Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes.

A 600-bp oriT-containing DNA fragment from the Rhodobacter sphaeroides 2.4.1 S factor (oriTs) (A. Suwanto and S. Kaplan, J. Bacteriol. 174:1124-1134, 1992) was shown to promote polarized chromosomal transfer when provided in cis. A Kmr-oriTs-sacR-sacB (KTS) DNA cassette was constructed by inserting oriTs-sacR-sacB into a pUTmini-Tn5 Km1 derivative. With this delivery system, KTS appeared to be randomly inserted into the genome of R. sphaeroides, generating mutant strains which also gained the ability to act as Hfr donors. An AseI site in the Kmr cartridge (from Tn903) and DraI and SnaBI sites in sacR-sacB (the levansucrase gene from Bacillus subtilis) were employed to localize the KTS insertion definitively by pulsed-field gel electrophoresis. The orientation of oriTs at the site of insertion was determined by Southern hybridization analysis. Interrupted mating experiments performed with some of the Hfr strains exhibited a gradient of marker transfer and further provided genetic evidence for the circularity and presence of two chromosomal linkage groups in this bacterium. The genetic and environmental conditions for optimized mating between R. sphaeroides strains were also defined. The results presented here and our physical map of the R. sphaeroides 2.4.1 genome are discussed in light of the presence of two chromosomes.     

Lectin Binding to Radopholus citrophilus and R. similis Proteins

Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r² = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes.     

Maternal effects on offspring growth and development depend on environmental quality in the frogBombina orientalis

Summary Differences in maternal investment and initial offspring size can have important consequences for offspring growth and development. To examine the effects of initial size variability in the frogBombina orientalis, we reared larvae (N=360) in one of two treatments representing different levels of environmental quality. We used snout-vent length at the feeding stage (stage 25, Gosner 1960) as a measure of maternal investment. In a “low quality” treatment, larvae were reared with two conspecific tadpoles and food was limited, whereas in a “high quality” treatment, larvae were reared individually and were fed ad libitum. Among tadpoles reared in the low quality treatment, individuals that were initially small had smaller body sizes through metamorphosis and longer larval periods than individuals that were initially large. Among tadpoles reared in the high quality treatment, initial size had only a weak influence on later larval size, and did not significantly affect metamorphic size of the duration of the larval period. This interaction between maternal investment and rearing conditions suggests that production of initially small offspring could be advantageous if these offspring develop in relatively benign environments, but disadvantageous if environments are more severe. These findings are discussed in light of previous studies that have demonstrated such interactions in organisms with complex life cycles.     

A Cyanobacterial Gene Encoding Peptidyl-Prolyl cis-trans Isomerase

The rotA gene encoding peptidyl-prolyl cis-trans isomerase has been identified, sequenced, and shown to be transcribed in the cyanobacterium Synechococcus sp. PCC7942. Inactivation of the gene by replacement of a region containing the open reading frame with a gene conferring kanamycin resistance resulted in merodiploids containing both the wild type and the modified genomic region. We were not able to isolate a kanamycin-resistant mutant in which all the genomic wild-type copies were substituted, which suggests that such replacement could have been lethal.     

Synthesis and evaluation of an inhibitor of carboxypeptidase A with a Ki value in the femtomolar range

Comparative studies among a series of tripeptide phosphonate inhibitors of the zinc peptidase carboxypeptidase A indicate that incorporation of the phosphonic acid analogue of valine at the P1 position results in significantly higher affinity than the glycine, alanine, or phenylalanine analogues. When applied to the tripeptide analogue Cbz-Phe-ValP-(O)Phe [ZFVP(O)F], determination of the inhibition constant Ki was complicated by the very slow rate of dissociation. The rate of exchange of [3H]ZFVP(O)F with enzyme-bound [14C]ZFVP(O)F was followed for periods of 3-4 months to measure dissociation rate constants in the range of (1.7-4.4) x 10(-9) s-1, corresponding to half-lives of 5-13 years. Although the on- and off-rate constants differ for different carboxypeptidase isozymes, their ratios, corresponding to the inhibition constants Ki, are consistently in the range of 10-27 fM. Both the inhibition constants and the dissociation rate constants appear to be the lowest values yet determined for an enzyme-small inhibitor interaction.     

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.