Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.     

Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin

Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle.     

Individualgeschichte und Gesangsauspr?gung beim Star (Sturnus vulgaris) in einer Kolonie

Zusammenfassung Die Lautäußerungen der Stare können in Gesangsstrophen und Pfeiflaute unterteilt werden. Pfeiflaute sind kurz (bis 2 s) und einfach gebaut, während die Strophen wesentlich länger andauern (meist über 20 s) und sich aus zahlreichen komplex gebauten Untereinheiten zusammensetzen. In einer Kolonie bei Frankfurt am Main, in der die Individuen durch Farbringe gekennzeichnet sind, wurde der individuelle Variationsspielraum der Lautäußerungen analysiert. In den Feinstrukturen der Pfeifthemen glichen sich die Koloniemitglieder weitgehend aneinander an. In den Feinstrukturen der Motivtypen der Gesangsstrophen traten dagegen große individuelle Unterschiede auf. , die während Jahren in der gleichen Kolonie brüteten, sangen nur sehr wenige gemeinsame Motivtypen. Die Repertoiregröße variierte zwischen 17–39 Motivtypen. Im Herbstgesang traten 5 bisher noch nicht beobachtete Motivtypen auf. Es gibt somit deutliche Hinweise darauf, daß auch der adulte Star neue Laute erlernen kann.

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Individual history and song structure of Starlings (Sturnus vulgaris) living in a colony

Summary Starling's song can be divided into warbling and whistles. Whistles have a short and simple structure, whereas warbling is sung in long sequences (most often more than 20 s). In the warbling one can distinguish many different subunits (repetition of motifs). The individual variability of songs was studied in a colony near Frankfurt/Main in West Germany, where starlings were colour banded since 1969. The following results were obtained:Birds from the same colony uttered nearly identical or very similar microstructures in the species-specific whistle themes. Different to the whistle themes very distinct individual differences characterized the warbling. Only a few motif types were common among colony members. The individual repertoire size ranged from 17 to 39 motif types. Comparison of the song of one individual between the breeding season and the following autumn shows that motifs, which were not observed during spring occurred in the autumn. This observation is a hint that starlings are capable to learn new motif types even as adults.

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Stipendium der Alexander-von-Humboldt-Stiftung     

Are prealbumin plasma levels linked to amino acid supply from peripheral tissues in liver cirrhosis?

Summary Prealbumin plasma level is considered a good index of liver function in liver cirrhosis. However, plasma protein levels depend not only on liver function, but also on amino acid supply which is consequent to nutritional status.In 12 cirrhotics we measured prealbumin plasma levels and the lower limb venous-artero difference of amino acid plasma levels in blood samples taken from femoral vein and femoral artery in post-absorptive conditions considered as a direct index of protein release from peripheral tissues and an indirect index of protein nutritional status.In arterial and in venous plasma amino acid sum was 1.86±0.40 (mean + sd) and 2.00 ± 0.04 mMol/l respectively.Prealbumin plasma levels were found directly correlated with the venousartero difference of amino acid plasma levels (r = 0.57p < 0.05) and of glutamate + glutamine levels (r = 0.73p < 0.007).In conclusions, these data suggest that prealbumin plasma levels are linked to amino acid supply from peripheral tissues in cirrhotics.     

Cerebrovascular smooth muscle culture. II. Characterization of adrenergic receptors linked to adenylate cyclase

Cultured and propagated smooth muscle cells contain adenylate cyclase (AC) responsive to catecholamines and their analogues. Isoproterenol and zinterol were the most effective stimulants of AC activity with EC50 = 8.5 X 10(-8)M. They were followed by epinephrine, phenylephrine and norepinephrine (EC50 = 7.5 X 10(-7)M, 6.5 X 10(-6)M and 4 X 10(-6)M, respectively). When the selective antagonists for beta 1 and beta 2 receptors (beta 1-type practolol and atenolol, beta 1/beta 2-type propranolol and beta 2-type butoxamine) were tested against isoproterenol, epinephrine and norepinephrine stimulation of AC activity, the beta 1 in contrast to beta 2 antagonists were found ineffective. The alpha-blockers (phentolamine alpha 1/alpha 2-type antagonists) and yohimbine (alpha 2-type antagonist) alone or in the presence of propranolol did not significantly inhibit the catecholamine-induced enhancement of cAMP formation. On the other hand, prazosine (alpha 1-type antagonist) blocked the stimulatory effect of epinephrine and norepinephrine on AC system. Similarly, the alpha 2-agonist, clonidine, did not affect the catecholamines' stimulated AC activity while alpha 1 agonist, phenylephrine, induced an additive enhancement of norepinephrine production of cAMP. The findings of beta-2- and alpha-1-type adrenergic receptors in the cultured cerebrovascular smooth muscle provide additional support for the implicated involvement of adrenergic innervation in the regulation of cerebral blood flow and/or systemic blood pressure.     

Proteolytic activity and general characteristics of a marine bacterium, Aeromonas proteolytica sp. N

Merkel, Joseph R. (Fort Johnson Marine Biological Laboratory, College of Charleston, Charleston, S.C.), Eugene D. Traganza, Barid B. Mukherjee, Travis B. Griffin, and J. M. Prescott. Proteolytic activity and general characteristics of a marine bacterium, Aeromonas proteolytica sp. n. J. Bacteriol. 87:1227-1233. 1964.-A highly proteolytic bacterial species was isolated from the alimentary canal of the marine borer, Limnoria. The morphological and biochemical characteristics of the organism indicated that it was a new Aeromonas species, for which the name A. proteolytica is proposed. When freshly isolated, the organism required seawater for growth; but, upon prolonged culture in the laboratory, it was able to grow in media of greatly reduced salt concentration, provided that relatively large amounts of peptone were supplied. Peptone or hydrolysates of casein were capable of supplying all organic nutrients required for growth and proteinase production. Certain individual amino acids were also able to furnish all energy, carbon, and nitrogen requirements. Inorganic nitrogen was utilized in the presence of citrate, but could not serve as the only source of nitrogen in the presence of glucose. The organism was facultatively anaerobic, but best growth and proteinase production occurred only with vigorous aeration. The amount of growth obtained in 24 hr increased rapidly as the incubation temperature was increased up to a maximum of 40 C, but no growth occurred at 42 C.     

Structure of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12

narL and narX mediate nitrate induction of nitrate reductase synthesis and nitrate repression of fumarate reductase synthesis. We report here the nucleotide sequences of narL and narX. The deduced protein sequences aid in defining distinct subclasses of regulators and sensors in the family of two-component regulatory proteins.     

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.