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Biomedical subjects

A Bhattacharjee

Publications and source records attributed to A Bhattacharjee.

At least 37 records · Page 2Linked to original sources
PubMed2000

Altered hepatic mRNA expression of apoptotic genes during dimethylnitrosamine exposure.

The role of TNFalpha in regulating apoptotic signaling was investigated during subacute, low-dose (5.0 mg/kg) dimethylnitrosamine (DMN)-induced hepatotoxicity. In TNFalpha receptor (TNFR) intact (wild-type, WT) mice following 4 and 7 DMN exposures, hepatic transcripts for TNFalpha and TNFR-1 were elevated as compared to vehicle controls. DMN hepatotoxicity in WT and TNFR-1/TNFR-2 double knockout (DKO) mice were then compared over a 7-d exposure period. Liver RNA was isolated to measure hepatic expression of TNFalpha/Fas-related genes and the Bcl-2 family of genes that impact apoptosis. Hepatic mRNA levels for Fas, the apoptosis-promoting gene Bax, and the anti-apoptotic gene, Bcl-X(L), were up regulated following 4 and 7 DMN exposures in both WT and TNFR DKO mice as compared to vehicle controls. Notably, hepatic transcript levels for Bax were higher in TNFR DKO mice treated with DMN compared to identically treated WT mice. However, we detected approximately equal DMN-induced apoptotic degradation of liver DNA following 1, 4, and 7 exposures in WT and TNFR DKO mice. Taken together, these data show DMN-induced hepatic TNFalpha expression and suggest that TNFR-1 signaling may be up regulated following 4 and 7 daily DMN exposures. However, TNFalpha is not required for apoptotic signaling at the mRNA transcript level within the liver and instead may actually decrease Bax production.

Animals↗
PubMed2000

Cloning of a T-type Ca2+ channel isoform in insulin-secreting cells.

The T-type Ca2+ channel is an important determinant of electrical activity and of Ca2+ influx in rat and human pancreatic beta-cells. We have identified and sequenced a cDNA encoding a T-type Ca2+ channel alpha1-subunit derived from INS-1, the rat insulin-secreting cell line. The sequence of the cDNA indicates a protein composed of 2,288 amino acids that shares 96.3% identity to alpha1G, the neuronal T-type Ca2+ channel subunit. The transmembrane domains of the protein are highly conserved, but the isoform contains three distinct regions and 10 single amino acid substitutions in other regions. Sequencing rat genomic DNA revealed that the alpha1-subunit we cloned is an alternative splice isoform of alpha1G. By using specific primers and reverse transcription-polymerase chain reaction, we demonstrated that both splice variants are expressed in rat islets. The isoform deduced from INS-1 was also expressed in brain, neonatal heart, and kidney. Functional expression of this alpha1G isoform in Xenopus oocytes generated low voltage-activated Ba2+ currents. These results provide the molecular biological basis for studies of function of T-type Ca2+ channels in beta-cells, which is where these channels may play critical roles in diabetes.

Alternative Splicing↗
PubMed2000

A mibefradil metabolite is a potent intracellular blocker of L-type Ca(2+) currents in pancreatic beta-cells.

It has been shown that mibefradil (Ro 40-5967) exerts a selective inhibitory effect on T-type Ca(2+) currents, although at higher concentrations it can antagonize high voltage-activated Ca(2+) currents. The action of mibefradil on Ca(2+) channels is use- and steady-state-dependent and the binding site of mibefradil on L-type Ca(2+) channels is different from that of dihydropyridines. By using conventional whole-cell and perforated patch-clamp techniques, we showed that mibefradil has an inhibitory effect on both T- and L-type Ca(2+) currents in insulin-secreting cells. However, the effect on L-type Ca(2+) currents was time-dependent and poorly reversible in perforated patch-clamp experiments. By using mass spectrometry, we demonstrated that mibefradil accumulates inside cells, and furthermore, a metabolite of mibefradil was detected. Intracellular application of this metabolite selectively blocked the L-type Ca(2+) current, whereas mibefradil exerted no effect. This study demonstrates that mibefradil permeates into cells and is hydrolyzed to a metabolite that blocks L-type Ca(2+) channels specifically by acting at the inner side of the channel.

Calcium Channel Blockers↗
PubMed1999

Dexamethasone-induced hypertrophy in rat neonatal cardiac myocytes involves an elevated L-type Ca(2+)current.

The mechanism responsible for dexamethasone-induced hypertrophy in infants has not been defined. In this study, we have investigated the role of L-type Ca(2+)currents in the development of dexamethasone-induced hypertrophy in rat neonatal cardiac myocytes. Using cytoplasmic membrane capacitance measurements, we have shown that the size of the cells treated with dexamethasone were larger than those of the control cells. In addition, treating the cells with 1 microM dexamethasone for 48 h increased L-type Ca(2+)current density significantly, without affecting the voltage-dependent activation and steady state inactivation of the current. The increase in current density was associated with an elevation of the mRNA transcript encoding the L-type Ca(2+)channel subunit alpha(1)C. Dexamethasone treatment also resulted in an increase in the peak amplitude of the intracellular Ca(2+)transient measured by fura-2/epifluorescence. Finally, we have demonstrated that the hypertrophic effect of dexamethasone, characterized by the ratio of protein content per cell, was blocked by the L-type specific antagonist, nifedipine. In conclusion, an elevation of L-type Ca(2+)current is involved in the process of dexamethasone-induced cardiac myocyte hypertrophy in neonatal rats.

Animals↗
PubMed1999

A low voltage-activated Ca2+ current mediates cytokine-induced pancreatic beta-cell death.

Insulin-dependent diabetes mellitus is characterized by the selective destruction of pancreatic beta-cells. Chronic treatment with cytokines induced a low voltage-activated (LVA) Ca2+ current in mouse beta-cells. The concomitant increase in the basal cytoplasmic free Ca2+ concentration ([Ca2+]i) was associated with DNA fragmentation and cell death. Antagonists of LVA Ca2+ channels prevented this elevation of basal [Ca2+]i and DNA fragmentation and reduced the percentage of cell death. Exposure to cytokines did not affect the profile of Ca2+ currents or basal [Ca2+]i in glucagon-secreting alpha-cells. An increased Ca2+ signal through LVA Ca2+ channels may thus be a key feature in cytokine-induced beta-cell destruction.

Animals↗
PubMed1998

Molecular dissection of dimethylnitrosamine (DMN)-induced hepatotoxicity by mRNA differential display.

Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to catalogue altered hepatic transcript expression during dimethylnitrosamine (DMN) exposure in vivo. Mice were administered DMN (1.5 or 5 mg/kg) or vehicle (phosphate-buffered saline) i.p. once daily for up to 7 days, and livers were collected 6 h post-injection. Total RNA was reverse transcribed and cDNA subsets were selectively amplified by PCR. DDRT-PCR products were fractionated on denaturing polyacrylamide gels, and differentially expressed bands were excised, reamplified, and subsequently cloned into a plasmid vector. This study identified 23 cDNAs that were induced and 25 cDNAs that were suppressed during DMN exposure. Altered expression during DMN exposure for cDNA clones was confirmed by Northern blotting, RNase protection, or in situ hybridization analyses. DNA sequence information indicated that four cDNAs suppressed during DMN exposure encode cytochrome P450 isoenzyme-cholesterol 7 alpha-hydroxylase (CYP7), a monokine, a myeloid cell differentiation protein, and mouse major urinary protein (MUP). We further observed a DMN-induced increase in transcripts for complement factor 3 (C3) and serum amyloid A (SAA). In contrast, the remaining differentially expressed transcripts detected by DDRT-PCR during DMN exposure demonstrated no similarity to sequences present in Genbank, suggesting that they may encode previously unreported gene products. In situ hybridization showed MUP transcripts to be expressed by hepatic centrilobular areas that undergo necrosis during subchronic DMN exposure. Thus, the utilization of DDRT-PCR has identified several differentially expressed hepatic mRNAs associated with various doses and stages of DMN exposure.

Alkylating Agents↗
PubMed1998

Recombinant human interleukin-11 in experimental Pseudomonas aeruginosa sepsis in immunocompromised animals.

The therapeutic potential of recombinant human interleukin-11 (rhIL-11) was tested in a neutropenic rat model that mimics the clinical consequences of myelosuppressive chemotherapy complicated by Pseudomonas aeruginosa sepsis. rhIL-11-treated animals (150 micrograms/kg intravenously every 24 h for 3 days) had reduced endotoxin levels (P < .05) and less pulmonary edema fluid (P < .001) and were protected (P < .01) against thinning and necrosis of the intestinal mucosa compared with the control group. The survival rate in rhIL-11-treated animals was 40% (19/47), whereas it was 0 (0 of 19) in the control group (P < .01). The addition of ciprofloxacin (10 mg/kg every 12 h) resulted in a survival rate of 9 (60%) of 15, while the combination of rhIL-11 and ciprofloxacin resulted in 100% survival (15/15; P < .05). These results indicate that rhIL-11 supports mucous membrane integrity of the alimentary tract and decreases the systemic inflammatory response to experimental gram-negative infection in immunocompromised animals.

Animals↗
PubMed1998

Outcome of prenatally diagnosed anterior abdominal wall defects.

One hundred consecutive cases of confirmed anterior abdominal wall defect, identified prenatally in the Oxford Prenatal Diagnosis Unit over 11 years, were studied. Fifty nine per cent of cases were suspected omphaloceles and 41% suspected gastroschisis. Fifty four per cent of omphaloceles were accompanied by other defects compared with 5% of those with gastroschisis. Overall, 29% of fetuses with omphalocele had an abnormal karyotype, and of those with another abnormality identified on scan (excluding four cases with no karyotype performed), 54% had an abnormal karyotype. Of the 27 cases with suspected isolated omphalocele, 14 were live born, all of whom have survived. If the 11 whose parents opted for termination of pregnancy are excluded, survival to birth was 88%. Six of the suspected isolated omphaloceles have Beckwith Wiedemann syndrome (BWS). Eight (57%) of the live born babies with omphaloceles had major problems up to the age of 2, but only one (7%) has long term major problems. This child has BWS and is deaf. Of the 39 cases of suspected isolated gastroschisis, 33 (85%) pregnancies resulted in live birth and one in neonatal death after surgery. Survival rate (excluding terminated pregnancies) was 97%. Gastroschisis was associated with a younger maternal age than omphalocele (p < 0.001) and lower birthweight centile (p < 0.01). Fifteen per cent of the gastroschisis babies had major problems up to the age of 2 years and 12% long term developmental problems. Ninety three per cent of the omphalocele babies and 88% of those who had gastroschisis have no long term problems. Over the study period there have been major changes in scanning equipment and expertise. Since 1991 no woman with a suspected isolated lesion has opted for termination of pregnancy.

Abnormalities, Multiple↗
PubMed1997

T-type calcium channels facilitate insulin secretion by enhancing general excitability in the insulin-secreting beta-cell line, INS-1.

The present study addresses the function of T-type voltage-gated calcium channels in insulin-secreting cells. We used whole-cell voltage and current recordings, capacitance measurements, and RIA techniques to determine the contribution of T-type calcium channels in modulation of electrical activity and in stimulus-secretion coupling in a rat insulin secreting cell line, INS-1. By employing a double pulse protocol in the current-clamp mode, we found that activation of T-type calcium channels provided a low threshold depolarizing potential that decreased the latency of onset of action potentials and furthermore increased the frequency of action potentials, both of which are abolished by administration of nickel chloride (NiCl2), a selective T-type calcium channel blocker. Moreover application of high frequency stimulation, as compared with low frequency stimulation, caused a greater change in membrane capacitance (deltaCm), suggesting higher insulin secretion. We demonstrated that glucose stimulated insulin secretion in INS-1 is reduced dose dependently by NiCl2. We conclude that T-type calcium channels facilitate insulin secretion by enhancing the general excitability of these cells. In light of the pathological effects of both hypo and hyperinsulinemia, the T-type calcium channel may be a therapeutic target.

Action Potentials↗
PubMed1996

Abnormally expressed low-voltage-activated calcium channels in beta-cells from NOD mice and a related clonal cell line.

A macroscopic low-voltage-activated (LVA) inward current was found in pancreatic beta-cells isolated from NOD mice. However, this current was not present in nondiabetic prone mouse (e.g., Swiss-Webster) pancreatic beta-cells. We performed pharmacological analyses on this current in NOD insulinoma tumor cells (NIT-1). This cell line was developed from pancreatic beta-cells of a transgenic NOD mouse. The sodium-channel blocker, tetrodotoxin (TTX; 2 micromol/l) had no effect on this LVA current. The amplitudes of currents elicited by a -20 mV test pulse retained similarity when the extracellular sodium concentration was increased from 0 to 115 mmol/l; when the extracellular calcium concentration was decreased from 10 to 2 mmol/l, there was an approximate 50% reduction of this current elicited by a -30 mV test pulse. Neither the L-type calcium-channel blocker, nifedipine (3 micromol/l), nor the N-type calcium-channel blocker, omega-CgTx-GVIA (1 micromol/l), at -30 mV produced an appreciable effect. The T-type calcium-channel blockers, nickel (3 micromol/l) and amiloride (250 micromol/l), effectively reduced the peak of this current. In 2 mmol/l calcium external solution, the threshold of voltage-dependent activation of this calcium current was approximately -65 mV, and the peak current occurred at -20 mV. Half-maximum steady-state inactivation was around -43 mV. The mean time constant of slow deactivating tail currents generated by a preceding 20 mV pulse was 2.53 ms. The intracellular free calcium concentration was two- to threefold higher in NOD mouse pancreatic beta-cells compared with Swiss-Webster pancreatic beta-cells. We concluded that there are LVA calcium channels abnormally expressed in NOD mouse beta-cells. This LVA calcium channel may be factorial to the high cytosolic free calcium concentration observed in these cells, and thereby may contribute to the pathogenesis of NOD mouse beta-cells.

Amiloride↗