Human glioma cells from a long-term cell line were selected for their ability to migrate on a glioma-derived extracellular matrix. When tested over 28 serial passages, the migration-selected strain showed a genetically stable, enhanced migration rate compared with the parental cells. Proliferation studies demonstrated that the growth rate of migration-selected cells was slightly arrested. Both the selected strain and the parental culture showed anchorage-independent growth in soft agarose and were tumorigenic in athymic mice. Using molecular genetic strategies' display to isolate genes expressed differentially between the 2 populations, a 300-bp sequence homologous to thromboxane synthase was upregulated in the migration-selected cells relative to the parental cells. Expression levels of thromboxane synthase were highly elevated in the migration-selected cells when assessed by RNAse-protection assay and by flow cytometry. Two specific thromboxane synthase inhibitors, Dazmegrel and Furegrelate, reduced the migration rate of the migration-selected cells to a rate equal to or less than the rate exhibited by the parental cells, respectively. The inhibitors effect on the parental cells was inconsequential. These results suggest that aberrations in the regulation of thromboxane synthase expression or activity may influence the motility of human glioma cells.
We report on the use of an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. The assay targets seven genes: the H1, H3, H5, N1, and N2 genes of influenza A virus; the matrix protein M1 gene of influenza A virus; and the nonstructural protein (NS) gene of influenza B virus. By combining a two-step reverse transcription-multiplex PCR with typing and subtyping on the electronic microarray, the assay achieved an analytical sensitivity of 10 2 to 10 3 copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 15 different influenza virus isolates, including two influenza B virus, five A/H1N1, six A/H3N2, and two A/H5N1 isolates. In addition, the assay correctly identified 8 out of 10 diluted, archived avian influenza virus specimens with complete typing and subtyping information and 2 specimens with partial subtyping information. In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The assay is a rapid, accurate, user-friendly method for simultaneously typing and subtyping influenza viruses.
The NanoChip400 system uses multiplex PCR chemistry and electronic microarray detection of influenza A and B viruses; respiratory syncytial viruses A and B; and human parainfluenza virus types 1, 2, and 3. The results obtained with the NanoChip 400 system were compared with those obtained by direct fluorescent-antibody staining (DFA) and real-time PCR with 122 and 130 specimens, respectively. Concordance between DFA and NanoChip 400 system was obtained for 106 of 122 (86.9%) specimens. On the basis of discrepancy analysis with specimens available for confirmatory real-time PCR testing, the sensitivity and specificity of the NanoChip 400 were 98.6% and 100%, respectively. With respect to specimens previously tested by real-time PCR, the NanoChip 400 system demonstrated a sensitivity of 91.1% and a specificity of 100%. The NanoChip 400 system provides clinical laboratories with a practical, rapid, and sensitive method for the detection of common respiratory viruses.There has been a considerable effort over the past two decades or more to develop methods for the rapid detection of viral agents. Although enzyme immunoassay and lateral flowbased antigen detection are the most frequently used methods for the rapid detection of respiratory viruses, the most reliable antigen detection method for respiratory viruses is considered to be direct fluorescent-antibody staining (DFA), based on extensive studies conducted over the past 20 years (4, 9). While DFA has generally shown good performance in comparison to viral culture, the actual sensitivity is probably lower than that in published reports of studies that used viral culture as the "gold standard," given the lability of respiratory viruses.While viral culture is considered the gold standard, specimen handling is a major factor in the recovery of respiratory viruses; furthermore, even with shell vial cultures, the clinical utility of viral culture has limitations, as the results do not have an immediate effect on clinical as well as infection control decisions (11).Screening for multiple viral agents by DFA is extremely useful for patient management as well as infection control. While the cost-effectiveness and cost-benefit of a rapid antigen detection infection control program for the reduction of nosocomial respiratory virus transmission have been demonstrated (1, 6, 13), the lack of trained personnel on off-shifts often limits the use of DFA to a single shift in many health care settings. While real-time PCR methods are available as analyte-specific reagents, real-time PCR is limited to the simultaneous detection of three respiratory viruses.DFA requires highly trained clinical laboratory scientists and is subject to nonspecific staining with specimens containing yeasts, certain bacteria, leukocytes, or mucus as well as because of binding to FC receptors (7,8,12). The specimen type and the collection technique are also important factors. Furthermore, it has been reported that approximately 5% of respiratory specimens submitted for immunofluorescence examination are ...
We have analyzed expression of a receptor protein tyrosine phosphatase (RPTPzeta/beta) in tissue samples from 23 human gliomas. Using the reverse transcription-polymerase chain reaction (RT-PCR) technique, we assayed for the presence or absence of mRNA transcripts encoding the intact receptor and 2 alternatively spliced forms of RPTPzeta/beta. Transcripts encoding the intact and truncated receptors were expressed in all of the lower grade gliomas (WHO grade 1-3) analyzed, but not in 55% of the grade 4 glioblastomas multiforme (GBM). However, this subset of GBMs did express an alternatively spliced secreted form comprised of only the RPTPzeta/beta extracellular domain. Our data suggests there may be a correlation between the loss of transcripts encoding the receptor forms of RPTPzeta/beta and progression from low to high grade gliomas. This work provides additional evidence for the importance of phosphatase isoform expression in human tumors.
Abstract. Type-l-protein phosphatase (PP-1) activity is reduced in skeletal muscle from human subjects with insulin resistance (Kida et al. 1990). This reduced phosphatase activity probably leads to the abnormal insulin action for glucose storage observed in insulin-resistant subjects. In the present study, a human homolog of rat liver PP-171 cDNA was isolated from human skeletal muscle. The nucleotide sequence contains a 957-nucleotide open reading frame encoding an amino acid sequence identical to that encoded by rat liver PP-1y1 cDNA. Northern blot analysis shows PP-171-specific mRNA is expressed in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. PP-171 was localized to human Chromosome 12.
BACKGROUND The authors previously demonstrated the presence of cells in primary human malignant gliomas that intrinsically are resistant to carmustine (BCNU). Numerous studies have identified mechanisms of therapy resistance in these cells; however, the authors' work and that of others suggest that additional mechanisms of resistance exist. METHODS The authors identified a glioma cell line that lacks detectable methylguanine methyltransferase expression and does not alter its expression of glutathione‐S‐transferase‐π in response to BCNU chemotherapy. This cell line was used in mRNA differential display experiments to identify genes involved in what to the authors' knowledge were previously undescribed mechanisms of resistance. RESULTS The overexpression of the gene encoding the transforming growth factor latency binding protein was demonstrated in glioma cells selected for resistance to BCNU, compared with their parental unselected cells. CONCLUSIONS Transforming growth factor‐β1 has pleiotropic functions in transformed and normal cells. Although activation of TGF‐β1 does not appear to be a causative factor in BCNU resistance in the current study, it may be involved in the growth of these resistant cells. Cancer 2000;89:850–62. © 2000 American Cancer Society.
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