Biomedical subjects

P Roy

Publications and source records attributed to P Roy.

At least 55 records · Page 3Linked to original sources

PubMed2001

Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization.

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.

Animals↗

PubMed2001

Human immunodeficiency virus type 1 env sequences from Calcutta in eastern India: identification of features that distinguish subtype C sequences in India from other subtype C sequences.

India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viral env from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype C env, and one had viruses with a subtype A env. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate C(IN). Overall, C(IN) lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for C(IN) sequences (K340E, K350A, and G429E), 56% of the C(IN) sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-C(IN) sequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in India.

Adult↗

PubMed2001

RGD tripeptide of bluetongue virus VP7 protein is responsible for core attachment to Culicoides cells.

Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts. The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.

Amino Acid Motifs↗

PubMed2001

Increased dopamine d(2) receptor occupancy and elevated prolactin level associated with addition of haloperidol to clozapine.

OBJECTIVE: The authors added haloperidol, a potent D(2) blocker, to ongoing treatment with clozapine in patients with schizophrenia to determine the effects of this combination on dopamine D(2) receptor blockade, prolactin level, and extrapyramidal side effects. METHOD: At baseline and 4-8 weeks after the addition of haloperidol (4 mg/day) to ongoing clozapine treatment, five patients were examined for prolactin elevation, extrapyramidal side effects, drug plasma levels, and D(2) receptor occupancy measured with [(11)C]raclopride and positron emission tomography imaging. RESULTS: Adding haloperidol significantly increased D(2) receptor occupancy, from a mean of 55% to 79%, and significantly increased the prolactin level. One patient developed akathisia, and another manifested mild extrapyramidal side effects. CONCLUSIONS: Adding a modest dose of haloperidol to clozapine results in the high D(2) receptor occupancy and sustained prolactin elevation usually associated with typical antipsychotics. These findings suggest that the lack of prolactin elevation associated with clozapine derives mainly from low D(2) receptor occupancy and not from the medication's effects on other receptors.

Adult↗

PubMed2001

Application of rat hepatocyte culture to predict in vivo metabolic auto-induction: studies with DFP, a cyclooxygenase-2 inhibitor.

The drug candidate DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] is a selective cyclooxygenase-2 inhibitor under evaluation for analgesic and anti-inflammatory therapy. The in vitro metabolic pathways (rat microsomes) involve hydroxylation of the isopropyl side chain at either of two positions, the methyl or the methine, thus producing a hydroxylated metabolite (DFHP) or a dealkylated metabolite (DFH). DFH formation was the major pathway. Using hepatic microsomes from rats treated with agents that induce specific CYP isozymes, it was shown that the dexamethasone-inducible rat CYP3A isozyme(s) play a major role in DFH formation. The roles of CYP3A1 and -3A2 were confirmed with genetically engineered rat CYP enzymes. The potential for induction of rat CYP3A by DFP was evaluated by incubating DFP in rat hepatocyte cultures and measuring the CYP3A levels. Both CYP3A immunoreactive protein and enzyme activity were induced in a dose-dependent manner. The induction was confirmed in vivo by dosing rats with DFP at 100 mg/kg for 4 days. Microsomes prepared from the excised livers showed that DFP gave approximately 55% of the induction observed with dexamethasone, as determined by Western blot. In vitro metabolic auto-induction of DFP was assessed by measuring the metabolism of DFP in hepatocytes treated with DFP. DFH formation was significantly enhanced in the DFP-treated cells. In vivo, treating rats with DFP at doses of 10 to 100 mg/(kg.day) for 13 weeks indicated that DFP induced its own metabolism. The C(max) and plasma drug area under the curve values during the thirteenth week were significantly lower than that on the first day, and the effect was dose-dependent.

Alkylation↗

PubMed2001

Antimicrobial effect of protein(s) isolated from a marine mollusc Telescopium telescopium.

Ammonium sulfate precipitated protein (SF-50) isolated from the spermatheca gland of Telescopium telescopium, an invertebrate marine snail, showed antimicrobial effect on Escherichia coli. The antimicrobial effect varied with the concentration of "SF-50" used and the effect was found to be comparable to antibiotics like amikacin, contrimoxazole and gentamycin in disc diffusion test. The "SF-50" was devoid of erythrocyte haemolysis property.

Animals↗

PubMed2001

[Local control in the management of cancer: a few thoughts].

Cancer is still considered from a physiopathological point of view as a disease of the cell. This concept is underlying the idea of cure. Treatment with curative intent should aim at eradicating all the tumoral cells. Local control is mandatory and essential in cancers localized in organ with vital function. In breast cancer a complex and controversial relationship exists between local relapse and increased risk of fatal distant metastasis. In case of organ preserving treatment, a complete local control is necessary from the start. If a too high risk of local relapse is foreseable, conservative treatment should not be recommended.

Breast Neoplasms↗

PubMed2001

Effect of flow on the detoxification function of rat hepatocytes in a bioartificial liver reactor.

Ethoxyresorufin-o-deethylation (EROD) can be used as a sensitive measure of hepatic detoxification function. In this study, we employed a fluorescence assay based on EROD to study the effect of varying Peclet number (or flow) on hepatic function in a microchannel flat-plate bioartificial liver (BAL) reactor containing a coculture of hepatocytes and fibroblasts. Static culture and reactor flow experiments established that: 1) a pseudo-steady-state detoxification rate could be attained at each Peclet number, 2) the steady-state detoxification rate increased nonlinearly with Peclet number (ranging from 167 to 2500), 3) the uptake rate of substrate was a linear function of cell surface substrate concentration (<1 microM), and 4) a shear stress of 10 dyne/cm2 did not adversely affect hepatic function for at least 12 h. A convection-diffusion-reaction model supports the conclusion that increased convective mass transfer of substrate to the cell surface is the primary cause of the observed increase in EROD rate with Peclet number. Our results suggest that detoxification rates can be enhanced by an order of magnitude by choosing an appropriate Peclet number. For our bioreactor configuration, this optimum corresponds to a Peclet number range of 1000-2000 at a Damkohler number of 0.55. The usefulness of the mathematical model is discussed in the context of scale-up to a clinical BAL reactor for human application.

Animals↗

PubMed2001

Changing pattern of biotypes, phage types & drug resistance of Salmonella typhi in Ludhiana during 1980-1999.

BACKGROUND & OBJECTIVES: Ludhiana, an industrial city of Punjab, has a large floating population where typhoid has become endemic. A retrospective study was carried out over a period of 20 years (1980-1999) at Ludhiana on the biotyping, phage typing and drug resistance pattern of Salmonella typhi. METHODS: Of a total of 1697 S. typhi isolates obtained, phage typing and biotyping were done of only 1243 isolates. Antimicrobial susceptibility pattern of these isolates was also studied. RESULTS: Of the 1243 S. typhi isolates, 963 (77.5%) and 280 (22.5%) were of biotype I and biotype II respectively. Twenty four different S. typhi phage types were prevalent in Ludhiana in the past two decades. Between 1980 and 1989, more prevalent phage types were phage type A (35%), O (17.6%) and E1 (15.1%). During 1990-1999, there was a considerable increase in the incidence of phage type E1 (48.1%). The cumulative analysis of past two decades revealed that the incidence of phage type E1 (38.8%) was most predominant. In the past one decade (1990-1999), 412 S. typhi isolates of 13 different phage types exhibited multidrug resistance (MDR) pattern ACCoT (resistant to ampicillin, chloramphenicol, co-trimoxazole and tetracycline). High chloramphenicol resistance (74.7%) and MDR pattern ACCoT (68.2%) was shown by phage type E1 of S. typhi. INTERPRETATION & CONCLUSION: An association was observed between drug resistance and phage type pattern of S. typhi as 70 per cent isolates of S. typhi phage types E1 and O exhibited ACCoT multidrug resistant pattern. Reemergence of chloramphenicol susceptibility in the last decade emphasizes the need for regular antimicrobial surveillance to minimize the misuse of these drugs.

Anti-Bacterial Agents↗

PubMed2001

Efficacy of topical and systemic itraconazole as a broad-spectrum antifungal agent in mycotic corneal ulcer. A preliminary study.

PURPOSE: To evaluate the efficacy of topical (1%) and systemic itraconazole against common fungi such as Aspergillus and other filamentous fungi that cause mycotic corneal ulcer. METHODS: A prospective randomised, controlled study was done in 54 clinically suspected cases of fungal keratitis of which 44 were culture proven. Half the cases (n=27) with superficial involvement were treated with only topical itraconazole (1%) and the other half were treated with both topical and systemic itraconazole. RESULTS: Aspergillus, Penicillium and Fusarium were the most common fungi isolated. The ulcer resolved in 42 eyes (77%) and 12 eyes (23%) did not respond well to treatment. Four of 12 non-responding eyes were caused by Fusarium species. CONCLUSION: Itraconazole, given either topically or systemically, is effective in treating mycotic corneal ulcers.

Administration, Oral↗

PubMed2000

Synthesis, characterization, and activity of metabolites derived from the cyclooxygenase-2 inhibitor rofecoxib (MK-0966, Vioxx).

Metabolites of the COX-2 inhibitor rofecoxib (MK-0966, Vioxx) were prepared by synthetic or biosynthetic methods. Metabolites include products of oxidation, glucuronidation, reduction and hydrolytic ring opening. Based on an in vitro whole blood assay, none of the known human metabolites of rofecoxib inhibits COX-1 nor contributes significantly to the inhibition of COX-2.

Animals↗

PubMed2000

Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide.

The anticancer alkylating agents cyclophosphamide (CPA) and ifosfamide (IFA) are prodrugs that undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic (N-dechloroethylated) metabolites. Whereas the human liver microsomal P450 catalysts of CPA and IFA 4-hydroxylation are well characterized, the P450 enzyme catalysts of the alternative N-dechloroethylation pathway are poorly defined. Analysis of a panel of fifteen human P450 cDNAs in the baculovirus expression system ('Supersomes') demonstrated that CYP3A4 exhibited the highest N-dechloroethylation activity toward both CPA and IFA, whereas CYP2B6 displayed high N-dechloroethylation activity toward IFA, but not CPA. The contributions of each human P450 to overall liver microsomal N-dechloroethylation were calculated using a recently described relative substrate-activity factor method, and were found to be in excellent agreement with the results of inhibition studies using the CYP3A inhibitor troleandomycin and an inhibitory monoclonal antibody to CYP2B6. With CPA as substrate, CYP3A4 was shown to catalyze >/=95% of liver microsomal N-dechloroethylation, whereas with IFA as substrate, CYP3A4 catalyzed an average of approximately 70% of liver microsomal N-dechloroethylation (range = 40-90%), with the balance of this activity catalyzed by CYP2B6 (range = 10-70%, dependent on the CYP2B6 content of the liver). Because CYP2B6 can make a significant contribution to human liver microsomal IFA N-dechloroethylation, but only a minor contribution to IFA 4-hydroxylation, the selective inhibition of hepatic CYP2B6 activity in individuals with a high hepatic CYP2B6 content may provide a useful approach to minimize the formation of therapeutically inactive but toxic N-dechloroethylated IFA metabolites.

Anti-Bacterial Agents↗

PubMed2000

Aromatic residues mediate the pressure-induced association of digoxigenin and antibody 26-10.

We have previously found that the complex between fluorescently labeled digoxigenin and the monoclonal antibody 26-10 forms with a decrease in volume of approximately 30 ml/mol, leading to increased association of these species under applied hydrostatic pressure. In the present study, we have utilized a panel of mutant antibodies and Fab fragments, previously characterized for their importance in the binding affinity of digoxin:26-10, to probe the molecular basis of pressure sensitivity in this complex, as measured by fluorescence polarization spectroscopy. Several mutations that result in marked decreases in affinity exerted little or no significant effect on the association volume. Mutation at any of several key aromatic residues of the 26-10 Fab heavy chain led to a decrease in the pressure-induced association, and two mutants with Trp-->Arg mutations at heavy chain residue 100 exhibited pressure-induced dissociation. The effect of charged groups was found to depend on their proximity to contacting aromatic groups. The ability to understand and control the pressure sensitivity of antigen-antibody complexes has numerous potential applications in immunoseparations and immunosensors.

Amino Acids↗

PubMed2000

Spectral distribution of infrared synchrotron radiation by an insertion device and its edges: A comparison between experimental and simulated spectra

The first measurements of the spectral distribution of infrared radiation emitted by an undulator are reported. They are compared with calculations including both velocity and acceleration terms. Measurements have been performed at the beam line SIRLOIN (Spectroscopie en InfraRouge Lointain). The agreement between the observations and this first exact numerical solution shows that the inclusion of the velocity term in the submillimeter frequency range is necessary. Moreover, structures due to undulator edges are observed in the far infrared and mid-infrared range, while the interference pattern due to redshifted harmonics of the undulator is dominating in the mid-infrared to near infrared.

Journal Article↗

PubMed2000

Gonadotropin-releasing hormone stimulates thyroid activity in a freshwater murrel, Channa gachua (ham.), and Carps, Catla catla (ham.) and Cirrhinus mrigala (ham.).

Injections of mammalian GnRH (mGnRH), salmon GnRH (sGnRH), and a homologous murrel, Channa punctatus, GnRH (cGnRH) to a murrel, Channa gachua, and a carp, Catla catla, at a dose of 1 microg/250 g body wt significantly increased plasma thyroxine (T4) levels above control. Piscine GnRHs (sGnRH and cGnRH) had significantly greater stimulatory effects compared with mGnRH. To observe whether this stimulatory effect by GnRHs is direct or indirect, thyroid follicles were isolated from hypobranchial muscles of freshwater murrel, C. gachua and incubated (1 x 10(6) follicles/well) in vitro at 30 degrees C for 2 h without hormone and for 3 h with hormones. Addition of these three GnRHs separately at a concentration of 1 microg/well stimulated T4 secretion; sGnRH and cGnRH caused greater secretion of T4 into the medium compared with mGnRH. Specificity of GnRH action in vitro was assessed by using anti-GnRH antibody which significantly (P < 0.01) inhibited GnRH-augmented T4 secretion. To gain further insight, 125I uptake by thyroid follicles and formation of [125I]T4 from this radioiodine pool was monitored in the presence or absence of sGnRH. sGnRH greatly augmented 1251 uptake by the follicles which resulted a fourfold increase in [125I]T4 formation out of this pool of 125I compared with the control. The results indicate GnRH stimulation of thyroid hormone formation and release in these teleosts suggesting a possible different mode of regulation of thyroid hormone secretion in teleosts.

Animals↗

PubMed2000

Predicting haloperidol occupancy of central dopamine D2 receptors from plasma levels.

Positron emission tomography (PET) is increasingly being used to study dopamine receptor occupancy and the clinical effects of antipsychotic medication. Dopamine D2 receptor occupancy has been shown to predict several clinical effects of antipsychotic medication including therapeutic response, motor and endocrine side-effects. Plasma levels may be used as a surrogate marker for central occupancy if the relationship between these two measures may be accurately described. This study was designed to test the capacity of a previously derived relationship equation (%D2 occupancy=plasma level/ED50+plasma level, where ED50= 0.40 ng/ml) to predict striatal D2 occupancy from plasma level. Twenty-one patients receiving treatment with low dose haloperidol underwent a 11C-raclopride PET scan to measure D2 occupancy. The D2 occupancy levels were accurately predicted by use of the previously generated equation with only a small degree of error (3.89% CI 0.45-7.33). Predicted and measured D2 occupancy values correlated closely (Pearson's r=0.864, P=0.003). The study indicates that reliable prediction of D2 occupancy from plasma levels is possible. This provides a potentially useful surrogate measure of D2 occupancy for research and possibly clinical practice, as the routine use of PET to measure occupancy levels is not feasible.

Adult↗

PubMed2000

Salicylic acid-independent induction of pathogenesis-related gene expression by fusicoccin.

Treatment of tomato plants (Lycopersicon esculentum Mill.) with fusicoccin (FC), an activator of the plasma-membrane H+-ATPase which maintains an electrochemical gradient across the plasma membrane, resulted in a dose-dependent accumulation of transcripts for intra- and extracellular pathogenesis-related (PR) proteins. The accumulation of PR protein transcripts was paralleled by an increase in leaf salicylic acid (SA) content. Transcripts of PR proteins and SA started to accumulate 3 h after FC treatment. 2-Aminoindan-2-phosphonic acid, an inhibitor of SA synthesis, was used to assess the role of SA in FC-mediated induction of PR gene expression. 2-Aminoindan-2-phosphonic acid was found to suppress the accumulation of SA but not the induction of PR gene expression in response to FC treatment. Furthermore, in transgenic tobacco plants overexpressing a bacterial salicylate hydroxylase gene (nahG-tobacco), PR transcripts accumulated after FC treatment to levels similar to those observed in control tobacco plants. The data indicate a role for the proton gradient across the plasma membrane in the SA-independent induction of PR gene expression.

Glycosides↗

PubMed2000

Nonmalignant obstruction is a common problem with metal stents in the treatment of esophageal cancer.

BACKGROUND: The use of metal stents for the treatment of dysphagia due to esophageal malignancy is an important advance because of ease of delivery and their self-expandable property. Obstruction due to tumor overgrowth is a recognized complication, but nonmalignant obstruction in patients with metal stents is rarely reported. METHODS: Database records of patients who had esophageal cancer and underwent metal stent insertion were reviewed. RESULTS: A total of 116 patients were seen between October 1993 and October 1997. Four types of metal stents had been used (Ultraflex, Z Stent, Wallstent, and Esophacoil). Detailed follow-up information was available for 81 patients, who constitute the study sample. Forty-nine (60%) stent obstructions were reported, 26 of the 49 (53%) were due to tumor overgrowth and 23 (47%) were not associated with malignancy. Histologic analysis of the nonmalignant obstructing tissue showed granulation tissue (56%), reactive hyperplasia (22%) and fibrosis (22%). CONCLUSIONS: Nonmalignant obstruction is a common although infrequently reported complication after placement of metal stents for esophageal cancer. The tissue response of the esophageal mucosa occurred with all 4 types of stents used. No specific characteristic of the stent or prior treatment seems to be related to obstruction of the stent in patients with either nonmalignant obstruction or tumor overgrowth.

Adenocarcinoma↗