Bovine babesiosis: failure to induce interferon gamma production in response to Babesia bovis antigens in cattle.

The immune response to Babesia bovis infection or vaccination was evaluated by measuring antibody and interferon gamma (IFN-gamma) production to protective recombinant and crude native B. bovis antigens. Cells from vaccinated or infected cattle failed to produce detectable IFN-gamma when stimulated with B. bovis antigens in vitro. In contrast, antibody was induced by protective recombinant B. bovis antigens. These findings are consistent with the argument that immunity to B. bovis infection is correlated most strongly with humoral rather than cell-mediated immune responses.     

Plasmid transduction using the D3112DeltaH miniphage in Pseudomonas aeruginosa cells]

Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.     

Fc epsilon receptor mediated Ca2+ influx into mast cells is modulated by the concentration of cytosolic free Ca2+ ions.

The relationship between the Fc epsilon receptor mediated stimulation of mast cells and the Ca2+ signal it induces were studied using thapsigargin (TG), a blocker of the endoplasmic reticulum Ca2+ pump. TG induced, in mucosal mast cells (RBL-2H3 line), a dose-dependent and an InsP3-independent increase in [Ca2+]i (from resting levels of 83-150 nM to 600-680 nM), and a secretory response amounting to 30-50% of that observed upon Fc epsilon RI clustering. The TG induced rise of [Ca2+]i is most probably provided by both arrest of its uptake by the endoplasmic reticulum and influx from the medium. Thus, Ca2+ influx in mast cells may be modulated by the [Ca2+]i level.     

Reduced puromycin sensitivity of translocated polysomes after the addition of elongation factor 2 and non-hydrolysable GTP analogues.

Treatment of reticulocyte polysomes with elongation factor eEF-2 and GTP led to an increased sensitivity of peptidyl-tRNA for puromycin as a result of the translocation from the ribosomal A-site to the P-site. Upon addition of an excess of the non-hydrolysable GTP analogue, GuoPP[CH2]P, the puromycin sensitivity decreased rapidly. The decrease in sensitivity required high concentrations of eEF-2 with half maximal effect at an eEF-2 concentration of around 1 microM. The data suggest either that peptidyl-tRNA had re-translocated back to the A-site due to the higher affinity of eEF-2 for the pre-translocation than for the post-translocation ribosome, or that the eEF-2-GuoPP[CH2]P complex blocks the peptidyl-transferase activity.     

Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N.

In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme.     

NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae.

The yeast nuclear gene NAM7 was previously isolated within a genomic fragment of 7.7 kb (1 kb = 10(3) bases or base-pairs), having the ability to suppress mitochondrial intronic mutations defective in RNA splicing. We have identified and sequenced the region on the insert corresponding to the NAM7 gene. A long open reading frame has been revealed which could code for a protein of 971 amino acids. Comparison of the NAM7 putative protein with data libraries did not reveal any strong similarity with known proteins. However, the NAM7 protein contains several motifs typical for proteins interacting with nucleic acids: (1) five motifs diagnostic for a superfamily of helicases appear in the same order and with similar distances; (2) the N-terminal portion possesses potential Zn-ligand structures belonging to the C chi superfamily. Deletion of the chromosomal copy of NAM7 gene leads to a partial impairment in respiratory growth that is particularly striking at low temperature. Southern blot analysis of DNA extracted from a nam7 :: URA3 deleted strain revealed the presence of a second gene whose sequence is related to that of the NAM7 gene and which could participate in the same process.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Activation of microcarrier-attached lymphocytes in microgravity

A technology has been developed to achieve optimal attachment of adhesion-independent lymphocytes to microcarrier beads. The activation of T-lymphocytes by concanavalin A was tested under microgravity conditions in an experiment carried out in space during the first Spacelab Life Science Mission. Activation, measured as the synthesis of deoxyribonucleic acid (DNA) and the production of interferon-gamma, more than doubled in attached lymphocytes in microgravity. The depression of the activation discovered in previous space experiments is due to an impairment not of the lymphocyte but of the macrophage function. The system described here may be useful for radiobiological investigations on the effect of high-energy particles and for testing the efficiency of the immune system in humans during the long-duration space flight planned in the future. The biotechnological significance of the increased lymphokine production in space remains to be assessed.     

Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.     

Effects of cadmium, copper and metallothionein synthesis inhibiting and stimulating compounds on zinc uptake and accumulation in rat hepatoma HTC cells.

Experiments were carried out to investigate the uptake and accumulation of Zn in rat hepatoma HTC cells, as affected by interfering metals (Cd, Cu), metallothionein synthesis inhibiting compounds (Actinomycin D, cycloheximide) and metallothionein synthesis stimulating compounds (dexamethasone, dibu-cAMP). Cell viability was tested under all experimental conditions by the measurement of LDH leakage, K+ uptake and total cell protein. Determinations of Zn were performed by AAS (total Zn) or by gamma-ray spectrometry (65Zn). Metallothionein analysis was carried out by Cd-saturation tests. The results indicate that cellular responses in rat hepatoma HTC cells with respect to the uptake and accumulation of 65Zn are fully comparable with literature data existing for 65Zn accumulation in rat hepatocytes, under all experimental conditions applied. Cu2+ and dibutyryl-cAMP did not significantly affect rates of 65Zn accumulation. Cd2+, Actinomycin D and cycloheximide reduced 65Zn uptake, but dexamethasone additions resulted in increased 65Zn accumulation in the cells. Effects on 65Zn were shown both in cytosolic and in the membranes/organelles cell fractions. HPLC chromatography in control cells suggested that newly accumulated cytosolic 65Zn was predominantly MT-associated. Dexamethasone-induced 65Zn accumulation could not be related to elevated cellular MT levels, nor were the total cytosolic Zn levels significantly affected. Non-specific attenuations in MT levels (Actinomycin D, cycloheximide and dibu-cAMP) yielded linear relations between cytosolic 65Zn and MT levels, without any change in cytosolic Zn (AAS). Combined addition of Cd and dexamethasone yielded elevated MT levels, but severely reduced total cytosolic Zn and 65Zn concentrations. The results further indicate the non-Zn-specific nature of dexamethasone-action and suggest the relatively easy Zn-complexing and Zn-release of MT. The simultaneous determinations of total cytosolic zinc and cytosolic 65Zn levels showed that the application and sole measurement of radiotracers may yield only one-sided views of what is actually present or occurring in the cells.