Isolation and Characterization of Thermophilic Bacterial Strains with Inulinase Activity

Nine bacterial strains growing on inulin as the sole carbon and energy source were isolated from soil samples by enrichment culture on a mineral medium. Four of the strains were thermophilic and belong to the genus Bacillus. The thermophilic strains synthesized a β-fructosidase that was active on both inulin and sucrose. The presence of inulin in the culture medium is necessary for enzyme synthesis. Most of the activity on inulin was recovered in the culture medium, and the enzyme was synthesized during cell growth.     

Isolation of Thy-1 caps and analysis of their phospholipid composition in mouse T-lymphoma cells

In this study we have used a density perturbation method to isolate anti-Thy-1 antibody-induced Thy-1 caps from mouse T-lymphoma cells in the absence of detergents, and then compared the phospholipid composit on of these capped membranes with that of uncapped membranes. Initial phospholipid analysis by two-dimensional thin layer chromatography (2-D TLC) reveals a significant increase in the amount of ³²P-labeled phosphatidylcholine in the Thy-1 capped membrane. In contrast, no significant changes are observed in the labeling of phosphatidylserine, phosphatidylethanolamine, or the sphingomyelins. Therefore, it is suggested that phosphatidylcholine may be involved in the organization and/or regulation of Thy-1 antigen redistribution. The composition of phosphoinositide in uncapped and capped membranes was analysed separately using one-dimensional thin layer chromatography (1-D TLC) to resolve phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4, 5-bisphosphate (PIP₂) from all other phospholipids. This analysis reveals a significant reduction in levels of PIP and PIP₂, but not PI, in Thy-1 caps. Through the use of ion exchange column chromatography, we have found an increased production of all three species of inositol phosphates during anti-Thy-1 antibody-induced capping. Inositol 1, 4, 5 -triphosphate (IP₃) shows the most significant increase, compared to the much smaller increases in inositol 4, 5-bisphosphate (IP₂) and inositol monophosphate (IP). These results suggest that the binding of anti-Thy-1 antibody to Thy-1 antigen activates phospholipase C which, in turn, initiates polyphosphoinositide turnover and IP₃ production. It is proposed that these observed effects are the result of early signal transducing events which are prerequisite steps in Thy-1 receptor cap formation.     

A CD44-like endothelial cell transmembrane glycoprotein (GP116) interacts with extracellular matrix and ankyrin.

We used complementary biochemical and immunological techniques to establish that an endothelial cell transmembrane glycoprotein, GP116, is a CD44-like molecule and binds directly both to extracellular matrix components (e.g., hyaluronic acid) and to ankyrin. The specific characteristics of GP116 are as follows: (i) GP116 can be surface labeled with Na 125I and contains a wheat germ agglutinin-binding site(s), indicating that it has an extracellular domain; (ii) GP116 displays immunological cross-reactivity with a panel of CD44 antibodies, shares some peptide similarity with CD44, and has a similar 52-kDa precursor molecule, indicating that it is a CD44-like molecule; (iii) GP116 displays specific hyaluronic acid-binding properties, indicating that it is a hyaluronic acid receptor; (iv) GP116 can be phosphorylated by endogenous protein kinase C activated by 12-O-tetradecanoylphorbol-13-acetate and by exogenously added protein kinase C; and (v) GP116 and a 20-kDa tryptic polypeptide fragment of GP116 from the intracellular domain are capable of binding the membrane-cytoskeleton linker molecule, ankyrin. Furthermore, phosphorylation of GP116 by protein kinase C significantly enhances GP116 binding to ankyrin. Together, these findings strongly suggest that phosphorylation of the transmembrane glycoprotein GP116 (a CD44-like molecule) by protein kinase C is required for effective GP116-ankyrin interaction during endothelial cell adhesion events.     

Magnesium and Manganese Content of Halophilic Bacteria

Magnesium and manganese contents were measured by atomic absorption spectrophotometry in bacteria of several halophilic levels, in Vibrio costicola, a moderately halophilic eubacterium growing in 1 M NaCl, Halobacterium volcanii, a halophilic archaebacterium growing in 2.5 M NaCl, Halobacterium cutirubrum, an extremely halophilic archaebacterium growing in 4 M NaCl, and Escherichia coli, a nonhalophilic eubacterium growing in 0.17 M NaCl. Magnesium and manganese contents varied with the growth phase, being maximal at the early log phase. Magnesium and manganese molalities in cell water were shown to increase with the halophilic character of the logarithmically growing bacteria, from 30 mmol of Mg per kg of cell water and 0.37 mmol of Mn per kg of cell water for E. coli to 102 mmol of Mg per kg of cell water and 1.6 mmol of Mn per kg of cell water for H. cutirubrum. The intracellular concentrations of manganese were determined independently by a radioactive tracer technique in V. costicola and H. volcanii. The values obtained by ⁵⁴Mn loading represented about 70% of the values obtained by atomic absorption. The increase of magnesium and manganese contents associated with the halophilic character of the bacteria suggests that manganese and magnesium play a role in haloadaptation.     

Screening of white-rot fungi on (14C)lignin-labelled and (14C)whole-labelled wheat straw

Summary 74 Basidiomycetes have been tested for ligninolytic capability on (¹⁴C)lignin-labelled wheat straw. Fifteen strains were selected and rested more accurately for ligninolytic activity and the capacity to degrade wheat straw. The asymptote, inflexion point and degradation rate were determined using a model approach. The fungi exhibited very different responses with respect to lignin biodegradation: high asymptote for Pleurotus ostreatus (77%), low inflexion points for Sporotrichum pulverulentum Nov. (6.1 days) and Pycnoporus spp. (2.7 to 4.7 days) with high and slow degradation rates, respectively (0.91% and 0.45% of ¹⁴CO₂ release/day). Degradation values for (¹⁴C)whole-labelled wheat straw exhibited less variation. Finally, the strains Pleurotus ostreatus, Dichomitus squalens and Bjerkandera adusta showed the highest selectivity of lignin removal.     

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.