosine triphosphate, glycogen, and protein concentration (mg/g wet wt) all significantly decreased following 90 days of hindlimb immobilization, but these three values returned to control levels by the 60th recovery day. Similarly, soleus muscle wet weight and protein content (mg protein/muscle) returned to control levels by the 14th recovery day. In contrast, maximal isometric tension did not return to normal until the 120th day. These results indicate that following muscular atrophy, which was achieved through 90 days of hindlimb immoblization, several biochemical and physiological values in skeletal muscle are recovered at various times after the end of immobilization.


Isocapnic hyperpnea in awake ponies during inspiration of 4% CO2.

Unanesthetized ponies were given 4% CO2 (inspired CO2 pressure = 28 Torr) to breathe at two levels of arterial O2 pressure (PaO2): 1) near 75 Torr and 2) near 200 Torr. During 4% CO2 breathing, at either level of PaO2, the mean arterial CO2 pressure (PaCO2) was unchanged from control measurements (control measurements were made at the same PaO2, but with no CO2 in inspired air), suggesting that awake ponies can "clear" 4% CO2. The ability of individual ponies to clear 4% CO2 was quite variable: some ponies did not clear 4% CO2 and others cleared 4% CO2 on one day but not on the following day. Based on the average of 20 experiments, however, PaCO2 was unchanged from 40 Torr during inspiration of 4% CO2. Direct measurement of chemical stimuli to breathing in arterial blood and cisternal cerebrospinal fluid indicate that ventilation increased during CO2 breathing even though PaCO2, pHa, PaO2, and CSF pH were not changed in a direction that might explain the accompanying change in ventilation. The authors suggest that stimuli to receptors other than peripheral or medullary chemoreceptors may be responsible for the reported "isocapnic hyperpnea."


A technique for measuring frequency response of pressure, volume, and flow transducers.

A device and methodology is presented for testing the frequency response of pressure, volume, or flow transducers. Also reported are responses of selected transducers of all three types over the range of 2--120 Hz. Several pressure transducers tested had good frequency response when connected to the test system with a minimum of interconnecting fittings; others did not. Use of additional connectors degraded the response as did the addition of air-filled catheters. The frequency response of the pneumotachometers tested were influenced largely by the response characteristics of the associated pressure transducer and interconnecting fittings. These results emphasize the need to test the response characteristics of any transducer with specific connectors and fittings that are to be used to make the actual measurements of pressure, volume, or flow.


Light and electron microscopic observations on the development of the blood vascular system of the human brain.

The development of the blood vascular system of the human fetal brain was examined by both light and electron microscopy. By light microscopy the brains of human embryos and fetuses ranging in size from 8.5 to 70 mm crown-rump length have been studied in serial sections, usually of the whole embryo or fetus. In the smallest specimens the whole brain was encapsulated by a very dense vascular plexus. From this, perforating offshoots passed to or from the substance of the brain, while other connexions were effected with neighbouring arterial and venous channels. These latter vessels were never more mature than capillaries, and their status only recognizable by their location and ultimately by their connexions with the heart. The neopallial part of the cerebral hemisphere was later than all other parts of the brain in receiving vessels perforating its substances. There is some evidence, however, that the cellular basis of a blood vascular supply was present in this part of the brain before lumina and blood cells appeared. The cerebral cortex of fetuses ranging from 50 to 100 mm crown-rump length was examined. Both the extrinsic and intrinsic vessels of the cortex were never more mature than capillaries; occasionally a capillary was seen to penetrate the cortex from the surrounding pial investment. Within the developing cerebral cortex blind ending solid endothelial sprouts were identified, as well as 'seamless' capillaries.


Development of mucosa of the human ileum.

Normal histology, glycogen and alkaline phosphatase have been studied in the mucosa of human fetal ileum between 28 and 140 days of gestation. From 112 days onwards, the perinuclear, supranuclear and infranuclear vacuolation, striate border, and basement membrane of the columnar epithelial cells of the villi, and the very close proximity of the latter to blood vessels, all support the idea that the cells are actively engaged in transport of materials. Alkaline phosphatase and glycogen become conspicuous in the cells at this stage. It is argued that by 112 days the epithelial cells of the ileum are absorbing glucose amniotic fluid as well as secreting mucus.


Myotube formation in skeletal muscle regeneration.

Muscle fibre regeneration in the latissimus dorsi of adult rats has been studied by light and electron microscopy. Both 'continuous' and 'discontinuous' modes of regeneration occur. In 'continuous' regeneration sarcoplasmic buds grow from the damaged muscle fibres, their nuclei apparently originating from the healthy portion of the muscle fibre. In 'discontinuous' regeneration mononucleated cells accumulate inside intact but vacated, sarcolemmal tubes. They differentiate into myofibril-containing myoblasts which later fuse. 'Continuous' regeneration occurs when the sarcolemmal sheaths are disrupted; 'discontinuous' regeneration when the sarcolemmal sheaths remain intact.


The anterior fibres of the levator ani muscle in man.

The anatomy of the anterior portion of the levator ani muscle in studied in 26 adult human cadavers of both sexes. This portion of the muscle is found to consist of three layers of muscle fibres. The three layers are: 1. The pelvic layer. Its fibres (1) are attached to the capsule of the prostate or adventitia of the lateral wall of vagina, (2) intermingle with and supplement the longitudinal muscle layer of the anal canal, and (3) are continuous with the fibres of the opposite side behind the recto-anal junction. 2. The middle layer. The most anterior fibres are twisted on themselves to form the round free border of the muscle that bounds the levator hiatus. The majority of the muscle fibres of this layer proceed backwards to cover and blend with the deep part of the external anal shincter, partly joining the anococcygeal ligament. 3. The perineal layer. These fibres surround the superficial part of the external anal sphincter. A respectable bundle of muscles fibres unites with that of the opposite side in from  of the lower part of the anal canal. Remaining fibres terminate in perianal skin or anococcygeal ligament. The role of the anterior portion of the levator ani in fixation and prevention of prolapse of the pelvic viscera is stressed.


Inguinal anatomy.

The flat muscles of the anterior abdominal wall pass down well in front of the pectineal ligament of Astley Cooper on the summit of the superior pubic rumus, thus providing space for the spermatic cord and the great vessels of the lower limb to leave the abdomen. The abdominal wall layers turn back lower down to close the gap on the medial side of the external iliac vein. The external oblique aponeurosis turns back to form a strong inguinal ligament, and the attached fascia lata turns back as the lacunar ligament to meet and fuse with the pectineus muscle fascia 1-1.5 cm below Cooper's ligament. This leaves a deep trough, between Cooper's ligament behind and the posterior inguinal wall in front, which houses the transversely placed femoral canal. The transversalis fascia of the floor of the femoral canal turns down to form the medial wall of the venous compartment of the femoral sheath, and has the support of the curved edge of the lacunar ligament which effectively bars the femoral canal from entering the thigh. It is usually taught, however, that the lower end of the femoral canal passes unprotected into the thigh as the medial or third compartment of the femoral sheath, to lie between the femoral vein in its sheath and the curved edge of the lacunar ligament. If this were the case there would be no hindrance to the descent of a process of parietal peritoneum containing omentum or gut into and distending the femoral canal in the thigh. Would such a swelling, before the process of peritoneum had broken through the walls of the canal into the subcutaneous tissues of the groin, pass for a femoral hernia? Fortunately this problem does not arise. A study of the posterior aspect of the anterior abdominal wall, removed with the attached anterior half of the bony pelvis, reveals many details of the design and attachments of the deep inguinal ring, the femoral trough and canals and the femoral sheath.


Radiographic profile of the first cervical vertebra.

Lateral cephalographs from 220 normal adolescent white orthodontic patients, and of individuals with craniofacial dysostosis and oculodento-osseous dysplasia were studied. Variations were observed in the morphology of the posterior margins of the superior articular processes, vertebral artery canals being present in 8%. Posterior arch dehiscence was found in eight individuals, including seven cases in the midline. Three patients had accessory ossicles above the posterior arch of the atlas, and two showed evidence of fusion of the second and third cervical vertebrae. The profile of the atlas was well outside the normal range in both syndromes studied.


The seminiferous tubules of the testis before, around and after birth.

A study has been carried out on the width of the seminiferous tubules, and the proportion of pre-germinal cells, in 174 stillborn children between 24 and 40 weeks of gestation, and in 146 children dying between the ages of birth and six years. There was a general diminution in the diameter of the seminiferous tubule throughout the last trimester and immediately before birth, followed by a slow and steady increase in the diameter of the tubule throughout childhood. The germinal cells within the seminiferous tubules appear to be under some slightly increased stimulatory influence during the last few weeks prior to delivery, and under considerable stimulation in the first 4 or 5 weeks after birth. Following this these cells appear to diminish in size and become hidden among the Sertoli cells, to be re-stimulated around the time of puberty.


Observations on the primordial oocyte of the bandicoot Isoodon macrourus (Peramelidae, Marsupialia).

An ultrastructural study of bandicoot primordial follicles and oocytes was undertaken, as information on this subject is lacking in marsupials. Conspiculous features of the ooplasm are a paranuclear complex (PNC), a vesicle-microtubule complex (VMC) and an aggregate of tubular cisternae (ATC). The PNCappears as one or, more rarely, several homogeneous eosinophil bodies at the light microscope level. Ultrastructurally it is particulate, consisting of five distinct types of bodies, most of which are composed of concentric fibrillar whorls, but others appear homogeneous, granular or crystalline. Embedded among the particles is a group of Golgi-like vesicles. The bandicoot PNC-unlike similar structures found in the ooplasm of a variety of vertebrates, and known variously as "Balbiani body", "yolk nucleus", etc.-totally lacks nitochondria. The VMC consists of vesicle-like organelles which may be drawn out into tubular extensions, while the bounding membrane may be decorated with granules. Bundles of microtubules ramify between the vesicles, from which they appear to originate. The vesicles contain a matrix similar to the ooplasm. The ATC contains a homogeneous substance more electron-dense than the surrounding ooplasm. 'Dense bodies' occur in the cytoplasm of both the follicle cells and the oocytes. These are elongate membrane-bound organelles, circular in cross section. An electron-dense core is separated from the membrane by a narrow, less dense zone. The genesis and morphogenetic significance of these various organelles is unknown.


Herbimycin, a new antibiotic produced by a strain of Streptomyces.

Herbimycin, a new antibiotic, was isolated from the fermentation broth of Streptomyces hygroscopicus strain No. AM-3672, a soil isolate. The molecular formula of herbimycin was determined to be C30H42N2O9. Herbimycin was found to have potent herbicidal activity against most mono- and di-cotyledonous plants, especially against Cyperus microiria STEUD. However, Oryza sativa showed strong resistance to herbimycin.


PS-5, a new beta-lactam antibiotic. I. Taxonomy of the producing organism, isolation and physico-chemical properties.

Antibiotic PS-5 is a new beta-lactam antibiotic isolated from fermentation broths of Streptomyces sp. strain A271. The strain was considered to be a new subspecies of Streptomyces cremeus and the name, Streptomyces cremeus subsp. auratilis, was proposed. Fermentative production, isolation and physico-chemical properties of PS-5 are described.


Olivanic acids, a family of beta-lactam antibiotics with beta-lactamase inhibitory properties produced by Streptomyces species. I. Detection, properties and fermentation studies.

The screening of soil actinomyces for beta-lactamase inhibitors is described. Using a plate test a number of strains of Streptomyces were found to produce beta-lactamase inhibitory activity designated olivanic acid complex. Factors affecting the production of this complex by Streptomyces olivaceus ATCC 21379 are reported. The complex showed antibacterial activity and also inhibited a number of different types of beta-lactamase in a progressive manner. Certain ampicillin-resistant bacteria are rendered sensitive to ampicillin in the presence of olivanic acid complex at a concentration which alone did not inhibit growth.


Olivanic acids, a family of beta-lactam antibiotics with beta-lactamase inhibitory properties produced by Streptomyces species. II. Isolation and characterisation of the olivanic acids MM 4550, MM 13902 and MM 17880 from Streptomyces olivaceus.

The olivanic acids MM 4550, MM 13902 and MM 17880 are members of a new family of beta-lactam antibiotics. An isolation and purification process utilising ion-par extraction and ion-exchange chromatography is described and the metabolites are characterised by physico-chemical and biological properties.


The structures of tridecaptins B and C (studies on antibiotics from the genus Bacillus. XXV).

On examining the structures of the antibiotics tridecaptins B and C, the constituent amino acids were separated and their chiralities were determined. The constituent fatty acids were identified by gas chromatography and mass spectrometry. Cleavage with N-bromosuccinimide and sequential analysis by EDMAN degradation demonstrates the sequence of the C-terminal side of tridecaptins B and C. Deacylation with polymyxin acylase of tridecaptin B and successive EDMAN degradation revealed the sequence of the N-terminal side of tridecaptin B. Finally partial acid hydrolysis on tridecaptin C clarified the sequence of the N-terminal side of tridecaptin C.


Resolution of peptide antibiotics, cerexins and tridecaptins, by high performance liquid chromatography (studies on antibiotics from the genus Bacillus. XXVI).

By high performance liquid chromatography, cerexin B was separated into four components (B1, B2, B3 and B4), cerexin D into four components (D1, D2, D3 and D4), tridecaptin A into components (A alpha and A beta), tridecaptin B into four components (B alpha, B beta, B gamma and B delta) and tridecaptin C into three components (C alpha 1, C alpha 2 and C beta 1). All components were preparatively isolated, and their fatty acid and amino acid compositions determined for structural elucidation.


Studies on auromomycin.

A new antitumor antibiotic, named auromomycin, was isolated from the culture broth of Streptomyces macromomyceticus, a macromomycin-producing strain. The antibiotic was recovered from the culture filtrate by salting out with ammonium sulfate and further purified by successive application of ion-exchange chromatography on Amberlite IRA-93 (Cl form) and DEAE-Sephadex (OH form), Gel filtration on Sephadex G-50 and hydrophobic chromatography on Octyl-Sepharose CL-4B. The antibiotic is an acidic polypeptide with a molecular weitht of 12,500 and an isoelectric point of pH 5.4 and consists of 16 different amino acids. It has characteristic absorption maxima at 273 nm and 357 nm in the ultraviolet spectrum and two minima at 280 nm and 350 nm in the optical rotatory dispersion spectrum. Auromomycin exhibits antibacterial activity not only against Gram-positive bacteria, but also Gram-negative bacteria. Antitumor activities of auromomycin were revealed against EHRLICH ascites carcinoma, ascites sarcoma 180, L1210 leukemia and LEWIS lung carcinoma. Auromomycin was found to be converted into macromomycin by adsorption chromatography on Amberlite XAD.


Biological activity of macromomycin.

Macromomycin (MCR) is a polypeptide antimuor antibiotic isolated from the culture broth of Streptomyces macromomyceticus. Antitumor activities of MCR were examined against three different tumor system, i.e., EHRLICH ascites carcinoma, L1210 leukemia and LEWIS lung carcinoma. Daily intraperitoneal treatment with MCR for 5 days showed a strong inhibition against EHRLICH ascites carcinoma. Both single and repeated intraperitoneal injections of MCR were effective over a wide dose range against intraperitoneally inoculated L1210 leukemia and MCR intravenously administered was also active against intravenously inoculated L1210 leukemia. Daily local subcutaneous injections of MCR produced the prolongation of life span of mice to which LEWIS lung carcinoma was subcutaneously inoculated with some cured mice, but daily intraperitoneal injections of MCR showed no activity. Single intravenous administration of MCR inhibited early LEWIS lung carcinoma, but not advanced LEWIS lung carcinoma. The combination of MCR with aracytidine, or cyclophosphamide showed a synergistic activity against L1210 leukemia. MCR was not inactivated by treatment with serum, although neocarzinostatin was markedly inactivated by the same treatment.


Antitumor activity of prumycin.

The antifungal antibiotic, prumycin, was studied for antitumor activity against several tumor systems. It was found to possess potential antitumor activity against a well-established mouse mammary adenocarcinoma in C3H/He mice. It was also active in prolongation of the lifespan of mice bearing P-388 lymphocytic leukemia. Moreover, prumycin did not depress the white blood cell counts in the mouse peripheral blood. However, severe alopecia was observed in mice treated with this agent at dosage level near the LD50.


13C-NMR studies on 1-N-[(S)-4-amino-2-hydroxybutyl]kanamycin A (butikacin) and related aminoglycosides.

The 13C-NMR spectra of 1-N-[(S)-4-amino-2-hydroxybutyl]kanamycin A (butikacin) (Fig. 1, 1b) and some of its related compounds have been recorded and are tabulated, assigned and discussed. The chemical shifts of many of the carbon nuclei are shown to be reasonably invariant amongst this series of compounds. A procedure is described for the determination of the (R,S)-epimer ratio of kanamycin A derivatives which have N-substituted groups containing a chiral centre.


Chemical modification of fortimicins: preparation of 4-N-substituted fortimicin B.

Among the new aminoglycoside antibiotic family for fortimicins, components A, C and D have higher activity compared to their 4-N-deacylated components B and KE. Synthesis and antibacterial activities of 4-N-acyl- and 4-N-alkyl-fortimicin B derivatives are described. 4-N-Acylfortimicin B's, which are relatively unstable in alkaline conditions, were converted to stable 4-N-alkyl derivatives with diborane. The activity is greatly affected by the 4-N-substituents, and the presence of hydrophilic group(s) is necessary to confer activity on the derivatives. 4-N-(2-Aminoethyl)-, 4-N-(4-amino-2-hydroxybutyl)- and 4-N-(2-hydroxy-4-methylaminobutyl)-fortimicin B are the most potent compounds among them.


Congeners of etamycin produced by Streptomyces griseoviridus.

Streptomyces griseoviridus produces in addition to etamycin several related compounds which can be separated by partition chromatography. One of these has been characterized by amino acid analysis and mass spectrometry and shown to have the same structure as etamycin except for replacement of the hydroxyproline residue by proline. Evidence was obtained for additional congeners similarly related to etamycin by amino acid exchange. The relative proportions of such congeners produced by S. griseoviridus depends upon the medium in which the culture is grown. Certain amino acids support good yields of the metabolites and the culture appears to be steered towards the synthesis of congeners containing such amino acids.


Effect of primycin on monovalent cation transport of erythrocyte membrane and lipid bilayer.

The effects of primycin were investigated on the alkali-cation transport of human erythrocytes and on the electric conduction of bimolecular lipid membranes. In the concentration range of 3.10(-6) approximately 10(-5) M primycin increased the permeability of erythrocytes to alkali-cations according to the sequences Cs+ greater than Rb+ approximately K+ greater than Na+, while the conductance of the negatively charged phosphatidylserine bimolecular lipid membrane increased by 2 approximately 3 orders of magnitude. The resistance-lowering effect of primycin strongly depended on the cationic species applied and a selectivity order Na+ greater than K+ greater than Rb+ greater than Cs+ was found. A possible mechanism of the primycin-membrane interaction is suggested on the basis of experimental data.


Holomycin and an antibiotic (MM 19290) related to tunicamycin, metabolites of Streptomyces clavuligerus.

Streptomyces clavuligerus produces penicillin N, several cephalosporins and the beta-lactamase inhibitor clavulanic acid. The detection, isolation and properties of further metabolites of this culture, MM 21801 and MM 19290, are described. MM 21801 was identified as the antibiotic holomycin. MM 19290 was shown to be related to tunicamycin, an antibiotic complex obtained from cultures of Streptomyces lysosuperificus.


The structure of nocamycin, a new antitumor antibiotic.

The structure of nocamycin, a new antitumor antibiotic, has been elucidated with the aid of mass- and PMR-spectroscopic investigation of the antibiotic and its various chemical transformation products. Nocamycin is structurally related to tirandamycins.


3-Hydroxyterphenyllin, a new metabolite of Aspergillus candidus. Structure elucidation by H and C nuclear magnetic resonance spectroscopy.

A new compound isolated from cultures of Aspergillus candidus Link is shown to be 3-hydroxyterphenyllin. The structure was assigned by comparing the 1H and 13C nmr spectra of the metabolite and its acetate derivative with those of terphenyllin and terphenyllin triacetate.


The structure of a novel sugar component of polyene macrolide antibiotics: 2,6-dideoxy-L-ribohexopyranose.

A novel carbohydrate has been isolated after acidic hydrolysis of nystatin A3, candidinin and polyfungin B and its structure established as 2,6-dideoxy-L-ribohexopyranose.


The identification of depsipeptides by chemical ionisation mass spectroscopy.

The chemical ionisation mass spectra of seven naturally occurring depsipeptides and some of their permethylated derivatives have been measured. The primary ionisation process involves an ester group and not an amide or other functionality. It probably occurs randomly when the molecule contains more than one ester link. Unlike electron impact mass spectra, those obtained under chemical ionisation conditions gave sequence information for all of the depsipeptides examined or their permethylated derivatives. A mechanism for the primary fragmentation is proposed.


Controlled biosynthesis of neoviridogriseins, new homologues of viridogrisein II. Production, biological properties and structure of neoviridogrisein II.

Our isolate of Streptomyces griseoviridus, which produced three minor factors in addition to viridogrisein and griseoviridus, which produced three minor factors in addition to viridogrisein and griseoviridin, was more sensitive to the addition of L-proline than the type culture of Streptomyces griseoviridus (NRRL2427). Yield improvement of these minor factors was attempted by the so-called controlled biosynthesis with L-proline. Addition of L-proline increased the production of neoviridogrisein II in which allo-hydroxy-D-proline in viridogrisein was replaced by D-proline.


Bioconversion and biosynthesis of 16-membered macrolide antibiotics. XIII. Regulation of spiramycin I 3-hydroxyl acylase formation by glucose, butyrate, and cerulenin.

The effects of glucose, butyrate, and cerulenin on the formation of spiramycin I 3-hydroxyl acylase were investigated by using the cell-free extract prepared from the spiramycin-producing strain of Streptomyces ambofaciens KA-1028. Glucose induced the formation of the acylase, and this induction was remarkably repressed by butyrate. Cerulenin, on the other hand, not only cancelled the repression by butyrate but also stimulated the formation of the acylase. The unsuccessful trapping of spiramycin I as an intermediate during the bioconversion from neospiramycin I to spiramycin III in the presence of cerulenin was due to the rapid acylation of spiramycin I caused by the acylase induced by cerulenin.


Genetics and biochemical studies of chloramphenicol-nonproducing mutants of Streptomyces venezuelae carrying plasmid.

Chloramphenicol-nonproducing and plasmid-less mutants obtained previously by treatment with acriflavine still produced a small amount of chloramphenicol in a medium. To study the role of plasmid in chloramphenicol production, 70 chloramphenicol-nonproducing mutants were isolated by acriflavine treatment, high-temperature incubation, UV-irradiation or nitrosoguanidine treatment, starting from a producer (SVM2). Most of them did not produce any amount of chloramphenicol. One mutant, SVM2-2A7 was found to produce 1-deoxychloramphenicol instead of chloramphenicol. The mutations (cpp) affecting chloramphenicol production were analyzed by crosses with a producing strain carrying the complementing auxotrophic markers. Except for the plasmid-less strains, all Cpp mutations including the 1-deoxychloramphenicol-producing mutation were mapped between met and ilv on the chromosome. Additional crosses indicated that these chromosomal cpp mutants still carried the plasmids which had a role in increasing chloramphenicol production. Therefore, it can be concluded that the structural genes for all or most steps of chloramphenicol biosynthesis including the 3-hydroxylation of p-aminophenylalanine are located between met and ilv on the chromosome of S. venezuelae and that the plasmid plays an important role in increasing the chloramphenicol production. The activity of arylamine synthetase involved in the initial step of the chloramphenicol biosynthesis was unrelated to the presence or absence of plasmid. Moreover, the presence of plasmids was not required for host resistance to chloramphenicol.


CI-867, a new broad-spectrum semisynthetic penicillin.

The synthesis and antimicrobial activity of a new semisynthetic penicillin are described. Both in vitro and in vivo, the compound shows promising antibacterial activity when compared with piperacillin and ticarcillin. High activity is shown against Pseudomonas and other Gram-negative bacteria.


Antibacterial activity of heneicomycin.

Heneicomycin is structurally similar to efrotomycin, mocimycin (kirromycin) and X-5108 (goldinomycin). Comparisons were limited because of the small supplies available. All antibiotics show the same in vitro antibacterial spectrum although some test cultures were less sensitive to efrotomycin. Heneicomycin compared favorably with efrotomycin when given subcutaneously or per os against infections with Moraxella bovis and Streptococcus pyogenes. The raid elimination of heneicomycin observed following oral administration may account for its poor activity against a Bordetella bronchiseptica infection where efrotomycin is effective. It appears more like X-5108 than efrotomycin biologically. The disaccharide on efrotomycin may account for the difference observed.


Studies on inhibition of adenosine deaminase by isocoformycin in vitro and in vivo.

Isocoformycin is a structural isomer of coformycin which has been demonstrated to be a potent inhibitor of adenosine deaminase. Isocoformycin showed a weaker inhibition of this enzyme than coformycin; the binding of coformycin to enzyme was irreversible, but isocoformycin inhibition was competitive with substrate. The Ki value of isocoformycin was 4.5 approximately 10 X 10(-8) M. Following intraperitoneal injection of isocoformycin in mice, the adenosine deaminase activity of homogenates of several organs was determined and the following ED50 values (50% inhibition doses) were observed: 29 mg/kg for thymus, 13 mg/kg for spleen, 80 mg/kg for liver and 20 mg/kg for kidney. The inhibition of adenosine deaminase in rabbit blood in vitro was also tested in comparison with coformycin.


Nicotine fading and self-monitoring for cigarette abstinence or controlled smoking.

This study compared four treatment approaches to cigarette smoking: (1) a nicotine fading procedure, in which subjects changed their cigarette brands each week to ones containing progressively less nicotine and tar; (2) a self-monitoring procedure in which subjects plotted their daily intake of nicotine and tar; (3) a combined nicotine fading/self-monitoring procedure; and (4) a slightly modified American Cancer Society Stop Smoking Program. Thirty-eight habitual smokers were assigned to one of the treatment groups. The study had two goals: (1) to achieve a clinically significant percentage of abstinence, and (2) to reduce the nonabstainers' smoking to a "safer" level by having them smoke low tar and nicotine cigarettes. The 18-month followup results showed that the nicotine fading/self-monitoring group was the most successful: 40% were abstinent and all who had not quit were smoking cigarettes lower in tar and nicotine than their baseline brands. Half the nonabstainers had decreased their rate of smoking relative to baseline while the other half had increased. Furthermore, the fading/self-monitoring group achieved the largest reductions from baseline in daily nicotine and tar intake (61% and 70% respectively). The results suggest that the study's goals were achieved and that the nonaversive combined procedure could be used to treat not only habitual smokers but also smokers with severe cardiovascular and respiratory problems, because it does not have some of the inherent limitations of the successful aversive smoking cessation procedures.


Biofeedback and rational-emotive therapy in the management of migraine headache.

Twenty-four migraine patients were randomly assigned to one of four conditions: (a) self-monitoring of headache activity (waiting list), (b) frontalis EMG biofeedback, (c) digit temperature biofeedback, and (d) digit temperature biofeedback plus Rational-Emotive Therapy (RET). Bidirectional control over the target physiological response was assessed through a reversal design in each session. Following at least a four-week baseline, the three biofeedback groups received 8 to 10, 30-minute sessions of bidirectional biofeedback training, scheduled twice a week. Subjects in the combined digit temperature biofeedback plus RET group received three 40-minute sessions of RET as an addition to the third, fifth, and seventh biofeedback sessions. Records of daily home practice were kept throughout treatment and three-month followup. Subjects on the waiting list monitored headaches for at least five months, corresponding to "baseline", "treatment", and three-month followup. Digit temperature biofeedback alone and in conjunction with RET did not prove to be more effective than the control conditions. All the EMG subjects reduced headache activity to two-thirds or less of the baseline level by the third month of followup. Bidirectional digit temperature performance did not improve with training, was demonstrated in only 33% of the biofeedback sessions, was not maintained over time, and was unrelated to improvement in headache activity. EMG subjects reported biofeedback performance to be an easier task and met the performance criterion on 85% of the sessions. The frequency of home practice contributed over 55% of the variance in retrospective estimates of headache improvement but was not related to changes in daily records of headache activity.


Comparison of established and innovative weight-reduction treatment procedures.

A central component of a commonly used weight-loss procedure developed by Stuart (1967), which emphasizes situational engineering (SE), was compared to eating behavior control (EBC), a treatment employing reinforcement of changes in eating habits. A third treatment encouraged development of individuals' own applications of a model of self-control and attempted to provide effective feedback regarding progress toward weight-loss goals. This self-initiated treatment (SI) was designed to be less directive than many behavioral treatment packages and to be more realistic about the expectations of subjects and the actual eating habits of overweight and normalweight people. Following baseline, treatment lasted six weeks and followup lasted 16 weeks. While all treatments produced weight losses, EBC surpassed SE during treatment. SI losses during treatment did not differ from SE or EBC, but SI was the only group that lost weight throughout followup, indicating the possible utility of more subtle, less directive interventions than those that have often been associated with behavioral approaches. The results of SE question the applicability to clinical procedures of the external cue hypersensitivity theory of obesity (Schachter and Rodin, 1974), to which SE is closely related.


Theoretical, practical, and social issues in behavioral treatments of obesity.

Although showing superior maintenance, behavioral treatments of obesity typically produce small weight losses at a decelerating rate. Rather than reflecting poor compliance with treatment, these findings are consistent with known compensatory metabolic changes that operate to slow weight loss and promote regain. Other problems associated with dieting include failure of caloric regulation, hyper-responsivity to food palatability, and hunger, which is greater under conditions of moderate restriction and unpredictability of access to food. The inevitability of treatment failure in many instances must be faced and efforts made to prevent further worsening of the obese patient's self-esteem. Prognosis and treatment planning may be aided by consideration of the historical difficulties of weight loss, the degree of hunger experienced on diets, which may reflect important physiological differences among individuals, and the use of food to optimize arousal level. Full involvement of the patient in setting goals and planning treatment is recommended.


Behavioral management of exercise: contracting for aerobic points.

Behavioral contracting was used to encourage physical exercise among college students in a multiple-baseline design. Subjects deposited items of personal value with the experimenters, which they could earn back on fulfillment of two types of contract contingencies. Subjects selected weekly aerobic point criteria, which they could fulfill by exercising in the presence of other subjects. In addition, subjects contracted to observe and record the exercise of other subjects and to perform an independent reliability observation once each week, with both of these activities monitored by the experimenters. Results indicated that the contract contingencies produced increases in the number of aerobic points earned per week for seven of eight subjects, that the aerobic point system possesses several advantages as a dependent variable for behavioral research on exercise, and that inexperienced observers could be quickly trained to observe exercise behavior and to translate those observations into their aerobic point equivalents. Finally, in a followup questionnaire completed 12 months after the end of the study, seven of the eight subjects reported that they were earning more aerobic points per week than had been the case during the baseline condition of this experiment.


A social ecology of hyperactive boys: medication effects in structured classroom environments.

Hyperactive boys on methylphenidate (Ritalin), hyperactive boys on placebo, and comparison boys were observed in quasi-naturalistic classroom settings. Ambient stimulation (quiet versus noisy conditions) and source of regulation (self-paced versus other-paced activities) were varied in a 2 x 2 design. Compared to their peers, hyperactive boys on placebo showed lower rates of task attention and higher  rates of gross motor movement, regular and negative verbalization, noise-making, physical contact, social initiation, disruption, and acts that were perceived as energetic, inappropriate, or unexpected. Self-paced activities resulted in increased rates of verbalization, social initiation, and high-energy episodes. High ambient noise levels reduced task attention and increased the rates of many other behaviors including verbalization, physical contact, gross motor movement, and high-energy acts. Medication-by-situation interactions emerged for both classroom dimensions, with hyperactive boys on placebo being readily distinguishable from their peers under some classroom conditions and indistinguishable under other conditions. Moderate relationships were found between teacher ratings and many individual behavior categories. Discussion focused on (a) the merits and limitations of a social ecological research perspective, and (b) the implications of these findings for the design of intervention strategies.


Behavioral assessment for pediatric intensive care units.

Two studies were conducted to analyze behaviors of staff and patients on a Pediatric Intensive Care Unit (PICU). In the first study, behavioral observation procedures were employed to assess patient state, physical position, affect, verbal behaviors, visual attention and activity engagement, and staff verbal behavior. On the average, one-third of the patients were judged to be conscious and alert but markedly nonengaged with their environment. In the second study, a member of the hospital staff provided alert patients with individual activities to determine whether a simple environmental manipulation could positively affect behavior of children in intensive care. Employing a reversal design, the activity intervention was found to increase attention and engagement and positive affect, and to decrease inappropriate behavior. Both studies demonstrate that behavioral assessment procedures can provide an empirical basis for designing PICU routines affecting children's psychosocial status, and, thus, complement current procedures designed to provide quality medical care.


Evaluation of biofeedback in the treatment of borderline essential hypertension.

Direct biofeedback of blood pressure was compared with frontal EMG biofeedback and with self-instructed relaxation for the treatment of essential hypertension in a controlled group outcome study. Patients were followed up for four months after the end of treatment. Generalization of treatment effects was assessed through pre- and posttreatment measurements of blood pressure under clinical conditions in a physician's office. There were no significant reductions in diastolic blood pressure. The systolic blood pressure (SBP) of the patients receiving blood pressure biofeedback decreased 8.1 mm mercury (p = 0.07) and the SBP of the patients in the relaxation condition decreased 9.5 mm mercury (p = 0.05). In the generalization measures, there were significant reductions in SBP for the relaxation group. The results are discussed in terms of the general lack of replicability within the area of biofeedback treatment of hypertension.


Personality, body weight and ultimate outcome in anorexia nervosa.

The Eysenck Personality Inventory, a highly standardized self-rating questionnaire, allows 2 measures of psychosocial functioning on independent scales entitled "emotionally" and "extroversion/introversion." A population of severely ill, low body weight anorectics revealed a high degree of "introversion" and variable amounts of "emotionality" on these measures. Low amounts of "emotionality" were associated with abnormally high "lie" scores. Following restoration of body weight to full matched population mean levels within an intensive treatment program, significant changes occurred on these measures--increased extroversion scores, decreased emotionality and lie scores. However, possibly qualitative changes had occurred in the state of emotionality since high levels were now related to good clinical outcome many years later. The nature of this phenomenon is discussed.


Racing thoughts in depressed patients.

Patients with racing thoughts and depression may have atypical features that suggest a schizophrenic or borderline state and make diagnosis problematic; clinical management may be difficult because of failure to respond to standard treatments. These patients may have variants of affective illness; and may respond favorably to lithium carbonate.


Psychiatric illness in a general urban emergency room: daytime versus nighttime population.

Two hundred adults presenting to the emergency room of an urban general hospital were interviewed by a standardized technique to evaluate the existence of a current or previous psychiatric illness. Half of the subjects presented between 8:00 a.m. and 4:00 p.m. (daytime group), and 100 presented between 12:00 a.m. and 8:00 a.m. (nighttime group). In the nighttime population 65% were judged to have current or past psychiatric illnesses. In the daytime population only 36% had current or past psychiatric illnesses. The differences between the 2 populations was highly significant. Fewer than 10% of the 200 patients presented with neuropsychiatric symptoms in their chief complaints.


Correlates of clinicians' perceptions of patients in alcoholism treatment.

Previous research has shown that clinicians' perceptions of alcoholic patients' interpersonal attractiveness exert an influence on clinical decision processes. The present study investigated the relationship between clinicians' perceptions of alcoholic patients, as measured by Byrne's Interpersonal Judgment Scale, and patients' personality, cognitive, and demographic characteristics. The results suggest that the male alcoholic patient is more likely to be perceived favorably if he is young and exhibits behaviors that correspond to clinicians' treatment expectations. Overall, the measured personality characteristics of patients accounted for only small proportions of the variance in clinician's ratings of patients attractiveness. Alternative interpretations of these data are discussed with reference to subsequent research efforts.


Female orgasm: role of pubococcygeus muscle.

An examination is made of the role of the pubococcygeus muscle in relation to female orgasm in 281 women. A statistically significant difference is reported between orgasmic and anorgasmic women and the physiological state of the pubococcygeus muscle as measured using a pressure sensitive device inserted in the vagina. These data suggest the pubococcygeus muscle plays an important part in the pathophysiology of female orgasm.


Post-parathyroidectomy psychosis: clinical and research implications.

The case of a 52-year-old female who suffered a post-parathyroidectomy pyschosis is presented and similar cases from the literature are reviewed. Studies of calcium and mangnesium metabolism in mental illness are reviewed, as fluctuations of these ions are felt to be involved in the pathogenesis of this condition. A definable syndrome of post-parathyroidectomy psychosis may exist and the implications of such a syndrome are discussed.


Hyperaldosteronism (Conn's disease) presenting as depression.

A case of muscle weakness with hypertension is presented. The patient had symptoms of depression. Diagnosis of hyperaldosteronism was suspected because of a low serum potassium and confirmed by discovery of an adrenal adenoma. The role of hypokalemia in mental disturbances is reviewed. Emphasis is placed on possible metabolic etiologies when mood changes, muscle paresis and hypertension coexist.


MMPI and drug treatment in alcohol withdrawal.

Thirty-four inpatients in a mild phase of alcohol withdrawal, exhibiting anxious and depressive symptoms, were administered an MMPI before and immediately after a 4-week drug treatment period. A double-blind design was used; 17 patients were treated with chlordiazepoxide and 17, with thioridazine. The pretreatment MMPI profile was typical of alcoholic patients; namely, peaks on scales 4 and 2. After the fourth week, the MMPI indicated that thioridazine was significantly more effective than chlordizaepoxide. Decreases in 7 of the 10 clinical scales averaged 7.29 T-score points, whereas only scale 6 was significantly decreased for the patients treated with chlordiazepoxide.


Detection of gastrin-specific mRNA using oligodeoxynucleotide probes of defined sequence.

Three oligodeoxynucleotides have been chemically synthesized and used as hybridization probes to detect gastrin-specific mRNA within an heterogeneous population of RNA molecules. Using these oligonucleotides whose nucleotide sequences were deduced from the amino acid sequence of the hormone, 0.2 fmol of gastrin mRNA can be detected/microgram of poly(A)-RNA from hog antrums. The 32P-labeled oligonucleotides hybridize specifically to RNA covalently coupled to DBM-paper (Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354). Labeled oligonucleotides also hybridize specifically to RNA separated by gel electrophoresis in the presence of CH3HgOH and covalently coupled to DBM-paper. Using this procedure, we have found that the mRNA coding for gastrin contains about 620 nucleotides, which is in agreement with results obtained using gastrin cDNA to determine mRNA size.


A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.


The reduction of methemerythrin by e-aq and CO2- from pulse radiolysis studies.

The rate constants for reduction of methemerythrin from Phascolopsis gouldii and Themiste pyroides by hydrated electrons are 2.0 and 3.9 x 10(9) M(-1)s(-1), respectively, at pH 8.2, I = 0.03 M, and 25 degrees C. There is only a small increase in rate when the pH is lowered to 6.3 and a very small decrease when the ionic strength is raised to 0.1 M. Adding azide ion (to form the met-azide adduct) has little effect on the reactivity towards e-aq. For the monomer form, metmyohemerythrin from T. pyroides, the reaction rate constant is 4.5 x 10(9) M(-1)s(-1). Methemerythrin from T. pyroides reacts with CO2- with a rate constant 6.8 x 10(7) M(-1)s(-1). The reactivity sequence e-aq greater than CO2- greater than SO2- (from dithionite reduction) towards methemerythrin is the same as that observed with reduction of heme proteins but the rate constants are some 10 to 100 times smaller for the former. Only 10 to 20% of the e-aq or CO2- radicals generated effect reduction of the iron centers in methemerythrin.


Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.


The structure of mouse testicular lactate dehydrogenase isoenzyme C4 at 2.9 A resolution.

The structure of lactate dehydrogenase isoenzyme C4 from mouse testes was solved at 2.9 A resolution using the technique of molecular replacement. The electron density map revealed a ternary-like configuration of the flexible loop peptide although density corresponding to the coenzyme and substrate molecules was not present. Apparently the apo-lactate dehydrogenase molecule in solution is in a dynamic equilibrium between the O (loop open as found in dogfish apo-lactate dehydrogenase M4) and C (loop closed as found in a variety of ternary complexes) conformations. During crystallization of the apoenzyme one or the other conformers is selected. The apparent stability of the closed conformation for the apo-lactate dehydrogenase C4 molecule may in part explain the low catalytic turnover number of the C isoenzyme. A possible substitution of an arginine residue at position 30 may also be a contributing factor as well as allowing NADP to act as coenzyme.


The tentative amino acid sequencing of lactate dehydrogenase C4 by X-ray diffraction analysis.

A tentative amino acid sequence of mouse testicular lactate dehydrogenase C4 was deduced from an electron density map and comparison with five other known lactate dehydrogenase sequences. The amino acid composition determined by chemical analysis agrees reasonably well with the present results. Necessary changes in amino acids were largely conservative and confined to the external portions of the molecule. Residues in the Q and P subunit contact regions were particularly well conserved as were most internal residues. The minimum base change/codon was similar between the C and H isoenzymes and between the C and M isoenzymes.


Lima bean proteinase inhibitor. Origins of circular dichroism bands and modification by Br2- and (CNS)2-.

Origins of CD bands in lima bean proteinase inhibitor were deduced from an acetylation-deacetylation study of the sole tyrosyl residue in the protein (Tyr 69), and by analogy with Bowman-Birk soybean proteinase inhibitor, a homologous protein with similar spectral properties. Tyr 69 is relatively inaccessible to N-acetylimidazole; 100-fold molar excess of the reagent in the presence of 6 M guanidine hydrochloride elicited about 70 to 80% O-acetylation. A broad negative CD band centered around 280 nm arises mainly from the longest wavelength transition of cystinyl side chains (epsilon L--epsilon R approximately equal to -0.8 M-1 cm-1 per disulfide). The second cystinyl transition gives rise to a positive CD band of a comparable intensity at 247 nm. The Lb vibronic transition of Tyr 69 has negative CD around 280 nm, contributing approximately 10% of the total CD intensity at 278 nm (epsilon L--epsilon R approximately equal to -0.5 M-1 cm-1). The 232 nm positive shoulder is from the La vibronic transition of Tyr 69. Radical anions, Br2- and (CNS)2-, generated by the irradiation of N2O-saturated inhibitor solutions containing KBr or KCNS, reduced tyrosyl CD without affecting disulfide CD bands, indicating that the radical anions damaged Tyr 69 without altering protein conformation. The inhibitor modified at Tyr 69 by Br2- and (CNS)2- retained full activity toward trypsin and chymotrypsin. The irradiation of the inhibitor in the air-saturated solution led to loss in tyrosyl as well as cystinyl CD bands and decline in both antiproteinase activities.


Purification and chemical characterization of papain-solubilized histocompatibility-2 antigens from mouse liver.

A large scale purification of histocompatibility-2 (H-2) antigens from mouse liver is described. The antigens were solubilized by a limited papain digestion of a crude preparation of liver membranes (strain A/J) and purified by ion exchange chromatography, gel filtration, affinity chromatography, and isoelectric focusing. The overall degree of purification of H-2Kk was 1,300-fold and that of H-2Dd was 1,500-fold; approximately 8 mg of purified H-2a antigens were obtained from 1 kg of liver. The purification was followed by a sensitive radioimmunoassay in which H-2a-containing fractions were used to inhibit the binding of 125I-labeled H-2a to appropriate antisera. H-2Dd and H-2Kk co-purified through all the steps but the concentration of H-2Kk was 2- to 3-fold higher than that of H-2Dd in the liver homogenate as well as in the purified H-2 preparation. beta 2-microglobulin was initially present in a 3- to 10-fold excess over H-2 in the liver homogenate, but the purified H-2 preparation contained approximately 2 mol of alloantigenic heavy chain/mol of beta 2-microglobulin. Isoelectric focusing and disc-gel electrophoresis showed a charge heterogeneity of H-2, with a mean isoelectric point of pH 4.9. Electrophoresis on sodium dodecyl sulfate gels showed one band. Denaturing conditions were required to remove beta 2-microglobulin and small amounts of impurities from H-2. The amino acid sequence of the first 27 residues of the isolated heavy chains was determined.


Role of eukaryotic initiation factor 5 in the formation of 80 S initiation complexes.

The function of eukaryotic initiation factor 5 (eIF-5) from rabbit reticulocyte lysate has been studied by sucrose gradient preparation of 40 S and 80 S initiation complexes. eIF-5 is required for transfer of initiator tRNA from 40 S preinitiation complexes to puromycin-reactive 80 S complexes. The transfer is dependent upon GTP hydrolysis and is associated with release of eIF-2 and eIF-3 from the 40 S subunit. The GTP-dependent loss of eIF-2 and eIF-3 is catalyzed by eIF-5 in the absence of 60 S subunits or when subunit joining is prevented by edeine, but not when GTP is replaced by GuoPP(NH)P. Unstable 40 S subunit . Met-tRNAf complexes generated by eIF-5 can form puromycin-reactive 80 S complexes when 60 S subunits are added in the absence of added GTP. In addition, kinetic evidence is presented that indicates GTP hydrolysis occurs prior to 80 S complex formation.


Thermodynamics of the interaction of insulin with its receptor.

Insulin binding to its cellular receptors is markedly dependent on the temperature. The thermodynamic parameters for the reaction of insulin with the high affinity state of its receptor have been evaluated with equilibrium studies at multiple temperatures between 5 degrees and 37 degrees C. The thermodynamics of the insulin-receptor interaction is not classical. The van't Hoff plot is not linear. Both the enthalpy and entropy changes, due to the formation of the hormone . receptor complex, decrease markedly with temperature, corresponding to a large heat capacity change of -766 cal/(mol deg) at 25 degrees C. The reaction is endothermic and entropically driven at low temperature and exothermic and enthalpically driven at higher temperature. This thermodynamic behavior is suggestive of a hydrophobic reaction and supports Blundell's concept that the loss of non-polar surface residues in the formation of the hormone . receptor complex is an important driving force of the reaction. Alternatively, this nonclassical behavior may indicate that the reaction of insulin with its receptor involves more than one step.


Interaction of membranous enzymes with membranous lipid substrates. Hydrolysis of diacylglycerol by lipase in rat brain microsomes.

The phospholipids in rat brain microsomes were labeled with tritium by intracerebral administration of radioactive fatty acids and converted to diacylglycerol with phospholipase C. The latter lipid was hydrolyzed in situ at pH 4.8, to monoacylglycerol and fatty acid by the endogenous microsomal lipase. This paper provides an experimental approach to determine whether the lipid was degraded by enzyme molecules residing in its own membrane (intramembrane interaction) or an adjacent membrane (intermembrane interaction). Direct interaction between separate membranes containing enzyme or substrate showed the existence of the inter-membrane route while dilution experiments provided evidence for the presence of the intramembrane interaction as well. A probable difference in the mechanisms of these two interactions is suggested by different shapes of the curves that describe the reaction rate as a function of the endogenous substrate. The curve resulting from the intermembrane interaction was hyperbolic while that representing the intramembrane route was of a parabola-like shape. Competition experiments suggested that when given a choice between the two, the enzyme utilized preferentially the substrate molecules in its own membrane.


Stimulation and inhibition of cellular functions by glucocorticoids. Correlations with rapid influences on chromatin structure.

The effect of media conditions on the glucocorticoid response has been examined in three types of cultured cells. In rat pituitary tumor cells (GC cells) growth hormone production was stimulated by glucocorticoids provided fresh culture media was present (enriched media conditions). In contrast, dexamethasone either failed to induce or deinduce growth hormone synthesis if added to cultures which had not received fresh media for 3 days (depleted media condition). With human skin fibroblasts, cortisol stimulated [3H]thymidine incorporation in the enriched condition but inhibited this response in the depleted condition. In mouse lymphoma (S49) cells the enriched media conditions significantly delayed the killing response to glucocorticoids (20% killing after 24 h versus 90% killing after 24 h for the depleted condition). Thus, the magnitude and in some cases, the direction of the glucocorticoid response are sensitive to the conditions to which the cells are exposed. In all three cell types the steroid also rapidly (detectable by 15 min, maximal by 2 h) altered chromatin structure as detected by a change in the number of initiation sites for Escherichia coli RNA polymerase assayed under cell-free conditions. This early nuclear response could be in a positive or negative direction and was also affected by the culture conditions; enriched media favored a positive or less negative effect on the initiation sites by the steroid, while depleted media favored a steroid-induced inhibition of this chromatin function. In S49 and GC cells the kinetics and magnitude of the change in chromatin closely followed receptor . glucocorticoid complex binding to nuclei while removal of dexamethasone from the culture media resulted in a rapid (t 1/2 = approximately 20 min) disappearance of the effect which paralleled loss of bound hormone from the nucleus. The glucocorticoid effect on chromatin was not observed in two lines of glucocorticoid-resistant mutant S49 cells. One line (R-) lacks detectable glucocorticoid receptors; the other line (Nti) has receptors that bind the hormone normally, but the receptor . glucocorticoid complexes bind more avidly to the nucleus. These results suggest that the receptor is involved in both the stimulatory and the inhibitory effects on chromatin. The findings in the Nti cells and of a slight lag between nuclear binding of receptors and initiation site alteration implies that some receptor property, in addition to nuclear binding per se, is responsible for the influence on chromatin. These results are discussed in terms of a model in which steroid hormones initiate their actions by influencing a reaction that modifies chromatin structure. The direction and magnitude of the reaction, and its effect on the expression of specific genes, are dictated by the metabolic state and differentiation of the cell.


Purification and characterization of two phospholipid exchange proteins from bovine heart.

Two phospholipid exchange proteins from bovine heart have been purified approximately 2000-fold and judged greater than 90% pure. The proteins are similar in molecular weight (both 33,400 by polyacrylamide gel electrophoresis and 23,500 by gel filtration), in amino acid composition, and in specificity, although they differ in isoelectric points, 5.3 and 5.6. The transfer of phospholipids between artificial membranes is catalyzed by these proteins at the following relative rates: 100 for phosphatidylinositol, 35 for phosphatidylcholine, 5 for sphingomyelin, and 0.1 for phosphatidylethanolamine. The use of these exchange proteins in the study of mixed phospholipid vesicle structure is demonstrated. The purified proteins catalyze the substitution of one membrane phospholipid species for another at a rate comparable to true exchange. The phospholipid exchange activity is inhibited by the presence of sphingomyelin, and also by reagents which react with sulfhydryl groups. Evidence is presented for two sites of N-ethylmaleimide binding on these exchange proteins. Reaction with one site has little effect on activity and occurs in the absence of membranes. Reaction with the second site occurs in the presence of phospholipid vesicles and leads to complete, irreversible inhibition of exchange activity.


Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus.

The vaccinia virus-induced DNA polymerase has been purified about 500-fold from a cytoplasmic extract of vaccinia-infected HeLa cells. Analysis of the purified fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide of 110,000 daltons, which is greater than 95% pure. This polypeptide co-sediments with polymerase activity through a glycerol gradient. The sedimentation coefficient of the enzyme is 6.3 S, and its Stokes radius is 4.6 nm. The molecular weight of the native enzyme derived from these values is 115,000. Vaccinia polymerase is therefore a single large polypeptide of 110,000 to 115,000 daltons. The purified fraction has no significant endonuclease activity, but a strong exonuclease activity co-purifies with polymerase activity through every step in the isolation. The polymerase and exonuclease activities are inactivated at 45 degrees C at the same rate. It is likely, therefore, that both activities are catalyzed by the same polypeptide. The exonuclease hydrolyzes DNA predominantly in the 3' leads to 5' direction, to produce 5' mononucleotides. The exonuclease degrades single-stranded DNA more rapidly than duplex DNA, and the rate of digestion of both single-stranded and double-stranded DNA increases as the size of the substrate decreases. Single-stranded circular DNA is a potent inhibitor of the exonuclease activity, but duplex circular DNA has no significant effect on its activity.


Action of Arthrobacter ureafaciens sialidase on sialoglycolipid substrates. Mode of action and highly specific recognition of the oligosaccharide moiety of ganglioside GM1.

A new bacterial sialidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) isolated from the culture filtrate of Arthrobacter ureafaciens was characterized in detail with respect to its action on sialoglycolipids. Strong electrolytes had a reversible inhibitory effect on the action of the enzyme on brain gangliosides in accordance with Debye-Hückel effect of ionic environment on ionic activity, and resulted in an acidic shift and a broadening of the pH optimum. Both ionic and non-ionic detergents markedly enhanced the enzymic activity on the gangliosides, and caused an acidic shift on the pH optimum of this enzyme. Sulfhydryl groups seemed to be involved in its active site. This enzyme had a highly specific action on sialidase-resistant ganglioside GM1, showing about 100-fold higher activity on GM1 than Clostridium perfringens sialidase, the only sialidase so far reported to cleave the lipid substrate in the presence of bile salts. In the absence of detergents, the activity of A. ureafaciens sialidase on GM1 was very low. Ganglioside GM1 in either the monomeric or micelar form was hydrolyzed to asialo-GM1 by A. ureafaciens sialidase most efficiently in the presence of sodium cholate of about three times the GM1 molar concentration. The presence of detergents increased both the Km and Vmax values for ganglioside GM1. The oligosaccharide prepared from GM1 by ozonolysis was cleaved well by this sialidase in the absence of detergents, and no detergent was found to affect the hydrolysis. The Km value for the sugar substrate was about two orders of magnitude greater than that for the corresponding lipid substrate. It is suggested that the hydrophobic ceramide moiety increases affinity of the lipid substrate to the enzyme, but inhibits hydrolysis of the substrate, possibly due to its hydrophobic interaction with hydrophobic portions of the enzyme molecule (resulting in lower Km and Vmax for lipid substrates). This inhibition may be released by detergent due to formation of mixed micelles of sialoglycolipid and detergent molecules. It is also indicated that recognition of the specific saccharide structure of GM1 by individual sialidases is essential for release of the resistant sialyl residue, and that A. ureafaciens sialidase seemed to have an isoenzymic or oligomeric structure.


Modulation of the phosphorylation state of rat liver pyruvate kinase by allosteric effectors and insulin.

The regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone. In this case, flux through pyruvate kinase was increased. The increase in flux correlated with an elevation in fructose bisphosphate levels but not with P-enolpyruvate levels which were unchanged. Pyruvate kinase was activated and its affinity for P-enolpyruvate was increased 7-fold in hepatocyte homogenates. Precipitation of the enzyme from homogenates with ammonium sulfate removed fructose 1,6-bisphosphate and activation was no longer observed. These results indicate that flux through and activity of pyruvate kinase can be controlled by the intracellular level of fructose 1,6-bisphosphate. The effect of elevated fructose 1,6-bisphosphate levels on the ability of glucagon to inactivate pyruvate kinase was also studied where only covalent enzyme modification is observed. Inactivation by maximally effective hormone concentrations was unaffected by elevated levels of fructose 1,6-bisphosphate, but the half-maximally effective concentration was increased from 0.3 to 0.8 nM. Activation of the cyclic AMP-dependent protein kinase by 0.3 nM glucagon was unaffected, but the initial rate of pyruvate kinase inactivation was suppressed. These results suggest that alterations in the level of fructose 1,6-bisphosphate can affect the ability of physiological concentrations of glucagon to inactivate pyruvate kinase by opposing phosphorylation of the enzyme. Consistent with this view was the finding that physiological concentrations of fructose 1,6-bisphosphate inhibited in vitro phosphorylation of purified pyruvate kinase. Inactivation of pyruvate kinase by 0.3 nM glucagon or 1 microM phenylephrine was also suppressed by 10 nM insulin. Insulin did not act by increasing fructose 1,6-bisphosphate levels. The antagonism to glucagon correlated well with the ability of insulin to suppress activation of the cyclic AMP-dependent protein kinase. However, no such correlation was observed with phenylephrine in the absence or presence of insulin. Thus, insulin can enhance pyruvate kinase activity by both cyclic AMP-dependent and independent mechanisms.


Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural analysis of novel hydroxylated compounds.

Arachidonic acid was incubated with rabbit peritoneal polymorphonuclear leukocytes (glycogen-induced) and compounds obtained from ether extractions were fractionated by silicic acid column chromatography. A fraction containing several unidentified metabolites of arachidonic acid was analyzed by reversed phase-high pressure liquid chromatography. The metabolites were esterified and further purified by silicic acid high pressure liquid chromatography. The structures of the pure compounds were elucidated by infrared and ultraviolet spectrometry, ozonolysis, and gas chromatography-mass spectrometry. The following novel compounds were identified: Compound 1, 5S, 12R-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 2, 5S, 12S-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 3, 5, 6-dihydroxy-7,9,11,14-eicosatetraenoic acid; Compound 4, a diastereoisomer of the latter. Evidence for the occurrence of the delta-lactone forms of the 5,12-dihydroxy acids is also presented.


Determination of the ADP concentration available to participate in energy metabolism in an actin-rich cell, the platelet.

Almost all cells contain actin, which in its polymerized form, F-actin, binds 1 molecule of ADP/monomer. Little is known about the availability to metabolism of this bound ADP. A comparison was therefore made between perchloric acid and EDTA/ethanol extracts of human blood platelets. When the cells were extracted under conditions where the ATPase activity was negligible, the ethanol extracts had a 75% higher ATP/ADP ratio and a higher adenylate energy charge than perchloric acid extracts. The methods differed in that a considerable portion of protein-bound ADP was not extracted by ethanol. This bound ADP behaved as though it were unavailable to energy metabolism and should thus be considered as a compartment separate from the bulk metabolic pool of extragranular platelet adenine nucleotides. These results suggest that the level of ADP obtained with the common acid extraction overestimates the level available to participation in metabolism.


Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.


Genetic polymorphism in normal human fibroblasts as analyzed by two-dimensional polyacrylamide gel electrophoresis.

Two dimensional gel electrophoresis has been used to measure the degree of genetic polymorphism among the proteins of normal human fibroblasts. Autoradiographic analysis of the gel protein profiles from radioactively labeled cells allowed comparison of as many as 300 discrete polypeptides at a time. In addition, a newly developed technique for double label autoradiography was used to increase the sensitivity of the system for detection of small differences in the protein profiles of different cell lines. Only about 1.2% of the proteins of different cell lines were found to differ in their electrophoretic mobility. This corresponds to an average heterozygosity of approximately 0.6%. Previous studies of genetic polymorphism using different methods of one-dimensional electrophoretic analysis have estimated the average heterozygosity of the human population at about 6.7%. Detailed mathematical analysis shows the variation of the observed from the expected number of differences to be statistically highly significant. While the reasons for this difference are not clear, the observation of low levels of genetic polymorphism on two-dimensional gels should enhance the usefulness of this technique for detection of altered proteins in inherited disease.


Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines.

An improved method of two-dimensional gel electrophoresis has been developed which produces high resolution, reproducible images suitable for computer analysis. In the images that are presented, more than 800 proteins have been resolved without significant overlap, and many more proteins can be detected after longer exposures. To establish the usefulness of such methods for detailed quantitative comparisons of cultured cells, extensive controls have been carried out to test the reproducibility of the electrophoretic procedures, the sample preparation procedures, and the cell culture conditions. A computerized scanning system has been developed which can automatically detect and integrate the densities of the spots on a two-dimensional fluorogram or autoradiogram. The corresponding proteins from two or more samples can then be matched and their intensities compared. Several types of graphic output have been used to display the number and magnitude of the differences between the compared samples. These methods were used to study the patterns of protein synthesis in the nerve cell line B103 and the glial cell line B9. Both exponentially dividing and stationary cultures were analyzed and the relative rates of synthesis of approximately 300 proteins were compared by computer. For each cell line, no major qualitative differences were found between dividing and stationary phase cells although numerous quantitative differences of up to 15-fold were detected. The proteins that were increased or decreased in rate of synthesis as B103 cells became confluent were in general not the same proteins that were increased or decreased in rate of synthesis as B9 cells reached confluence, indicating that most of the changes do not reflect growth control responses common to all cells. When the two cell lines were analyzed in the same state of growth and compared by computer, qualitative differences were found in approximately 5% of the proteins analyzed, and at least 40% of the shared proteins were involved in quantitative differences of 2-fold or more. The rates of synthesis of the shared proteins were more divergent between the two cell lines than between dividing and stationary phase cells of either line. These studies show, therefore, that these cell lines can be distinguished, regardless of growth state, by their cell-specific proteins and by their characteristic rates of synthesis of many of the shared proteins.


Purification of RNA-DNA hybrids by exclusion chromatography.

A simple method for selection of RNA-DNA hybrids has been developed and applied to the purification of adenovirus-specific messenger RNA. Cytoplasmic RNA prepared from adenovirus type 2 (ad2)-infected HeLa cells or from an ad2-transformed rat cell line was hybridized in solution to the complementary strands of ad2 DNA. The hybridization mixture was subsequently fractionated by chromatography on a Sepharose 2B column. The intact probe DNA as well as the RNA-DNA hybrids are excluded from the gel matrix and elute with the void volume. Nonhybridized RNA, in contrast, is included into the gel matrix and elutes as a broad peak well separated from the excluded fractions. Fractions corresponding to the void volume, were collected and the RNA-DNA hybrids were denatured in 90% formamide. The selected RNA was separated from the DNA by affinity chromatography on poly(U)-Sepharose. Restriction endonuclease fragments of DNA with a large enough size to make them excluded from the agarose column were also used for hybridization. In these experiments hybridizations were carried out under conditions which would allow R-loop formation (Thomas, M., White, R.L., and Davis, R.W. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2294-2298) and the hybridized RNA was separated from unhybridized RNA by Sepharose chromatography. The validity of the method was demonstrated by programming an in vitro protein-synthesizing system with selected RNA.


Differential dependence of reovirus-associated enzyme activities on genome RNA as determined by psoralen photosensitivity.

Irradiation with long wavelength UV light in the presence of 4'-substituted psoralens abolished the in fectivity of reovirus and reovirus subviral particles Photoreaction of intact virions, cores, or genome RNA resulted in the formation of psoralen monoadducts and cross-links, demonstrating that the viral genome RNA in situ is double-stranded. Virus-associated RNA polymerase activity was lost following psoralen photoreaction, consistent with dependence on the genome RNA as template for viral mRNA synthesis. By contrast, reovirus guanylyl transferase and methyl transferase activities that catalyze the formation of mRNA 5'-terminal cap, m GpppG, were relatively unaffected by photoreaction. Cations that inhibit psoralen binding to nucleic acids also protected against loss of RNA polymerase activity. Partially photoinactivated viral cores produced decreased amounts of full length viral mRNAs. The absence of prematurely terminated transcripts indicates that template RNA modification by psoralen adducts has an all-or-none effect on initiation of mRNA synthesis by the core-associated RNA polymerase.


Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.


The isolation and amino acid/sugar composition of human fibroblastoid interferon.

Human fibroblastoid interferon produced from an established human cell line was purified by controlled-pore glass and concanavalin A-Sepharose column chromatography followed by preparative two-dimensional gel electrophoresis. The purification procedure provided a 10% recovery of pure interferon with good reproducibility. The purified protein was homogeneous with respect to its molecular weight of 20,000 and net electrical charge at pH 2.5. Interferon of high specific activity of 5 x 10(8) units/mg of protein was directly demonstrated in the polyacrylamide gel before staining with Coomassie brilliant blue. Parallel purification of a sham-induced interferon preparation did not yield an equivalent product indicating the purified interferon is not derived from uninduced cells or from the fetal calf serum of the tissue culture growth medium. Pure interferon was radioiodinated by Bolton-Hunter reagent. Amino acid analysis of the pure preparation shows interferon to be a leucine-rich glycoprotein containing a high percentage of glutamic/glutamine residues and that disulfide bridges(s) are important for its biological activity.


Nonidentity of the 48,000-dalton protein of mRNA-protein particles and the beta subunit of eukaryotic initiation factor 2.

The beta subunits of the GTP-dependent initiator methionyl-tRNA-binding protein and the 48,000-dalton protein, which is one of the two major proteins present in isolated mRNA . protein particles, have been shown to share a number of similar structural and mRNA-binding properties. The nonidentity of these two proteins is now established by documentation of differing molecular weights, isoelectric focusing points, and products of protease or cyanogen bromide cleavage.


An alternating conformation characterizes the phosphodiester backbone of poly(dA-dT) in solution.

A highly homogeneous 145-base-pair fragment of double helical poly(dA-dT) . poly(dA-dT) was obtained by micrococcal nuclease digestion of a semisynthetic chromatin prepared from the nucleosome core histones (H2A, H2B, H3, H4) and the synthetic polydeoxyribonucleotide. In contrast to higher molecular weight alternating copolymers, this fragment displayed two resolved 31P NMR signals, separated by 24 Hz at 10.93 MHz. The two signals were of equal intensity at all temperatures less than the Tm for the fragment. Analyses of the possible origins for the two reasonances leads to the conclusion that the phosphodiester backbone of this DNA contains two distinct phosphorus environments, probably in an alternating array. We suggest that this may indicate the presence of sequence-dependent local variation in the helical structure of DNA in general.


Active transport of thallous ions by Streptococcus lactis.

Tl+ ions have been shown to mimic or compete with K+ in a number of membrane systems. We confirmed that in starved, valinomycin-treated cells of Streptococcus lactis 7962, Tl+ ions distributed themselves across the bacterial membrane in response to the potassium diffusion potential. In glucose-energized cells, however, Tl+ was taken up by a system specifically stimulated by sodium salts. The intracellular levels of Tl+ exceeded those attained by [3H]triphenylmethylphosphonium ion, a lipophilic cation which accumulates in response to the membrane potential. The uptake of Tl+ by (Na+ and glucose)-stimulated cells was strongly inhibited by potassium salts. These experiments suggest that metabolic energy is coupled to Tl+ transport by means of a high energy phosphate compound and that Tl+ ions are actively transported by a membrane carrier whose normal substrate is K+. The uptake of Tl+ is not a valid method for determining the streptococcal membrane potential.


Similarity of the structure of ferritin and iron . dextran (imferon) determined by extended X-ray absorption fine structure analysis.

Ferritin, a natural complex of iron oxide encased in protein, and iron . dextran, a synthetic complex of iron oxide coated with dextran, have the similar properties of maintaining high concentrations of iron in solution at physiological pH and releasing iron relatively slowly in vivo. Extended x-ray absorption fine structure (EX-AFS) analysis was performed on each complex and compared to see if the structures of the iron cores were similar. The results obtained from the extended x-ray absorption fine structure technique show that the near-neighbor environment around the average iron atom in ferritin and iron . dextran is identical, within experimental uncertainty, for the first three shells. The similarity of the iron cores in both complexes may explain the similarity of iron release in vivo. Ferritin has a protein coat which is composed of 24 subunits arranged in a hollow sphere with six channels through which the iron may move during deposition and release. However, little is known about the requirements of the protein structure in ferritin for the maintenance of high concentrations of iron in a soluble, nontoxic form or about the role of the protein in the release of iron from ferritin. The results suggest that iron . dextran will be a useful model compound in studies of the relation of the iron core and protein in ferritin to function.


A high molecular weight protease in liver cytosol.

A high molecular weight (greater than 400,000) protease active with [3H]leucine-labeled globin has been found in the postmicrosomal fraction of mouse kidney, brain, heart, spleen, and tumor cells and is most active in liver. The presence in liver was unexpected because liver cytosol is very ineffective in the breakdown of endogenous, labeled proteins. The enzyme has a number of properties that distinguish it from known cathepsins in addition to its high molecular weight. It is most active at pH approximately 7.5. When purified, it is unstable above 20 degrees C and is stabilized by metal chelating agents such as citrate, creatine-P, and glycerate-3-P. It is an -SH protease, but its thermal instability is not affected by 1 mM dithiothreitol. The enzyme is not lysosomal.


One-electron reduction of flavodoxin. A fast kinetic study.

The reaction kinetics of fully oxidized flavodoxin from Clostridium MP with the hydrated electron have been investigated by the pulse radiolysis method. Four spectrally distinct processes have been observed with the ultimate formation of the singly reduced flavin form of the protein. The last two species obtained in the reaction sequence are spectrally similar, and are connected through a reaction which is first order. It is proposed that this reaction involves a protein conformational alteration.


Enoyl coenzyme A reduction by bovine mammary fatty acid synthetase. Specificity and other characteristics.

The NADPH-dependent enoyl coenzyme A reductase activity of bovine mammary fatty acid synthetase has been characterized with regard to substrate specificity and the product formed. A relatively high specificity for an unsubstituted, four-carbon, 2,3-enoyl chain in trans configuration is obtained. Reduction of trans-crotonyl-CoA results in butyrate, 50% of which is coenzyme A-bound. The reaction is subject to product inhibition, specifically by butyryl-CoA and NADP. Free coenzyme A, on the other hand, is an activator. The pH profile, susceptibility to inhibition by -SH reagents, the results of the relative activities obtained with substrate analogues and homologues, and the ready use of crotonyl-CoA as a primer in fatty acid synthesis are consistent with a mechanism in which the crotonyl group is transferred to an -SH group, is reduced, and then is either transferred back to CoA or hydrolyzed.


alpha Chain mutations with opposite effects on the gelation of hemoglobin S.

The preparation of three hemoglobin tetramers containing the hemoglobin S mutation at beta 6 and an additional one at alpha 6, alpha 47, and alpha 75 is described. The effect of the substitutions in the alpha chains on polymerization was investigated by the equilibrium solubility of the gels as well as the abrupt change in oxygen affinity associated with the onset of gelation. Substitution of a histidine for aspartic acid at alpha 47 causes a marked inhibition of polymerization. This inhibition probably results from tetramers which carry the two substitutions on the same alpha beta dimer. By contrast, the introduction of a tyrosine at alpha 75 and an alanine at alpha 6 have the opposite effect and are the first examples of alpha chain mutations which potentiate the gelation of Hb S. The molecular mechanisms responsible for the effects of the mutations on the self-association of Hb S are discussed.


Isolation and characterization of gangliosides with a new sialosyl linkage and core structures. II. Gangliosides of human erythrocyte membranes.

Eight monosialosylgangliosides, G1 to G8, have been isolated from human erythrocyte membranes and their structures have been determined. Gangliosides G4 and G7 have been characterized by having 2 leads to 6-linked sialic acid to galactose at their termini. Ganglioside G5 was a positional isomer of a brain ganglioside GM1 as to the linkage of sialic acid. Ganglioside G8 was characterized as a branched chain ganglioside similar to a fucoganglioside previously isolated but devoid of fucose, and it showed a strong blood group I activity. Structures of these four new gangliosides are shown below: (formula: see text).