Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5′ phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-β-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation.     

Molecular analysis of nondisjunction in mice heterozygous for a Robertsonian translocation

A Robertsonian translocation results in a metacentric chromosome produced by the fusion of two acrocentric chromosomes. Rb heterozygous mice frequently generate aneuploid gametes and embryos, providing a good model for studying meiotic nondisjunction. We intercrossed mice heterozygous for a (7.18) Robertsonian translocation and performed molecular genotyping of 1812 embryos from 364 litters with known parental origin, strain, and age. Nondisjunction events were scored and factors influencing the frequency of nondisjunction involving chromosomes 7 and 18 were examined. We concluded the following: 1. The frequency of nondisjunction among 1784 embryos (3568 meioses) was 15.9%. 2. Nondisjunction events were distributed nonrandomly among progeny. This was inferred from the distribution of the frequency of trisomics and uniparental disomics (UPDs) among all litters. 3. There was no evidence to show an effect of maternal or paternal age on the frequency of nondisjunction. 4. Strain background did not play an appreciable role in nondisjunction frequency. 5. The frequency of nondisjunction for chromosome 18 was significantly higher than that for chromosome 7 in males. 6. The frequency of nondisjunction for chromosome 7 was significantly higher in females than in males. These results show that molecular genotyping provides a valuable tool for understanding factors influencing meiotic nondisjunction in mammals.     

The roles of juvenile hormone and biogenic amines on pheromone response plasticity and diapause termination in male Caloptilia fraxinella

In insects that exhibit a period of delayed reproduction, the timing of mating and reproduction is controlled by environmental conditions that regulate endogenous factors such as hormones and biogenic amines (BAs). Caloptilia fraxinella (Ely) (Lepidoptera: Gracillariidae) undergoes a 9‐month reproductive diapause from adult eclosion in the summer until diapause termination the following spring when adults mate. Male response to female sex pheromone is plastic, and is most acute when moths are reproductively active. The aim of this study is to further elucidate the mechanisms involved in the regulation of male response to pheromone in C. fraxinella, and to test whether the application of BAs with and without a juvenile hormone analogue (JHA) to males in different physiological states impacts pheromone responsiveness, as measured by electroantennogram and wind tunnel bioassays. Treatment of male C. fraxinella in reproductive diapause with one application of a JHA induces the highest subsequent pheromone response in the fall, but does not alter pheromone response earlier in reproductive diapause in the summer. The JHAs methoprene and pyriproxyfen similarly enhance pheromone response in the fall. Treatment with methoprene alone or in combination with one of the BAs octopamine, dopamine or serotonin increases male pheromone responsiveness in the fall. The increase in pheromone response can be attributed to methoprene only, as treatment with any of the BAs alone does not enhance male response to pheromone. Biogenic amine treatment lowers male responsiveness to pheromone in some experiments, indicating that there may be a role for BAs in maintaining low pheromone response during reproductive diapause in this species.     

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor α in the monocyte cell line THP-1

The induction of tumour necrosis factor (TNF)-α from the monocytic cell line THP-1 by the streptococcal antigen I/II from Streptococcus mutans serotype f (protein I/IIf) was studied by use of recombinant polypeptides containing discrete domains of the protein. The derivatives carrying the N-terminal alanine-rich region (A region) and the adjacent variable region (extended V region) of the protein bound to THP-1 cell extracts in a saturable fashion, and one derivative lacking both the A and the extended V regions was not able to bind monocyte cell extracts, suggesting that the domains responsible for the binding of protein I/IIf to monocytes were the A and the extended V regions. Sodium metaperiodate pretreatment of THP-1 cell extracts, tunicamycin pretreatment of monocyte cells or competition with N-acetyl neuraminic acid (NANA) and fucose resulted in a 45–70% reduction in binding activity of the derivatives carrying the extended V region, demonstrating the lectin-like mode of recognition of the monocytic receptor by the extended V region and the role of NANA and fucose in this recognition process. Besides, the stimulation of monocytes to release TNF-α by the derivatives containing the A region and the extended V region was effective and was not affected by the addition of polymyxin B or vitamin D₃, suggesting that CD14 does not play the role of receptor in stimulation of monocytes by protein I/IIf to release TNF-α.     

Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism

Summary We have analysed the segregation of a TA-repeat polymorphism in intron 17b of the cystic fibrosis transmembrane conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the F508 mutation (i.e. with at least one parent not carrying F508) or closely linked DNA markers. At least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. Of the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were performed using this polymorphism.     

RAC1 expression and role in IL-1β production and oxidative stress generation in familial Mediterranean fever (FMF) patients

Objectives

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder. The caspase-1-dependent cytokine, IL-1β, plays an important role in FMF pathogenesis, and RAC1 protein has been recently involved in IL-1β secretion. This study aims to investigate RAC1 expression and role in IL-1β and caspase-1 production and oxidative stress generation in FMF.

Materials and methods

The study included 25 FMF patients (nine during attack and remission, and 16 during remission only), and 25 controls. RAC1 expression levels were analyzed by real-time PCR. Ex vivo production of caspase-1, IL-1β, IL-6 and markers of oxidative stress (malondialdehyde, catalase, and glutathione system) were evaluated respectively in supernatants of patients’ and controls’ PBMC and PMN cultures, in the presence and absence of RAC1 inhibitor.

Results

RAC1 gene expression and IL-1β levels were increased in patients in crises compared to those in remission or controls. RAC1 expression levels were correlated with MEFV genotypes, patients carrying the M694V/M694V genotype having a two-fold increase in the expression levels compared to those carrying other genotypes. Caspase-1 levels were higher in LPS-induced PBMC of patients in remission than controls. Spontaneous and LPS-induced IL-1β production were comparable in patients in remission and controls, whereas LPS-induced IL-6 production was enhanced in patients, compared to controls. RAC1 inhibition resulted in a decrease in caspase-1 and IL-1β, but not IL-6, levels. Malondialdehyde levels produced by LPS-stimulated PMNs were increased in patients in remission compared to those in controls, but decreased following RAC1 inhibition. Catalase and GSH activities were reduced in unstimulated PMN culture supernatants of patients in remission compared to controls and were increased in the presence of RAC1 inhibitor.

Conclusion

These results show the involvement of RAC1 in the inflammatory process of FMF by enhancing IL-1β production, through caspase-1 activation, and generating oxidative stress, even during asymptomatic periods.

     

NMR investigation of Tyr105 mutants in TEM-1 beta-lactamase: dynamics are correlated with function

The existence of coupled residue motions on various time scales in enzymes is now well accepted, and their detailed characterization has become an essential element in understanding the role of dynamics in catalysis. To this day, a handful of enzyme systems has been shown to rely on essential residue motions for catalysis, but the generality of such phenomena remains to be elucidated. Using NMR spectroscopy, we investigated the electronic and dynamic effects of several mutations at position 105 in TEM-1 beta-lactamase, an enzyme responsible for antibiotic resistance. Even in absence of substrate, our results show that the number and magnitude of short and long range effects on (1)H-(15)N chemical shifts are correlated with the catalytic efficiencies of the various Y105X mutants investigated. In addition, (15)N relaxation experiments on mutant Y105D show that several active-site residues of TEM-1 display significantly altered motions on both picosecond-nanosecond and microsecond-millisecond time scales despite many being far away from the site of mutation. The altered motions among various active-site residues in mutant Y105D may account for the observed decrease in catalytic efficiency, therefore suggesting that short and long range residue motions could play an important catalytic role in TEM-1 beta-lactamase. These results support previous observations suggesting that internal motions play a role in promoting protein function.     

Centrosomal latency of incoming foamy viruses in resting cells

Completion of early stages of retrovirus infection depends on the cell cycle. While gammaretroviruses require mitosis for proviral integration, lentiviruses are able to replicate in post-mitotic non-dividing cells. Resting cells such as naive resting T lymphocytes from peripheral blood cannot be productively infected by retroviruses, including lentiviruses, but the molecular basis of this restriction remains poorly understood. We demonstrate that in G0 resting cells (primary fibroblasts or peripheral T cells), incoming foamy retroviruses accumulate in close proximity to the centrosome, where they lie as structured and assembled capsids for several weeks. Under these settings, virus uncoating is impaired, but upon cell stimulation, Gag proteolysis and capsid disassembly occur, which allows viral infection to proceed. The data imply that foamy virus uncoating is the rate-limiting step for productive infection of primary G0 cells. Incoming foamy retroviruses can stably persist at the centrosome, awaiting cell stimulation to initiate capsid cleavage, nuclear import, and viral gene expression.