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.     

Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988     

Ketone Body Utilization for Lipid Synthesis in the Murine Sciatic Nerve: Alterations in the Dysmyelinating Trembler Mutant

This work demonstrates that in vitro sciatic nerves of normal and trembler adult mice can use ketone bodies (beta-hydroxybutyrate and acetoacetate) and butyrate for lipid synthesis. In normal sciatic nerves, beta-hydroxybutyrate is incorporated in total lipids to a larger extent than acetoacetate (141% and 33%, respectively, of acetate incorporation), whereas for trembler sciatic nerves, these percentages are only 69% and 27%. Incorporation of ketone bodies is greater into sterols than into other lipids. Lipid metabolism of ketone bodies in trembler nerves is altered and could reflect a process similar to Wallerian degeneration: a dramatic decrease of sterol and free fatty acid synthesis and an increased synthesis of triglycerides. Moreover, differences seen in precursor incorporation into lipids between normal and trembler sciatic nerves suggest that their lipid metabolism is not the same.     

Resumption of cellular activity induced by cytokinin and gibberellin treatments in tomato flowers targeted for abortion in unfavorable light conditions

Mitotic activity and nuclear DNA synthesis in tomato ( Lycopersicon esculentum Mill., cv. King plus) flowers targeted for abortion under unfavorable light conditions are completely stopped 6 days after macroscopic appearance of the inflorescence. Ovular cells are arrested at the G₁ (80%) and G₂ (20%) stages of the cell cycle. Exogenous applications of a mixture of N⁶-benzyladenine (BA) and gibberellins A₄₊₇ (GA) directly on the inflorescence may prevent its failure. Nuclear DNA synthesis and mitoses resume in ovules of the flower 16 to 20 h after the BA+GA treatment. When applied alone, BA and GA are able to mimic the effect of the mixture upon the progression of ovular cells through their cycle. Sporogenesis processes are also set in motion by the exogenous plant growth regulators. The mechanism of action of cytokinins and gibberellins in the control of floral development is discussed.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

Phytanic acid alpha-oxidation and complementation analysis of classical Refsum and peroxisomal disorders

Summary We have measured the production of ¹⁴CO₂ from exogenous [1-¹⁴C] phytanic acid in fibroblast monolayers from patients with classical Refsum's disease and peroxisomal disorders. Activities in the different disorders were (percentage of control): classical Refsum's disease (5%), isolated peroxisomal acyl-CoA oxidase deficiency (75%), Zellweger syndrome (4%), neonatal adrenoleukodystrophy (5%), and rhizomelic chondrodysplasia punctate (3%). Absence of complementation was demonstrated between Zellweger syndrome and infantile Refsum's disease lines after polyethylene glycol fusion, with decreases of average activity of 11% relative to unfused cell mixtures. Classical Refsum's disease, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy lines all complemented one another, and Zellweger syndrome or infantile Refsum's disease lines, with average activity increases of 522%–772%. No intragenic complementation was observed within either group. Four complementation groups were detected suggesting that at least four genes are involved in phytanic acid -oxidation: one gene for the enzyme phytanic acid -hydroxylase (probably mitochondrial); one gene for a regulatory factor for the expression of phytanic acid -decarboxylation activity and two membrane-bound peroxisomal enzymes involved in the synthesis of plasmalogens; two genes for the assembly of functional peroxisomes and/or import of proteins into peroxisomes.