Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Prognostic factors in human primary breast cancer: comparison of c-myc and HER2/neu amplification.

Amplification of oncogenes in primary tumours may have prognostic and/or therapeutic significance for patients with breast cancer. We have studied HER2/neu and c-myc amplification together with steroid receptors in human primary breast tumours and related the outcome with (relapse-free) survival. A strong inverse correlation was found between HER2/neu amplification and the presence of oestrogen and progesterone receptors. Actuarial 5-years survival showed that breast cancer patients with c-myc amplification in their primary tumours experience a shorter relapse-free survival, especially in node-negative and in receptor-positive tumours, whereas HER2/neu amplification may be of prognostic value for overall survival in receptor-negative tumours. Overall, in our hands, c-myc amplification appeared to be a more potent prognosticator than HER2/neu amplification in human primary breast cancer.     

Genetic variability in Plantago species in relation to their ecology

Summary A survey of enzyme variability in several populations of Plantago major in the Netherlands has been made. Nine of 36 loci were found to be polymorphic. The most extensively studies population showed 7 polymorphic loci (19%). The average heterozygosity was 0.005, a low value since P. major is predominantly inbreeding; a first estimate of the outcrossing rate is only 10%. All nine variable loci show simple Mendelian inheritance, seven of them could be placed into four different linkage groups. Marked differences in allele frequencies were found between two subspecies: ssp. major and ssp. pleiosperma. Two enzyme loci possess subspecies-specific alleles, Pgm-1 and Got-1. The most likely explanation of this phenomenon is the existence of fitness differences, caused either by the enzyme loci themselves or by linked loci.Grassland Species Research Group Publication no. 14     

The molten globule intermediate for protein insertion or translocation through membranes

Insertion of some protein toxins into membranes proceeds through an unfolding step. The unfolding trigger can be the low pH in endosomes, exposure to body temperature, reduction of disulphide bonds or proteolytic cleavage occurring at the membrane surface. The insertion intermediates are not fully unfolded but have the features of a 'molten globule state' that is also observed at early stages of polypeptide folding. In this article, we review the evidence supporting these ideas and speculate about the implications of the molten globule intermediate for understanding the general mechanisms of protein insertion and translocation across membranes.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.     

Numerical simulation of tissue differentiation around loaded titanium implants in a bone chamber

The application of a bone chamber provides a controlled environment for the study of tissue differentiation and bone adaptation. The influence of different mechanical and biological factors on the processes can be measured experimentally. The goal of the present work is to numerically model the process of peri-implant tissue differentiation inside a bone chamber, placed in a rabbit tibia. 2D and 3D models were created of the tissue inside the chamber. A number of loading conditions, corresponding to those applied in the rabbit experiments, were simulated. Fluid velocity and maximal distortional strain were considered as the stimuli that guide the differentiation process of mesenchymal cells into fibroblasts, chondrocytes and osteoblasts. Mesenchymal cells migrate through the chamber from the perforations in the chamber wall. This process is modelled by the diffusion equation. The predicted tissue phenotypes as well as the process of tissue ingrowth into the chamber show a qualitative agreement with the results of the rabbit experiments. Due to the limited number of animal experiments (four) and the observed inter-animal differences, no quantitative comparison could be made. These results however are a strong indication of the feasibility of the implemented theory to predict the mechano-regulation of the differentiation process inside the bone chamber.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Differential expression of mycothiol pathway genes: are they affected by antituberculosis drugs?

Mycothiol (MSH) is the major cellular thiol in Mycobacterium tuberculosis (M.tb). We hypothesize that the mycothiol-dependent detoxification pathway may serve an important role during oxygen stress management in M. tuberculosis, derived from normal aerobic metabolism, the macrophage environment and through the action of anti-tubercular antibiotics, such as Isoniazid (INH). Total mRNA and DNA were isolated from M. bovis BCG at different stages of growth in 7H9 mycobacterial medium. Three genes involved in mycothiol metabolism and encoding the enzymes mycothiol S-conjugate amidase (Mca, Rv1082), NADPH dependent mycothiol reductase (mtr, Rv2855), and N-Acetyl-1-D-myo-Inosityl-2-Amino-2-Deoxy-alpha-D-Glucopyranoside Deacetylase (GlcNAc-Ins deacetylase, Rv1170 or mshB) were investigated for genomic rearrangements and expression. The results show that the genomic domains of the genes remain conserved in evolutionary diverse and unrelated M. tuberculosis isolates. The genes encoding enzymes implicated in mycothiol reduction, mtr (Rv2855) and the mycothiol-dependant detoxification of electrophilic agents, Mca (Rv1082), are shown to be actively transcribed during logarithmic M. bovis BCG growth. The gene encoding GlcNAc-Ins deacetylase (the rate limiting mycothiol biosynthesis step) shows induction in the presence of INH. Antisense oligonucleotides to both GlcNAc-Ins deacetylase (Rv1170) and mtr (Rv2855) mRNA affect mycobacterial growth. In conclusion the results presented here suggest that these enzymes are sensitive to free radical generating antituberculosis drugs and may be useful targets for new drug development.     

The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense

The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors predominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. Together, these findings indicate an essential role for CD97 in the migration of neutrophils.