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PubMed2001

Detection of West Nile virus in mosquitoes by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay employing < > detection technology was developed to identify West Nile virus in experimentally infected mosquitoes. The specificity of the assay was evaluated with the following viruses: eastern equine encephalitis, Ilheus, West Nile and yellow fever viruses. The limits of detection were determined using West Nile viral RNA extracted from serial dilutions of virus culture in infected mosquitoes. Limit of detection was 5 PFU from extracted mosquitoes. We were able to detect the presence of one infected mosquito in a pool of 50 repeatedly. When the RT-PCR was used with coded samples of intrathoracically-infected and uninfected mosquitoes, the assay detected the virus in all infected mosquitoes. Analytic sensitivity and specificity were 100%. This assay offers an efficient and rapid method of identifying West Nile virus in infected mosquitoes or cell culture.

Animals↗
PubMedDate not supplied

Characteristics of growth, morphology, contractility, and protein expression of fibroblasts derived from keloid.

Phenotypic alterations of keloid-derived fibroblasts were characterized by comparison with the phenotypes of normal fibroblasts from the same patient. Explant cultures of keloids showed unique features. Keloid explants contracted considerably and reduced their size during culture, whereas the size of normal skin explants remained unchanged. Enlarged cells were found among fibroblasts which had grown out of all the explants and were morphologically distinct from fibroblasts; however, keloid explants produced many more of them than did the normal tissues. The growth rate of fibroblast colonies formed from normal explants was five times higher than keloid explants. Keloid fibroblasts which had been serially cultivated contracted lattices of collagen gels at a rate similar to normal fibroblasts. Proteins extracted from serially cultivated fibroblasts were mapped on polyacrylamide two-dimensional electrophoretic gels. No significant qualitative alterations in protein expression in keloid cells were found as compared with normal fibroblasts. However, some quantitative changes were found between the two. A computer-assisted image analyzer detected 151 polypeptide spots--50 spots (33%) of which increased their amounts in keloid cells, whereas 34 spots (22.5%) decreased in comparison with normal fibroblasts. Sixteen major polypeptides were identified as known proteins with the aid of time-of-flight mass spectrometry. The level of expression of these identified proteins was similar between normal and keloid cells, except stathmin whose expression was suppressed in keloid fibroblasts.

Journal Article↗
PubMed1995

Induction and intracellular localization of 90-kilodalton heat-shock protein in rat kidneys with acute gentamicin nephropathy.

BACKGROUND: We previously reported the induction of 73-kilodalton heat-shock protein (HSP73) in injured tubular epithelial cells in rat kidneys with gentamicin-induced acute renal failure. In the present study, we examined serial expression of 90-kilodalton heat-shock protein (HSP90), another major HSP, in this animal model. EXPERIMENTAL DESIGN: Sprague-Dawley rats received gentamicin (80 mg/kg/day) for 14 days and developed acute proximal tubular injury. Serial immunohistochemical localization of HSP90 was observed at both light microscopic and electron microscopic levels, using a specific antibody against HSP90. In addition, serial renal extracts were analyzed by immunoblot. RESULTS: On light microscopy, HSP90 was induced in injured proximal tubular epithelial cells and accumulated in fine granules 36 hours after the gentamicin exposure. The size and number of these granules gradually increased to Day 12 and decreased from Day 18, and the granules disappeared on Day 27. Electron microscopy showed the accumulation of HSP90 in the swollen lysosomes and the nucleoli of the injured proximal tubular epithelial cells. On serial immunoblot analysis of renal extracts, increased amounts of HSP90 were found in association with the induction of HSP90 in injured cells. Furthermore, on immunoblot of nuclear fractions from kidneys at Days 0 and 6, HSP90 was detected in the fraction at Day 6. CONCLUSIONS: These results demonstrate that HSP90 is induced in the lysosomes and the nucleoli of damaged cells during the course of gentamicin-induced acute tubular injury. HSP90 may have roles in the disposition of degenerated proteins and in the new protein synthesis for the protection and repair of target cells from gentamicin nephrotoxicity.

Acute Kidney Injury↗