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Biomedical subjects

D Sarkar

Publications and source records attributed to D Sarkar.

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

The small DNA binding domain of lambda integrase is a context-sensitive modulator of recombinase functions.

lambda Integrase (Int) has the distinctive ability to bridge two different and well separated DNA sequences. This heterobivalent DNA binding is facilitated by accessory DNA bending proteins that bring flanking Int sites into proximity. The regulation of lambda recombination has long been perceived as a structural phenomenon based upon the accessory protein-dependent Int bridges between high-affinity arm-type (bound by the small N-terminal domain) and low-affinity core-type DNA sites (bound by the large C-terminal domain). We show here that the N-terminal domain is not merely a guide for the proper positioning of Int protomers, but is also a context-sensitive modulator of recombinase functions. In full-length Int, it inhibits C-terminal domain binding and cleavage at the core sites. Surprisingly, its presence as a separate molecule stimulates the C-terminal domain functions. The inhibition in full-length Int is reversed or overcome in the presence of arm-type oligonucleotides, which form specific complexes with Int and core-type DNA. We consider how these results might influence models and experiments pertaining to the large family of heterobivalent recombinases.

Bacteriophage lambda↗
PubMed2001

Splenectomy and splenic slice grafting in the management of thalassemia.

Over the last decade and a half, we performed a splenectomy along with slice grafting of the spleen in 170 (beta28 and Ebeta 142) transfusion-dependent, high-risk thalassemic patients. Of these, 17 were selected for study of the fate of the grafts and their immune status before and after operation. The procedure had equally good advantages of splenectomy and immunoconservation as partial splenectomy, partial splenic embolization, or partial splenic dearterialization, if not better, but none of the disadvantages of the other procedures.

Child↗
PubMed2001

The cAMP-binding proteins of Leishmania are not the regulatory subunits of cAMP-dependent protein kinase.

The most commonly used method to determine the cAMP binding activity in cytosolic extracts of promastigotes of Leishmania spp. underestimated by approximately 11.5-fold the total amount of [(3)H]cAMP bound, when compared with results obtained by the modified Millipore filter technique. Three cAMP-binding proteins (BPI, BPII and BPIII) were partially purified and characterized. The native molecular masses of BPI, BPII and BPIII were estimated to be 105, 155 and 145 kDa, respectively. The binding of [(3)H]cAMP to these proteins was affected to different extents by several cAMP analogues. Antibodies directed against the types I and II regulatory subunits of PKA did not cross-react with the leishmanial extract. Photoaffinity labeling of the cytosolic extracts with 8-N(3)-[(32)P]cAMP specifically labeled a band of M(r) 116000 and a band of M(r) 80000 partially saturable by cAMP. From these results, it is concluded that the leishmanial cAMP-binding proteins appear to belong to a different class distinct from the regulatory subunits of cAMP-dependent protein kinases.

Animals↗
PubMed2001

Nitric oxide: not just a negative inotrope.

Nitric oxide (NO) appears to play a role in modulating cardiac function in both health and disease. Early studies in isolated rodent cardiac myocytes demonstrated a depressant effect of NO supplied by NO donors (exogenous) as well as NO generated within myocytes (endogenous). There is increasing evidence for a functional NO generating system within the human myocardium, which appears upregulated in certain disease states. Induction of the high output nitric oxide synthase isoform (iNOS) has been demonstrated in the failing myocardium, though its functional significance remains unproven. More recently published data have contradicted the notion that NO acts solely as a negative inotrope demonstrating positive inotropy in both isolated rodent and human ventricular myocytes in response to a range of NO donors. Different NO donors have different NO release kinetics and generate a range of NO species (NO., NO+ and NO-) which may interact at a number of subcellular targets. The observed response of any cardiac preparation to an NO donor represents the net effect of activation of different effector targets and may explain the contradictory reported effects of NO. To realise the therapeutic potential of NO will require specific targeting at a subcellular level.

Depression, Chemical↗
PubMed2001

Expression of adrenocorticotropin receptor gene in adrenocortical adenomas from patients with Cushing syndrome: possible contribution for the autonomous production of cortisol.

OBJECTIVE: To examine whether inhibition of endogenous adrenocorticotropin (ACTH) secretion in patients with Cushing syndrome affects the expression of the ACTH receptor (ACTH-R) gene in adrenocortical adenoma and attached atrophic normal gland. SUMMARY BACKGROUND DATA: ACTH increases adrenal cell growth and steroidogenesis by means of ACTH-R. In vivo and in vitro studies have shown that expression of ACTH-R is upregulated by its own ligand ACTH in several species. In patients with Cushing syndrome resulting from adrenocortical adenoma, there is autonomous production of cortisol from the adenoma. This strongly inhibits endogenous ACTH secretion, giving rise to the speculation that the expression of the ACTH-R gene in these patients is also suppressed. However, previous studies have shown that administration of exogenous ACTH to these patients leads to a further increase in the production of cortisol, suggesting the expression of functional ACTH-R in the adenoma. The authors, therefore, examined the expression of the ACTH-R gene in these patients. METHODS: Fourteen patients with Cushing syndrome were studied. Glucocorticoid excess resulting from autonomous production from the adenomas was ascertained, and unilateral adrenalectomy was performed. The levels of ACTH-R and cytochrome P450 side chain cleavage enzyme (P450scc) mRNAs were determined by Northern blot analysis. The entire coding region of the ACTH-R gene in these patients was sequenced. RESULTS: ACTH-R mRNA abundance in the attached atrophic normal adrenals was suppressed and invariably less than that in the normal gland obtained from a patient with renal cancer. However, the expression of ACTH-R mRNA was not suppressed in any of the adenomas. Expression of ACTH-R mRNA in the adenomas was four- to sixfold greater than that in the attached atrophic gland. No mutation in the coding sequence of the ACTH-R gene in the adenoma was detected in any of the patients. The mRNA in the adenomas appeared to be translated into functionally active receptor because intramuscular administration of ACTH resulted in significant increases in plasma cortisol before surgery but not 3 months after surgery. In addition, there was a positive linear correlation between the expressions of ACTH-R and P450scc mRNAs in the adenoma tissue. CONCLUSIONS: Suppressed ACTH secretion in patients with Cushing syndrome results in reduction of the ACTH-R mRNA expression in nonneoplastic adrenocortical cells. However, the regulatory mechanism of ACTH-R expression might be different in adenoma. Persistent expression in the adenoma of ACTH-R alone, even in the absence of ACTH, might result in increased basal adenyl cyclase activity, as observed in the case of thyroid-stimulating hormone receptor, and thereby might play a role in the autonomous production of cortisol.

Adenoma↗
PubMed2001

The human homolog of Diminuto/Dwarf1 gene (hDiminuto): a novel ACTH-responsive gene overexpressed in benign cortisol-producing adrenocortical adenomas.

To elucidate the molecular mechanism of the pathogenesis of benign functioning adrenocortical adenomas causing Cushing's syndrome, we employed suppression PCR-based cDNA subtractive hybridization to identify novel genes that are differentially expressed in the adenoma. In this report we describe the adenoma-specific overexpression of the human homolog of the Diminuto/Dwarf1 (hDiminuto) gene. Northern blot analysis revealed that hDiminuto mRNA was overexpressed in the adenoma tissue of 14 patients with Cushing's syndrome in comparison to the adjacent nontumorous adrenal gland. In situ hybridization using hDiminuto cRNA probe showed its abundant expression in the tumor cells, whereas the nontumorous cells showed a low level of expression. As the atrophic adjacent gland may not represent the normal architecture, we examined the expression pattern of hDiminuto mRNA in normal human adrenal cortex. In situ hybridization revealed that it was expressed in all layers of the normal adrenal cortex. In situ apoptosis detection by the TUNEL method revealed that a low level of hDiminuto expression in the atrophic, adjacent gland was associated with numerous TUNEL-positive cells in all layers of cortex. In contrast almost no apoptotic cell was detected in the tumor or in the normal adrenal cortex where hDiminuto expression was abundant. These results are compatible with a recent report that hDiminuto acts as an antiapoptotic factor in neurons. The expression of hDiminuto in the normal adrenal cortex was most abundant in the zona fasciculata, suggesting its possible regulation by ACTH/cAMP. Indeed, forskolin treatment of H295R human adrenocortical cells resulted in a significant induction of the mRNA in a time- and dose-dependent manner. To further demonstrate the physiological regulation, an in vivo experiment was carried out in dexamethasone-treated rats. ACTH administration to these rats increased the mRNA expression. These results led us to speculate that the overexpression of hDiminuto in the adenoma could be due to the abundant expression of ACTH receptor, as we previously described. Diminuto is involved in steroid synthesis and cell elongation in plants. We, therefore, hypothesize that hDiminuto might be involved in the molecular events of adrenocortical tumorigenesis by facilitating steroid synthesis and cell growth.

Adenoma↗
PubMed2001

Overexpression of glutathione-S-transferase A1 in benign adrenocortical adenomas from patients with Cushing's syndrome.

Benign adrenocortical adenoma is a major primary cause of Cushing's syndrome. Although numerous studies have been performed, the molecular mechanism of adrenocortical adenoma is yet to be elucidated. In this study we endeavored to identify genes differentially regulated in adrenocortical adenoma by suppression PCR-based complementary DNA (cDNA) subtractive hybridization. The cDNA population in atrophied nontumorous adrenal gland adjacent to the adenoma was subtracted from that in the adenoma. Then adenoma-specific cDNAs were amplified by PCR. We cloned several cDNAs that are selectively up-regulated in the adenoma, one of which was identified to encode glutathione-S-transferase A1 (GSTA1). Northern blot analysis revealed that GSTA1 messenger ribonucleic acid was abundantly expressed in the adenoma compared with that in the adjacent atrophied nontumorous gland. Western blot analysis and immunohistochemistry showed high expression of GSTA1 also at the protein level. In concordance with this finding, GST activity was significantly higher in the adenoma than in the adjacent atrophied nontumorous gland. To clarify the role of GSTA1 in adrenocortical cells, GST activity in the H295R human adrenocortical cell line was inhibited by ethacrynic acid. Inhibition of GSTs interfered with proliferation of the cells. We, therefore, hypothesize that overexpression of GSTA1 in adrenocortical adenomas might be involved in the growth of tumor cells. We also speculate that this overexpression might be an adaptive response to excess cortisol production.

Adenoma↗
PubMed2000

Scientific, ethical, and logistical considerations in introducing a new operation: a retrospective cohort study from paediatric cardiac surgery.

OBJECTIVE: To review the initial impact on mortality of infants with congenital heart disease of a new surgical technique that is now taken for granted. DESIGN: Retrospective cohort study. SETTING: A tertiary paediatric cardiology centre. SUBJECTS: 325 consecutive neonates with simple transposition of the great arteries admitted before, during, and after the preferred management changed from the Senning operation to the arterial switch (1978-98); and 100 consecutive neonates requiring a different neonatal open heart operation that did not change in that period. MAIN OUTCOME MEASURES: Mortality before and early after operation reconstructed sequentially as the series evolved and retrospectively once the series was complete; actuarial survival associated with the different treatment strategies. RESULTS: For both the transposition and the comparison group, early mortality in 1998 was lower than in 1978. During that period, however, there was a phase temporally related to the adoption of the switch operation in which early mortality for transposition increased. Actuarial survival of recent patients with "intention to treat" with arterial switch is superior to those with intention to treat with the Senning operation, as predicted when the switch operation was first adopted. CONCLUSIONS: A period of increased hazard for individual patients may occur when a specialist community, a particular unit, and an individual surgeon are all learning a new technique concurrently. Obtaining informed consent during this time of uncertainty is helped by clarity about the objectives of treatment and availability of relevant local and international data.

Child↗
PubMed2000

Positive inotropic effects of NO donors in isolated guinea-pig and human cardiomyocytes independent of NO species and cyclic nucleotides.

OBJECTIVE: To characterise the inotropic response of isolated myocytes to a range of structurally unrelated NO donors and to assess the role of NO release kinetics, NO species and cyclic nucleotides in mediating the observed changes. METHOD: Guinea-pig (GP) and human myocytes were prepared by enzymatic digestion. Paced contractile amplitude was recorded at 37 degrees C. NO release was measured by reduction of oxyhaemoglobin and using an NO electrode. Cyclic nucleotides were measured using a tritium labelled competitive binding assay. RESULTS: The NO donors S-nitrosoglutathione (GSNO) and diethylamine/NO (DEA/NO) produced positive inotropic effects in GP myocytes at (10(-5) M) (25 and 111% increases of contraction amplitude). The response to GSNO was significantly enhanced in the presence of a low concentration of isoprenaline (3x10(-10) M). Positive inotropy was observed with a range of both thiol and non-thiol donors, amongst which a fast rate of NO release was associated with positive inotropy. The response to GSNO was abolished by the free NO scavenger oxyhaemoglobin, but not by ODQ (soluble guanylyl cyclase [sGC] inhibitor), Rp-cAMPS (protein kinase A inhibitor) or thapsigargin (sarcoplasmic reticulum Ca(2+) uptake blocker). Direct measurement of cyclic nucleotides showed a rise in cGMP but not cAMP. Human ventricular myocytes showed a significant increase of contraction with GSNO (48+/-15.8%, n=7, P<0. 05) in the presence of isoprenaline and a marked response to DEA/NO alone. CONCLUSIONS: Isolated GP and human myocytes show a positive inotropic effect with certain NO donors. This is independent of sGC and cAMP. The rate of NO release from donors appears important in mediating the effect.

Animals↗
PubMed2000

Culture in vector-averaged gravity under clinostat rotation results in apoptosis of osteoblastic ROS 17/2.8 cells.

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity environment. We then evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6-24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell-surface integrin beta1 and disorganization of actin cytoskeleton. These results suggest that vector-averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.

Actins↗
PubMed2000

Effects of glucocorticoids on tumor necrosis factor alpha-dependent activation of nuclear factor kappaB and expression of the intercellular adhesion molecule 1 gene in osteoblast-like ROS17/2.8 cells.

Recently, we showed that tumor necrosis factor alpha (TNF-alpha) stimulates expression of the intercellular adhesion molecule 1 (ICAM-1) and interleukin-6 (IL-6) genes through activation of p65-p50 heterodimer nuclear factor KB (NF-kappaB) in rat osteoblast-like ROS17/2.8 cells. In the present study, we investigated effects of a synthetic glucocorticoid, dexamethasone (Dex), on TNF-alpha-dependent activation of NF-kappaB and expression of the ICAM-1 gene. ROS17/2.8 cells were pretreated with Dex for 6 h and then exposed to TNF-alpha. Electrophoretic mobility shift assay (EMSA) revealed that TNF-alpha-dependent activation of NF-kappaB was almost completely suppressed by Dex treatment. Increase in ICAM-1 messenger RNA (mRNA) level by TNF-alpha also was markedly suppressed by Dex. Western blot and immunocytochemical analyses showed that Dex attenuated the TNF-alpha-induced nuclear translocation of p65. Treatment with protein synthesis inhibitor cycloheximide (CHX) reversed the Dex effect, indicating that Dex requires de novo protein synthesis for its action. Northern blot analysis revealed that Dex increased IkappaB-alpha mRNA level synergistically with TNF-alpha, whereas it decreased p65 mRNA level. The p105 and IkappaB-beta mRNA levels were not altered by Dex. Consistent with the mRNA level, Dex increased the amount of IkappaB-alpha protein in the cytoplasm in either the presence or the absence of TNF-alpha. Considering a role of IkappaB to sequester NF-kappaB in the cytoplasm, it was suggested that an increase in IkappaB-alpha protein and the concomitant decrease in p65 synthesis account for the Dex-induced suppression of NF-kappaB activation in osteoblastic cells.

Animals↗
PubMed2000

Involvement of AP-1 and steroidogenic factor (SF)-1 in the cAMP-dependent induction of human adrenocorticotropic hormone receptor (ACTHR) promoter.

Adrenocorticotropic hormone receptor (ACTHR) is expressed predominantly in the adrenal glands, and its expression is upregulated by its own ligand, ACTH, via a cAMP-dependent pathway. In the present study, we characterized the 5'-regulatory region of human ACTHR gene to elucidate the molecular mechanisms underlying its adrenal-specific and ACTH/cAMP-dependent expression. The promoter region (-1017/+47 when the transcription start site is regarded as + 1) and its serial 5'-deletions (-764/+47, -503/+-47, -214/+47 and -56/+47) were ligated into the upstream of a luciferase (luc) reporter gene. These constructs were transfected into adrenocortical Y1 cells or non-adrenal JEG3 and Cos-1 cells. In all the cell lines, the luc activity gradually increased with serial 5'-deletions and the maximum activity was conferred by - 56/+ 47. However, the magnitude of luc activity of each deletion construct in non-adrenal cells was much less than that in Y1 cells, suggesting that the promoter functions in an adrenal-specific manner. We identified two Steroidogenic Factor (SF)-1-binding sites at -209 and -35. Electrophoretic mobility shift assay (EMSA) demonstrated that both sites bind to SF-1. Mutation of both sites significantly decreased the activity of -214/+47 promoter in Y1 cells. Transfection of SF-1-expressing plasmid into non-adrenal cells significantly increased the promoter activity, suggesting that SF-1 plays a role in the tissue-specific expression of human ACTHR gene. We identified the region, -764 to -503, that was required for the forskolin/cAMP responsiveness of the promoter. This region contains one AP-1 site. EMSA revealed that the binding of AP-1 to this site increased significantly upon treatment of Y1 cells with forskolin. Mutation of the site abolished the forskolin-responsiveness. In non-adrenal cells, the forskolin-responsiveness was observed only when SF-1-expressing plasmid was cotransfected. This is the first demonstration that both AP-1 and SF-1 are required for the cAMP-dependent induction of human ACTHR gene.

Animals↗
PubMed2000

Evaluation of oral hypo-osmolar glucose-based and rice-based oral rehydration solutions in the treatment of cholera in children.

UNLABELLED: In a randomized controlled clinical trial, the efficacy of a low-sodium low-glucose oral rehydration solution (ORS) and a low-sodium rice-based ORS was compared with standard WHO glucose ORS in the treatment of severe cholera in children aged 2-10y. In total, 120 children were evaluated for the study, of whom 58 patients were positive for Vibrio cholerae and were included in the study. Of these 58 cases, 19 received rice-based hypo-osmolar ORS, 20 received WHO-ORS and 19 received glucose-based hypo-osmolar ORS. The clinical characteristics (age, preadmission duration of diarrhoea, frequency of stool before admission, incidence of vomiting, body weight and volume of initial fluid requirement) were comparable in the three treatment groups. All patients received tetracycline in a dose of 50 mg/kg/d of body weight in 4 divided doses for 3 d. CONCLUSIONS: Patients who received rice-based hypo-osmolar ORS had subsequently reduced (p < 0.05) stool output, ORS consumption and diarrhoea duration than the patients who received either WHO-ORS or glucose-based hypo-osmolar ORS.

Child↗
PubMed2000

Rotation in clinostat results in apoptosis of osteoblastic ROS 17/2.8 cells.

Clinostat is an effective, ground-based tool which can be used to verify data from space flight, and to test hypotheses and experimental conditions for eventual space flights. Rotation in clinostat appears to mimic the microgravity environment by nulling the gravitational vector by continuous averaging. In the present study, we exposed osteoblast-like ROS 17/2.8 cells to a vector-averaged gravity environment in a clinostat and found that the cells undergo apoptotic death during the first 24 hr of clino-rotation. We suggest that apoptosis might be one of the mechanisms for reduced bone formation as observed in actual space flights.

Apoptosis↗
PubMed1999

Expression of E16/CD98LC/hLAT1 is responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We employed cDNA representational difference analysis to identify new genes that are upregulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a human hepatoblastoma cell line HepG2. We isolated several TCDD-responsive cDNAs. Sequence analysis revealed that one of them encodes E16/CD98LC/hLAT1, an integral membrane protein involved in multiple cellular functions including cellular transport of L-type amino acids. Northern blot analysis confirmed the TCDD-dependent upregulation of the mRNA. Induction of E16/CD98LC/hLAT1 mRNA by TCDD did not require de novo protein synthesis as revealed by the experiment using cycloheximide. Consistent with the changes at mRNA level, the transport of 3H-leucine into HepG2 cells was significantly increased by TCDD treatment. These findings provide a novel aspect of biological effects of TCDD on human hepatocytes.

Amino Acid Transport Systems↗
PubMed1999

Complementary role of two fragments of domain V of 23 S ribosomal RNA in protein folding.

We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298).

Animals↗
PubMed1999

Comparison of long-term outcomes of atrial repair of simple transposition with implications for a late arterial switch strategy.

BACKGROUND: We report the single-institution, long-term results of 358 patients with simple transposition of the great arteries surviving >30 days after a Mustard (n=226, 1965 to 1980) or Senning (n=132, 1978 to 1992) procedure. METHODS AND RESULTS: Outcome measures included late death, reintervention, ECG and ambulatory ECG rhythm, new arrhythmia, and functional status. Average follow-up was 13.4 (range 0.32 to 17.9) years for the Senning group and 11.7 (range 0.04 to 23.9) years for the Mustard group. The Senning group had a better survival rate at 5, 10, and 15 years (95% versus 86%, 94% versus 82%, and 94% versus 77%, respectively). In both groups, the majority of late deaths were sudden, without preceding ventricular dysfunction. Survival and survival free of reintervention were significantly better in the Senning group (relative risk [RR] 0.34, P=0.06 versus RR 0.39, P=0.027). Loss of sinus rhythm was comparable and unrelated to death. After era correction, the incidence of atrial flutter was similar and strongly associated with late death in both groups. Clinical systemic ventricular failure was uncommon, and at last follow-up, 92% of the Senning group and 89% of the Mustard group were in New York Heart Association class I. In a model exploring the implications of elective arterial switch conversion, this would only be beneficial if the hazard late after switch was markedly reduced and/or the hazard after the Senning procedure increased with time. CONCLUSIONS: Late outcomes after the Senning procedure are superior to those after the Mustard procedure. Both groups had late sudden deaths that were not associated with clinical systemic ventricular failure. Good functional status after the Senning procedure suggests that a strategy of elective switch conversion cannot be justified for patients with isolated transposition.

Cardiac Surgical Procedures↗