Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona

Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.     

Expression of GM-CSF receptors in male germ cells and their role in signaling for increased glucose and vitamin C transport

We studied the expression and function of the granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in male germ cells. RT-PCR showed expression of mRNAs encoding the alpha- and beta-subunits of the GM-CSF receptor in human testis, and the presence of the alpha- and beta-proteins was confirmed by immunoblotting with anti-alpha and anti-beta-antibodies. Immunolocalization studies showed the level of expression of GM-CSF alpha- and beta-subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM-CSF revealed that bull spermatozoa have about 105 high-affinity sites with a K(d) of 222 pM and approximately 1100 low-affinity sites with a K(d) of 10 nM. GM-CSF signaled, in a time- and dose-dependent manner, for an increased uptake of glucose and vitamin C.     

Evaluation of the pituitary-testicular function during experimental nephrosis

To investigate the pituitary-testicular function in nephrotic rats, a sequence of experiments was undertaken in adult male rats after a single dose of puromycin aminonucleoside (PAN). Endocrine modifications were evaluated chronologically throughout the experimental disease in order to determine the appearance of hormone alterations which lead to the axis dysfunction. Serum concentration of LH, FSH, androstenedione, total and free testosterone, estradiol as well as urine testosterone were measured by specific RIAs on days 3, 7 and 10 after treatment on nephrotic and control groups. Prolactin was also evaluated on day 10. Likewise, total weight of various androgen responsive tissues from both groups was recorded, and the number of androgen receptor (AR) binding sites were determined. To know the functional status of the hipophyseal-testicular unit, groups of nephrotic and control rats were stimulated with LHRH (300 ng/100 g b.w.) or with one or four doses of hCG (8 UI), respectively. Additionally, the relative in vitro biological activity of FSH from nephrotic and control rats before and after LHRH stimulus was determined. The results from the hormonal profile revealed clear endocrine disorders characterized by a progressive diminution of all serum hormones except prolactin and urine testosterone, which remained unmodified. The weight of the main androgen responsive tissues, the ventral prostate and the seminal vesicle, decreased parallelly to androgen diminution. The binding analysis of AR shows a significant elevation of the available androgen sites in all analyzed tissues except kidney and hypothalamus. The secretion of LH and FSH from nephrotic animals after LHRH administration was lower than that from intact animals at the registered times. Interestingly, the biological activity of FSH from nephrotic rats was not detectable at both, before and after LHRH administration. Testicular response to hCG stimuli, in terms of testosterone synthesis was not significantly different in the two groups analyzed with respect to the intact animals. By contrast, no response was observed in terms of estradiol production at either one or four doses of hCG. On the whole, the results presented herein allow us to conclude that experimental nephrosis has a harmful effect on the pituitary-testicular axis, and strongly suggests that the endocrine dysfunction is initiated at the hypophyseal level; even though a specific testicular damage is also present.     

Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.     

Efficient removal of hexavalent chromium by a tolerant Streptomyces sp. affected by the toxic effect of metal exposure

AIMS: To isolate and analyse chromium-resistant micro-organisms suitable for bioremediation. METHODS AND RESULTS: Strain CG252, with a minimal inhibitory concentration of 500 microg ml(-1), was isolated from contaminated soils and identified as a Streptomyces sp. by 16S rDNA sequence analysis. Assays carried out at various Cr(VI) concentrations indicated that chromium removal was more efficient at lower concentrations and that this activity resulted in accumulation of Cr(III). Atomic adsorption analysis indicated that the chromium removed was not associated with cell mass and activity assays showed that the capacity to reduce Cr(VI) was most probably due to a soluble cytosolic enzyme. Cells grown as biofilms showed enhanced removal of Cr(VI) with respect to planktonic cells, while analysis of growth and colony morphology indicated that Cr(VI) had a toxic effect on this strain. CONCLUSIONS: Streptomyces sp. CG252 tolerated heavy metals and elevated levels of chromium, despite its negative effect on growth and development, and was efficient at removing Cr(VI) by promoting reduction to Cr(III). SIGNIFICANCE AND IMPACT OF THE STUDY: Strain CG252's capacity to tolerate heavy metals and to reduce Cr(VI) to the less toxic Cr(III), especially when forming biofilms, makes it a promising candidate for detoxification of sites containing this heavy metal.     

Identification of the ligands of protein interaction domains through a functional approach

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.