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Biomedical subjects

A Sharma

Publications and source records attributed to A Sharma.

At least 163 records · Page 9Linked to original sources
PubMed2001

Transfection of mGSTA4 in HL-60 cells protects against 4-hydroxynonenal-induced apoptosis by inhibiting JNK-mediated signaling.

The mammalian alpha-class glutathione S-transferase (GST) isozymes mGSTA4-4, rGSTA4-4, and hGSTA4-4 are known to utilize 4-hydroxynonenal (4HNE) as a preferred substrate. During the present studies, we have examined the effect of transfecting human myeloid HL-60 cells with mGSTA4, on 4-HNE-induced apoptosis and the associated signaling mechanisms. Results of these studies show that treatment of the wild-type or vector-only-transfected HL-60 cells with 20 microM 4-HNE caused apoptosis within 2 h. The cells transfected with mGSTA4 did not undergo apoptosis under these conditions even after 4 h. In the wild-type and vector-transfected cells, apoptosis was preceded by JNK activation and c-Jun phosphorylation within 30 min, and an increase in AP-1 binding within 2 h of treatment with 20 microM 4-HNE. In mGSTA4-transfected cells, JNK activation and c-Jun phosphorylation were observed after 1 h, and increased AP-1 binding was observed after 8 h under these conditions. In the control cells, 20 microM 4-HNE caused caspase 3 activation and poly(ADP-ribose) polymerase cleavage within 2 h, while in mGSTA4-transfected cells, a lesser degree of these effects was observed even after 8 h. Transfection with mGSTA4 also provided protection to the cells from 4-HNE and doxorubicin cytotoxicity (1.6- and 2.6-fold, respectively). These results show that 4-HNE mediates apoptosis through its effects on JNK and caspase 3, and that 4-HNE metabolizing GST isozyme(s) may be important in the regulation of this pathway of oxidative-stress-induced apoptosis.

Aldehydes↗
PubMed2001

Effects of orally active taxanes on P-glycoprotein modulation and colon and breast carcinoma drug resistance.

BACKGROUND: The taxane paclitaxel (Taxol) is often of limited efficacy in chemotherapeutic regimens because some cancer cells express high levels of the efflux pump, P-glycoprotein (Pgp), which removes the drug from the cells. The orally active paclitaxel analog IDN-5109 has been reported to overcome Pgp-mediated drug resistance. We tested whether IDN-5109 acts by modulating Pgp activity. METHODS: Human MDA435/LCC6mdr1 and MDA435/LCC6 breast carcinoma cells, which express and do not express Pgp, respectively, were incubated with [3H]IDN-5109 and paclitaxel to determine intracellular drug accumulation. Flow cytometry was used to analyze intracellular retention of two Pgp substrates, rhodamine 123 (Rh-123) and doxorubicin, in both breast carcinoma cell lines and in human colon carcinoma cells (SW-620, DLD1, and HCT-15, whose Pgp levels vary) treated with different taxanes. The effects of IDN-5109 and paclitaxel on tumor growth in vivo were studied with the use of tumors established through xenografts of Pgp-expressing SW-620 and DLD1 cells in severe combined immunodeficiency mice. All statistical tests were two-sided. RESULTS: Pgp-expressing cells treated with IDN-5109 or with the taxane-based drug resistance reversal agent tRA96023, which blocks Pgp activity, retained 8.1- and 9.4-fold more Rh-123 (P =.0001), respectively, and 1.7- and 1.9-fold more doxorubicin (P =.001), respectively, than cells treated with paclitaxel. Non-Pgp-expressing cells treated similarly demonstrated no increased retention of either substrate. MDA435/LCC6mdr1 cells retained 5.3-fold more [3H]IDN-5109 than [3H]paclitaxel after 2 hours (P =.01). IDN-5109 showed statistically significantly higher tumor growth inhibition than paclitaxel against the SW-620 xenograft (P =.003). CONCLUSIONS: IDN-5109 modulates Pgp activity, resulting in superior tumor growth inhibition against Pgp-expressing tumors as compared with paclitaxel. IDN-5109 may broaden the spectrum of taxane use to include colon tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress.

It is known well that activation of the hexosamine pathway causes insulin resistance, but how this activation influences pancreatic beta-cell function remains unclear. In this study, we found that in isolated rat islets adenovirus-mediated overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the first and rate-limiting enzyme of the hexosamine pathway, leads to deterioration of beta-cell function, which is similar to that found in diabetes. Overexpression of GFAT or treatment with glucosamine results in impaired glucose-stimulated insulin secretion and reduction in the expression levels of several beta-cell specific genes (insulin, GLUT2, and glucokinase). Additionally, the DNA binding activity of PDX-1, an important transcription factor for these three genes, was markedly reduced. These phenomena were not mimicked by the induction of O-linked glycosylation with an inhibitor of O-GlcNAcase, PUGNAc. It was also found that glucosamine increases hydrogen peroxide levels and that several hexosamine pathway-mediated changes were suppressed by treatment with the antioxidant N-acetyl-l-cysteine. In conclusion, activation of the hexosamine pathway leads to deterioration of beta-cell function through the induction of oxidative stress rather than O-linked glycosylation. Thus, the hexosamine pathway may contribute to the deterioration of beta-cell function found in diabetes.

Animals↗
PubMed2001

Templating of thin films induced by dewetting on patterned surfaces.

The instability, dynamics, and morphological transitions of patterns in thin liquid films on chemically heterogeneous striped surfaces are investigated based on 3D nonlinear simulations. The film breakup is suppressed on some potentially destabilizing nonwettable sites when their spacing is below a characteristic length scale of the instability, lambda(h). The thin film pattern replicates the substrate surface energy pattern closely only when (i) the periodicity of substrate pattern lies between lambda(h) and 2lambda(h), and (ii) the stripe width is within a range bounded by a lower critical length, below which no heterogeneous rupture occurs, and an upper transition length above which complex morphological features unlike the substrate pattern are formed.

Journal Article↗
PubMed2001

Enhanced maturation of porcine neonatal pancreatic cell clusters with growth factors fails to improve transplantation outcome.

BACKGROUND: Porcine neonatal pancreatic cell clusters (NPCC) are a potential source of islet tissue for clinical transplantation. They can normalize glycemia after transplantation, although after a relatively long (several weeks) period of time, possibly due to the immaturity of the tissue. METHODS: One week after isolation NPCCs were immobilized in alginate hydrogel to be cultured for 2 more weeks in the presence of different growth factors, which were applied individually or in various combinations. Their effect was assessed by measuring DNA and insulin content, and expression of islet genes by reverse transcriptase-polymerase chain reaction. Enhanced maturation of NPCCs was also evaluated after transplantation in streptozotocin-diabetic mice. RESULTS: A combination of fetal calf serum, insulin-like growth factor-I, nicotinamide and sodium butyrate in NPCCs media from day 7 to day 21 resulted in increased insulin/DNA content and higher expression of insulin, somatostatin, GLUT2 and Nkx6.1 genes. NPCCs cultured under the same conditions from day 3 to day 12 were transplanted into diabetic mice. Control mice were transplanted with NPCCs cultured in parallel in the presence of nicotinamide, but with no serum, insulin-like growth factor-I or butyrate. Normoglycemia was achieved at the same rate in both groups. Plasma porcine C-peptide (week 6) and graft insulin content (week 20) were also similar in both groups. CONCLUSIONS: Increased insulin content of NPCCs was achieved in vitro by addition of fetal calf serum, insulin-like growth factor-I, nicotinamide, and sodium butyrate, but this increase did not translate into a faster achievement of normoglycemia after transplantation, which suggests that there is a time frame required for complete maturation that is difficult to alter.

Animals↗
PubMed2001

Effect of 2',4'-dichlorobenzamil hydrochloride, a Na(+)-Ca(2+) exchange inhibitor, on human spermatozoa.

The present study is aimed to investigate the contact spermicidal efficacy of 2',4'-dichlorobenzamil hydrochloride (DBZ), a Na(+)-Ca(2+) exchange inhibitor, on ejaculated human spermatozoa. The drug produced a dose- and time-dependent spermicidal action on human spermatozoa. A concentration of 4 mM produced total loss of sperm viability within 1 min of addition to total semen. On the other hand, a similar action on spermatozoa separated from semen was noted at 0.5 mM concentration. The loss of spermatozoal viability was accompanied with an increase in intracellular Ca(2+). Sperm revival testing with glucose suggested a spermicidal rather than a spermiostatic action.

Amiloride↗
PubMed2001

Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets. METHODS: Using semiquantitative multiplex reverse-transcribed polymerase chain reaction we determined the gene expression of VEGF and its receptors in cultured and transplanted rat islets. RESULTS: After exposure of islet cells to hypoxia in vitro, increases were found in the gene expression of the VEGF120 and VEGF164 isoforms, with simultaneous increases in VE-cadherin, Flk-1/KDR, and Flt-1. In vivo studies consisted of analysis of islet grafts transplanted into both normal and diabetic recipients. Expression of both VEGF120 and VEGF164 in grafts was up-regulated for the first 2-3 days after transplantation, with the response being more prolonged in the diabetic rats. These increases were followed by reduced expression of VEGF on days 5, 7, and 9. Increases in the expression of VE-cadherin in islet grafts in normal and diabetic recipients tended to parallel VEGF expression, with the increases in both probably being caused by hypoxia. The early increases of VEGF expression were followed by a rise in the expression of VEGF receptors, which probably represents the early stages of angiogenesis. Graft expression of Flk-1/KDR and Flt-1 was enhanced at 3 and 5 days in the normoglycemic recipients, while in the diabetic recipients increases were found later on days 5, 7, and 14. CONCLUSIONS: The delayed expression of VEGF receptors in the diabetic recipients could reflect impaired angiogenesis caused by the diabetic milieu; this delay could contribute to the less outcomes of grafts transplanted into a hyperglycemic environment.

Animals↗
PubMed2001

Liver irradiation: a potential preparative regimen for hepatocyte transplantation.

Advances in the understanding of hepatocyte engraftment and repopulation of the host liver have already led to the use of hepatocyte transplantation (HT) with some success in the treatment of inherited and acquired liver diseases. Wider application of HT is severely limited by the unavailability of large number of transplantable hepatocytes and difficulties associated with transplanting an adequate number of cells for achieving therapeutically satisfactory levels of metabolic correction. Therefore, there is a need for preparative regimens that provide a growth advantage to the transplanted (healthy) hepatocytes over the host's own (diseased) hepatocytes so that the former can repopulate the host liver. We have recently shown that when the liver of recipient rats was subjected to radiotherapy and partial hepatectomy before HT, the transplanted hepatocytes engrafted in and massively repopulated the liver, and also ameliorated the adverse clinical and histopathological changes associated with hepatic irradiation. This protocol was then used as a preparative regimen for transplanting normal hepatocytes into jaundice mutant rats (Gunn strain), which lack hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase and is a model of Crigler-Najjar syndrome Type I. The results showed long-term correction of the metabolic abnormality, suggesting that the transplanted hepatocytes repopulated an irradiated liver and were metabolically functional. This strategy could be useful in the treatment of various genetic, metabolic, or malignant diseases of the liver.

Animals↗
PubMed2001

Transcription factors recognizing overlapping C1-A2 binding sites positively regulate insulin gene expression.

Transcription factors binding the insulin enhancer region, RIPE3b, mediate beta-cell type-specific and glucose-responsive expression of the insulin gene. Earlier studies demonstrate that activator present in the beta-cell-specific RIPE3b1-binding complex is critical for these actions. The DNA binding activity of the RIPE3b1 activator is induced in response to glucose stimulation and is inhibited under glucotoxic conditions. The C1 element within the RIPE3b region has been implicated as the binding site for RIPE3b1 activator. The RIPE3b region also contains an additional element, A2, which shares homology with the A elements in the insulin enhancer. Transcription factors (PDX-1 and HNF-1 alpha) binding to A elements are critical regulators of insulin gene expression and/or pancreatic development. Hence, to understand the roles of C1 and A2 elements in regulating insulin gene expression, we have systematically mutated the RIPE3b region and analyzed the effect of these mutations on gene expression. Our results demonstrate that both C1 and A2 elements together constitute the binding site for the RIPE3b1 activator. In addition to C1-A2 (RIPE3b) binding complexes, three binding complexes that specifically recognize A2 elements are found in nuclear extracts from insulinoma cell lines; the A2.2 complex is detected only in insulin-producing cell lines. Furthermore, two base pairs in the A2 element were critical for binding of both RIPE3b1 and A2.2 activators. Transient transfection results indicate that both C1-A2 and A2-specific binding activators cooperatively activate insulin gene expression. In addition, RIPE3b1- and A2-specific activators respond differently to glucose, suggesting that their overlapping binding specificity and functional cooperation may play an important role in regulating insulin gene expression.

Animals↗
PubMed2001

Pattern Formation in a Thin Solid Film with Interactions.

We investigate a new type of surface instability of a thin elastic film subjected to surface interactions such as van der Waals and electrostatic forces from another solid surface in its vicinity. It is found that a sufficiently soft (shear modulus <10 MPa) and nearly incompressible film deforms to form an undulating pattern without any mass transport. A novel feature is that the characteristic length scale of the pattern is nearly independent of the nature and magnitude of the external force, but varies linearly with the film thickness. These results explain some recent experiments and are applicable to problems such as adhesion and friction at soft solid interfaces, peeling of adhesives, patterning of solid surfaces, etc.

Journal Article↗
PubMed2001

Burkitt's lymphoma of stomach: A case report and review of literature.

Primary gastrointestinal non-Hodgkin's lymphoma is a distinct clinicopathological entity. This is the commonest site of all extranodal lymphomas. Non-endemic Burkitt's lymphoma (high-grade, small non-cleaved lymphocytic type) is rare in non-HIV adult population. We hereby report Burkitt's lymphoma of stomach in a non-HIV adult. Gastric and lymph node biopsy confirmed the diagnosis. Patient was treated using a third generation chemotherapy protocol without any surgical resection. The patient relapsed within 3 months of completion of primary treatment and died of progressive disease.

Adult↗
PubMed2001

Crossreactivity of human anti-dsDNA antibodies to phosphorylcholine: clues to their origin.

The presence of anti-double stranded DNA (dsDNA) antibodies is a serological diagnostic feature of systemic lupus erythematosus (SLE), an autoimmune rheumatic disorder. Studies by several investigators have suggested that a response to a microbial antigen can lead to the induction of SLE-like autoimmunity, in both humans and mice, since anti-dsDNA antibodies have been shown to crossreact with foreign antigens. In particular, anti-DNA antibodies have been shown to crossreact with phosphorylcholine (PC), a dominant epitope on pneumococcal cell wall polysaccharide. We have investigated the binding characteristics of human polyclonal anti-DNA antibodies from the sera of SLE patients. In this study we show that the DNA binding of polyclonal serum derived antibodies can be partially inhibited by phosphorylcholine (PC). The binding of affinity-purified anti-DNA antibodies from the sera of patients with SLE was also found to be inhibited by PC. We further demonstrated that the serum IgG1 (T dependent) anti-DNA response was more likely to crossreact with PC than the IgG2 (T independent) response to DNA. The studies suggest there may be a T dependent and T independent response to DNA with the T dependent response displaying more crossreactivity with microbial antigen.

Antibodies, Antinuclear↗
PubMed2001

Subcutaneous epinephrine vs nebulized salbutamol in asthma.

OBJECTIVE: This study was conducted to compare the efficacy of the subcutaneous epinephrine with nebulized salbutamol. METHODS: Fifty asthmatic children in the age range of 6-14 years were divided into two equal groups. Group I children were given subcutaneous epinephrine and Group II were nebulized with salbutamol. Patients were observed at 15, 20, 30, 60, 120, 180 and 240 minute intervals. Both the groups had comparable mean increase in peak expiratory flow rate (PEFR %) (Group I 27.7 +/- 0.7; Group II 28.8 +/- 0.06, p >0.05). RESULT: In Group I there was significant increase in systolic blood pressure, 30 minutes after the start of treatment, however it settled on its own by 60 minutes. CONCLUSION: Both the groups had satisfactory improvement in clinical parameters which continued upto 4 hours after start of treatment. Subcutaneous epinephrine can be safely used if nebulizers are not available.

Acute Disease↗
PubMed2001

Quantifying the 5-HT1A agonist action of buspirone in man.

RATIONALE: Buspirone is used as a neuroendocrine challenge in which the increase of circulating prolactin is taken as a measure of the sensitivity of central serotonergic (5-HT(1A)) pathways. Interpretation of the test is complicated, however, by the fact that buspirone possesses D(2) antagonist and 5-HT(1A) agonist activity, both of which will result in the release of prolactin. To understand the significance of prolactin secretion in response to buspirone, it is important to measure the differential actions of the two controlling pathways. OBJECTIVE: To characterise the dual action of buspirone in stimulating the secretion of prolactin by blocking the 5-HT(1A) action with the 5-HT1A antagonist action of pindolol. METHODS: Healthy male subjects (n=35) received buspirone (0.5 mg x kg bw(-1) orally) with and without pre-treatment with the 5-HT(1A) receptor antagonist pindolol (40 mg over 2 days, 0.5 mg x kg bw(-1) on test day). Nine subjects underwent two additional trials in which they received a placebo with and without pre-treatment with pindolol. RESULTS: Pindolol alone caused a small but significant reduction (18%) in the tonic release of prolactin. Buspirone alone produced a robust prolactin response which was reduced to approximately half by pindolol pre-treatment. Pindolol pre-treatment also, on average, delayed the onset and peak of the prolactin response. There was wide variation among individuals both in the absolute response to buspirone and in the proportion that could be attributed to the non-serotonergic agonist action of buspirone (22-82% IQ range). CONCLUSIONS: Our results indicate that while serotonergic pathways play a minor role in the tonic release of prolactin, the response to a buspirone challenge alone cannot be used as a simple index of central serotonergic activity. However, if two challenges are carried out, one with buspirone and the other with buspirone plus pindolol, quantitative measures can be made of the sensitivity of both the 5-HT(1A) and the putative D(2) pathways controlling prolactin release.

Adolescent↗
PubMed2001

Keratonconjunctivitis sicca is not uncommon in children with juvenile rheumatoid arthritis.

Keratoconjunctivitis sicca (KCS), characterised by aqueous tear deficiency, is the most common ocular complication in adult rheumatoid arthritis (RA). In juvenile rheumatoid arthritis (JRA), however, it remains under-reported. For this prospective study, 50 children with JRA were examined clinically and underwent tests for KCS (Schirmer's I and rose bengal tests, fluorescein staining, and tear film breakup time). Six children (12%) with two or more abnormal tests were diagnosed as having definite KCS, while one child with only one abnormal test was labelled with probable KCS. Five of the six (83.3%) with definite KCS were males, and three (50%) had a pauciarticular form of the disease. Two children (33.3%) with definite KCS had no ocular symptoms, five were receiving only nonsteroidal anti-inflammatory drugs, and one was additionally on methotrexate. Keratoconjunctivitis sicca appears to be a common ocular complication and all children with JRA should be screened for it with a comprehensive battery of tests.

Adolescent↗