Zebrafish embryology and neural development

The zebrafish is rapidly increasing in popularity with developmental biologists. Driving this interest are the elegant methods for in vivo observations and recovery of early developmental mutations. The past year has seen the introduction of additional methods for in vivo manipulation of identified cells and the application of these methods to mutant analysis.     

Synergism within polyhexamethylene biguanide biocide formulations

Polyhexamethylene biguanides (PHMB) are mixtures of polymeric biguanides with an average polymer length (n) of 5, but containing high (n greater than 15, mol. wt 3300) and low molecular weight material (n = 2, mol. wt 400). Studies involving discrete molecular weight fractions of PHMB have shown that antimicrobial activity of PHMB increases with increasing polymer length. Cell suspensions which had not been subjected to centrifugation and/or washing during their preparation were employed. Whilst activity was still observed to increase with n, the trend was much reduced as n exceeded six. Centrifugation and washing of cells markedly increased the activity of high but not low molecular weight materials and corresponded to losses upon centrifugation of envelope lipopolysaccharide (LPS). Such envelope LPS represented high affinity binding sites on the surfaces of the cells. Combinations of various molecular weight fractions of PHMB were evaluated against filter-washed cells and revealed a profound synergy between extremes of polymer length.     

Novel cloning vectors for Bacillus thuringiensis.

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Injury and recovery of Bacillus megaterium from mild chlorhexidine treatment

Treatment of Bacillus megaterium cell suspensions with 12 /μmol/1 chlorhexidine diacetate for 5 min led to an approximate 50%, reduction in viability when plated onto tryptone soya agar (TSA). Fifty percent of the surviving fraction were unable to form colonies on TSA containing 5.5% w/v KCI. Such loss of KCl tolerance is indicative of membrane damage, and was recovered within 30 min of incubation in tryptone soya broth (TSB). Multiplication of the damaged organisms did not recommence in this medium until after 60 min. Inclusion of inhibitors of respiration, and of protein, RNA and DNA synthesis in the TSB recovery medium did not significantly affect either the rate or extent of the recovery of KCl tolerance by damaged organisms.     

Comparative evaluation of macrophage stimulation and depression on tumor growth and macrophage content and function in mice

Summary In a syngeneic murine adenocarcinoma model, the administration of glucan, an RE stimulant, inhibited tumor growth and increased tumor macrophage populations. Conversely, the administration of methyl palmitate, an RE depressant, potentiated tumor growth and decreased the number of tumor macrophages. Glucan and methyl palmitate also produced diverse alterations in serum lysozyme levels that reflected their contrasting influences on RE functional status, thus supporting the role of serum lysozyme as an index of macrophage function. The diverse results produced by macrophage stimulation or depression in regard to tumor growth, tumor macrophage population, and serum lysozyme concentration indicate that a relationship may exist between macrophage functional activity and host resistance to neoplasia.     

Dimeric epoxy fatty acid methyl esters: formation,chromatography and mass spectrometry

The formation of dimers is reported from the thermal treatment of a series of epoxy fatty acid methyl esters. These compounds were isolated from the reaction mixture by steric exclusion chromatography and were subsequently characterised by their high resolution electron impact and ammonia chemical ionisation mass spectra. The spectra were consistent in each case with the presence of a mixture of four possible positional isomers each containing an ether bridge linking a pair of fatty acid methyl esters across the carbon chains, with a keto group on a carbon adjacent to the bridge on one of the esters.     

Trichinella spiralis: Generation in the presence of rat serum of factors chemotactic for rat cells

Chemotaxis of rat peritoneal cells, of which the eosinophil was the predominant migratory cell type, toward incubates of Trichinella spiralis was studied using a modified Boyden chamber. Excysted muscle larvae, preadults, and adults were incubated in a buffered medium for 20 hr at 37 C. Worms were incubated alone or with serum or spleen cells, or both, from immune and nonimmune rats. Incubates of worm stages alone possessed no chemotactic activity as compared with incubation medium as a negative control and zymosan-activated serum as a positive control. Both normal and immune sera tested alone stimulated cell migration to the same degree. Incubates of spleen cells from either normal or immunized hosts did not show chemotactic activity. Chemotaxis caused by normal and immune sera were not altered by incubation with homologous spleen cells. Addition of larva, preadults, and adult worms to sera, however, enhanced chemotactic activity over sera alone. Chemotaxis caused by larvae plus immune sera was significantly greater than that stimulated by larvae plus normal sera. This difference decreased when preadults were substituted for larvae and was not observed when adult worms were used. Reversal of the chemical gradients showed that active cell migration caused by various incubates was due to Chemotaxis.     

DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.