Fibrinogen and fibrin in strong magnetic fields. Complementary results and discussion

When fibrin polymerizes in a strong magnetic field, it can be highly oriented. The structural diffraction study of the oriented polymer becomes thus possible. The magnetic birefringence can also be used to study the development of the polymer Fibrinogen in solution is weakly oriented in high magnetic fields. In this work we present complementary results and discussion. The validity of the comparison of the orientation parameters of fibrinogen and fibrin with those of other orientable known biological structures is discussed. The orientation of fibrin formed from fibrin monomer solution is compared to that of fibrin formed by the action of thrombin on fibrinogen. The conditions to obtain highly oriented fibrin gels suitable for three dimensional structure studies are also briefly discussed.     

Influence of long term treatment with 3-methylcholanthrene or carbaryl on mixed-function oxidase activity in 3T3 fibroblasts: Studies by microspectrofluorimetry on single cells

Using benzo(a)pyrene (BaP) as a probe for aryl hydrocarbon hydroxylase (AHH) activity, differences in mixed-function oxidase (MFO) activity were observed using microspectrofluorimetry in single living cells during long term treatment with 3-methylcholanthrene (3-MC) or carbaryl. Although these two compounds differ in chemical structure, similar effects were observed in 3T3 cell populations. The results suggest that the two compounds activate the same enzymatic system and that individual cells of a supposed homogeneous cell population are not equally sensitive to xenobiotics, i.e. subpopulations were observed which have differences in AHH activity.     

Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane,with special reference to the ω-hydroxylase

The subcellular localization of the ω-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeast. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractioned by density gradient centrifugation. The distribution of ω-hydroxylase and 15 other constituents chosen as possible markers of its subcellular membranes has been established. ω-Hydroxylase resulted in being bound to a membrane that containes also cytochrome P-450 and NADPH-cytochrome c reductase. This membrane clearly differs from five other subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K⁺-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline phosphatase, 5′-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.     

Cryotolerance and Cold Adaptation in Lactococcus lactis Subsp. lactis IL1403

The physiology of the cold-shock response in Lactococcus lactis subsp. lactis IL1403 at a subzero temperature, and cold-induced adaptation to heat shock, were investigated. Preincubation of cells at 8°C led to the development of cryotolerance, i.e., an enhanced capacity to survive exposure to freezing temperature (-20°C). Pretreatment with chemicals considered to be chaotropic agents did not induce cryotolerance or, in contrast, led to a decrease in survival capacity at -20°C. Interestingly, preincubation at 8°C led also to thermololerance to a 52°C challenge, but preincubation of cells at 42°C for 30 min did not improve their capacity to survive freezing-thawing exposure. These results demonstrate that cold- and heat-shock responses are physiologically linked by a complex relation. Furthermore, food processing at low temperature before subzero or heat treatment may need to be reconsidered.     

The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution.

The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods. The enzyme is a typical two-domain protein. One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet. The active-site serine residue (Ser-48) is within a cleft located between the two domains.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Enzymatic Protection Against Peroxidative Damage in Isolated Brain Capillaries

The content of polyunsaturated fatty acids, the activities of superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase, and the concentration of reduced glutathione were measured in cerebral microvessels isolated from rat brain. Polyunsaturated fatty acids, mainly arachidonic, linoleic, and docosahexaenoic acids, accounted for 32% of total fatty acids in cerebral microvessels. Whereas total SOD activity in the microvessels was slightly lower than that found in cerebrum and cerebellum, glutathione peroxidase and glutathione reductase activities were twice as high and catalase activity was four times higher. Glutathione peroxidase in microvessels is active on both hydrogen peroxide and cumen hydroperoxide, and it is strongly inhibited by mercaptosuccinate. After several hours of preparation, the concentration of reduced glutathione in isolated microvessels was 0.7 mumol/mg of protein, which corresponds to a concentration of approximately 3.5 mM. Our results indicate that the blood-brain barrier contains large amounts of peroxide-detoxifying enzymes, which may act, in vivo, to protect its highly polyunsaturated membranes against oxidative alterations.     

The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.

Fragments of the lipophosphoglycan of Leishmania donovani were generated by phospholipase C digestion and mild acid hydrolysis. The fragments were purified and examined for inhibitory activity on protein kinase C isolated from rat brains. On a molar basis, the 1-O-alkylglycerol portion of LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not as effective. In addition, several glycolipid antigens from L. major, which contain short carbohydrate chains attached to phosphatidylinositol, were also efficient inhibitors of the enzyme. These results are consistent with the hypothesis that protein kinase C may be a key target for the parasites to overcome within host macrophages.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).