Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Somatic mutations in the retinoblastoma-1 gene (RB1) and loss of RB1 protein function have been implicated in a number of human malignancies, but the role of RB1 gene and protein abnormalities in ductal pancreatic cancer (DPCA) is virtually unknown. We therefore analyzed expression of the RB1 protein immunohistochemically and/or by western blotting in a total of 54 sporadic and eight familial cases of archival and frozen DPCA and in 18 pancreatic carcinoma cell lines by using the antibodies RB-WL-1, 84-B3-1, and PMG3-245. Mutations in the RB1 promotor region and exons 13-21 of the RB1 gene were likewise examined by single-strand conformation polymorphism (SSCP) analyses and DNA sequencing of genomic DNA from 30 microdissected primary pancreatic tumors and the pancreatic carcinoma cell lines. Moreover, amplification and expression of a major regulatory component of RB1 function, cyclin D1, were assessed by southern and immunohistochemical analyses, respectively. The DPCAs were heterogeneous in both the intensity of RB1 nuclear staining and the percentage of immunoreactive cells. The tumors often had areas where RB1 staining was weak or absent adjacent to normal pancreatic tissue; however, only two of 32 archival cases and one of 30 frozen cases of DPCA completely lacked RB1 nuclear staining. Immunohistochemical and western blot analyses of 18 pancreatic carcinoma cell lines demonstrated the absence of RB1 expression in only two cell lines, Capan-1 and QGP-1. Analyses of the RB1 gene and promotor region by SSCP and DNA sequencing largely confirmed the immunochemical findings. Three of 30 primary carcinomas had abnormalities revealed by SSCP analyses. In one case a single base-pair deletion was confirmed in exon 18 and resulted in premature termination and the absence of detectable RB1 protein. A second case had TAC-->TTC missense mutation in exon 13. The third primary carcinoma could not be reliably sequenced because it had a low percentage of epithelial cells. The cyclin D1 gene was not amplified in any of the primary pancreatic tumors or cell lines examined. These immunochemical and molecular analyses of the RB1 tumor suppressor gene and cyclin D1 proto-oncogene in a large series of human pancreatic cancers and cell lines indicate that RB1 and cyclin D1 alterations occur during the development of some human DPCAs. Nevertheless, it is probable that alterations in cell-cycle regulation in DPCAs more frequently involve pathways other than those involving RB1 and cyclin D1.
Mol Carcinog 1996 Feb
PMID:Molecular and immunochemical analyses of RB1 and cyclin D1 in human ductal pancreatic carcinomas and cell lines. 859 83

Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the alpha-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire alpha-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.
Mol Gen Genet 1996 Aug 27
PMID:Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica). 880 4

Androgen insensitivity syndrome (AIS) is associated with a wide range of quantitative or qualitative defects in the androgen receptor (AR). In some patients with AIS, however, no defects are detectable in the ligand-binding properties of the AR. We have analyzed the ARs of two unrelated patients with this category (termed 'receptor-positive type') of AIS. Sequence analysis of these patients' AR gene revealed single amino acid substitutions (579Cys(TGC)-->Phe(TTC) and 582Phe(TTC)-->Tyr(TAC)) in exon B encoding the first zinc finger of the DNA-binding domain of the AR. These mutations have not been previously reported. Moreover, cotransfection assays and mobility shift assays revealed that these patients' mutant ARs had defective transcriptional activity of the target gene because of impaired DNA-binding ability to the androgen-responsive element. These findings strongly indicate that these mutations are responsible for the pathogenesis of AIS in these patients.
Mol Cell Endocrinol 1996 Jun 18
PMID:Androgen insensitivity syndrome due to new mutations in the DNA-binding domain of the androgen receptor. 880 34

Homology-based PCR was used to isolate angiotensin II type 2 (AT2) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe133 as TTC and Gln326 as CAG; base substitutions are in bold-italics), the AT2 receptor protein was identical to other reported murine AT2 clones. When transfected into COS-1 cells, the expressed AT2 receptor displayed high affinity for AngII and for AT2-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT2 receptors, defined as peak I and peak III receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT2 receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT2 receptors failed to immunoreact to either peak III receptors or cloned AT2 receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT2 receptor is identical to peak III receptors from N1E-115 cells and that a novel AT2 receptor (peak I) remains to be cloned.
Brain Res Mol Brain Res 1997 Apr
PMID:Cloning and expression of angiotensin II type 2 (AT2) receptors from murine neuroblastoma N1E-115 cells: evidence for AT2 receptor heterogeneity. 910 76

This study tests the hypothesis that increased levels of plasma lipids can accelerate accumulation of myocardial triacylglycerols in post-ischemic but viable myocardium. Two groups of dogs underwent 90 min of left anterior descending coronary artery (LAD) occlusion followed by 240 min of reperfusion. The first group of saline-treated dogs (n = 7) had physiological levels of plasma lipids during reperfusion: a second group treated with Liposyn and heparin (n = 5) experienced increased plasma lipids during reperfusion. The transmural content of triacylglycerols was determined during ischemia and reperfusion using 1H NMR one-dimensional chemical shift imaging (1D CSI), and at the end of reperfusion using Oil Red-O staining and chemical assay. TTC staining was used to identify the extent of irreversibly injured myocardium. Subepicardial and plasma triacylglycerol content, measured both by 1D CSI and chemically, did not change during reperfusion in saline-treated dogs. Infusing dogs with Liposyn and heparin for 90 min during reperfusion transiently elevated their plasma triacylglycerols, which returned to normal levels following Liposyn wash-out. During Liposyn wash-out, myocardial triacylglycerols measured by 1D CSI preferentially increased in the subepicardium of area-at-risk myocardium (P < 0.05). Triacylglycerol content, measured chemically, also increased in area-at-risk compared to non-ischemic subepicardium (P < 0.001). Significant endocardial damage occurred in both groups, but elevated levels of plasma lipids did not increase the size of the area-at-risk. Therefore, elevated plasma lipids caused a preferential accumulation of triacylglycerols in area-at-risk myocardium during reperfusion without exacerbating irreversible ischemic injury. These results are consistent with either inhibited fatty acid oxidation or mis-matched fatty acid extraction and oxidation in area-at-risk myocardium.
J Mol Cell Cardiol 1997 Feb
PMID:1H NMR measurement of triacylglycerol accumulation in the post-ischemic canine heart after transient increase of plasma lipids. 914 Aug 7

In rabbits, inhibition of either protein kinase C or protein tyrosine kinase abolishes the infarct size reduction achieved by ischemic preconditioning. In pigs, however, inhibition of protein kinase C does not attenuate ischemic preconditioning. The present study tested whether inhibition of protein tyrosine kinase alone or in combination with inhibition of protein kinase C interferes with ischemic preconditioning in pigs. In 29 enflurane-anesthetized pigs, the LAD was cannulated and perfused from an extracorporeal circuit. Protein tyrosine kinase and protein kinase C were inhibited by continuous intracoronary infusion of genistein (5x10(-6) mol/l) and staurosporine (10(-7) mol/l), respectively. Subendocardial blood flow (ENDO) was measured with microspheres. Infarct size was analysed by TTC staining (% of LV area at risk) following 90 min low-flow ischemia and 120 min reperfusion. In the presence of genistein, 90 min ischemia at an ENDO of 0.06+/-0.01 (+/-s.e.m.) ml/min/g resulted in an infarct size of 16.7+/-4.2% (n=8). With genistein, ischemic preconditioning by 10 min ischemia and 15 min reperfusion still reduced infarct size to 6.5+/-2.7% (ENDO: 0.05+/-0. 01 ml/min/g, n=7, P<0.05). In the presence of both genistein and staurosporine, infarct size following 90 min ischemia was 14.1+/-3. 6% (ENDO: 0.06+/-0.01 ml/min/g, n=7). With genistein and staurosporine, ischemic preconditioning no longer reduced infarct size significantly (11.5+/-3.1%, ENDO: 0.06+/-0.01 ml/min/g, n=7). The effective attenuation of ischemic preconditioning only by simultaneous inhibition of both, protein kinase C and protein tyrosine kinase, suggests a complex signal cascade involving both protein kinases.
J Mol Cell Cardiol 1998 Feb
PMID:Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinase in pigs. 951 96

In a previous report, we have demonstrated that simultaneous inhibition of nucleoside transport and adenosine deaminase accumulates endogenous adenosine and protects the myocardium against stunning. The differential cardioprotective effects of erythro-9(2-hydroxy-3-nonyl)-adenine (EHNA), a potent inhibitor of adenosine deamination but not transport, and p-nitrobenzylthioinosine (NBMPR), a selective blocker of adenosine and inosine transport, are not known. Thirty-seven anaesthetized adult dogs were instrumented to monitor left ventricular performance using sonomicrometery. Dogs were randomly assigned into four groups. The control group (n = 8) received only the vehicle solution. Treated groups received saline containing 100 microM EHNA (EHNA-group, n = 7), 25 microM NBMPR (NBMPR-group, n = 7), or a combination of 100 microM EHNA and 25 microM NBMPR (EHNA/NBMPR-group, n = 10). Hearts were subjected to 30 min of normothermic global ischaemia and 60 min of reperfusion while on bypass. Adenine nucleotides, nucleosides, oxypurines and NAD+ were determined in extracts of transmural myocardial biopsies using HPLC. TTC staining revealed the absence of necrosis in this model. Drug administration did not affect myocardial ATP metabolism and cardiac function in the normal myocardium. Ischemia caused about 50% ATP depletion and accumulation of nucleosides. The ratio between adenosine/inosine at the end of ischemia was 1:10, 1:1, 1:1 and 10:1 in the control, EHNA-, NBMPR- and EHNA/NBMPR-group, respectively. Upon reperfusion, both nucleosides washed out from the myocardium in the control and EHNA-group while retained in the myocardium in the NBMPR and EHNA/NBMPR groups. Ventricular dysfunction 'stunning' persisted in the control group (52%) and in the EHNA-treated group (32%) after 30 min of reperfusion. Significant improvement of function was observed in the EHNA group only after 60 min of reperfusion. LV function recovered in the NBMPR- and EHNA/NBMPR-treated groups during reperfusion. ATP recovery occurred only when animals were pretreated with the combination of EHNA/NBMPR and remained depressed in the control group and EHNA and NBMPR-treated groups. At post mortem, TTC staining revealed the absence of myocardial necrosis. Superior myocardial protection was observed with inhibition of nucleoside transport by NBMPR alone or in combination with inhibition of adenosine deaminase by EHNA. Selective blockade of nucleoside transport by NBMPR is more cardioprotective than inhibition of adenosine deaminase alone in attenuating myocardial stunning. It is not known why EHNA partially inhibit adenosine deaminase, in vivo.
Mol Cell Biochem 1998 Mar
PMID:Differential cardioprotection with selective inhibitors of adenosine metabolism and transport: role of purine release in ischemic and reperfusion injury. 954 45

Expansions of the triplet repeat, GAA/TTC, inside the first intron of the frataxin gene causes Friedreich's ataxia (FRDA). It was of interest to us to examine whether the FRDA repeat forms an unusual DNA structure, since formation of such structure during replication may cause its expansion. Here, we show that the FRDA repeat forms a triplex in which the TTC strand folds on either side of the same GAA strand. We have determined the high-resolution NMR structures of two intramolecularly folded FRDA triplexes, (GAA)2T4(TTC)2T4(CTT)2 and (GAA)2T4(TTC)2T2CT2(CTT)2 with T.A.T and C+.G.C triads. T4 represents a synthetic loop sequence, whereas T2CT2 is the natural loop-folding sequence of the TTC strand. We have also made use of site-specific 15N-labeling of the cytosine residues to investigate their protonation status and their interaction with other protons. We show that the cytosine residues of the Hoogsteen C+.G pairs in this triplex are protonated close to physiological pH. Therefore, it appears that the triplex formation offers a plausible explanation for the expansion of the GAA/TTC repeats in FRDA.
J Mol Biol 1999 Feb 05
PMID:The high-resolution structure of the triplex formed by the GAA/TTC triplet repeat associated with Friedreich's ataxia. 992 83

The linear regression analysis of infarct size (IS) v ischemic myocardial blood flow (MBF) does not account for the heterogeneity of MBF and infarcted tissue; moreover, it cannot assess a blood flow threshold for infarction (MBFT) accurately, as with ischemic preconditioning (IP) the close relationship between ischemic MBF and IS otherwise observed is lost. Finally, the impact of resting blood flow on myocardial infarction cannot be considered in such analysis. Therefore, in a retrospective data analysis of 32 enflurane-anaesthetized swine undergoing 90 min severe ischemia and 120 min reperfusion without (CON, n = 12) or with IP induced by either 3 (IP3, n = 8) or 10 min ischemia (IP10, n = 12) and 15 min reperfusion, a MBFT was assessed by logistic regression (LR) in individual tissue pieces. MBFT was arbitrarily defined as that ischemic MBF (microspheres) at which infarct probability was 0.2, derived from the ratio of infarcted (n = 141, TTC) to all tissue samples (n = 684). The duration of the preconditioning ischemia and MBF both at rest and during the sustained ischemia were significant predictors of infarct probability. Ischemic MBFT at an infarct probability of 0.2, was 0.089 +/- 0.023 ml/min/g in CON. MBFT was decreased to 0.051 +/- 0.03 ml/min/g with IP3 (P < 0.05 v CON) and further to 0.004 +/- 0.037 ml/min/g with IP10 (P < 0.05 v CON, IP3). Corresponding to the leftward shift of MBFT, the relationships between infarct probability and MBF were shifted in parallel by IP with no change in their slopes.
J Mol Cell Cardiol 1998 Dec
PMID:Impact of resting and ischemic blood flow on infarct probability in ischemic preconditioning--a new approach to infarct size-blood flow data by logistic regression. 999 May 42

A novel DNA structure, sticky DNA, is described for lengths of (GAA.TTC)n found in intron 1 of the frataxin gene of Friedreich's ataxia patients. Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) triplexes in negatively supercoiled plasmids at neutral pH. An excellent correlation was found between the lengths of (GAA.TTC) (> 59 repeats): first, in FRDA patients, second, required to inhibit transcription in vivo and in vitro, and third, required to adopt the sticky conformation. Fourth, (GAAGGA.TCCTTC)65, also found in intron 1, does not form sticky DNA, inhibit transcription, or associate with the disease. Hence, R.R.Y triplexes and/or sticky DNA may be involved in the etiology of FRDA.
Mol Cell 1999 Apr
PMID:Sticky DNA: self-association properties of long GAA.TTC repeats in R.R.Y triplex structures from Friedreich's ataxia. 1023 Mar 99


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