The pericellular boundary layer modulates acetylcholine receptor stability in cultured myotubes

Degradation of acetylcholine receptors in cultured chicken myotubes was measured by release into the medium of radioactivity from 125I-labeled alpha-bungarotoxin. Disturbance of the pericellular boundary layer by stirring of the culture medium shortened the half-life of receptor in the membrane from 24 to 12 h. The effect could not be explained by dissociation of toxin-receptor complexes or by conditioning of the bulk phase of the medium. The rates of synthesis and degradation of total cell protein and the degradation of lactoperoxidase-iodinated surface protein were not affected by medium stirring. The loss of glucosamine-labeled material from the cells was enhanced by stirring, however, and this resulted entirely from the increased shedding of high molecular weight glycosubstances from the cells. Cells in stirred cultures contained lower levels of surface coat material stainable with colloidal thorium. These results indicate that glycosubstances of the pericellular matrix protect ACh receptors from degradation.     

Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis

The Bacillus subtilis gene encoding CheB, which is homologous to Escherichia coli CheY, the regulator of flagellar rotation, has been cloned and sequenced. It has been verified, using a phage T7 expression system, by showing that a small protein, the same size as E. coli CheY, is actually made from this DNA. Despite the fact that the two proteins are 36% identical, with many highly conserved residues, they appear to play different roles. Unlike CheY null mutants, which swim smoothly, CheB null mutants tumble incessantly. However, a CheB point mutant swims smoothly, even in the presence of a plasmid bearing cheB, which restores the null mutants to wild type. Expression of CheB in wild type B. subtilis makes the cells exhibit more tumbling. Since both absence of CheB and presence of high levels of CheB cause tumbling, CheB appears to be required, in certain circumstances, for both smooth swimming and tumbling. Expression in wild type E. coli makes the cells smooth swimmers and strongly inhibits chemotaxis.     

Activation of human basophils through the IL-8 receptor.

IL-8 and its structural analogs derived from blood platelets have been proposed as stimuli of IgE-independent basophil activation. In order to clarify the mechanism of action of these peptides, we examined the effects of pure IL-8, connective tissue-activating peptide III (CTAP-III), neutrophil-activating peptide 2 (NAP-2), and platelet factor 4 (PF-4) on blood basophils with and without pretreatment by IL-3, which modulates mediator release. After pretreatment with IL-3, significant histamine release was observed with 10(-8) M and 10(-7) M IL-8 and 10(-7) M NAP-2, but not with the other peptides. At higher concentrations (10(-6) M), however, all IL-8 analogs, as well as the unrelated cationic peptides poly-D-lysine, histone VS, and lysozyme, induced histamine release to variable degrees. Binding and competition studies with [125I]IL-8 revealed specific IL-8R on basophils from a patient with chronic myelogenous leukemia and normal individuals. From 3500 to 9600 receptors with a mean Kd value of 0.15 nM were found on average per chronic myelogenous leukemia and normal basophil, respectively. NAP-2 weakly competed for IL-8 binding. IL-8 and, to a lesser extent, NAP-2 led to a transient rise of cytosolic free calcium concentration ([Ca2+]i), which was independent of a preexposure to IL-3. IL-8 prevented the [Ca2+]i rise induced by NAP-2, but did not influence [Ca2+]i responses to other agonists, e.g. C5a, C3a, or platelet-activating factor. IL-8 induced [Ca2+]i changes and histamine release in IL-3-primed basophils were pertussis toxin sensitive. CTAP-III or PF-4 did not compete for IL-8 binding, did not induce [Ca2+]i changes, and did not influence the [Ca2+]i response to IL-8 and NAP-2. This study shows that IL-8 and NAP-2 activate human basophils by a receptor-mediated mechanism similar to that operating in neutrophils. At high concentrations histamine release can also be induced by cationic peptides by a mechanism that does not involve the IL-8R, and probably depends on cationic interactions.     

Short-term effects of wildfire on microbial biomass and abundance in black pine plantation soils in Turkey

Measurement of soil microbial biomass and abundance offers a means of assessing the response of all microbial populations to changes in the soil environment after a fire. We examined the effects of wildfire on microbial biomass C and N, and abundance of bacteria and fungi 2 months after a fire in a pine plantation. Soil organic carbon (C_org), total nitrogen (N_tot), and electrical conductivity (EC) increased following the fire. In terms of microbial abundance, the overall results showed that burned forest soils had the most bacteria and fungi. Microbial biomass C and N from soil in the burned forest were not significantly different from their unburned forest counterparts. However, microbial indices indicated that fire affects soil microbial community structure by modifying the environmental conditions. The results also suggested that low-intensity fire promotes microorganism functional activity and improves the chemical characteristics of soils under humid climatic conditions.     

A satellite cell mitogen from crushed adult muscle

Single fiber-satellite cell units from skeletal muscle of adult rats were used to study the regulation of satellite cell proliferation. The satellite cells remained quiescent during culture in serum-containing medium but could be induced to enter the cell cycle by exposure to a saline extract of crushed adult muscle. The activity in the extract has a molecular weight greater than 30K and is heat and trypsin sensitive. The mitogenic activity does not result from transferrin. Little or no activity was obtained from crushed extracts of heterologous tissues. Proliferation of myogenic cells from rat embryos was also stimulated by the muscle mitogen but growth of muscle fibroblasts was not enhanced. The time response of satellite cell proliferation after exposure to the muscle mitogen showed that the cells enter DNA synthesis after a lag period of 18 hr and proliferate with a generation time of 12 hr. This confirms that satellite cells in adult muscle are in G0, or an extended G1. The mitogen is also effective in stimulating muscle growth and myoblast fusion in vivo when injected into 1-week-old rat pups. These experiments suggest that muscle regeneration is initiated by the release of an endogenous mitogen from traumatized muscle.     

Contrasted levels of genetic diversity in a benthic Mediterranean octocoral: Consequences of different demographic histories?

Understanding the factors explaining the observed patterns of genetic diversity is an important question in evolutionary biology. We provide the first data on the genetic structure of a Mediterranean octocoral, the yellow gorgonian Eunicella cavolini, along with insights into the demographic history of this species. We sampled populations in four areas of the Mediterranean Sea: continental France, Algeria, Turkey, and the Balearic and Corsica islands. Along French coasts, three sites were sampled at two depths (20 and 40 m). We demonstrated a high genetic structure in this species (overall F_ST = 0.13), and most pairwise differentiation tests were significant. We did not detect any difference between depths at the same site. Clustering analyses revealed four differentiated groups corresponding to the main geographical areas. The levels of allelic richness and heterozygosity were significantly different between regions, with highest diversity in Algeria and lowest levels in Turkey. The highest levels of private allelic richness were observed in Algeria followed by Turkey. Such contrasted patterns of genetic diversity were not observed in other Mediterranean octocorals and could be the result of different evolutionary histories. We also provide new empirical evidence of contrasting results between tests and model‐based studies of demographic history. Our results have important consequences for the management of this species.     

Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase

We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.