Early Interactions of Rhizobium leguminosarum bv. phaseoli and Bean Roots: Specificity in the Process of Adsorption and Its Requirement of Ca(sup2+) and Mg(sup2+) Ions

Roots of Phaseolus vulgaris L. were incubated with dilute suspensions (1 x 10(sup3) to 3 x 10(sup3) bacteria ml(sup-1)) of an antibiotic-resistant indicator strain of Rhizobium leguminosarum bv. phaseoli in mineral medium and washed four times by a standardized procedure prior to quantitation of adsorption (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:371-376, 1986). The population of rhizobia remaining adsorbed on roots after washing was homogeneous, as indicated by the first-order course of its desorption by hydrodynamic shear. Rhizobia were maximally active for adsorption in the early stationary phase of growth. The process leading to adsorption was rapid, without an initial lag, and slowed down after 1 h. Adsorption of the indicator strain at 10(sup3) bacteria ml(sup-1) was inhibited to different extents in the presence of 10(sup3) to 10(sup8) antibiotic-sensitive competitor rhizobia ml(sup-1). After a steep rise above 10(sup4) bacteria ml(sup-1), inhibition by heterologous competitors in the concentration range of 10(sup5) to 10(sup7) bacteria ml(sup-1) was markedly less than by homologous strains, while at 10(sup8) bacteria ml(sup-1) it approached the high level of inhibition by the latter. At 10(sup7) bacteria ml(sup-1), all of the heterologous strains tested were consistently less inhibitory than homologous competitors (P < 0.001). These differences in competitive behavior indicate that in the process of adsorption of R. leguminosarum bv. phaseoli to its host bean roots, different modes of adsorption occur and that some of these modes are specific for the microsymbiont (as previously reported for the alfalfa system [G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-381, 1986]). Moreover, whereas the nonspecific process occurred either in the absence or in the presence of Ca(sup2+) and Mg(sup2+) ions, expression of specificity was totally dependent on the presence of those cations. R. leguminosarum bv. phaseoli bacteria adsorbed in the presence of Ca(sup2+) and Mg(sup2+) were more resistant to desorption by shear forces than were rhizobia adsorbed in their absence. These results indicate that (i) symbiotic specificity in the P. vulgaris-R. leguminosarum bv. phaseoli system is expressed already during the early process of rhizobial adsorption to roots, (ii) Ca(sup2+) and Mg(sup2+) ions are required by R. leguminosarum bv. phaseoli for that specificity, and (iii) those cations cause tighter binding of rhizobia to roots.     

Reproductive disorders in dairy cattle: I. Respective influence of herds, seasons, milk yield and parity

This study was performed in two large dairy units (with 130 and 213 calving cows each) during one year. The objectives were to investigate 1) epidemiological patterns of main post-partum reproductive disorders (metritis, post-partum and post-service anestrus, repeat breeding and embryonic death) and 2) the impact of herd, calving season, milk yield and parity on these patterns. Approximately 20% of the cows in both herds were not affected by any of the disorders. Prevalence of metritis was high (32 to 44%) and appeared influenced by the herds' conditions interacting with calving months and milk yield effects. Cyclic post-partum anestrus incidence was also essentially affected by the herd effect with an added seasonal interaction. Other disorders in both herds were also primarily subjected to the seasonal effect. Individual milk yield and parity only marginally affected the epidemiological patterns. We concluded that even in similar environmental conditions, no general patterns of incidence of reproductive disorders can be drawn and that they are essentially dependent on the characteristics of each herd management.     

A new class of potent gastrin antagonists

The hexapeptide Z-Tyr(SO-3)-Met-Gly-Trp-Met-Asp-NH2, from the natural sequence of C-terminal cholecystokinin was found to be a competitive antagonist of cholecystokinin receptors, in vitro. In the present study, we report that this peptide inhibits gastrin-induced acid secretion in vivo, (ED50 = 1.5 mumol . kg-1), without agonist activity. Desulfation of the tyrosine residue slightly altered this effect. The tripeptide Boc-Trp-Met-Asp-NH2 showed similar effects, but had lower potency (ED50 = 12 mumol . kg-1). From these preliminary results, it can be concluded that removal of the phenylalanine residue from the C-terminal sequence of CCK or gastrin, leads to an antagonist of the natural hormones and that C-terminal phenylalanine residue is important for agonist activity. As compared with proglumide, a well known gastrin receptor antagonist, these peptides were 20-200 times more potent as inhibitors on the same model.     

Protoplast production from filamentous fungi with their own autolytic enzymes

Abstract Production of protoplasts in different genera of filamentous fungi with their own lytic enzymes obtained from autolyzed cultures, as well as the regeneration of these protoplasts, has been studied. The results support the idea that the use of these autolytic enzymes could be a general method of production of protoplasts from filamentous fungi.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Three new species of penicillium

Three new species of microfungi belonging to the genus Penicillium Link ex Fries are described and illustrated. All but one have been isolated from the atmosphere of las Palmas, capital city of the island of Gran Canaria (Canary Islands, Spain). They clearly differ from all species of the genus described so far and are, therefore, described and proposed as new species: Penicillium hispanicum sp. nov., Penicillium grancanariae sp. nov., and Penicillium palmensis sp. nov.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

A sensitive fluorimetric microassay for the determination of glutathione peroxidase activity. Application to human blood platelets

The method proposed for measuring glutathione peroxydase (GSH-Px) activity is based on the determination of oxidized glutathione (GSSG) using o-phtalaldehyde (OPT) as a fluorescent reagent. This method makes it possible to study the kinetics of both substrates (peroxide and reduced glutathione, GSH), and allosteric kinetics were found for GSH, with human platelets as the source of GSH-Px. Different methods for platelet disruption were compared. The reference values obtained for GSH-Px activity in human blood platelets by this fluorimetric procedure and the conventional enzymatic method were very similar and significantly higher than those previously reported; the reasons for this difference are discussed.     

Transferrin, somatomedins and growth

Plasma transferrin is significantly positively correlated to the growth velocity in normal children and children with various growth disorders. In hypopituitary dwarfs, both plasma transferrin and somatomedin are significantly lower than in controls. Acute variations of the plasma transferrin levels could be involved in the regulation of the GH secretion by a feedback mechanism. Yet, transferrin is neither a somatomedin or a somatomedin-binding protein, and the mechanisms relating growth to transferrin remain unknown.