The ets genes in cells and viruses: implications for leukemias and other human diseases.
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Alpharetrovirus↗
Biomedical subjects
N K Bhat
Publications and source records attributed to N K Bhat.
Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I.
An in vitro system reconstituted with mouse liver polysome translation products was used to study the nature of polypeptide species imported into mitochondria from different mouse tissues such as liver, kidney, brain, and heart, as well as from Ehrlich ascites, Novikoff hepatoma, and Morris hepatoma 3924A tumor lines. Mouse hepatic mitochondria import a number of proteins including 160-kilodalton (kDa) carbamoyl-phosphate synthetase I (CPS-I). Two other proteins of 63 and 57 kDa of unknown function are also imported as major components by mouse liver mitochondria. Under these in vitro conditions, however, mitochondria from non-CPS-I expressing tissues such as brain, kidney, and heart failed to import and process the precursor forms of CPS-I (pCPS-I). Furthermore, mitochondria from three different tumor lines (Novikoff hepatoma, Morris hepatoma, and Ehrlich ascites) containing negligible CPS-I activity were also unable to import and process pCPS-I to any significant level. Similarly, the 63-kDa protein was selectively transported into liver and kidney mitochondria and also into Ehrlich ascites mitochondria at reduced levels, but not into mitochondria from heart and brain. Nevertheless, the 57-kDa protein and a number of proteins of less than 45 kDa are transported efficiently by all of the mitochondrial types studied. These results provide evidence for tissue- or cell-specific selectivity at the mitochondrial membrane level for the transport of some proteins. The transports of 63- and 57-kDa proteins are differentially inhibited by mouse liver mitochondrial matrix and membrane fractions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome.
The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The transport and processing of carbamyl phosphate synthetase-I in mouse hepatic mitochondria.
Carbamyl phosphate synthetase-I (CPS-I)2, purified from mouse hepatic mitochondria consists of electrophoretically homogeneous polypeptide species of 160 kilodaltons molecular weight (Kd). Monospecific antibody to CPS-I immunoprecipitated a putative precursor of 165Kd protein from in vitro translation products programmed with mouse liver free polysomes or poly(A) RNA. Isolated mitochondrial particles can take up and process pCPS-I into mature CPS-I of 160Kd in an in vitro transport system. The in vitro transport of CPS-I is energy dependent and requires intact mitochondria. The processing of pCPS-I appears to involve a single endoproteolytic cleavage.
Animals↗
Percutaneous fulgarization of vas.
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Electrocoagulation↗
Qualitative and comparative nature of mitochondrial translation products in mammalian cells.
A method has been described for the efficient incorporation of [35S]methionine into isolated mitochondrial particles from various mammalian tissues. The method involves the incubation of digitonin-treated mitochondrial particles (mitoplasts) in a low sucrose medium. Electrophoretic analysis of 35S-labeled products on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions shows that mitoplasts from Ehrlich ascites cells, mouse liver, and rat liver synthesize 19-24 polypeptide species including some high molecular weight components in the size range of 1.0 X 10(5). The polypeptide species synthesized in the mitoplast system resemble the cycloheximide-resistant products synthesized in the intact cells with respect to size distribution and total number, although significant quantitative differences between the two systems are observed. Experiments on pulse--chase analysis of 35S-labeled mitochondrial products and the effects of protease inhibitors on the electrophoretic profiles suggest no significant proteolytic degradation during the incubation or analysis. Further, control experiments with nuclease-treated mitoplasts and use of specific protein synthesis inhibitors show that all of the labeled polypeptides are the intramitochondrial translation products. Extensive comparison between the products synthesized in Ehrlich ascites and mouse and rat liver mitochondria, using one- and two-dimensional gels under denaturing conditions, shows striking variations, suggesting possible heterogeneity.
Animals↗
Preferential attack of mitochondrial DNA by aflatoxin B1 during hepatocarcinogenesis.
Administration of the hepatic carcinogen aflatoxin B1 to experimental animals results in covalent binding to liver mitochondrial DNA at concentrations three to four times higher than nuclear DNA. The concentration of carcinogen adducts in mitochondrial DNA remains unchanged even after 24 hours, possible because of lack of excision repair. Similarly, mitochondrial transcription and translation remain inhibited up to 24 hours suggesting long-term effects of aflatoxin B1 on the mitochondrial genetic system.
Aflatoxin B1↗
Inhibition of mitochondrial protein synthesis during early stages of aflatoxin B1-induced hepatocarcinogenesis.
Experiments were designed to determine the in vivo effects of a single 6-mg/kg dose aflatoxin B1 on rat liver mitochondrial transcription and translation processes. With the use of intact hepatocytes and also a highly active mitoplast system for incorporation, it was observed that both mitochondrial transcription and translation activities are inhibited progressively even after 24 hr of carcinogen administration. Electrophoretic patterns of mitochondrial translation products show some qualitative changes during early periods of carcinogen administration. At later stages (greater than 12 hr), however, there is a general inhibition of many of the products, although by this time there is a qualitative and quantitative recovery in the synthesis of mitochondrial proteins imported from the cytoplasm. These results, along with the data showing considerably high levels of aflatoxin B1 binding to mitochondrial DNA suggest that mitochondrial genetic system is one of the direct targets during experimental carcinogenesis.
Aflatoxin B1↗
The complexity of mitochondrial translation products in mammalian cells.
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Animals↗
Modulation of hepatic transcription and translation during early stages of aflatoxin B1 carcinogenesis.
Isolated rat hepatocytes have been used to study the transcription and translation processes at varied time intervals after administration of a single dose (6 mg/kg) of hepatocarcinogen aflatoxin B1 (AFB1). The effects of AFB1 during the first 24 h of drug treatment show a characteristic inhibition followed by periods of rapid recovery and also a hyperactive state when both heterogeneous nuclear RNA (HnRNA) transcription and cytoplasmic translation processes reach about 200% of control. Analysis of [3H]orotic acid pulse-labeled HnRNA and cytoplasmic polyadenylic acid(poly (A)) containing mRNA on sucrose gradients under conditions which prevent aggregation show a stepwise reduction in the total isotopic incorporation as well as in the size distribution during the first 9 h of AFB1 treatment. Between 9 and 24 h after AFB1 treatment, there is a recovery in the total incorporation as well as in the size of HnRNA and poly (A) containing mRNA. Analysis of translation products by a two-dimensional procedure shows loss of some high molecular weight products in AFB1-treated cells. At 24 h after AFB1 treatment (hyperactive stage) there is a total recovery of activity. Also, several new translation products not detected in the control hepatocytes are synthesized at this stage. The hyperactive stage might represent gene-derepression or reprogramming of gene expression.
Aflatoxin B1↗
Immunologic detection of protein antigens after phenol-chloroform denaturation.
The study of gene expression in cells and tissues often begins with phenol-chloroform extraction of the biologic material of interest for the isolation of intact mRNA. In most cases, the proteins denatured by phenol-chloroform are discarded. However, we found that the proteins recovered from phenol-chloroform extractions maintain their antigenicity. Therefore a method was developed for recovering the proteins from phenol-chloroform-denatured extracts that could be saved in lyophilized form until immunologic analysis. In this way, the RNA and the protein analysis can utilize exactly the same sample, and the biologic material can be saved. This is important because often these materials are available only in limited quantities. The method has been used to examine the sea urchin ets-related antigen and sea urchin ets-2 mRNA.
Animals↗
Influence of antioxidants and the CYP1A1 isoleucine to valine polymorphism on the smoking--lung cancer association.
To evaluate the association between CYP1A1 genotype and lung cancer risk and to assess the effect of CYP1A1 genotype and antioxidant supplementation on the smoking--lung cancer relationship we conducted a case-control study nested within a large cancer prevention trial cohort. Controls (n = 324) were matched to cases (n = 282) on age (+/- 5 years), intervention group and study clinic in a 1:1 ratio, using incidence density sampling. Genotype was determined by a PCR-based method and logistic regression was used to calculate relative risk estimates. Overall, we found no association between CYP1A1 genotype and lung cancer risk. CYP1A1 genotype did not modify the effect of smoking on lung cancer risk. However, in an examination of subgroups defined by randomized intervention assignment our findings suggest that alpha-tocopherol supplementation may reduce the risk of lung cancer associated with cumulative smoking exposure regardless of CYP1A1 genotype with the greatest effect seen among those with the variant CYP1A1 allele.
Aged↗
In vivo modulation of ETS genes induced by electromagnetic fields.
We have previously shown that electromagnetic field (EMF) exposure induces ETS1 oncogene overexpression in different cell lines. In order to investigate in vivo EMF effects, BALB/c mice were exposed at different times to 50 MHz radiation, modulated (80%) at 16 Hz. The exposed and control animals were sacrificed and the spleen excised for rt-pcr and western blot analysis. We observed an increase in ETS1 mRNA and protein expression, but a decrease in ETS2 protein levels. Preliminary results from this experimental model show in vivo evidence of the effect of EMF on ETS oncogene expression.
Animals↗