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Biomedical subjects

S Chakrabarti

Publications and source records attributed to S Chakrabarti.

At least 145 records · Page 8Linked to original sources
PubMed1999

Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein.

Metallothionein (MT) has been shown to protect cells from free radical induced DNA damage after exposure to copper, hydrogen peroxide and also radiation. In order to study the role of MT in radiation induced apoptosis, age-matched male control mice (C57BL/6J), MT-I overexpressing (MT-I*) and MT-null transgenic mice were exposed to whole body cobalt 60 gamma-irradiation at 0, 5, or 10 Gy, and their thymus were removed 24 h later. The basal levels of MT and zinc concentrations in the thymus were measured by 109Cadmium-heme assay and atomic absorption spectrophotometry, respectively. The MT expression after radiation was determined by immunohistochemical staining using a polyclonal antibody to MT. The extent of apoptosis in thymocytes was determined by histology (H&E stain). DNA was isolated from the thymus, and DNA fragmentation was determined by agarose gel electrophoresis. The results showed that the basal level of MT protein in MT-I* thymus was 2.4-fold higher than control mice, and that MT was inducible in both MT-I* and control C57BL6 thymus after radiation exposure. Minimal MT protein was detected in MT-null mice thymus before or after radiation, while, a significantly higher number of apoptotic cells and DNA fragmentation were found in MT-null thymus after whole body irradiation. These data demonstrated a protective role for MT in radiation-induced apoptosis in mouse thymus.

Animals↗
PubMed1999

Novel non-isotopic detection of MutY enzyme-recognized mismatches in DNA via ultrasensitive detection of aldehydes.

A highly sensitive method to detect traces of aldehyde-containing apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based on this method, a novel approach to detect DNA base mismatches recognized by the mismatch repair glycosylase MutY is demonstrated. Open chain aldehydes generated in nucleic acids due to spontaneous depurination, DNA damage or base excision of mismatched adenine by MutY are covalently trapped by a new linker molecule [fluorescent aldehyde-reactive probe (FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing AP sites is FARP-trapped, biotinylated and immobilized onto neutravidin-coated microplates. The number of FARP-trapped aldehydes is then determined via chemiluminescence using a cooled ICCD camera. AP sites induced in plasmid or genomic calf thymus DNA via mild depurination or by simple incubation at physiological conditions (pH 7, 37 degreesC) presented a linear increase in chemiluminescence signal with time. The procedure developed, from a starting DNA material of approximately 100 ng, allows detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7) bases, and extends by 1-2 orders of magnitude the current limit in AP site detection. In order to detect MutY-recognized mismatches, nucleic acids are first treated with 5 mM hydroxylamine to remove traces of spontaneous aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides engineered to have a centrally located A/G mismatch demonstrate a strong chemiluminescence signal. Similarly, single-stranded M13 DNA that forms mismatches via self-complementation (average of 3 mismatches over 7429 bases) and treated with MutY yields a signal approximately 100-fold above background. No signal was detected when DNA without mismatches was used. The current development allows sensitive, non-isotopic, high throughput screening of diverse nucleic acids for AP sites and mismatches in a microplate-based format.

Aldehydes↗
PubMed1999

Prevalence of the C282Y mutation of the hemochromatosis gene in liver transplant recipients and donors.

Hemochromatosis heterozygotes may be predisposed to end-stage liver disease from other causes. The aims of this study were to determine the prevalence of the hemochromatosis mutation, C282Y, in end-stage liver disease and to determine if transplantation of C282Y heterozygous livers adversely affected survival. The C282Y status of patients who underwent hepatic transplantation and, whenever possible, their donors, was determined and correlated with hepatic iron status. Survival was compared in patients who received livers from heterozygotes and normals. Prevalence of C282Y in recipients was compared with 5,211 voluntary blood donors. Twenty-six C282Y heterozygotes were detected among 304 transplant recipients (8.6%) compared with a prevalence of 8.4% in blood donors. Six of 26 heterozygous recipients (23%) had >/=2+ iron staining in their explanted livers compared with 40 of 277 wild-type livers (14%) (P = ns). There was no significant difference in mean hepatic iron concentration between C282Y heterozygotes and wild-type explanted livers with >/=2+ iron staining. Seven of 31 patients (23%) with alcoholic liver disease were C282Y heterozygotes. Twenty-four heterozygotes were detected in 141 donors (17.0%). Survival did not differ between recipients who received heterozygous or normal livers. The prevalence of C282Y heterozygotes in patients requiring liver transplantation does not differ significantly from the general population. Heterozygotes are not at increased risk of developing end-stage liver disease. Transplantation of C282Y heterozygous livers is a safe, effective practice.

Adolescent↗
PubMed1999

Alternate splicing produces a soluble form of the hereditary hemochromatosis protein HFE.

HFE is a non-typical MHC class 1-type protein that is mutated in hereditary hemochromatosis. The purpose of this study was to identify possible splice variants of HFE mRNA and investigate the regulation of these isoforms in duodenum and liver of patients with normal and altered iron stores. RT-PCR was performed using HFE specific primers and duodenal RNA obtained from patients with hemochromatosis, iron deficiency, secondary iron overload and normal controls. The reaction products were visualized by Southern blot and identified by DNA sequence analysis. Additional studies were performed on RNA isolated from liver and a range of human tissues. A truncated (soluble) form of HFE protein was identified that lacks the transmembrane domain and occurs as a result of alternative splicing. Soluble HFE was found predominantly in the duodenum, spleen, breast, skin and testicle. In hereditary hemochromatosis full length HFE was the predominant isoform present in the duodenum similar to iron deficiency. Alternate splicing produces soluble HFE that may have a unique function to regulate cellular iron transport.

Alternative Splicing↗
PubMed1999

Leukocytes utilize both alpha4 and alpha5 integrins for intraislet infiltration in non-obese diabetic mice.

The loss of insulin-producing cells during the development of type 1 diabetes is dependent on leukocyte infiltration of beta-islets in the pancreas. Injection of antibodies to integrins and their ligands has been shown to prevent the development of diabetes in non-obese diabetic (NOD) mice. However, little is known about the progression of infiltration by leukocytes after their homing and extravasation into the pancreas. In the present study, the effect of monoclonal antibodies (mAbs) to alpha4 and alpha5 integrins on leukocytes that had infiltrated the islets was characterized in NOD mice at 10 weeks of age when insulitis was in progress, or in mice with recent onset of diabetes. Injection of mAbs to either alpha4 or alpha5 integrins had little effect on the extent of leukocyte infiltration in 10-week-old or diabetic mice. In contrast, leukocyte infiltration was significantly reduced upon injection of mAbs to both alpha4 and alpha5 integrins. The reduction in leukocyte infiltration was due to decreases in the percentage of islets with intraislet infiltration. However, the observed effect of mAbs to alpha4 and alpha5 integrins was reversible, since intraislet infiltration resumed upon termination of antibody treatment. Results suggest that after homing to the pancreas, leukocytes utilize both alpha4 and alpha5 integrins for intraislet infiltration.

Animals↗
PubMed1999

Diabetes-induced vascular dysfunction in the retina: role of endothelins.

AIMS/HYPOTHESIS: Vasoactive factors like endothelins, by virtue of the microvascular regulation as well as by other effects, possibly play important parts in the pathogenesis of diabetic retinal microangiopathy. We investigated retinal vascular dysfunction in streptozotocin-induced diabetes and its relation with endothelins in short- and long-term diabetes. METHODS: Diabetic rats with or without an endothelin receptor antagonist (bosentan) treatment were investigated after 1 month and 6 months of follow-up. Retinal blood flow was measured and compared with age- and sex-matched non-diabetic control animals. Retinal tissues were analysed for endothelin-1, endothelin-3, endothelin A and endothelin B mRNA. Distribution of endothelin-1 and endothelin-3 was investigated by immunocytochemistry and that for endothelin receptors by ligand binding and autoradiography. RESULTS: Diabetic animals showed hyperglycaemia, glycosuria, elevated glycated haemoglobin values and reduced body weight gain. Retinal blood flow showed an increased resistivity index, an indicator of vasoconstriction, after 1 month of diabetes which was prevented by treatment with bosentan. This functional change in diabetes was eliminated after 6 months of follow-up. The retina from the diabetic animals showed increased mRNA expression for endothelin-1, endothelin-3 and endothelin A after one month. In addition, endothelin B mRNA expression was increased after 6 months. Furthermore, endothelin-1 and endothelin-3 immunoreactivity and endothelin receptor concentrations were increased in the retina of diabetic rats. CONCLUSION/INTERPRETATION: The results from this study indicate that the endothelin system is of importance in mediating retinal changes in diabetes although mechanisms of the endothelin system alteration as well as their effects might vary depending on the duration of diabetes. [Diabetologia (1999) 42: 1228-1234]

Animals↗
PubMed1999

Solution stability of linear vs. cyclic RGD peptides.

Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2-12 at 50 degrees C. Reversed-phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-first-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.

Chromatography, High Pressure Liquid↗
PubMed1999

Fulminant adenovirus hepatitis following unrelated bone marrow transplantation: failure of intravenous ribavirin therapy.

Fulminant hepatic failure due to adenovirus infection is a rare complication following stem cell transplantation. We report this complication in an unrelated bone marrow transplant recipient 30 weeks post-transplant. Treatment with intravenous ribavirin was started within 36 h of admission, but he succumbed to unusually fulminant hepatic failure. Adenovirus type 2 was isolated from stool surveillance samples and from post-mortem liver samples. Adenovirus DNA was detected by PCR in blood and sputum samples at admission and was identified in post-mortem liver tissue by electron microscopy. Implications of the failure of ribavirin therapy are discussed.

Adenoviridae Infections↗
PubMed1999

ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.

Adenosine Triphosphatases↗
PubMed1999

Fluorescent labelling of closely-spaced aldehydes induced in DNA by bleomycin-Fe(III).

PURPOSE: To test the ability of FARPhC and FARP, two novel fluorescent reagents, to label aldehyde-containing sites (principally abasic sites) generated in DNA by the radiomimetic drug bleomycin, and to use fluorescent energy transfer from FARPhc (donor) to FARP (acceptor) to quantitate such closely-spaced sites. MATERIALS AND METHODS: FARPhc, 7-hydroxycoumarin-3-carboxylic acid (((((amino-oxymethyl) carbonyl) hydrazino) carbonylethyl) amide) was synthesized with a protocol similar to the one recently reported for FARP (a fluorescein-based probe). RESULTS: Both FARPhc and FARP form stable oxime bonds with the open-chain aldehydes generated upon acidic depurination of DNA. Plasmid DNA exposed to bleomycin-Fe(III)-ascorbate undergoes extensive strand breakage, and upon subsequent reaction with FARPhc and/or FARP it becomes fluorescently labelled, indicating the generation of aldehyde-containing sites. The binding of the probes to calf thymus or plasmid DNA results in significant fluorescent energy transfer among closely-spaced fluorophores, as revealed by the fluorescence increase following digestion of fluorescently labelled samples with nuclease P1. The fluorescent quenching is most evident when both FARPhc and FARP are used simultaneously to trap aldehyde sites. When single-stranded oligonucleotides engineered to contain either one or two closely spaced bleomycin binding sites are exposed to bleomycin and then fluorescently labelled, the oligonucleotides demonstrate significantly increased fluorescent energy transfer with two binding sites indicating a dependence of aldehyde site generation and clustering on the local sequence of a single strand. CONCLUSIONS: A new detection method for DNA damage induced by bleomycin following fluorescent labelling of aldehyde group-containing sites (FLAGS) and their clustering via fluorescent energy transfer is demonstrated. The method is applicable to any form of DNA. This work may lead to a general approach for the quantification of multiply damaged sites in DNA, a subset of DNA lesions that may have major biological significance.

Aldehydes↗
PubMed1999

Use of PCR to identify enteroaggregative Escherichia coli as an important cause of acute diarrhoea among children living in Calcutta, India.

The importance of enteroaggregative Escherichia coli (EAggEC) as a possible aetiological agent of acute diarrhoea among children in Calcutta, India, was investigated. Simultaneously the use of a previously described PCR diagnostic system was assessed for its ability to identify EAggEC infection. E. coli strains isolated during a 1-year case-control study from faecal samples of 388 children aged <5 years, with or without diarrhoea, were examined for EAggEC by HeLa cell adherence assay in parallel with a PCR assay with primers generated from an EAggEC DNA probe. A blind comparison was made between the two methods to determine their diagnostic potential. E. coli isolates that adhered to HeLa cells in an aggregative pattern were the sole isolates significantly more often in 254 cases (9%) than in 134 control (2%) children. Age stratification showed that EAggEC were isolated more frequently from children aged <36 months. The EAggEC isolates belonged to several O serogroups and showed multiple drug resistance. Both methods were positive for 26 samples, nine samples were positive by PCR alone and seven samples were positive by culture alone, thus indicating a 78% sensitivity and 97% specificity for the PCR assay. EAggEC is an important aetiological agent of acute diarrhoea among infants in and around Calcutta, and the PCR diagnostic system may be useful to identify such infection in epidemiological studies.

Acute Disease↗
PubMed1999

Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae.

The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. cholerae is ToxR, a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis and toxR transcriptional fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed from toxR. This may be due to increased heat shock induction in the dnaK mutant. In vivo, however, although expression from heat shock promoters in the dnaK mutant was similar to that observed in vitro, expression of both toxR and htpG was comparable to that by the parental strain. In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease in htpG expression was observed. These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro.

Animals↗
PubMed1999

Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans.

Epithelial cells lining the adult human colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, approximately one-third of human colon cancers and cancer cell lines have been shown to express GRP-binding sites. Because GRPR activation causes the proliferation of many cancer cell lines, GRP has been presumed to act as a clinically significant growth factor. Yet GRP has not been shown to be expressed by colon cancers in humans nor has the effect of GRP and/or GRPR coexpression on tumor behavior been investigated. We therefore determined GRP and GRPR expression by immunohistochemistry in 50 randomly selected colon cancers resected between 1980 and 1997, all 37 associated lymph node and liver metastases, and 20 polyps. Tumor sections studied were those that contained the margin and adjacent nonmalignant epithelium. Overall, 84% of cancers aberrantly expressed GRP or GRPR, with 62% expressing both ligand and receptor, whereas expression was not observed in adjacent normal epithelium. Consistent with the previously established mitogenic capabilities of GRP, tissues coexpressing GRP and GRPR were more likely to express proliferating cell nuclear antigen than tissues not expressing both ligand and receptor. Yet GRP/GRPR coexpression was seen with equal frequency in stage A as in stage D cancers and was only detected in 1 in 37 metastases. Furthermore, Kaplan-Meier analysis did not reveal any difference in patient survival between those whose tumors did or did not express GRP/GRPR. In contrast, GRP/GRPR coexpression was found in all well-differentiated tumor regions, whereas poorly differentiated tissues never coexpressed GRP/GRPR. Overall, these data indicate that, although GRP is a mitogen, it is not a clinically significant growth factor in human colon cancers. Rather, the strong association of GRP/GRPR coexpression with tumor differentiation raises the possibility that these proteins primarily act in vivo as morphogens.

Adult↗
PubMed1999

Orally administered NHE1 inhibitor cariporide reduces acute responses to coronary occlusion and reperfusion.

Na+/H+ exchange (NHE) mediates myocardial ischemic and reperfusion injury. We examined the effects of dietary administration of the potent and selective NHE1 inhibitor cariporide on acute responses to coronary artery ligation and reperfusion in the anesthetized rat. Male Sprague-Dawley rats received control rat chow or an identical diet containing 3 parts per million of cariporide for 1 wk before 225 min of occlusion of the left main coronary artery or 45 min of occlusion followed by 180 min of reperfusion. Hearts were excised and divided into left ventricle, right ventricle, and interventricular septum for analysis of NHE1 mRNA expression and apoptosis by staining with terminal deoxynucleotidyl transferase-mediated nick end labeling. Ischemia and reperfusion were associated with a threefold elevation in NHE1 mRNA expression in control animals that was significantly reduced in cariporide-fed rats. Cariporide reduced mortality from 26% of animals to 0%. The incidence of all arrhythmias was significantly reduced, including ventricular fibrillation (from 42 to 0%) and ventricular tachycardia (from 81 to 15%), as well as the number of ventricular premature beats (from 70 +/- 12 to 17 +/- 6). Cariporide moderately reduced apoptosis only in the reperfused left ventricle to values not significantly greater than those in sham-operated animals, and this was associated with a significantly higher ratio of Bcl-2 to Bax. This study suggests that NHE inhibition with dietary cariporide represents an effective management of acute postinfarction responses.

Administration, Oral↗
PubMed1999

Modulation of Na+/H+ exchange isoform 1 mRNA expression in isolated rat hearts.

Na+/H+ exchange (NHE) has been demonstrated to mediate myocardial ischemia and reperfusion injury as well as injury produced by hydrogen peroxide (H2O2) or lysophosphatidylcholine (LPC). However, changes in gene expression in response to injurious factors have not been extensively studied. We examined Na+/H+ exchange isoform 1 (NHE-1) expression using Southern detection of the RT-PCR product in response to 30 min of global ischemia with or without reperfusion in isolated rat hearts or to 30 min of exposure to either H2O2 (100 microM) or LPC (5 microM). We also determined whether ischemic preconditioning (2x 5-min ischemia) alters basal NHE-1 expression or the subsequent response to insult. Ischemia with or without reperfusion increased NHE-1 expression approximately sevenfold (P < 0.05), whereas either H2O2 or LPC increased expression approximately twofold. Preconditioning reduced NHE-1 message by approximately 70% (P < 0.05) and significantly attenuated the effects of ischemia, H2O2, or LPC. The internal standard, beta-globin was unaffected by any treatment. Our results indicate that NHE-1 expression is rapidly increased in response to ischemia with or without reperfusion as well as in response to H2O2 or LPC. In contrast, preconditioning was associated with downregulation of NHE-1. These results may be important in furthering our understanding of NHE-1 in cardiac disease states and suggest that the antiporter adapts rapidly to cardiac conditions associated with pathology.

Animals↗