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Biomedical subjects

S Sharma

Publications and source records attributed to S Sharma.

At least 343 records · Page 19Linked to original sources
PubMed2000

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution.

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment.

Acetonitriles↗
PubMed2000

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo.

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.

Animals↗
PubMed2000

Characterization of domains of the phosphoriboprotein P0 of Plasmodium falciparum.

Antibodies against the amino-terminal domain of the Plasmodium falciparum P0 phosphoriboprotein were detected extensively in immune people living in malaria endemic areas of India. It has been shown earlier that specific antibodies raised against the PfP0N domain (17-61 amino acid) of the PfP0 protein inhibit P. falciparum growth in vitro. To study the properties of the rest of the protein, the remaining 61-316 amino acids on the carboxy-side of the PfP0 protein were expressed as a glutathione-S-transferase fusion protein (PfP0C). Antibodies raised against PfP0C identified the 38 kDa P0 protein on a parasite Western blot analysis. An ELISA assay using both the PfP0N and PfP0C fusion proteins showed no reactivity with malaria patient sera samples, but showed extensive reactions with the immune sera. Antibodies against both the PfP0C and PfP0N domains were raised in rabbits and different inbred strains of mice. T-cells from immunized mice showed lymphoproliferation when presented with PfP0 protein domains. IgG from both anti-PfP0N and anti-PfP0C sera inhibited the growth of P. falciparum in vitro in a concentration dependent manner. The IgG did not show any significant effect on the growth of intraerythrocytic stages, but specifically inhibited re-invasion of red cells. Merozoites and sexual stages showed surface reactivity to both anti-PfP0N and anti-PfP0C antibodies in immunofluorescence assays. These properties strongly indicate PfP0 as a possible target for invasion-blocking antibodies.

Adult↗
PubMed2000

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum.

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2000

A spectrophotometric method for assay of tannase using rhodanine.

A method for assay of microbial tannase (tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Unlike the previous protocols, this method is sensitive up to gallic acid concentration of 5 nmol and has a precision of 1.7% (relative standard deviation). The assay is complete in a short time, very convenient, and reproducible.

Aspergillus niger↗
PubMed2000

Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication.

In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.

Adenoviridae↗
PubMed2000

Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis.

Cyclooxygenase-2 (COX-2), the enzyme at the rate-limiting step of prostanoid production, has been found to be overexpressed in human lung cancer. To evaluate lung tumor COX-2 modulation of antitumor immunity, we studied the antitumor effect of specific genetic or pharmacological inhibition of COX-2 in a murine Lewis lung carcinoma (3LL) model. Inhibition of COX-2 led to marked lymphocytic infiltration of the tumor and reduced tumor growth. Treatment of mice with anti-PGE2 mAb replicated the growth reduction seen in tumor-bearing mice treated with COX-2 inhibitors. COX-2 inhibition was accompanied by a significant decrement in IL-10 and a concomitant restoration of IL-12 production by APCs. Because the COX-2 metabolite PGE2 is a potent inducer of IL-10, it was hypothesized that COX-2 inhibition led to antitumor responses by down-regulating production of this potent immunosuppressive cytokine. In support of this concept, transfer of IL-10 transgenic T lymphocytes that overexpress IL-10 under control of the IL-2 promoter reversed the COX-2 inhibitor-induced antitumor response. We conclude that abrogation of COX-2 expression promotes antitumor reactivity by restoring the balance of IL-10 and IL-12 in vivo.

Adjuvants, Immunologic↗
PubMed2000

Utility values and age-related macular degeneration.

OBJECTIVE: To ascertain the utility values associated with age-related macular degeneration and varying degrees of visual loss. DESIGN: A cross-sectional study. PARTICIPANTS: Eighty white patients with unilateral or bilateral age-related macular degeneration in 1 or both eyes, and visual loss to a minimum of the 20/40 level in at least 1 eye. MAIN OUTCOME MEASURES: Utility values were measured in 5 groups according to the visual acuity in the better-seeing eye, 1 (20/20 to 20/25), 2 (20/30 to 20/ 50), 3 (20/60 to 20/100), 4 (20/200 to 20/400), and 5 (counting fingers to light perception), using the time trade-off and the standard gamble methods. Conventionally assigned anchor utility values were 1.0 for perfect health and 0.0 for death. RESULTS: The mean utility value for the total group with age-related macular degeneration was 0.72 (95% confidence interval [CI], 0.66-0.78) using the time trade-off method and 0.81 (95% CI, 0.76-0.86) using the standard gamble method. Using the time trade-off method correlated with the visual acuity in the better-seeing eye, the results were as follow: group 1, 0.89 (95% CI, 0.82-0.96), group 2, 0.81 (95% CI, 0.73-0.89), group 3, 0.57 (95% CI, 0.47-0.67), group 4, 0.52 (95% CI, 0.38-0.66), and group 5, 0.40 (95% CI, 0.29-0.50). Thus, those patients in group 1 were willing to trade 11% of their remaining lifetime in return for perfect vision in each eye, whereas those in group 5 were willing to trade 60% of their remaining lifetime in return for perfect vision in each eye. CONCLUSION: Age-related macular degeneration causes a substantial decrease in patient utility values and is highly dependent on the degree of visual loss in the better-seeing eye.

Aged↗
PubMed2000

Sonographic findings in grade III dengue hemorrhagic fever in adults.

PURPOSE: Sonography has been used to evaluate children with dengue hemorrhagic fever, but to our knowledge no such studies have been conducted with adults. We present the sonographic findings in 40 adults with severe (grade III) dengue hemorrhagic fever (DHF). METHODS: Forty patients (30 men and 10 women, aged 16-65 years) given a presumptive diagnosis of grade III dengue hemorrhagic fever during a documented regional epidemic underwent abdominal sonography. Ten also underwent chest radiography. Serologic confirmation was obtained in 5 patients, and in the rest the diagnosis was based on epidemiologic and clinical findings. RESULTS: Sonographic findings included pleural effusion in 21 patients (53%), thickening of the gallbladder wall in 17 (43%), and mild ascites in 6 (15%). These findings were similar to those of previous studies of severe DHF in children, although the incidence of pleural effusion and ascites was slightly lower in our series. Neither pleural effusion nor ascites was apparent on clinical examination. Of the 10 patients who underwent both sonography and chest radiography, sonography detected pleural effusion in all 10, whereas radiography detected it in only 3. CONCLUSIONS: Sonographic findings in DHF in adults (pleural effusions, ascites, and gallbladder wall thickening) were similar to those described for children but seem to be of lesser severity. Abdominal sonography is a useful diagnostic tool for confirming suspected cases of DHF. Sonography was found to be superior to chest radiography in detecting pleural effusions in the 10 patients in whom radiographs were available.

Adolescent↗
PubMed2000

Dual ultrastructural localization of mu-opiate receptors and substance p in the dorsal horn.

Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence-specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP-immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR-labeled dendrites that received any afferent input were contacted by at least one SP-containing axon or terminal. Although SP-immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP-containing spinal interneurons may be regulated by MOR ligands. A few SP-labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second-order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post- and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics.

Animals↗
PubMed2000

Percutaneous transluminal angioplasty of the aorta in children with nonspecific aortoarteritis: acute and follow-up results with special emphasis on left ventricular function.

Nonspecific aortoarteritis (NSAA) is a chronic disease of unknown etiology and may result in stenosing and obstructive lesions of the aorta. Transluminal balloon angioplasty was carried out in 17 children, 4-12 years of age (mean 8.9 +/- 2.7 years) with symptomatic stenosis of thoracic or abdominal aorta due to NSAA. Hypertension was present in 15 children, and 11 had significant left ventricular dysfunction. All children had a clinically inactive disease. The stenosis was in descending thoracic aorta in 10, thoracoabdominal in 3 and only abdominal in 4. Symptomatic improvement with relief of stenosis was seen in 16 of 17 children, although in 3, the results were suboptimal. The peak systolic gradient fell from 70.9 +/- 19.3 mmHg to 32.0 +/- 19.2 mmHg immediately after dilation (P < 0.0001). Angiographically measured luminal diameter stenosis also fell from 82.5 +/- 7.7% to 19.4 +/- 11.8% (P < 0.0001). Dissection flap was seen in 13 cases. It was large in cases with long, diffuse and eccentric stenosis of the thoracic or thoracoabdominal aorta. On follow-up (available for 14 of 16 cases for a period of 2-87 months mean 20.7 +/- 21.5 months, median 15.5 months) two patients developed restenosis. Left ventricular function improved in all 8 children that had sustained benefit of angioplasty. Blood pressure normalized in 9 of 13 children and the drug requirements fell in 4 others. Further remodeling of the lesion with decrease in gradients and luminal stenosis was observed in 11 children that have been restudied. Balloon angioplasty of aorta in children with NSAA is a feasible and safe procedure. Small intimal flap is seen in the majority, extensive dissection is more likely in the long diffuse lesions in descending thoracic aorta. Hypertension and left ventricular dysfunction improve after relief of obstruction.

Angioplasty, Balloon↗
PubMed2000

Characteristics of women with recurrent molar pregnancies.

OBJECTIVES: The aim of this study was to examine the incidence and characteristics of women who develop a second molar pregnancy after a previous episode of gestational trophoblastic disease. METHODS: A retrospective analysis was carried out on completed registration forms from referring hospitals in the North of England to the Sheffield Trophoblastic Screening Service over a 13-year period. All cases of second molar pregnancy were identified. Details of histology, blood group, ethnic origin, age, and subsequent pregnancies were examined. RESULTS: Between 1 January 1985 and 31 December 1997, 5030 patients were registered for follow-up and 275 (5.5%) required treatment for persistent disease. Thirty-five women had a subsequent molar pregnancy, a total of 0.70% of all registrations. There was no significant difference in age at first registration between those who were registered for one molar event and those who developed a subsequent molar pregnancy. The risk of a second molar event was highest in the second year after the initial diagnosis and reduced thereafter. There was a trend toward an increased risk of second molar pregnancy in Indian/Pakistani women when compared to Caucasian women (relative risk 2.4) but this was not significant at conventional levels. There was a significantly increased incidence of blood group B in patients that developed a molar pregnancy when compared to the normal population (P < 0.05), but there was no difference in distribution of blood group between those registered for their first molar event and those with two or more events. Patients who presented with a partial mole tended to have a partial mole as the second event but patients who presented with a complete mole were at risk of a subsequent complete mole, partial mole, or choriocarcinoma. Six percent of patients required chemotherapy for the second molar event, indicating no increase in aggressiveness in second moles. Two patients had three molar events. CONCLUSION: In the United Kingdom the risk of second molar pregnancy is less than 1%. There is an increased risk of molar pregnancy in women with blood group B and a trend toward an increased risk of second molar pregnancy in Indian/Pakistani women. Only 6% of patients required chemotherapy for the second mole; a second molar pregnancy is not an indication for chemotherapy.

ABO Blood-Group System↗
PubMed2000

Purification of wheat germ amylase by precipitation.

alpha-Amylase from various sources was found to bind alginate in free solution. The alginate-enzyme complex could be precipitated with Ca(2+). The enzyme activity could be recovered by dissolving the precipitate in 1 M maltose and precipitating alginate alone by addition of Ca(2+). Based upon these observations, alpha-amylase from wheat germ was purified with 68-fold purification and 72% recovery. The molecular weight estimated by SDS-PAGE was 18 kDa. The method also worked equally well with alpha-amylase for the whole wheat seed. The latter enzyme could be purified 54-fold with 70% activity recovery. The molecular weight of this second enzyme was estimated to be 45 kDa by SDS-PAGE.

Alginates↗
PubMed2000

Oxidative stress, phototherapy and the neonate.

Phototherapy is the most widely used form of therapy for unconjugated hyperbilirubinaemia. Its non-invasive nature and few side effects reported earlier have led to the assumption that it is innocuous. Recent research has revealed that phototherapy is a photodynamic stress and can induce lipid peroxidation. There is increasing evidence that many severe diseases of the neonate are caused by oxidative injury and lipid peroxidation. In the present communique, we review the oxidative susceptibility of the neonate and the evidence now available that phototherapy induces oxidative stress. Although intensive phototherapy (up to 40 mwatt/cm2/nm) has been reported to be increasingly effective, a little caution, we believe is warranted, till more definite data in the human neonate, help resolve the issue.

Bilirubin↗
PubMed2000

Isolated congenital coronary arteriovenous fistula.

During the last eight years a total of nine cases of isolated congenital coronary arteriovenous fistula (CAVF) have been diagnosed at our centre. Age of these patients ranged from 11 days to 22 years (mean 6.4 +/- 4.2 years). There were seven male and two female patients. Right and left coronary arteries were the sites of origin in seven and three cases respectively. One patient had two fistulae, one each originating from right coronary artery and obtuse marginal artery. The drainage sites were right ventricle in seven cases and right atrium in two. The pulmonary artery pressure was normal in eight cases and elevated in one case. The pulmonary artery to systemic flow ratio ranged from 1.1:1-2.4:1 in all except the 11 day-old-baby in whom it was 4:1. Pulmonary vascular resistance (PVRI) was below 3.5 in all cases (range 1.0-3.5 units). Surgical ligation of fistula was done in four cases and two patients underwent coil embolisation of fistula. Follow-up is available in five out of eight patients including three cases in whom CAVF was closed. Two other young patients are being followed-up conservatively and all the patients are asymptomatic. CAVF can present at any age; majority of the cases have small left to right shunts and more commonly CAVF arise from right coronary artery. Surgical ligation of fistula is safe in older patients, non-surgical embolisation using coils may be an alternative to surgery in selected cases.

Adolescent↗
PubMed2000

Hazard analysis and critical control points of weaning foods.

Hazards and critical control points were identified during the preparation, feeding and storage of weaning foods fed to the children (6-24 months) belonging to low income group (LIG) families using survey methods, spot and indepth observations and microbial analysis. High microbial counts were obtained for the weaning food samples procured from the families that were rated as poor for both personal hygiene and environmental sanitation. Food samples that were held at ambient temperature (25-35 degrees C) showed high counts of coliforms, AMCC, yeast and mold, Staphylococcus aureus and Bacillus cereus. Spice mixtures and mop samples also revealed high counts for all these organisms. Coliforms were not isolated from the water samples of all the 10 households. However, a high count of Bacillus cereus (10(3-5)/ml) was observed in water samples of two selected households. The hazards and critical control points identified were high initial contamination of raw foods, poor environmental sanitation and personal hygiene, feeding of overnight moist foods stored at ambient temperature and insufficient steaming of these foods.

Bacillus cereus↗