The protein kinases tightly bound to DNA are present in normal tissues and in regenerating liver, but strongly decreased in hepatomas

We have previously described in rat liver two protein kinases tightly bound to DNA, one is serine-specific, the other arginine-specific. In this work we show that both enzymes are present in various rat tissues and in liver from various species. Both kinase specific activities are strongly decreased in methyl-DBA-induced hepatomas and in HTC cells but not in regenerating liver after hepatectomy. This decrease is then not related to cell proliferation.     

The evolution of insecticide resistance: Have the insects won?

While insecticides have greatly improved human health and agricultural production worldwide, their utility has been limited by the evolution of resistance in many major pests, including some that became pests only as a result of insecticide use. Insecticide resistance is both an interesting example of the adaptability of insect pests, and, in the design of resistance management programmes, a useful application of evolutionary biology. Pest susceptibility is a valuable natural resource that has been squandered; at the same time, it is becoming increasingly expensive to develop new insecticides. Pest control tactics should therefore take account of the possibility of resistance evolution. One of the best ways to retard resistance evolution is to use insecticides only when control by natural enemies fails to limit economic damage. This review summarizes the recent literature on insecticide resistance as an example of adaptation, and demonstrates how population genetics and ecology can be used to manage the resistance problem.     

Kurloff cell proteoglycans: presence of two main size-populations of intracellular protease-resistant proteochondroitin sulphate. Effect of D-xyloside

This paper reports that the Kurloff cell sulphated and chondroitinase AC sensitive material previously described filtered on Sepharose CL4B columns as 2 main populations with Kav of 0.25 and 0.44. Its alkaline treatment resulted in the elution of 2 peaks with Kav of 0.52 and 0.78. Their reduction in size observed after alkaline treatment and the 6-fold increase in the (35S) sulphate incorporation after addition of 0.1 mM xyloside to the incubation medium indicate that these intracellular sulphated glycosaminoglycans exist in the form of proteoglycans. They were characterized by their resistance to degradation by pronase, papain or cathepsin D, as assessed by gel filtration chromatography on Sepharose CL6B or CL4B. After the glycosaminoglycans were digested with chondroitinase AC, thin-layer chromatography analysis indicated the presence of delta di-4S and delta di-6S in a ratio of 7:1. The presence of such protease-resistant proteochondroitin sulphate in intracytoplasmic granules of both Kurloff cells and other natural killer cell types is emphasized.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.     

Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

In vitro study of lipid metabolism in the mealworm larval fat body

Lipid metabolism in Tenebrio larval fat body has been studied in vitro. Lipid release required the presence of diluted hemolymph in the incubation medium. This time-dependent release of lipid was strongly stimulated in a dose-dependent manner by Tenebrio corpora cardiaca (CC) extracts or synthetic adipokinetic hormone (AKH I). Furthermore, some glycerol was released when larval fat body was incubated without hemolymph, and this phenomenon was also dose dependent for added CC extracts. Lipid synthesis was estimated in vitro by following the incorporation of radioactivity from [6-¹⁴C] glucose into fatty acids. Lipogenesis occurred in the absence of added carbohydrates in the medium, but it was stimulated by the addition of glucose, and especially trehalose (10 mg ml^?1). Intestinal insulin-like peptide (ILP) also stimulated in vitro lipogenesis in a dose-dependent fashion. We conclude that lipolytic and lipogenetic activities of larval mealworm fat body in vitro are effectively under hormonal control.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

Lethal activity of miconazole in Neisseria gonorrhoeae grown in pure and mixed cultures

Abstract Low concentrations (e.g. 2 × 10⁻⁶ M) of an imidazole derivative anti-fungal agent, miconazole, were lethal for the Gram-negative, facultative aerobic pathogen Neisseria gonorrhoeae grown either alone or in mixed culture with the yeast Candida albicans . Electron microscopic observation of Neisseria cells exposed to miconazole showed the presence of blebs in the outer wall and areas of separation between the wall and the cytoplasmic membrane. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell lysates did not reveal differences in major outer membrane proteins between the treated and the untreated cells of any one strain. Imidazole derivatives are frequently used in the treatment of candidiasis. Our in vitro results show that low concentrations of one of them, miconazole, can be bactericidal for N. gonorrhoeae , a bacterium that can colonise sites of the human body where Candida is often found.