Genetic diversity assessment of Tunisian <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> population isolated from cattle

Background

The genetic diversity of M. bovis in Tunisia is still underestimated despite the implementation of an eradication program. The lack of data about spatial distribution of the M. bovis population hinders the control of bovine tuberculosis (bTB) progress. This study represents the largest molecular analysis of M. bovis isolates in Tunisia. It is aimed to upgrade the understanding of bTB epidemiology and the geographical distribution of the infection. Tuberculosis research was performed in cattle (n?=?149) with TB-compatible lesions collected over 5 months from a slaughterhouse located in Sfax, Tunisia.

Results

Ninety-four animals were found to be infected by M. bovis and two others by M. caprae. Spoligotyping revealed twenty-five patterns, SB0120, SB0134, and SB0121 being the most prevalent profiles (36.4%, 11.4%, and 7.2%, respectively). Three new spoligotypes were detected: SB2345, SB2344 and SB2343. MIRU-VNTR analysis classified the isolates in seventy-three profiles and showed a large genotypic variety observed within the main spoligotype which was split into several MIRU-VNTR types: 29 in SB0120 (h?=?0.983), 10 in SB0134 (h?=?0.981) and 7 in SB0121 (h?=?1). Genotyping revealed a common pattern in different geographic regions. It also showed that Sfax, located in southern-Tunisia, represents a high-risk area with an elevated genetic diversity.

Conclusions

Spatial analysis may provide insights into disease transmission, which affects the effectiveness of eradication campaigns in cattle.

     

Bacteriological survey of sixty health foods.

A bacteriological survey was performed on 1,960 food samples encompassing 60 types of health foods available in the Baltimore-Washington, D.C., metropolitan area. No consistent bacteriological distinction (aerobic plate counts, total coliform and fecal coliform most probable numbers) was observed between foods labeled as organic (raised on soil with compost or nonchemical fertilizer and without application of pesticides, fungicides, and herbicides) and their counterpart food types bearing no such label. Types and numbers of samples containing Salmonella were: sunflower seeds, 4; soy flour, 3; soy protein powder, 2; soy milk powder, 1; dried active yeast, 1; brewers' years, 1; rye flour, 1; brown rice, 1; and alfalfa seeds,1. The occurrence of this pathogen in three types of soybean products should warrant further investigation of soybean derivatives as potentially significant sources of Salmonella.     

Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S-locusan S-RNase and a recently described pollen expressed S-haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21kb in total of the S-locus region in 3 different apricot S-haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunusspecies, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-boxand the S-RNase genes was determined exactly in the S ₂-haplotype (2.9kb) and inferred approximately in the S ₁-haplotype (< 49kb) confirming that these genes are linked. Sequence analysis of the 5 flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S ₁, S ₂ and S ₄) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB₁, SFB₂ and SFB₄) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S ₁- and S ₂-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB ₁ and SFB ₂ are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.     

Formation of Two Different Types of Ion Channels by Amphotericin B in Human Erythrocyte Membranes

The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (≤1.0 × 10⁻⁶ M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 × 10⁻⁶ M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K⁺ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft–associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.     

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.     

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.     

An inquiry into the selective protection of glycine during the radiolysis of glycine-alanine mixtures in aqueous solutions and its implications to the preservation of optically active amino acids in the early earth

Akaboshi et al. (1990) has found an unexpected protection of the achiral amino acid, glycine, towards ionizing radiation at the expense of the selective destruction of the chiral amino acids, alanine and aspartic acid. The present work examines the mechanism of this protection for the case of alanine. We have developed a computer model for the radiolysis of glycine, alanine and glycine-alanine mixtures in aqueous solution. It is established that this protection is due in part to the reaction of the α-radical of glycine with alanine to regenerate a more stable α-radical, according to the following reaction, $$ \cdot CH(NH_3^ + )CO_2^ - + CH_3 CH(NH_3^ + )CO_2^ - \to CH_2 (NH_3^ + )CO_2^ - + CH_3 \dot C(NH_3^ + )CO_2^ -$$ The rate constant of this reaction was estimated to be ≤10⁴M^-1s^-1. The implications for this selective protection of glycine are considered for a hypothetical case in which there would be an enrichment of about 10% ofL-alanine in the primitive ocean and taking the glycine/alanine ratios obtained in CH₄-and CO₂- dominated atmospheres using electric discharge experiments. It is predicted that alanine would be rapidly destroyed and radioracemized in spite of the fact that the concentration of alanine is equal or significantly lower than that of glycine. Assuming that chiral amino acids were a prerequisite for the origin of life, it can be deduced that life could have appeared in a relatively short period of time unless there was a constant supply of optical amino acids from extraterrestrial sources.