Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

Hemodynamic effects of anti-G suit inflation in a 1-G environment

This study evaluated effects of various anti-G inflation pressures on cardiac volumes and the relationship of these volume changes to mean arterial pressure changes. Ventricular volumes were calculated using two-dimensional echocardiography. An anti-G suit was inflated to 2, 4, and 6 psi in the standing and supine positions for 10 male subjects. In the supine position, mean arterial pressure increased from base line for all three inflation pressures (P = 0.05). The end-diastolic volume increased after 2-psi inflation (P = 0.03). Cardiac output or stroke volume did not change. After standing, mean arterial pressure (P = 0.002), end-diastolic volume (P = 0.002), and stroke volume (P = 0.05) fell after suit deflation. Peripheral vascular resistance fell in the 2- and 4-psi inflation profiles. In the standing protocol, mean arterial pressure, end-diastolic volume, stroke volume, and cardiac output rose with all three inflation pressures (P less than 0.05). After reclining, heart rate increased (P = 0.02) and mean arterial pressure fell (P less than 0.05) in the 4- and 6-psi inflation profiles after suit deflation. Increases in mean arterial pressure are caused by increases in cardiac preload and cardiac output after inflation of the anti-G suit while subjects were standing. Increased cardiac preload was not consistently seen after inflation while subjects were supine. Changes in end-diastolic volume and mean arterial pressure were dependent on the pressure used to inflate the anti-G suit.     

Preservation of ischemic myocardial tissue with an antagonist of vasoconstrictor eicosanoids

A new antagonist of the vasoconstrictor eicosanoids, L-640,035, was studied in a standardized model of myocardial ischemia (MI) in anesthetized cats. This eicosanoid antagonist was not found to exert any overt hemodynamic action in cats subjected to a sham myocardial ischemia protocol. However, the antagonist markedly reduced the S-T segment of the electrocardiogram when administered 30 min after permanent occlusion of the left coronary artery. Moreover, circulating activities of the marker enzyme creatine kinase (CK) were markedly attenuated by L-640,035 3-5 h after the onset of MI. This was verified by cardiac biopsies 5 h post-MI since myocardial CK activities decreased much less in treated MI cats than in MI cats receiving only the vehicle for L-640,035 (i.e., ethanol). The active metabolite of the antagonist in biological fluids (i.e., L-636,499) markedly antagonized the vasoconstrictor actions of endoperoxide and thromboxane analogs, but not of noneicosanoids in isolated perfused coronary arteries.     

Estimation of regional cutaneous cold sensitivity by analysis of the gasping response.

Regional cutaneous sensitivity to cooling was assessed in males by separately immersing four discrete skin regions in cold water (15 degrees C) during head-out immersion. The response measured was gasping at the onset of immersion; the gasping response appears to be the result of a nonthermoregulatory neurogenic drive from cutaneous cold receptors. Subjects of similar body proportions wore a neoprene "dry" suit modified to allow exposure to the water of either the arms, upper torso, lower torso, or legs, while keeping the unexposed skin regions thermoneutral. Each subject was immersed to the sternal notch in all four conditions of partial exposure plus one condition of whole body exposure. The five cold water conditions were matched by control immersions in lukewarm (34 degrees C) water, and trials were randomized. The magnitude of the gasping response was determined by mouth occlusion pressure (P0.1). For each subject, P0.1 values for the 1st min of immersion were integrated, and control trial values, although minimal, were subtracted from their cold water counterpart to account for any gasping due to the experimental design. Results were averaged and showed that the highest P0.1 values were elicited from whole body exposure, followed in descending order by exposures of the upper torso, legs, lower torso, and arms. Correction of the P0.1 response for differences in exposed surface area (A) and cooling stimulus (delta T) between regions gave a cold sensitivity index [CSI, P0.1/(A.delta T)] for each region and showed that the index for the upper torso was significantly higher than that for the arms or legs; no significant difference was observed between the indexes for the upper and lower torso.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant.

We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene is not essential. However, cells with deletions in skp1+ are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1+ specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery.     

Regeneration dynamics of alluvial gaps in a warm temperate rain forest in New Zealand

Gaps formed by stream laid gravels in a warm temperate Podocarp-Angiosperm rain forest in New Zealand were quickly invaded by 41 free-standing woody species. Height-age curves for the eight most frequent species were used to construct a 12 yr chronosequence which showed 18 species arrived by 0.9 yr and 33 by 1.9 yr. This rapid invasion was supplied from both gaps and mature forest and fitted the Initial Floristic Model coupled with a Facilitation Mechanism. Over the 12 yr period, stem density and births peaked at 0.9 yr and deaths at 1.9 yr with a natality of 39.5/m², a mortality of 38.5/m² and nearly 99% dying as seedlings. Plant height increased to 5.1 m at 12.1 yr with height increment peaking at 43 cm/yr at 7.3 yr. Maximum potential heights ranged from 4 to 60 m and were positively correlated with longevity which varied from 20 to 1150 yr for 19 known species. A decline in height increment to 0.9 yr showed a below-ground component in intraspecific competition with significant negative correlations between stem density and height indicating a later above-ground component. There was no significant height competition amongst the 11 most frequent species and only one significant negative interspecific association. This suggests high species coexistence for which Equal chance was probably the major mechanism. Given this coexistence, rapid species arrival, early sexual maturity and similar density and height-age patterns, the main dynamic variables may be maximum height, longevity and height increment, all of which are, partly, genetically programmed. Most dynamic affinities are with tropical forest, although height growth and species richness are warm temperate.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.