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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we describe the use of in situ PCR to detect a viral transgene in rat brain. Previously, we have reported in vivo gene transfer by using a defective herpes simplex viral vector in mammalian brain (Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S.P., Hanlon, B.A., Rabkin, S.D., Pfaff, D.W., Mol. Cell. Neurosci. 2 (1991) 320-330). For detection of the LacZ transgene, we have used histochemical staining for the protein product, beta-galactosidase, and in situ hybridization for its mRNA, but the DNA itself cannot be reliably detected with conventional methods. Therefore we have adapted the technique of in situ PCR, so that we may detect minute quantities of transgenic vector DNA following in vivo gene. The brain sections, prefixed, were treated with PBS-detergent before PCR amplification to increase permeability for peptides and oligonucleotides across cellular barriers in brain tissue. Pretreatment with detergent retained better brain morphology than the more widely used proteinase treatment. The PCR mixture containing dNTPs, primers, digoxigenin-dUTP (Dig-dUTP) and buffer was loaded onto each brain section. Slides containing brain sections were placed in an aluminum boat and then on the block of the thermal cycler. Temperature was brought to 82 degrees C before adding Taq polymerase ('hot start' method). Dig-labeled PCR amplified fragments were then detected by alkaline-phosphatase-linked anti-digoxigenin-antibody. Positive signals were seen within the nucleus of transduced neurons, indicating presence of viral DNA. Enhanced specificity was observed with the use of Dig-labeled primers which eliminates the possibility of non-specific viral DNA detection through primer-independent reactions. Overall, this technique can serve not only as an internal control for transgene presence during comparisons of experimental groups of animals, but may also have clinical applications including the detection of viral infection in human brain such as HIV in pathology specimens.
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PMID:In situ PCR for in vivo detection of foreign genes transferred into rat brain. 950 88

Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-V binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane's outer surface. Both methods were combined, in double-fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception.
Mol Hum Reprod 1998 Aug
PMID:Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. 973 32

Dendritic cells are antigen-presenting cells that constitutively express high levels of major histocompatibility complex class II (Ia) antigen on their plasma membrane. Previous studies have shown that the number of dendritic cells in the rat airway mucosa decreases rapidly after glucocorticoid treatment. We sought to determine whether apoptosis contributes to this steroid-induced cell decrease. Dendritic cells in tracheal whole mounts were revealed by immunoperoxidase staining using the OX-6 (anti-Ia) monoclonal antibody. In untreated rats, a dense network of Ia-immunoreactive (Ia+) cells with highly branched cytoplasmic processes was observed just beneath the tracheal epithelium (1,405 +/- 140 cells/mm2 mucosa; mean +/- SEM, n = 6). In rats treated with dexamethasone (10 mg/kg, intraperitoneally), four distinct changes in dendritic cell morphology were evident 4 to 8 h after injection: (1) appearance of large Ia+ granules in cytoplasmic processes, (2) narrowing of cytoplasmic processes, (3) loss of Ia immunoreactivity from the cell surface, and (4) fragmentation of cells into small Ia+ bodies. These changes accompanied a 56% decrease in the number of Ia+ cells over 8 h. The contribution of apoptosis to this decrease in Ia+ cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) of nucleosomal DNA fragments in histologic sections. The number of TUNEL+ bodies increased from a control value of 174 +/- 47 bodies/mm2 mucosa to 2,108 +/- 294 bodies/mm2 mucosa at 4 h and 936 +/- 343 bodies/ mm2 mucosa at 8 h (n = 4 rats per time point). The location of TUNEL+ bodies closely corresponded to that of Ia+ cells stained in adjacent histologic sections. We conclude that apoptosis contributes to the rapid decrease in airway dendritic cells after glucocorticoid treatment.
Am J Respir Cell Mol Biol 1998 Oct
PMID:Glucocorticoid-induced apoptosis of dendritic cells in the rat tracheal mucosa. 976 56

Prostaglandins and other arachidonic acid (AA) metabolites are synthesized by keratinocytes in response to tumor promoters and are produced at very high levels in tumors. After phorbol ester treatment, AA is hydrolyzed from keratinocytes primarily by the cytosolic form of phospholipase A2 (cPLA2), which exhibited a strong substrate preference for phosphatidylcholine over phosphatidylethanolamine and AA over other fatty acids. Phorbol esters increase cPLA2 activity but not the level of expression. To dissociate increased cPLA2 activity from other phorbol ester effects and thus determine the effects of altered AA release on cell growth, the murine keratinocyte cell line, HEL-30, was stably transfected with the sense or antisense cDNA for cPLA2. The resulting cell lines displayed corresponding over- or underexpression and up to 23-fold differences in cPLA2 activity between them. Phorbol ester caused a 15-fold difference in AA release between sense and antisense transfectants. Prostaglandin E2 levels correlated with AA release levels. The sense transfectants showed an enhanced proliferative capacity, based on increased cell number over time and [3H]thymidine incorporation. The antisense transfectants had significantly (>60%) reduced growth rates, compared with both parental cells and sense transfectants. The extent of apoptosis was determined in tumors from cell lines grown in graft chambers in vivo. The number of apoptotic cells was significantly greater in tumors from the sense transfectants, based on terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, compared with the parental or antisense lines. These data are in agreement with a recent study (M. C. Stern et al., Mol. Carcinog., 20: 137-142, 1997) showing a correlation between increased apoptosis and tumor progression in this model system. These results suggest that the elevated eicosanoid synthesis that is observed in skin carcinomas contributes to the growth and progression of these tumors.
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PMID:Suppression or elevation of cytosolic phospholipase A2 alters keratinocyte prostaglandin synthesis, growth, and apoptosis. 978 14

Neuropeptides such as gonadotrophin releasing hormone (GnRH) are presumed to play an important role in the regulation of the function and growth of human placenta. Knowledge about the placental site of GnRH expression and the eventual co-localization of its peptide with the GnRH receptor (GnRH-R) is crucial for a better understanding of possible autocrine/paracrine mechanisms. We therefore investigated these questions by use of in-situ reverse transcription-polymerase chain reaction (RT-PCR) alone or in combination with immunocytochemistry in human first and third trimester placentae. Paraffin-embedded placental sections (7 microm thick), or single trophoblasts in monolayer cultures for up to 3 days, were treated with proteinase K. Following RT with GnRH or GnRH-R specific oligoprimers, PCR was performed employing primers with exon-exon overlaps to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP and nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) for substrate visualization. The GnRH peptide was detected using a sandwich-antibody assay. GnRH and GnRH-R gene expression was found in all first and third trimester placentae, with abundant signals for the GnRH and GnRH-R message both in the cyto- and syncytiotrophoblasts. Single trophoblasts of different gestational ages in culture also displayed GnRH expression in individual cytotrophoblasts and in syncytiotrophoblast-like fusionates. Additional immunostaining revealed GnRH peptide to be co-localized with GnRH-R message in trophoblast layers. Since messages for GnRH and GnRH-R were found in virtually all trophoblasts, we infer that GnRH and GnRH-R are co-expressed in identical cells. These data strongly suggest that the trophoblasts are the source of GnRH, and that there is autocrine/ paracrine regulation by GnRH in human placenta.
Mol Hum Reprod 1998 Oct
PMID:Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in-situ reverse transcription-polymerase chain reaction. 980 83

Pathogenic Yersinia cause a systemic infection in mice that is dependent on the presence of a large plasmid encoding a number of secreted virulence proteins called Yops. We previously demonstrated that a plasmid-encoded Yop, YopJ, was essential for inducing apoptosis in cultured macrophages. Here we report that YopJ is a virulence factor in mice and is important for the establishment of a systemic infection. The oral LD50 for a yopJ mutant Yersinia pseudotuberculosis increases 64-fold compared with wild-type. Although the yopJ mutant strain is able to reach the spleen of infected mice, the mutant strain seldom reaches the same high bacterial load that is seen with wild-type Yersinia strain and begins to be cleared from infected spleens on day 4 after infection. Furthermore, when in competition with wild-type Yersinia in a mixed infection, the yopJ mutant strain is deficient for spread from the Peyer's patches to other lymphoid tissue. We also show that wild-type Yersinia induces apoptosis in vivo of Mac-1(+) cells from infected mesenteric lymph nodes or spleens, as measured by quantitative flow cytometry of TUNEL (Tdt-mediated dUTP-biotin nick-end labeling)-positive cells. The levels of Mac-1(+), TUNEL+ cells from tissue infected with the yopJ mutant strain were equivalent to the levels detected in cells from uninfected tissue. YopJ is necessary for the suppression of TNF-alpha production seen in macrophages infected with wild-type Yersinia, based on previous in vitro studies (Palmer, L.E., S. Hobbie, J.E. Galan, and J.B. Bliska. 1998. Mol. Microbiol. 27:953-965). We conclude here that YopJ plays a role in the establishment of a systemic infection by inducing apoptosis and that this is consistent with the ability to suppress the production of the proinflammatory cytokine tumor necrosis factor alpha.
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PMID:Yersinia-induced apoptosis in vivo aids in the establishment of a systemic infection of mice. 984 26

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10(3) copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100 microl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.
Mol Cell Probes 1998 Dec
PMID:Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma. 984 58

The oleic acid (OA) model of acute lung injury in rats is characterized by a massive and rapid influx of polymorphonuclear neutrophils (PMN) within 1 h, with a peak inflammatory response at 4 h and resolution by 72 h. We hypothesized that PMN apoptosis is involved in the resolution of OA-induced acute lung injury. To test this hypothesis, healthy adult Fischer 344 rats were given 30 microl OA in 0.1% bovine serum albumin (BSA) intravenously; controls were given BSA alone and killed at 1, 4, 24, and 72 h after OA to obtain bronchoalveolar lavage fluid (BALF) and lung tissue. Cell pellets from BALF and formalin-fixed, paraffin-embedded tissue section samples were processed for terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to identify apoptotic cells. Propidium iodide was used to counterstain nuclei. Percentage of nuclei undergoing apoptosis was counted under a fluorescent microscope. Control rats showed only resident alveolar macrophages (AM) in the BALF with no apoptosis. At the peak of injury, 1 h and 4 h after OA injection, we observed a massive PMN response without any evidence of apoptosis. At 24 h, when the OA injury is clinically and histologically in early resolution, we observed intense apoptosis of PMN nuclei along with evidence of apoptotic bodies in the cytoplasm of AM. Some of the AM also showed apoptotic nuclei at 72 h. Similar observations were made in the lung tissue sections. The results of the TUNEL assay were confirmed by DNA ladders and electron microscopy. We conclude that apoptosis of PMN and clearance by AM is an important mechanism in resolution of OA- induced acute lung injury.
Am J Respir Cell Mol Biol 1998 Dec
PMID:Neutrophil apoptosis during the development and resolution of oleic acid-induced acute lung injury in the rat. 984 20

Many chemotherapeutic agents are thought to exert their genotoxic effects through induction of programmed cell death (PCD) (apoptosis) in tumor cells. The bcl-2 is an anti-apoptotic oncoprotein and can confer a survival advantage to tumor cells by preventing apoptosis. Overexpression of bcl-2 may therefore be implicated in resistance to chemotherapy. We studied the significance of bcl-2 expression and the PCD index in pediatric acute lymphoblastic leukemia. Evaluation of bcl-2 by immunocytochemistry and PCD by an enzymatic end labelling technique using biotin-dUTP was carried out in a total of 55 cases and 40 controls. Bcl-2 was found to be expressed in 47% (26/55) of the acute lymphoblastic leukemia cases. The positive cells varied from 0-49% among individual samples. Pre-treatment (spontaneous) apoptosis was observed in 62% (34/55) cases. The mean pre-treatment PCD index was 8.27 1.3%, while the median PCD index was 5. The PCD value for the leukemic samples analyzed were then classified as either high apoptosis values ( 5) and low apoptosis values (<5). PCD index was high in 53% (29/55) and low in 47% (26/55). However, 23% (13/55) of cases did not show presence of either apoptosis or bcl-2. There was no association between clinical and laboratory parameters with the apoptotic index or bcl-2 protein expression. However, evaluation of apoptotic index and bcl-2 expression on day 7 of induction chemotherapy showed a borderline correlation between these markers and initial WBC count, presence of mediastinal mass and hepatosplenomegaly. Follow-up of these patients is being done to look for any association between treatment response and apoptosis.
Int J Mol Med 1998 Apr
PMID:Bcl-2 protein and apoptosis in pediatric acute lymphoblastic leukemia. 985 93

T cells that infiltrate the synovial lesions of rheumatoid arthritis may play a key role in its pathogenesis. To learn more about their functional nature, we determined the frequency of synovial T cells that harbored the TNF alpha and Fas ligand transcript by a technique, called Fluorescence In-Cell Labeling Polymerase Chain Reaction (FICL-PCR). The mRNA of interest was detected in fixed cells by the incorporation during PCR of a fluorescein-12-dUTP label following an initial reverse transcription PCR step. Using this technique the CD3 transcript was detected in the T leukemic cell line, MOLT-4, with calculated sensitivity and specificity values of 91% and 100%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells in peripheral blood mononuclear cells from 12 rheumatoid arthritis patients were 5.1+/-2.3% and 4.8+/-3.1%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells among synovial mononuclear cells from six rheumatoid arthritis patients was 16.8+/-8.3% and 10.8+/-1.8%, respectively. This result indicates that the cytotoxic T cells expressing TNF alpha accumulate in rheumatoid arthritic lesions where they may play a pathogenic role.
Mol Biol Rep 1998 Nov
PMID:Detection of TNF alpha and Fas ligand mRNA within synovial mononuclear cells by fluorescence in-cell labeling PCR (FICL-PCR). 987 Jun 11


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