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Biomedical subjects

R Sharma

Publications and source records attributed to R Sharma.

At least 91 records · Page 5Linked to original sources
PubMed1995

A genetically engineered fusion protein M4/TNF with increased bifunctional activity refolded in the presence of protein disulfide isomerase.

Recombinant DNA techniques were used to clone, to construct and to express a fusion protein M4/TNF in Escherichia coli. The fusion protein includes the chimeric F(ab')2 fragment (M4) recognizing the human tumor-associated TAG72 antigen and the tumor necrosis factor alpha (TNF) moiety. The M4/TNF purified from inclusion bodies of the bacteria homogenates was further solubilized in a denaturing buffer containing 6 mol l-1 guanidine and refolded in a refolding buffer. Our results showed that the M4/TNF refolded in a buffer containing 6 mmol l-1 oxidized glutathione (GSSG), 0.2 mmol l-1 dithioerythione (DTE) and 0.5 mumol l-1 protein disulfide isomerase (PDI) displayed a 4-fold higher anti-TAG72 immunoreactivity and a 5-fold higher TNF activity than that refolded in the same refolding buffer but without PDI. Our data thus indicates that the protein disulfide isomerase not only facilitates the correct formation of disulfide-bonds of the antibody molecule, but also the correct refolding of the TNF moiety in vitro.

Amino Acid Sequence↗
PubMed1995

Microcolpohysteroscopy compared with colposcopy in evaluation of abnormal cervical cytology.

Microcolpohysteroscopy (MCH) permits in vivo visualisation of cervical epithelium and is a useful procedure in the diagnostic workup of a patients with abnormal pap smear. Our study compared standard colposcopy with MCH in 45 patients with dysplasia reported on cytology. On comparing with final histopathological diagnosis colposcopy had an accuracy of 95.6 percent in diagnosing cervical intraepithelial neoplasia (CIN) within one grade. MCH had a diagnostic accuracy of 84.4 percent in predicting CIN changes. Further MCH was able to evaluate the endocervical squamocolumner junction in all cases of unsatisfactory colposcopy. Microcolpohysteroscopy is a useful adjunct to colposcopy and is also of value in locating the epicenter of lesions and is diagnosing HPV infection. MCH prior to conisation is of value in tailoring the biopsy to fully include the CIN lesion.

Adult↗
PubMed1995

Tissue magnesium content and histopathological changes in non-survivors of aluminium phosphide poisoning.

The study was conducted in 30 non-survivors of Aluminium Phosphide poisoning and similar number of age and sex matched controls (fatalities as a result of road side accidents, head injury, etc). Magnesium content was estimated in brain, heart, stomach, kidney, liver and lung using atomic absorption spectrophotometer. It was found that tissue magnesium levels were not significantly different (p = NS) when comparison was carried out between controls (Group II) and Patients who were not given magnesium as part of treatment (group IB). However magnesium levels in different organs of patients who received magnesium as part of treatment were found to be significantly higher (group IA)(P < 0.01) in comparison to controls as well as patient group not treated with magnesium sulphate. Significant histopathological changes were observed in almost all the organs. The changes seem to be the result of direct tissue damage by phosphine rather than shock and anoxia which occurred in all these cases.

Adolescent↗
PubMed1994

The discovery of novel, structurally diverse protein kinase C agonists through computer 3D-database pharmacophore search. Molecular modeling studies.

A computer protein kinase C (PK-C) pharmacophore search on 206,876 nonproprietary structures in the NCI 3D-database led to the discovery of five compounds which were found to possess PK-C binding affinities in the low micromolar range and six others having detectable, but marginal, binding affinities. Molecular modeling studies showed that in addition to the presence of the defined pharmacophore, hydrophobicity and conformational energy are the two other important factors determining the PK-C binding affinity of a compound. The modeling results were confirmed by synthetic modification of two inactive compounds, producing two active derivatives. These newly discovered, structurally diverse lead compounds are being used as the basis for further synthetic modifications aimed at more potent PK-C ligands that will compete with the phorbol esters.

Information Systems↗
PubMed1994

Comparison of a conformal nonaxial boost with a four-field boost technique in the treatment of adenocarcinoma of the prostate.

PURPOSE: A comparative study of a standard four-field boost technique and a customized nonaxial external beam technique for the treatment of adenocarcinoma of the prostate was performed to quantify rectal and bladder sparing. METHODS AND MATERIALS: Sixteen patients with localized carcinoma of the prostate who underwent simulation and treatment planning computed tomography formed the basis of this study. The prostate, seminal vesicles, lymph node, pelvic bones, rectum, bladder, and skin volumes were contoured from the CT slices to the three-dimensional treatment planning system. Each patient was planned for both standard four-field and nonaxial techniques to a boost treatment dose of 24 Gy to the prostate. Isodose plans were chosen on the basis of adequate tumor coverage and normal tissue sparing. RESULTS: The volumes of the prostate, rectum, and bladder ranged from 46-148 cc, 25-196 cc, and 50-378 cc, respectively. Data analysis demonstrated a statistically significant reduction (p < 0.01) in normal tissue irradiation with the nonaxial external beam technique at dose levels of 15 and 20 Gy. CONCLUSION: The nonaxial four-field external beam technique appears to be superior to the standard four-field approach in reducing the volume of high dose irradiation to the adjacent rectum and bladder.

Adenocarcinoma↗
PubMed1994

Life, death and genomic change in perturbed cell cycles.

HeLaS3 cells undergo apoptosis after 18-24 h of cell cycle stasis irrespective of the agent employed (colcemid, aphidicolin, cis-platin). At high drug concentrations apoptosis occurs in cells arrested in the cell cycle in which the drug is applied and at a cell cycle position dependent on the mechanism of drug action. At low concentrations (or short exposure times) cells undergo apoptosis after progressing through an aberrant mitosis and only after 18 h of cell cycle stasis in a 'pseudo G1/S' cell cycle position. Aberrent mitoses result in multipolar mitoses, chromosomal breakage and interchromosomal concatenation events. We propose that the ability of cells to delay progression into aberrent mitosis, as well as their propensity to undergo apoptosis, are important determinants of clinical cytotoxicity. We also suggest that apoptosis plays an important role in preventing the generation of aneuploidy and recombination and rearrangement events commonly associated with cancer.

Animals↗
PubMed1994

A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues.

A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the alpha-class isozymes of GST. It is characterized by kinetic properties intermediate between the alpha- and pi-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P., et al. (1992) FEBS Lett., 313, 173-176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other alpha-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with M(r) values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxides suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.

Amino Acid Sequence↗
PubMed1994

Estimation of genomic complexity, heterologous expression, and enzymatic characterization of mouse glutathione S-transferase mGSTA4-4 (GST 5.7).

We have previously isolated a cDNA clone for a unique mouse lung glutathione S-transferase, mGSTA4-4 (GST 5.7) (Zimniak, P., Eckles, M. A., Saxena, M., and Awasthi, Y. C. (1992) FEBS Lett. 313, 173-176). By genomic Southern blotting and polymerase chain reaction single strand conformation polymorphism analysis we have now demonstrated the presence of at least two mGSTA4-related genes in the mouse. The heterogeneity of mGSTA4-4 was further examined by comparing the structural and kinetic properties of mGSTA4-4 isolated from mouse lung with those of recombinant rec-mGSTA4-4 expressed in Escherichia coli. Except for the isoelectric point, the physical properties of the two proteins were indistinguishable. Western blots using antibodies against rec-mGSTA4-4 have shown selective expression of the enzyme in mouse tissues. Even though the substrate specificity profiles of the tissue-isolated and recombinant enzymes, which point to a role of mGSTA4-4 in the detoxification of lipid peroxidation products, were generally similar, significant differences were observed with selected substrates. The existence of functionally distinct forms of mGSTA4-4 and the presence of more than one gene strongly suggest that the previously observed differences in properties of mGSTA4-4 isolated from various mouse tissues (Awasthi, S., Singhal, S. S., Srivastava, S. K., and Awasthi, Y. C. (1993) Arch. Biochem. Biophys. 301, 143-150) may be due to tissue-specific expression of mGSTA4-related genes.

Animals↗
PubMed1994

Differentiation of malignant and degenerative bone lesions using dexamethasone interventional 3- and 24-hour bone scintigraphy.

Seventy-seven adult patients with suspected skeletal metastases were divided into two groups. In group A (n = 30), following intravenous administration of 20 mCi (740 MBq) of technetium-99m methylene diphosphonate (99mTc-MDP), 3- and 24-h scintigraphy of bone lesions was performed. The 24/3 h lesion to bone background radiouptake ratio (RUR) was calculated for each lesion. In group B (n = 47), the same procedure was followed with dexamethasone intervention (10 mg in 24 h) following the 3-h acquisition. In group A, after determination of the critical point, malignant and degenerative bone lesions could be separated with a sensitivity, specificity and accuracy of 0.76, 0.72 and 0.73, respectively. The mean RUR of the malignant lesions was 1.20 +/- 0.23, and that of the benign lesions, 0.95 +/- 0.15. In group B cases, significantly increased sensitivity, specificity and accuracy of 0.87, 0.94 and 0.92, respectively, were found (P < 0.001). The mean RUR of the malignant lesions was 1.48 +/- 0.34, and that of degenerative lesions, 0.88 +/- 0.19. Dexamethasone interventional bone scintigraphy seems to be a new cost-effective method for differentiating malignant from degenerative bone lesions using the RUR.

Bone Diseases↗
PubMed1994

Human glutathione S-transferases.

1. Multiple forms of glutathione S-transferase (GST) isoenzymes present in human tissues are dimers of subunits belonging to three distinct gene families namely alpha, mu and pi. Only the subunits within each class hybridize to give active dimers. 2. These subunits are differentially expressed in a tissue-specific manner and the composition of glutathione S-transferases in various tissues differs significantly. 3. Minor GST subunits not belonging to these three classes are also present in some tissues. 4. An ortholog of rat GST 8-8 and mouse mGSTA4-4 is selectively expressed in some human tissues including bladder, brain, heart, liver, and pancreas. This isoenzyme designated as GST 5.8 expresses several fold higher activity towards 4-hydroxy-2,3-trans-nonenal as compared to the routinely used substrate 1-chloro-2,4-dinitrobenzene.

Animals↗
PubMed1994

Mortality in affective disorder.

472 bipolar patients were followed up retrospectively over 17 years. The patients that died were compared with the general population and a control group. The former comparison showed greater mortality from suicide, cardiovascular and respiratory causes in the index population and the latter, that the deceased were more likely to have been unmarried, with greater frequency and duration of admissions, a shorter follow-up period and were less likely to have received lithium treatment. The suicides were significantly younger at onset and death than the index and control groups, and suicide was uncommon where follow-up extended over 10 years.

Adult↗
PubMed1994

The epidermal growth factor mitogenic signal is modulated by protein kinase C in T51B rat liver cells.

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12-O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.

Animals↗
PubMed1994

Plasmapheresis reduces proteinuria and serum capacity to injure glomeruli in patients with recurrent focal glomerulosclerosis.

To test the hypothesis that a circulating mediator is associated with recurrent idiopathic focal glomerulosclerosis (FGS), we studied the effect of plasmapheresis on reducing proteinuria in patients with the disease. An in vitro assay measured the capacity of sera before and after plasmapheresis to cause increased albumin permeability (P(albumin)++) in isolated rat glomeruli. Nine patients (five males aged 2 to 66 years) who underwent plasmapheresis for recurrent FGS were identified. Study variables included age, sex, time from diagnosis of recurrence to first pheresis, glomerular hyalinosis, complications, outcome, and proteinuria before and after plasmapheresis. Rat glomeruli were isolated in medium containing 4 g/dL bovine serum albumin, and P(albumin) was determined from the change in glomerular volume in response to an albumin gradient after incubation of the glomeruli in a 1:50 dilution of patient serum. Plasmapheresis reduced proteinuria from a mean of 12 +/- 7.46 g/24 hr to 5.1 +/- 7.39 g/24 hr (P = 0.03). Six patients in whom the diagnosis was made early in the course of the disease and in whom plasmapheresis was initiated immediately had lasting remissions. Preplasmapheresis biopsies in the patients who did not achieve remissions showed both epithelial foot process effacement and glomerular sclerosis. Serum samples were available from four patients for albumin testing in vitro. P(albumin)++ was reduced from a mean of 0.76 +/- 0.17 before pheresis to 0.18 +/- 0.31 after (P = 0.07). Therefore, the mechanism by which plasmapheresis reduces proteinuria in patients with recurrent FGS involves the decreased capacity of sera from these patients to injure the glomerular permeability barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Modulation of cisplatin cytotoxicity by sulphasalazine.

The efficacy of cisplatin [cis-diamminedichloroplatinum (II); DDP] is hampered by acquired or de novo resistance of malignant cells to its cytotoxic effects. We have previously reported that cisplatin resistance parallels glutathione S-transferase (GST) activity in several human small-cell lung cancer cell lines. In the presently described studies, we used sulphasalazine, an inhibitor of GSTs, to evaluate the relative role of GSTs in mediating cisplatin resistance in two human small-cell lung cancer cell lines, NCI H-69 and H-2496. The H-69 cell line, which contained relatively higher GST activity than the H-2496 cell line (317 +/- 7 vs 9 +/- 1 mU mg-1 protein respectively), also displayed a greater degree of cisplatin resistance (IC50 values of 25.0 +/- 3.9 vs 4.5 +/- 1.0 microM respectively). Western blot and Northern blot analyses of purified GSTs revealed the expression of only the pi-class GST in both cell lines. Sulphasalazine inhibited the purified GSTs (IC50 of 10 microM for H-69 and 12 microM for H-2496) from both lines in a competitive manner with similar Ki values (6.5 and 7.9 microM for the H-69 and H-2496 cell lines respectively). Cytotoxicity studies revealed that sulphasalazine increased the cytotoxicity of cisplatin towards both cell lines. Isobologram analysis showed that sulphasalazine synergistically enhanced the cytotoxicity of cisplatin towards both cell lines, the magnitude of synergy being remarkably higher in H-69 cells than in H-2496 cells. Our studies indicate that clinically achievable concentrations of sulphasalazine may be useful in modulating cisplatin resistance in malignancies with increased GST-pi content.

Carcinoma, Small Cell↗
PubMed1994

Polymorphonuclear leukocytes increase glomerular albumin permeability via hypohalous acid.

Acute glomerulonephritis is characterized by the presence of neutrophils within glomeruli and the generation of reactive oxygen species (ROS) by activated polymorphonuclear leukocytes (PMNs). Hydrogen peroxide (H2O2) and other ROS including hypothalous acids have been implicated in PMN mediated injury. To determine the role of specific ROS in PMN mediated glomerular injury, isolated rat glomeruli were incubated for 30 minutes at 37 degrees C with H2O2, with H2O2 and myeloperoxidase, or with activated PMNs. Scavengers of ROS were included in some experiments. PMNs were harvested from rat peritoneal cavity and activated with phorbol myristate acetate (PMA). Glomerular albumin permeability (Palbumin) was calculated from the volume response to an oncotic gradient. Palbumin of glomeruli incubated with H2O2 (10(-3) or 10(-1) M) was not increased, while Palbumin after incubation with H2O2 and MPO was markedly increased (0.94 +/- 0.004). Palbumin after incubation with PMA, or with non-activated PMNs was not different from that of control glomeruli, Palbumin of the glomeruli incubated with activated PMNs increased (0.85 +/- 0.01, P < 0.001). This increase in Palbumin was inhibited by superoxide dismutase, catalase, or taurine (Palbumin = 0.035 +/- 0.06, -0.39 +/- 0.10, 0.028 +/- 0.06, respectively) and ameliorated by sodium azide (Palbumin = 0.21 +/- 0.03). In contrast, dimethyl sulfoxide did not prevent the increase in Palbumin (Palbumin = 0.92 +/- 0.01). Our results show that the hypohalous acid derived from that of H2O2-MPO-halide system is capable of increasing Palbumin. We conclude that hypohalous acid may be the primary mediator of the immediate increase in glomerular protein permeability induced by PMNs.

Albumins↗
PubMed1994

Influence of certain essential oils on carcinogen-metabolizing enzymes and acid-soluble sulfhydryls in mouse liver.

The influence of essential oils from naturally occurring plant dietary items such as cardamom, celery seed, cumin seed, coriander, ginger, nutmeg, and zanthoxylum on the activities of hepatic carcinogen-metabolizing enzymes (cytochrome P450, aryl hydrocarbon hydroxylase, and glutathione S-transferase) and acid-soluble sulfhydryl level was investigated in Swiss albino mice. Each oil was fed by gavage at 10 microliters/day for 14 days, and then the animals were sacrificed and their hepatic enzyme activities and sulfhydryl levels were evaluated. Only nutmeg and zanthoxylum oils induced cytochrome P450 level significantly (p < 0.05), whereas cardamom oil caused a significant reduction in its activity (p < 0.05). Furthermore, aryl hydrocarbon hydroxylase activity was significantly elevated only by treatment with ginger oil (p < 0.01), whereas nutmeg oil caused a significant reduction in its activity (p < 0.01). The remaining oils did not significantly alter the level of cytochrome P450 and aryl hydrocarbon hydroxylase activity. Glutathione S-transferase activity was significantly elevated in all experimental groups (p < 0.1-p < 0.001) compared with controls. The acid-soluble sulfhydryl was significantly elevated only by the essential oils of cardamom (p < 0.05), nutmeg (p < 0.05), and zanthoxylum (p < 0.01). Our observations suggest that intake of essential oils affects the host enzymes associated with activation and detoxication of xenobiotic compounds, including chemical carcinogens and mutagens.

Animals↗
PubMed1994

Modulation of phenobarbitone-induced loss of gap junctional intercellular communication in hepatocyte couplets.

Phenobarbitone (PB) produced a dose- and time-dependent decrease in gap junctional intercellular communication (GJIC) (up to 25.0 +/- 5.3% inhibition) in rat hepatocyte couplets (4 h cultures). The effect was reversible and independent of protein synthesis. This inhibition was exacerbated (to 53.3 +/- 5.4% inhibition) by depletion of intracellular glutathione following pretreatment with diethylmaleate (0.5 microM, 15 min). Inhibition was also significantly enhanced by addition of the cytochrome P450 inhibitors SKF 525A (25 microM) and metyrapone (20 nM). In contrast, hepatocyte couplets derived from rats pretreated with PB (0.1% w/v in drinking water) for up to 28 days were fully functional regarding GJIC and were found to be refractory to the effects of PB added in vitro. This, coupled with the lack of effect of p-hydroxy-PB, suggests that an active metabolite of PB is not involved in the inhibition of GJIC which may, instead, be through an oxidative stress, which is prevented by glutathione.

Animals↗