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S Addya

Publications and source records attributed to S Addya.

At least 19 recordsLinked to original sources
PubMed2002

Four-laser scanning confocal system for microarray analysis.

We have constructed a confocal scanner suitable for routine microarray analysis from commercially available parts. We have outlined the details that should be considered when designing such an instrument and listed some of the specific components comprising the system [the full list of system components is available on CD from the corresponding author (D.J.G.) at no charge]. Here, we describe the methods used to test the linearity and sensitivity of the instrument. Performance was evaluated with two commonly used dyes, fluorescein and Cy5. While the instrument had a linear correlation between the dye concentration and fluorescence intensity, the observed deviation from a slope of 1.0 underscores the importance of running multipoint calibration experiments to obtain accurate dye quantitation over the full dynamic range of the scanner. This method has utility in testing commercial instruments in addition to the scanner described here. An array with over 300 spots dyed with Cy3 was scanned with our instrument and a high-end commercial instrument. The agreement between the two instruments was very good over a 1000-fold intensity range. Our scanner is a cost-effective alternative to more costly commercial scanners with similar capabilities.

Fluoresceins↗
PubMed2000

Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors.

Reperfusion of ischemic myocardium results in apoptotic cell death and DNA fragmentation. Several transcription factors are known to regulate the apoptotic cell death. This study sought to examine the regulation of cardiomyocyte apoptosis by these transcription factors. Isolated working rat hearts were divided into six groups: control, 15 min ischemia, 60 min ischemia, 15 min ischemia followed by 2 h reperfusion, ischemic stress adaptation by subjecting the hearts to four cyclic episodes to 5 min ischemia, each followed by 10 min of reperfusion, and adaptation followed by 15 min ischemia and 2 h reperfusion. Redox-regulated transcription factors, NF kappa B and AP-1 and the expression of two anti- and pro-apoptotic genes, Bcl-2 and p53 were determined. The results demonstrated NF kappa B and AP-1 progressively and steadily increased as a function of the duration of ischemia. In the adapted heart, NF kappa B binding remained high while AP-1 binding was lowered to almost baseline value. The anti-oxidant gene, Bcl-2 was downregulated in the ischemic/reperfused heart, but upregulated in the preconditioned myocardium. Significant induction of the expression of p53 occurred after ischemia and reperfusion. Apoptotic cells were barely detected in the adapted myocardium which was subjected to the same ischemia/reperfusion protocol. The results demonstrate for the first time differential regulation of cardiomyocyte apoptosis by pro- and anti-apoptotic transcription factors and genes as a function of different durations of ischemia and reperfusion.

Animals↗
PubMed1999

Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.

Animals↗
PubMed1999

15-Deoxy-delta(12,14) prostaglandin J2: a putative endogenous promoter of adipogenesis suppresses the ob gene.

Leptin is considered a key factor in the regulation of appetite and energy expenditure, but little is known about the control of its synthesis and release. Thiazolidinediones (TZDs) have recently been shown to downregulate leptin expression, and it has been speculated that downregulation of the ob gene occurs through activation of the transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma). However, there are no studies using an endogenous PPARgamma ligand. We examined the effect of 15-deoxy-delta(12,14) prostaglandin J2 (15d-PGJ2), a putative natural ligand of PPARgamma, on ob gene expression in fully differentiated 3T3-L1 adipocytes and compared its effect with that of two other PPARgamma activators, the TZD troglitazone (Trog) and indomethacin (Indo). 15d-PGJ2, Trog, and Indo all inhibited leptin expression at concentrations at which they activate PPARgamma. The inhibition of leptin expression of PPARgamma activators was surprising, since PPARgamma is known to induce adipogenesis during which the ob gene is expressed. To address the possibility that PPARgamma plays different roles before and after the induction of adipogenesis, we examined the effects of the three PPARgamma ligands on the expression of leptin and the glucose transporter protein GLUT4, both of which are expressed during differentiation of 3T3-L1 preadipocytes to adipocytes. In the absence of PPARgamma ligands, leptin and GLUT4 synthesis increased from day 3 to day 9 or 10 during differentiation. However, in the presence of any of the three PPARgamma ligands, GLUT4 expression was unaffected, while ob gene expression was inhibited. We hypothesize that PPARgamma may be essential for induction of adipocyte differentiation but then needs to be inactivated to allow expression of the ob gene.

Adipose Tissue↗
PubMed1998

Interaction of adrenodoxin with P4501A1 and its truncated form P450MT2 through different domains: differential modulation of enzyme activities.

Recently we showed that the beta-naphthoflavone-inducible liver mitochondrial P450MT2 consists of two N-terminal truncated forms of the microsomal P4501A1, termed P450MT2a (+5/1A1) and MT2b (+33/1A1) [Addya et al. (1997) J. Cell Biol. 139, 589-599]. In the present study, we demonstrate that intact P4501A1 and the major mitochondrial form, P450MT2b (routinely referred to as P450MT2), show distinct substrate specificities and preference for different electron transport proteins. Enzyme reconstitution and spectral studies show that the wild-type adrenodoxin (Adx), but not the mutant Adx, binds to P450MT2 in a functionally productive manner (Kd = 0.6 microM) and induces a characteristic high-spin state. Adx binding to intact P4501A1 or +5/1A1 is less efficient as seen from spectral shift patterns (Kd = 1.8-2.0 microM) and reconstitution of enzyme activity. Use of Adx--Sepharose affinity matrix yielded < 90% pure P450MT2 (specific activity: 13.5 nmol/mg of protein) starting from a partially purified fraction of 10-15% purity, further demonstrating the specificity of P450MT2 and Adx interaction. Chemical cross-linking studies show that the bovine Adx forms heteroduplexes with both P450MT2 and intact P4501A1, though at different efficiencies. Our results show that Adx interacts with P450MT2 through its C-terminal acidic domain 2, while interaction with intact P4501A1 likely involves the N-terminal acidic domain 1. These results point to an interesting possibility that different electron transfer proteins may differently modulate the enzyme activity. Our results also demonstrate for the first time as to how a different mode of Adx interaction differently modulates the substrate specificities of the two P450 forms.

Adrenodoxin↗
PubMed1997

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2.

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Amino Acid Sequence↗
PubMed1997

Localization of multiple forms of inducible cytochromes P450 in rat liver mitochondria: immunological characteristics and patterns of xenobiotic substrate metabolism.

Hepatic mitochondria contain inducible cytochromes P450 that cross-react with antibodies to P4501A1/2 and 2B1/2. In the present study, we present evidence for the occurrence of additional P450 forms in rat liver mitochondria that cross-react with antibodies to microsomal P4503A1/2 and 2E1. Protease protection and also immunoelectron microscopy studies were carried out to support the mitochondrial location of the immunoreactive P450s. The solubility of immunoreactive proteins in 0.1 M Na2CO3 suggests that the mitochondrial P450 forms tested are not membrane-integral proteins. The mitochondrial-associated P450 forms are capable of metabolizing resorufin derivatives, erythromycin, and p-nitrophenol in an adrenodoxin- and adrenodoxin reductase-supported system. Treatment of rats with phenobarbital (PB) resulted in the induction of mitochondrial pentoxyresorufin O-deethylase (PROD), benzoxyresorufin O-deethylase (BROD), and erythromycin N-demethylase (ERND) activities by 17-, 23-, and 2-fold, respectively. These activities were inhibited by 33 to 64% by antibodies to P4502B1/2 and P4503A1/2. The induction of the above monooxygenase activities correlated with the levels of mitochondrial proteins cross-reacting with antibodies to P4502B1/2 and P4503A1/2 in PB-treated livers. Similarly, administration of beta-naphthoflavone (BNF) resulted in a marked elevation of O-deethylation of ethoxy-, benzoxy-, and methoxyresorufins and a 2-fold increase in ERND activity. Immunoblot and immunoinhibition experiments using P4501A1/2, P4502B1/2, P4503A1/2, and P4502E1 antibodies revealed the presence of P450 forms closely related to the microsomal inducible forms. Results of immunoinhibition studies, using antibodies to adrenodoxin and reconstitution of enzyme activity with purified P450 forms, suggested a role for the mitochondrial P450 in the metabolism of xenobiotic substrates. The purified mitochondrial P450s also exhibited overlapping substrate specificities for resorufin derivatives and erythromycin.

Adrenodoxin↗
PubMed1995

Localization of a transcription promoter within the second exon of the cytochrome P-450c27/25 gene for the expression of the major species of two-kilobase mRNA.

The rat P-450c27/25 (CYP27) gene is expressed as two distinctly sized mRNAs of 2 and 2.3 kb (kilobase). The 2 kb mRNA is the predominant form in the liver with negligible 2.3 kb species. Rat kidney and hepatoma, on the other hand, contain significant levels of the 2.3 kb species. Rat CYP27 gene contains 11 exons of 80-415 nucleotides that are separated by 10 introns of 83 bases to approximately 10 kb. S1 nuclease protection and primer extension analyses using liver RNA showed a prominent 5' terminus 86 nucleotides downstream from the start of exon 2. This site, designated as +1, is the start site for the 2 kb mRNA. 5' RACE analysis of rat kidney and hepatoma RNAs showed the presence of a 5' extended mRNA with a sequence complementary to the Spi2 mRNA. A cryptic TATA box (TTTAAA) is located 24 nucleotides upstream of the 2 kb mRNA transcription initiation site at +1. A 106 bp DNA fragment (sequence -83 to +23) that houses the putative TATA motif forms three differently migrating complexes with nuclear extract from the murine 3T3 cells. DNAse I footprinting and competition with synthetic DNA showed that complex A represents the bound Sp1 factor and complexes B and C are due to unknown factors binding to the -83 to -71 and -20 to -12 sequences, respectively. In vivo transcription analysis using -840/+23 DNA and its 5' deletions cloned in a CAT reporter plasmid suggests that the basal promoter elements are located within sequence -45 to +23 of the gene. Finally, in vitro transcription analysis in HeLa cell nuclear extract showed that intact TTTAAA motif and complex C-forming sequence from this region are essential for transcription initiation at the +1 position of the promoter. Our results demonstrate that the 2 kb mRNA is transcribed as an independent transcript driven by an immediate upstream promoter located within exon 2.

Animals↗
PubMed1994

Purification and characterization of a hepatic mitochondrial glutathione S-transferase exhibiting immunochemical relationship to the alpha-class of cytosolic isoenzymes.

Hepatic mitochondria from different mammalian species contain varying levels of glutathione S-transferase (GST) activities. More than 70% of the activity detectable in the mouse liver mitochondria is associated with the soluble matrix. The mouse mitochondrial matrix GST was purified using a combination of (NH4)2SO4 fractionation, Sephadex gel filtration and affinity chromatography on glutathione (GSH) conjugated Sepharose. The purified GST comigrates with the mouse cytosolic MI (or alpha form), and exhibits an apparent molecular mass of 25 kD on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antibody to the purified mitochondrial GST cross-reacted with the similarly migrating cytosolic MI GST, suggesting extensive immunochemical relatedness between these two forms. As previously demonstrated for the cytosolic alpha form, the mitochondrial GST catalyzes aflatoxin B1-GSH conjugation (6.3 nmol/mg protein/min) and exhibits peroxidase activity (6.7 mumol/mg protein/min). The putative mitochondrial GST only in intact mitochondria, but not in sonic disrupted mitochondria, is resistant to proteolytic digestion with trypsin, demonstrating its intramitochondrial location. Isoelectric focusing on the flat bed polyacrylamide gel system resolves the mitochondrial GST into two distinct components with apparent pI of 9.9 and 9.7, both of which cross-react with polyclonal antibody to the mitochondrial GST. Under the identical conditions, the most cationic form of cytosolic GST cross-reacting intensely with the antibody resolves as a single component with an apparent pI of 9.4. Thus the mitochondrial GST resembles the alpha family of isoenzymes, though it appears to represent independent molecular species different from the cytosolic forms.

Aflatoxin B1↗
PubMed1991

Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone.

Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries.

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.

Amino Acid Sequence↗
PubMed1989

Effects of cadmium treatment in vitro on the uptake and release of [3H]serotonin by human blood platelets.

Effects of cadmium treatment on human platelets were studied with respect to uptake and release of 5-[3H]hydroxytryptamine (5-HT). The uptake of 5-[3H]HT in the presence of varying concentrations of CdCl2 (0.001-10 mM) was inhibited significantly with respect to control platelets and the inhibition was maximum at 1 mM CdCl2 concentration. From studies on the kinetics of 5-[3H]HT uptake a higher Km and significantly lower Vmax for CdCl2-treated platelets were observed. CdCl2 stimulated spontaneous release but inhibited thrombin-induced release of 5-[3H]HT. Spontaneous release of 5-[3H]HT induced by CdCl2 was not significantly altered in the presence of externally available CaCl2 (1 mM).

Blood Platelets↗
PubMed1988

Effects of cadmium treatment in vitro on the antioxidant protection mechanism and activation of human blood platelets.

Interactions of human platelets with cadmium in vitro were studied with respect to the platelet activation process as indicated by malondialdehyde (MDA) formation and also to the components of the cellular antioxidant defence system such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH), and reduced glutathione (GSH). Cadmium treatment stimulated platelet MDA formation after a lag phase of at least 15 min and this effect was completely blocked by either 1 mM aspirin or 1 mM CaCl2. Cadmium pretreated platelets also displayed a much higher (5 fold) MDA formation when stimulated by thrombin. Platelet catalase activity was decreased by almost 50% after incubation with cadmium. There was also a moderate decline in platelet GSH and GR activity along with a stimulation of GST and G6PDH activity. These results suggest: (1) the cadmium effect on platelets as observed by enhanced formation of MDA via the cyclooxygenase pathway involves intraplatelet accumulation of cadmium which is inhibited by calcium, (2) a modest decline in GSH, presumably due to the inadequacy of H2O2 detoxification mechanism, does not adversely affect platelet function because of the adaptive response of G6PDH, and (3) intracellular accumulation of cadmium may result in platelet hyperactivity through higher intraplatelet free calcium levels resulting directly through cadmium action or indirectly through higher H2O2 levels due to catalase inhibition.

Adult↗