Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

Receptor-mediated endocytosis of CC-chemokines

Chemokines are chemotactic proteins which play a central role in immune and inflammatory responses. Chemokine receptors are members of the seven transmembrane G-protein coupled family and have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. To study chemokine endocytosis in detail we have used novel site-specific chemistry to make a fluorescently labeled CC-chemokine agonist (rhodamine-MIP-1alpha) and antagonist (NBD-RANTES). We have also generated a CHO cell line stably expressing a hemagglutinin-tagged version of the CC-chemokine receptor 1 (CCR1), and using these reagents we have examined the receptor-mediated endocytosis of CC-chemokines by confocal microscopy. Our studies reveal that the agonist was internalized and accumulated in transferrin receptor-positive endosomes whereas the antagonist failed to internalize. However, receptor-bound antagonist could be induced to internalize by co-administration of agonist. Analysis of receptor redistribution following chemokine addition confirmed that sequestration was induced by agonists but not by antagonists.     

Isolated fractures of the lesser trochanter in children

In a retrospective study of 1126 children with fractures of the proximal third of the femur, three children were found to have isolated fractures of the lesser trochanter. This fracture occurred from a fall in one child and following sporting activities, without a history of injury, in the others. In the latter children, the clinical presentations were similar to those of children with transient synovitis of the hip or Perthes disease. In each child, plain radiographs showed an avulsion fracture of the bony portion of the lesser trochanter. Early and complete recovery followed symptomatic treatment even when there was marked proximal displacement of the avulsed segment of the lesser trochanter.     

The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase-2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial-to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4-MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen-induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen-stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.     

Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site

Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase's zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase is rapidly, site-specifically modified by fluorescein maleimide (FM) at this cysteine. Modification with FM at this residue does not lead to any activity loss in a coupled RNA/DNA synthesis assay, indicating that it is not a catalytically essential residue. In contrast, iodoacetamidefluorescein (IAF) modifies cysteine 39 more slowly and stoichiometrically inhibits activity. It was not clear why these two similar fluorescent dyes should have such different inhibitory effects when attached to the same cysteine. The IAF inhibition must be due to some property of the link between the fluorescein and the cysteine because that is how it differs from FM. The pKa's of the fluoresceins from both FM- and IAF-modified primase are strongly shifted to lower values (approximately 5.4) compared to free fluorescein. These results strongly suggest that the deprotonated form of the fluoresceins are stabilized on primase by a strong interaction with the adjacent zinc in the zinc finger motif. The ability to place a noninhibitory FM at this site will be of great assistance in fluorescence energy transfer studies since the distances established to cysteine 39 will reflect the distance to the essential zinc finger motif.     

The evolution of packages for monolithic microwave andmillimeter-wave circuits

The maturing of monolithic microwave integrated circuit (MMIC) technology has spawned a variety of new military and commercial applications. As-a result, there is an increased emphasis on the packaging of MMIC chips and MMIC-based components. Currently, the industry is applying a number of new assembly and packaging technologies to RF components and subsystems driven by the forces of performance, size and weight, and cost. This paper outlines the current evolution in microwave and millimeter-wave packaging using examples drawn from the area of active array antennas