Specific DNA probes for the identification of the fish pathogen,Renibacterium salmoninarum

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.The authors are with the instituto de Bioquímica, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile     

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 M GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0±7.0 nM VIP, whereas the maximal activity (at 1 M VIP)corresponded to an increase of about 140% with respect to basal values (7.5±0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED₅₀=1.8±1.4 nM)>VIP(ED₅₀=25.0±7.0 nM)>PHI (ED₅₀=725.0±127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of _s and _i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of¹²⁵I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd=0.43 M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.     

Gene expression associated with in vivo induction of early phase-long-term potentiation (LTP) in the hippocampal mossy fiber-Cornus Ammonis (CA)3 pathway.

Affymetrix microarray technology was used to characterize whole-hippocampus gene expression associated with in vivo N-methyl-D-aspartate (NMDA)-R-independent long-term potentiation (LTP) in the mossy fiber (MF)-Cornus Ammonis (CA)3 pathway of adult male F344 rats. Acute MF responses were evoked by stimulation of the MF bundle and recorded in stratum lucidum of CA3. Following recording of baseline responses at 0.05 Hz, animals received either CPP (NMDA-R antagonist, 10 mg/kg) or naloxone (opioid-R antagonist, 10 mg/kg). LTP was induced by two 100 Hz 1-sec trains at the intensity sufficient to evoke 50% of the maximal response. Responses were collected for an additional hour. In controls, MF responses were collected at 0.05 Hz for 1 hr, but 100 Hz trains were not delivered. Hippocampi were harvested prior to total RNA isolation. Fragmented cRNA was hybridized to a rat U34 neurobiology array. F344 rats exhibited characteristic LTP in the presence of CPP and LTP blockade in the presence of naloxone. As a result, genes associated with both NMDA-independent LTP and naloxone-induced blockade were identified. These include genes involved in transmitter transport, intracellular messengers, growth factors and ion channels. Up-regulated include NMDA-R2D, neuropeptide Y (NPY), proenkephalin, BDNF and NGFR. Down-regulated genes include IGF-1 and GABA-B.     

Chloride secretion in the submandibular gland of adult and early postnatal rats studied by X-ray microanalysis

Submandibular acinar cells of 1-day-old, 7-day-old, and adult rats were analyzed with X-ray microanalysis after stimulation with carbachol for different time periods (2–7 min). In unstimulated animals, marked differences in elemental content between compartments could be observed: secretory granules had a higher Ca and lower P and K content than other cell compartments. Comparison between different age groups showed significant differences for Ca, which increased with age in all compartments; Mg increased with age in the secretory granules and the apical cytoplasm. Only the glands from adult animals showed a significant effect of cholinergic stimulation: a transient decrease in Cl and K. The Cl concentration in the secretory granules decreased to 60% of the control value, which suggests that the granules release Cl upon stimulation. In young animals, no or little change in elemental distribution was observed after stimulation. This may indicate that Cl-secretion mechanisms are much less prominent in young animals. The ultrastructure of submandibular secretory granules depends on the preparation method: condensed and electrondense in freeze-substituted unfixed tissue, decondensed and more translucent in aldehyde-fixed tissue. This may indicate that the granules can transport water, and swell during the process of aldehyde fixation.     

Yeast aminopeptidase I is post-translationally sorted from the cytosol to the vacuole by a mechanism mediated by its bipartite N-terminal extension.

Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N-terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a pAPI-CUP1 chimera indicates that pAPI is transported to the vacuole by a post-translational mechanism. Deletion of the first 16 amino acids results in accumulation of the truncated protein in the cytosol, indicating that pAPI is actively transported to the vacuole. The chimera pAPI-myc, constructed by fusing a myc tag to the C-terminus of pAPI, was exploited to dissect the mechanism of pAPI transport. Cell fractionation studies show the presence of iAPI-myc and mAPI in a fraction of vacuoles purified by density centrifugation. This and the sequential conversion of pAPI-myc into iAPI-myc and mAPI lacking the myc tag is consistent with insertion of pAPI into the vacuolar membrane through its N-terminal extension. The specific mechanism of API sorting demonstrates a new pathway of protein transport in vacuolar biogenesis.     

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.     

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.