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Biomedical subjects

G Biswas

Publications and source records attributed to G Biswas.

At least 37 records · Page 2Linked to original sources
PubMed2001

Physical interaction and functional synergy between glucocorticoid receptor and Ets2 proteins for transcription activation of the rat cytochrome P-450c27 promoter.

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

3T3 Cells↗
PubMed2001

Congenital sternal cleft.

A cleft of the sternum is a rare congenital anomaly. We present a case of a sternal cleft in a 7-year-old boy. A split iliac bone graft covered with the sternocostal portion of a pectoralis major flap was used to reconstruct the defect.

Bone Transplantation↗
PubMed2001

The sandwich temporoparietal free fascial flap for tendon gliding.

Microsurgical transfer of the superficial and deep temporal fascia based on the superficial temporal vessels has been documented. This article analyzes the functional recovery when each layer of this facial flap is placed on either side of reconstructed or repaired tendons, to recreate a gliding environment. This fascial flap also provided a thin, pliable vascular cover in selected defects of the extremities. Six patients (four male and two female) with tendon loss and skin scarring of the hand (three dorsum, one palmar, and one distal forearm) and posttraumatic scarring of the ankle with tendoachilles shortening (one patient) underwent this procedure. No flap loss was witnessed. Good overall functional recovery and tendon excursion were observed. Complication of partial graft loss was observed in two patients.

Adult↗
PubMed2000

Accumulation of mitochondrial P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat brain during chronic treatment with beta-naphthoflavone. A role in the metabolism of neuroactive drugs.

The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to beta-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH(2) termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.

Animals↗
PubMed2000

IP(3), IP(3) receptor, and cellular senescence.

Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP(3)) receptor levels, reduced mitogen-evoked IP(3) formation and Ca(2+) release, and Ca(2+) store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% ("young") or between 53 and 58 MPDs (TI < 28%; "senescent")]. We found that the cytosolic Ca(2+) release triggered by either ionomycin or by several IP(3)-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca(2+) transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP(3) formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca(2+) release response to intracellularly applied IP(3). Finally, to compare IP(3) receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP(3) receptor antiserum, Ab(40). A approximately 260-kDa band corresponding to the IP(3) receptor protein was noted; its intensity was reduced by approximately 50% in senescent cells. Thus, we suggest that reduced IP(3) receptor expression, lowered IP(3) formation, and Ca(2+) release, as well as Ca(2+) store depletion, all contribute to the deficient Ca(2+) signaling seen in HDFs undergoing replicative senescence.

Bradykinin↗
PubMed2000

Novel biochemical and functional insights into nuclear Ca(2+) transport through IP(3)Rs and RyRs in osteoblasts.

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.

3T3 Cells↗
PubMed1999

Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at ser128.

We have investigated mechanisms of mitochondrial targeting of the phenobarbital-inducible hepatic mitochondrial P450MT4, which cross-reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N-terminus. Inducers of cAMP and protein kinase A-mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N-terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1-DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.

Amino Acid Sequence↗
PubMed1999

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

ADP-ribosyl Cyclase↗
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

Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis.

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.

Animals↗
PubMed1999

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle crosstalk.

We have investigated the mechanism of mitochondrial-nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Deltapsim) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+-responsive factors calineurin, calcineurin-dependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-kappaB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca2+]i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Adenosine Triphosphate↗
PubMed1999

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

3T3 Cells↗
PubMed1998

Primary cutaneous aspergillosis: our experience in 10 years.

We describe nine patients with primary cutaneous aspergillosis who were diagnosed during the 10-year period between 1987 and 1996. All except one were adults. Seven of those nine cases had an immunocompromised state. Among the immunocompromised patients, six had burn wounds and one was a renal transplant recipient. Aspergillus flavus was isolated from seven patients and A. fumigatus from one, and in one other case the species could not be determined. Three patients who had total burn surface area of more than 70% died. The other patients responded well to extensive surgical debridement with or without institution of oral itraconazole.

Adult↗
PubMed1998

Aflatoxin B1-induced micronuclei and cell cycle alterations in lung and bone marrow cells and their modulation by Piper argyrophyllum extract.

Aflatoxin B, (AFB1) is a clastogen that causes cellular damage by covalent modification of nucleic acids. In this investigation, male rats were injected i.p. with AFB1 (8 mg/kg b.w.) in DMSO and the same dose of AFB1 was also administered intratracheally (i.t.) to the animals separately. The animals were killed after 26 h of the carcinogen treatment, femur bone was removed, and bone marrow cells were isolated and stained with Mayer's hematoxylin and eosin. Micronuclei (Mn) were scored by using light microscopy. Bronchoalveolar lavage (BAL) was prepared from rats administered AFB1 i.t. A part of BAL was fixed with 70% ethanol, stained with the fluorochrome DAPI, and analysed for cell cycle variations; the other part of the lavage was used for making slides to record Mn with a fluorescent microscope. A significantly greater proportion of lung cells were found to enter cell cycle with extended S-phase due to AFB1 treatment. Mn were induced in polychromatic erythrocytes (PCE) as compared to normochromatic erythrocytes (NCE) in the bone marrow of AFB1-treated rats, where there was nearly a three-fold increase in the number of Mn of bone marrow cells. The administration of AFB1 resulted in a two-fold rise in the Mn in the lung cells. The effect of BSO, DEM, and PB, the modulators of AFB1 metabolism, was studied on AFB1-induced Mn formation. A significant increase in the Mn score in PCEs of BSO- and DEM-treated rats was noted, while a slight reduction in the Mn score was noted in the case of PB-treated rats. The administration of the methanol extract of the leaves of Piper argyrophyllum (taken up in DMSO) to rats for a week exhibited normalising effect on AFB1-induced Mn in bone marrow cells. These observations record the induction of Mn in lung cells due to AFB1 for the first time. We propose the utility of AFB1-induced Mn as a model for screening plant extracts as inhibitors of genotoxicity. Prevention of genotoxic changes described above by phytochemicals is being pursued in our Laboratories.

Aflatoxin B1↗
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↗
PubMed1996

Chemoprevention of carcinogen-DNA binding: the relative role of different oxygenated substituents on 4-methylcoumarins in the inhibition of aflatoxin B1-DNA binding in vitro.

Eighteen 4-methylcoumarins bearing methoxy/hydroxy/acetoxy functionalities have been reported to effectively inhibit the rat liver microsome-mediated aflatoxin B1-DNA binding in vitro. The contribution of functionality on coumarin nucleus towards the inhibition of AFB1-DNA binding is in the order acetoxy > hydroxy > methoxy. The results illustrate the structure-activity relationship.

Aflatoxin B1↗