Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.     

Density-dependent vital rates and their population dynamic consequences

We explore a set of simple, nonlinear, two-stage models that allow us to compare the effects of density dependence on population dynamics among different kinds of life cycles. We characterize the behavior of these models in terms of their equilibria, bifurcations, and nonlinear dynamics, for a wide range of parameters. Our analyses lead to several generalizations about the effects of life history and density dependence on population dynamics. Among these are: (1) iteroparous life histories are more likely to be stable than semelparous life histories; (2) an increase in juvenile survivorship tends to be stabilizing; (3) density-dependent adult survival cannot control population growth when reproductive output is high; (4) density-dependent reproduction is more likely to cause chaotic dynamics than density dependence in other vital rates; and (5) changes in development rate have only small effects on bifurcation patterns. Received: 12 April 1999 / Published online: 3 August 2000     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Running economy and maximal oxygen consumption after 4 weeks of oral Echinacea supplementation

The purpose of this investigation was to determine the effects of 4 weeks of oral Echinacea (ECH) supplementation on erythropoietin (EPO), red blood cell (RBC) count, running economy (RE), and VO2max. Twenty-four men aged 24.9 ± 4.2 years, height 178.9 ± 7.9 cm, weight 87.9 ± 14.6 kg, body fat 19.3 ± 6.5% were grouped using a double-blind design and self-administered an 8,000-mg·d(-1) dosage of either ECH or placebo (PLA) in 5 × 400 mg × 4 times per day for 28 days. Blood samples were collected and analyzed for RBCs and EPO using automated flow cytometery and enzyme-linked immunosorbent assay. Maximal graded exercise tests (GXTs) were administered to measure VO2max, RE, and heart-rate responses. Analysis of variance was used to determine statistically significant differences (P ≤ 0.05). The EPO increased significantly in ECH at 7 days (ECH: 15.75 ± 0.64, PLA: 10.01 ± 0.73 mU·ml(-1)), 14 days (ECH: 18.88 ± 0.71, PLA: 11.02 ± 0.69 mU·ml(-1)), and 21 days (ECH: 16.06 ± 0.55, PLA: 9.20 ± 0.55 mU·ml(-1)). VO2max increased significantly in ECH (ECH: 1.47 ± 1.28, PLA: -0.13 ± 0.52%). Running economy improved significantly in ECH as indicated by a decrease in submaximal VO2max during the first 2 stages of the GXT (stage 1: ECH -1.50 ± 1.21, PLA 0.60 ± 1.95%; stage 2: ECH -1.67 ± 1.43, PLA 0.01 ± 1.03%). These data suggest that ECH supplementation results in significant increases in EPO, VO2max, and running economy.