Impact of moisture on thermally induced denaturation and decomposition of lyophilized bovine somatotropin

The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via differential scanning calorimetry were evaluated with respect to moisture content. The transition peak temperature of rbSt decreased with increasing moisture from 161°C in the dry state to a plateau of 65°C at 28% moisture, which is similar to that of rbSt in solution. Using high performance liquid chromatography, this irreversible endothermic transition consisted primarily of unfolding, hydrophobic aggregation, and some covalent modifications. In the dry state, covalent modifications, including polymerization into compounds of higher molecular weight, were more prominent, while in the presence of moisture, hydrophobic aggregation was most prominent. The irreversibility and scan rate dependence of the endothermic phenomena supports the kinetic nature of the transition rather than a simple equilibrium between globular and unfolded states. The apparent activation energy, for the net transition (i.e., unfolding, hydrophobic aggregation, and covalent modifications) was 57 kcal/mol for rbSt at 9.9% moisture. The observed enthalpy of the transition increased, decreased, then approximately leveled off as a function of increasing moisture content. This can be explained by the increasingly significant contribution of the exothermic aggregation at higher moisture contents. © 1995 John Wiley & Sons, Inc.     

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA-encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin-layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A₂, which indicated that PE is also required for ShlA activity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A₂, which demonstrated that PE binds to the N-terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA-255 determined a molar mass that corresponded to that of unmodified ShlA-255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell-bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.     

Drosophila Immunity: Analysis of Larval Hemocytes by P-Element-Mediated Enhancer Trap

Our aim was to identify new genes involved in the cellular aspects of defense mechanisms of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for expression of the lacZ reporter gene in larval hemocytes at the end of the third instar. We have selected 21 lines in which we observed a reproducible lacZ expression in blood cells. These lines were classified according to the subsets of hemocytes in which lacZ was expressed, and we identified five lines that can be used as lamellocyte markers. Three lines were selected for further analysis. The first exhibited strong lacZ expression in all lamellocytes. The second expressed lacZ in plasmatocytes and lamellocytes, and exhibited a melanotic tumor phenotype in larvae homozygous for the insertion. A third line showed a striking insertion-linked phenotype of melanized lymph glands (the hematopoietic organ), which resulted in the total absence of circulating hemocytes in the mutant larvae. We anticipate that this mutation, which we named domino, will prove a useful tool in the analysis of the role of hemocytes during the various aspects of immune response and melanotic tumor formation.     

Immunohistochemical correlation of human adrenal nerve fibres and thoracic dorsal root neurons with special reference to substance P

Applying a double-labelling immunofluorescence technique, six types of substance P-containing nerve fibres were distinguished in the human adrenal gland according to the immunohistochemical colocalization of (I) calcitonin gene-related peptide (CGRP), (II) cholecystokinin, (III) nitric oxide synthase, (IV) dynorphin, (V) somatostatin, and (VI) vasoactive intestinal polypeptide. Fibre populations I to IV in their mediator content resembled the respective subpopulations of primary sensory neurons in human thoracic dorsal root ganglia, while populations V and VI revealed no correspondence with dorsal root neurochemical coding. Nerve fibres with the combination substance P/nitric oxide synthase occurred only in the adrenal cortex, whereas all other fibre types were present in both cortex and medulla. As revealed by immuno-electron microscopy, substance P-immunolabelled axon varicosities (a) exhibited synaptic contacts with medullary chromaffin cells or with neuronal dendrites, (b) were directly apposed to cortical steroid cells and (c) were separated from fenestrated capillaries only by the interstitial space. These findings provide immunochemical support for an assumed sensory innervation of the human adrenal gland, and additionally suggest participation of substance P in efferent autonomic pathways. Furthermore, the results are indicative for a differentiated involvement of substance P in the direct and indirect regulation of neuroneuronal and neuroendocrine interactions.     

Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1.25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 · 10^?5 M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in V_max of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50–70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

Procedure for the identification of Escherichia coli mutants affected in components containing glycerol derived from phospholipid turnover: Isolation of mutants lacking glycerol in membrane-derived oligosaccharides (MDO)

Abstract A mutant screening procedure is described which allows the identification of mutants carrying lesions in lipoprotein, membrane-derived oligosac-charides (MDO), and other compounds of the E. coli cell envelope containing glycerol derived from phospholipid metabolism. Two mutants lacking glycerol in MDO and one mutant devoid of lipoprotein demonstrate the usefulness of the procedure.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

Neutralizing antibodies to simian papovavirus SA12 in Old World primates in laboratory colonies: high prevalence in baboons

Sera from 517 laboratory-housed nonhuman primates representing five genera and from 13 laboratory workers were examined for the presence of neutralizing antibodies to SA12 virus. The antibody prevalences were as follows: baboons, 66%; patas and vervet monkeys, 24%; macaques, 8%, and chimpanzees, 2%. The serum of one laboratory worker had antibodies. These results suggest that SA12 virus is a common infection of nonhuman primates in laboratory colonies, especially baboons.