Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pigs are potential providers of donor tissues for xenotransplantation (e.g. of pancreatic islets) in Type 1 diabetes. In this context, our group has studied the use of islets from specific pathogen-free (SPF) pigs as a means of reducing the risks of "conventional zoonosis". Although this approach does not prevent the transmission of pig endogenous retrovirus (PERV) to humans, we attempted to determine the presence of C-type PERV mRNAs for gag, pol, and env subtypes as a first descriptive step in the retroviral characterisation of SPF pig tissues (especially pancreas). Using semiquantitative reverse-transcriptase polymer chain reaction with 18S rRNA and beta-actin as internal controls, PERV mRNA levels were compared in a large panel of tissues from SPF and conventional pigs. PERV mRNAs for gag, pol, env-A and env-B were present in all tissues studied from the nine SPF pigs tested. Signals for env-C mRNAs were of much lower intensity than those for env-A and B, and most often undetectable in pancreas. The mRNA levels for gag, pol, env-A, env-B and env-C mRNAs were lower in pancreas (p < 0.01) than in all other tissues. Among other porcine tissues likely to be grafted in man, the highest retroviral mRNA levels were detected in kidney (p < 0.01), followed by liver, lung and heart. Amplified PERV mRNA signals were about 17 times less frequent in pig pancreas than in the retroviral-producing porcine cell line G2, while kidney contained about 6 times more PERV mRNAs than pancreas. The levels of gag, pol, env-A, env-B, and env-C mRNAs also varied between tissues of conventional pigs: PERV mRNA levels were lowest in pancreas, and env-C mRNAs were most often undetectable. For all SPF tissues tested, pol, gag, env-A, env-B, and env-C mRNA levels were in the same range or slightly higher than in corresponding tissues of conventional pigs. In summary, this study of C-type PERV mRNAs in a large panel of tissues from SPF pigs, in the context of our strategy of quality assurance and sanitary control, indicated that PERV mRNA levels were in the same range in SPF and corresponding conventional pig tissues, confirming that the use of SPF pigs would not prevent the risk of PERV transmission to human recipients of xenografts. PERV-A and PERV-B may be mainly represented, and PERV-C much less, in these pig tissues (particularly pancreas). The fact that pancreas expressed the lowest PERV mRNA levels and kidney the highest, among porcine tissues likely to be grafted, could be of interest from a clinical point of view. Pig tissues may differ in their loads of PERV sequences, which could be a factor in the risk of PERV transmission during xenotransplantation.
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PMID:Porcine endogenous retroviral mRNAs in pancreas and a panel of tissues from specific pathogen-free pigs. 1063 79

Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been widely employed as an ultra-sensitive method for detection of micrometastases in patients with various types of malignancies. Messenger RNA of a specific marker gene is a target for RT-PCR amplification to examine the presence of micrometastases in body fluids or tissues obtained from human. We developed the RT-PCR assay specific for rat beta-actin mRNA, which cannot detect human counterpart and assessed how much contamination of rat tissues can influence the result of RT-PCR assay and how to avoid the influence of the contamination in RT-PCR assay.
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PMID:Prevention of cross-contamination during sampling procedure in molecular detection for cancer micrometastasis. 1077 38

Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P < or = 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 +/- 0.32 vs. 0.90 +/- 0.14 IL-1 copies/beta-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-beta were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 +/- 0.16 vs. 1.36 +/- 0.50 TGF-beta copies/beta-actin copies, P < 0.05). No differences in tumor necrosis factor-alpha, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and gamma-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.
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PMID:Early graft failure of xenogeneic islets in NOD mice is accompanied by high levels of interleukin-1 and low levels of transforming growth factor-beta mRNA in the grafts. 1111 99

Apoptosis of intestinal epithelial cells (EC) plays a role in total parenteral nutrition (TPN)-induced villus atrophy. Among the mediators of apoptosis in EC are some members of the Bcl-2 family of proteins. Bcl-2 members can either be anti- (Bcl-2, Bcl-x(L), Bcl-w) or pro-apoptotic (Bax, Bak, Bid, Bad, Bcl-x(S)). To determine whether the observed increase in apoptosis induced by TPN is associated with an alteration in these Bcl-2 members' mRNA expression, mice were randomized to either TPN or oral feeding (controls). Animals were killed after 7 days and the intestine was harvested. EC were purified with magnetic beads. Apoptosis was detected by cell-surface expression of phosphatidylserine using flow cytometry. EC mRNA expression was determined by reverse-transcriptase polymerase chain reaction. Results were expressed relative to beta-actin. TPN resulted in a significant ( P < 0.05, unpaired t-test) increase in apoptosis: TPN 29.4 +/- 11.3% versus control 14.4 +/- 5.1%. The expression of the pro-apoptotic members Bax, Bak, Bid, and Bcl-x(S) was significantly ( P < 0.05) decreased after TPN. In contrast, a significant increase was observed in the anti-apoptotic member Bcl-2. mRNA expression of Bcl-w, Bad, and Bcl-x(L) was not significantly different between the control and TPN groups. Thus TPN-induced apoptosis was associated with an increased expression of anti-apoptotic factors and a decrease in pro-apoptotic factors. This contrasts with other reports where these factors showed converse effects under apoptotic conditions. Our results may demonstrate a unique regulatory pathway that may counter the observed increase in TPN-induced EC apoptosis.
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PMID:Total parenteral nutrition-induced apoptosis in mouse intestinal epithelium: regulation by the Bcl-2 protein family. 1247 68

Tissue inhibitors of metalloproteinase (TIMP) are important participants in various physiological processes that involve tissues remodeling. They help maintain a delicate balance between physiological degradation and synthesis of the extracellular matrix. A better understanding of TIMP activity will be helpful in understanding the etiology of periapical lesions and their means of treatment. The fibroblast is a prominent cellular component of the periapical tissues. The potential implications of cytokine-mediated tissue destruction still remain to be elucidated. The purpose of this study was to determine the effects of interleukin (IL)-1alpha and transforming growth factor (TGF)-beta on the expressing of TIMP-1 by primary gingival fibroblast cultures. After exposure to cytokines for 8 h, total RNA in gingival fibroblasts was isolated and evaluated by reverse-transcriptase polymerase chain reaction. Densitometric analysis of the TIMP-1 mRNA gene expression, after normalization by beta-actin, demonstrated that exposure to IL-1alpha resulted in a decreased level of TIMP-1 mRNA compared with the control groups. However, the TIMP-1 mRNA was up-regulated by TGF-beta. In addition, when the cells were cultured in combination with TGF-beta (1 ng/ml) and IL-1alpha for 8 h, the level of TIMP-1 mRNA was dramatically reduced. These results demonstrated that in human periapical tissue cytokines differentially and specifically regulate expression of TIMP-1 mRNA. An understanding of the actions of cytokines on gingival fibroblasts may result in new therapies to augment current treatment of periapical lesions.
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PMID:Regulation of tissue inhibitors of metalloproteinase-1 gene expression by cytokines in human gingival fibroblasts. 1248 47

Due to the scarcity of available human livers, porcine hepatocytes are currently being evaluated as a xenogeneic cell source for extracorporeal bioartificial liver (BAL). Hypothermic storage of isolated porcine hepatocytes could support stocking of cell-loaded bioreactors for BAL use and may provide bioreactors ready to be used at the patient's bedside. For the development of this technology, it is of utmost importance to ensure cell viability and differentiated functions after low-temperature storage and following warm reperfusion. We compared cell viability, functional activity and apoptosis in isolated porcine hepatocytes which were perfused within a radial-flow bioreactor (RFB), stored at 4 degrees C and then reperfused at 37 degrees C. RFBs were loaded with 8 x 10(9), > or = 90% viable hepatocytes at 37 degrees C for 3 h. RFBs were then flushed with 4 degrees C University of Wisconsin solution (UW) and subsequently stored for 24 h or 48 h. RFBs were then reperfused for 8 h with recirculating medium plus serum at 37 degrees C . Cytochrome P450 (CYP) activity was studied before and after cold storage by means of monoethylglycinexylide (MEGX) detection in the effluent medium, after repeated lidocaine injections. After reperfusion experiments, hepatocytes were harvested for total RNA isolation. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used in order to amplify specific mRNAs for Bcl-2 and Bax genes, by using appropriate primers; beta-actin primers were used as control. Total RNA was extracted by northern blotting analysis and for Bcl-2, Bax and beta-actin RNA messenger detection, RT-PCR amplification was used. Freshly isolated hepatocytes perfused into the RFB showed a progressive increase of MEGX while a loss in Bax expression was paralleled by an increase in Bcl-2 expression, in comparison to starting hepatocytes. After 4 degrees C storage and warm reperfusion, MEGX production was preserved in 24 h- and 48 h-stored bioreactors as well as a sharp increase of Bcl-2 and a decrease of Bax mRNAs. Our study suggests that refrigeration of hepatocyte-bioreactors is a suitable strategy to maintain both viability and function of isolated hepatocytes, for up to 48 h a time-length that is compatible with long-distance delivery of ready-to-use bioreactors.
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PMID:Modulation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) gene expression in isolated porcine hepatocytes perfused within a radial-flow bioreactor after low-temperature storing. 1265 48

The surrounding extracellular matrix of airway wall tissues changes in response to mechanical stresses and hypoxia. The presence of matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), is correlated with collagen degradation and tissue repair in lung disorders. The aim of this study was to evaluate the expression of MMP-9 and TIMP-1 in the lung of fetal rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Administering 100 mg of nitrofen dissolved in 1 ml olive oil to pregnant Wistar rats on day 9 of gestation induced left-sided CDH in fetal rats. In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into two groups: normal controls (n = 10) and nitrofen-induced left-sided CDH (n = 10). Immunoreactivity of the staining for MMP-9 and TIMP-1 in the lung tissues was semiquantitatively analyzed using the staining scores. The relative amount of MMP-9 or TIMP-1 divided by the amount of beta-actin for each lung sample was measured by using the real-time reverse-transcriptase polymerase chain reaction. The immunoreactivity of MMP-9 was significantly increased in the CDH group (n = 5) compared with the control group (n = 5) (p = 0.031). On the other hand, the immunoreactivity of TIMP-1 in the two groups was not significantly different (n = 0.134). The relative amount of MMP-9 (or TIMP-1) in the CDH group (n = 5) does not differ significantly from that in the control group (n = 5) (p = 0.059, 0.596, respectively), but the relative amount of MMP-9 is higher in the CDH group, although it is not significantly higher. On the other hand, the ratios of MMP-9 to TIMP-1 were significantly higher in the CDH group (p = 0.028). In conclusion, fetal rats with nitrofen-induced CDH, a model of respiratory disorders, manifested the excess of MMP-9 activity due to the absence of TIMP-1 that would suggest a trend toward disruption of the extracellular matrix in the CDH lung tissues.
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PMID:Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1: expression in the lung of fetal rats with nitrofen-induced diaphragmatic hernia. 1272 18

Vascular endothelial growth factor-A (VEGF-A) is an important mediator of angiogenesis in normal and neoplastic tissues. Total VEGF-A levels have been associated with melanoma progression, but the relative contributions of each isoform is unknown. To determine whether differences in the production of any or all of the major VEGF-A isoforms are related to stage of progression, we compared message levels for the three major isoforms of VEGF in melanoma specimens from different stages of progression.Primary melanomas (N = 18), primary recurrences (N = 5), regional dermal metastases (N = 11), nodal metastases (N = 12), normal lymph nodes (N = 18), and distant metastases (N = 9) were prospectively collected. Samples from the horizontal and vertical growth phases of primary tumors were also collected from five additional patients. Message levels for the three major VEGF-A isoforms were measured using real-time quantitative reverse-transcriptase polymerase chain reaction and normalized to beta-actin mRNA levels. There was a marked increase in the expression of all three VEGF-A isoforms from the vertical growth phase tissue as compared with the horizontal growth phase tissue. Primary tumors, local recurrences, regional dermal metastases, nodal metastases, and distant metastases all produced more VEGF(121) and VEGF(165) than negative nodes. Nodal metastases produced the highest level of these two isoforms, higher even than distant metastases. There was no significant difference in VEGF(189) message among the groups. Melanomas in the vertical growth phase produce more VEGF-A (all isoforms) than in the horizontal growth phase. Nodal metastases produce the highest levels of VEGF(121) and VEGF(165), but not VEGF(189) as compared with other stages of progression. These data suggest that the soluble forms of VEGF-A might be an important factor in melanoma metastasis to regional lymph nodes.
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PMID:Differential expression of vascular endothelial growth factor-A isoforms at different stages of melanoma progression. 1294 96

Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system.
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PMID:Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle. 1524 Nov 86

Improved methods of bone regeneration are needed in the craniofacial rehabilitation of patients with significant bone deficits secondary to tumor resection, congenital deformities, and prior to prosthetic dental reconstruction. In this study, a gene-enhanced tissue-engineering approach was used to assess bone regenerative capacity of Sonic hedgehog (Shh)-transduced gingival fibroblasts, mesenchymal stem cells, and fat-derived cells delivered to rabbit cranial bone defects in an alginate/collagen matrix. Human Shh cDNA isolated from fetal lung tissue was cloned into the replication-incompetent retroviral expression vector LNCX, in which the murine leukemia virus retroviral LTR drives expression of the neomycin-resistance gene. The rat beta-actin enhancer/promoter complex was engineered to drive expression of Shh. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the transduced primary rabbit cell populations expressed Shh RNA. Shh protein secretion was confirmed by enzyme-linked immunosorbent assay (ELISA). Alginate/ type I collagen constructs containing 2 x 10(6) Shh-transduced cells were introduced into male New Zealand White rabbit calvarial defects (8 mm). A total of eight groups (N=6) were examined: unrestored empty defects, matrix alone, matrix plus the three cell populations transduced with both control and Shh expression vectors. The bone regenerative capacity of Shh gene enhanced cells was assessed grossly, radiographically and histologically at 6 and 12 weeks postimplantation. After 6 weeks, new full thickness bone was seen emanating directly from the alginate/collagen matrix in the Shh-transduced groups. Quantitative two-dimensional digital analysis of histological sections confirmed statistically significant (P<0.05) amounts of bone regeneration in all three Shh-enhanced groups compared to controls. Necropsy failed to demonstrate any evidence of treatment-related side effects. This is the first study to demonstrate that Shh delivery to bone defects, in this case through a novel gene-enhanced tissue-engineering approach, results in significant bone regeneration. This encourages further development of the Shh gene-enhanced tissue-engineering approach for bone regeneration.
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PMID:Sonic hedgehog gene-enhanced tissue engineering for bone regeneration. 1551 Jan 77


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