Prevalence of hypertension, its correlates and awareness among adult tribal population of Kerala state, India.

BACKGROUND: Increasing prevalence of hypertension is a public health problem in India. AIMS: To study prevalence, correlates, and awareness of hypertension among tribal adult population in Kerala. SETTING AND DESIGN: A community-based, cross-sectional study was carried out in tribal areas of Kerala by adopting multistage random sampling procedure. MATERIALS AND METHODS: Data was collected on socio-demographic and behavioral factors, and anthropometric measurements were carried out. Body mass index (BMI) was categorized using the classification recommended for Asians. Waist circumference ≥ 90 cm for men and ≥ 80 cm for women was used cut off for defining an abdominal obesity. Bivariate and multivariate analysis was carried out to study association of hypertension with socio-demographic variables, personal habits, and obesity. RESULTS: A total of 4,193 adults (men 1,891, women: 2,302) of ≥ 20 years of age were covered. The overall prevalence of hypertension was 40% (n=1671). The prevalence of hypertension increases with increase in age among both the genders. Regression analysis showed that the risk of hypertension was significantly (P<0.001) lower among educated and among higher socio-economic status group. Sedentary activity had 1.3 times (CI=1.09-1.60) and alcohol consumption had 1.4 (CI=1.17-1.73) times higher risk of hypertension. The risk of hypertension was 1.7 times higher among overweight/obese subjects. Overall, only 10% (n=164) of the adult population was aware of hypertension status, and about 8% (n=129) were on regular treatment. CONCLUSION: It was observed that the prevalence of hypertension was higher among tribal adult population of Kerala and was associated with age, gender, education, HHs wealth index, physical inactivity, alcohol consumption, and overweight/obesity.

Shorter and Rugged Analytical GC Method for Residual Solvents Content in Active Pharmaceutical Ingredient.

This work describes the highly selective and sensitive robust analytical method (gas chromatography) for the qualitative and quantitative analysis of commonly used residual solvents (like methanol, ethanol, acetonitrile, dichloromethane, methyl tert-butyl ether, n-hexane, 1-propanol, ethyl acetate, tetrahydrofuran, N,N-diisopropylethylamine (DIPEA) and dimethylformamide), in an active pharmaceuticals ingredients. The developed method was optimized with good resolution (>1.9 between close eluting solvents) and a shorter run time (20 min). All the solvents were separated using GC column DB-624, 30-m length, 0.32-mm diameter, 1.8-µm particle size, nitrogen as a carrier gas and recorded the signals using flame ionization detector (FID). Further, analytical method validation has been performed for the developed analytical method and the validation results demonstrated its routine application for the determination of residual solvents content by GC-head space (HS). The practical application was demonstrated by the suitable API batch analysis (having sample Con. 40 mg/mL). The present developed method has more advantages than the other methods for the qualitative and quantitative applications, such as shorter runtime, selective for multiple solvents (11 organic solvents) analysis at a time, highly sensitive at ppm levels. This GC-HS method could be used for the qualitative and quantitative determination of the residual solvents content in APIs.

Development and validation of GC-MS method for the determination of methyl methanesulfonate and ethyl methanesulfonate in imatinib mesylate.

A gas chromatography-mass spectrometry (GC-MS) method has been developed for the identification and determination of two carcinogenic and genotoxic mesylate esters viz. methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) in imatinib mesylate (INM). The method was optimized based on the peak shapes and resolution of MMS and EMS. The method was validated as per International Conference of Harmonization (ICH) guidelines in terms of limits of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy, specificity and robustness. The LOD and LOQ values were found to be 0.3 and 1.0 microg/ml, respectively. The method is linear within the range of 1-15 microg/ml for both the compounds. These mesylate esters were not found in three different batches of pure and pharmaceutical formulations of INM.

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.

Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value.

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 microg cloprostenol 36 h before sponge removal and LOPU. Following in vitro maturation (IVM), oocytes were fertilised in vitro with frozen-thawed semen produced using the egg yolk-free Bioxcell extender (IVM, L'Aigle, France). The average number of follicles aspirated (17.9 +/- 8.0 per goat), oocytes recovered (15.7 +/- 8.4 per goat) and cleavage after IVM/in vitro fertilisation followed by a short 24-h in vitro culture in modified synthetic oviduct fluid medium (72 +/- 7%) were similar to results reported previously by our group and others in younger goats. A total of 296 embryos was transferred into 50 heat-synchronised recipients, of which 40 became pregnant (80%) and 38 progressed all the way to term, delivering 86 live kids. The present study indicates that LOPU-IVEP can be used successfully to extend the reproductive life of valuable goats that have acquired difficulties becoming pregnant by artificial insemination after multiple kiddings.

Quantitative histology and MGF gene expression in rats following SSC exercise in vivo.

PURPOSE: We investigated the effects of muscle length during stretch-shortening cycles (SSC) in vivo on changes in MGF gene expression and quantitative morphometry in rat skeletal muscle. METHODS: Dorsiflexor muscles of male Sprague-Dawley rats were exposed to seven sets of 10 SSC at 500 degrees .s(-1). Animals were randomly assigned to a long muscle length injury group (L-inj), short muscle length injury group (S-inj), or isometric group (Iso), with recoveries examined at 6 or 48 h post-injury for each group. Following exposure, animals were euthanized, and the tissue was prepared for either histology (quantitative morphometry) or RNA isolation, followed by quantitative real-time reverse transcriptase polymerase chain reaction. mRNA levels were measured for mechano-growth factor (MGF), while 18S ribosomal RNA served as the internal reference sample. RESULTS: Stereological measures indicative of edema and myofiber degeneration were significantly increased in the L-inj SSC group at 48 h when compared with the S-inj or Iso group. MGF mRNA was increased transiently at 6 h in the isometric group. In contrast, MGF mRNA was increased at 48 h in the S-inj, but was not increased at either time point in the L-inj group. CONCLUSION: These data strongly indicate that exposure to SSC at longer muscle lengths result in greater morphometric indices of inflammation and degeneration than SSC conducted at a shorter muscle lengths or isometric contractions, at the same time that the adaptation to SSC was prolonged and, apparently, not resolved in the L-inj group that was manifested by the lack of up-regulation in MGF mRNA.

Actin changes in normal human and rat leukocytes and in transformed human leukocytic cells.

The DNase I inhibition assay was used for the determination of the relative amounts of monomeric and total actin in normal human peripheral blood lymphocytes, normal Sprague-Dawley rat peritoneal leukocytes, and 6 transformed human cell lines of lymphoid and myeloid origins. In the normal lymphocytes and leukocytes, greater than 50% of the total actin was monomeric. In contrast, only 1 of the 6 transformed lines had greater than 50% of its actin in the monomeric form. In the other 5 lines, the percentage of actin in the monomeric form ranged from 23 to 39%. The normal lymphocytes, peritoneal leukocytes, and 2 of the transformed cell lines (CEM and K562) were examined in greater detail. The total actin (as a percentage of the total protein) was found to be much lower in the transformed cell lines than it was in the normal lymphocytes. The total amount of actin in the normal rat leukocytes was very similar to the amount in the normal human lymphocytes. In addition to these differences between the normal and transformed human cells, treatment of the normal lymphocytes with mitogenic doses of concanavalin A was found to significantly reduce the amount of monomeric actin in the cells. Similar treatment of the transformed cells produced no significant reduction in the monomeric actin.

Lack of correlation between induction of chemotactic peptide receptors and stimulus-induced actin polymerization in HL-60 cells treated with dibutyryl cyclic adenosine monophosphate or retinoic acid.

We used the promyelocytic leukemic cell line HL-60 to explore the molecular mechanisms regulating stimulus-induced actin polymerization in myeloid cells. HL-60 cells express very few chemotactic peptide receptors in their undifferentiated state and fail to undergo actin polymerization when stimulated with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). However, when the cells were induced to differentiate with dibutyryl cyclic AMP (dbcAMP) or retinoic acid, they acquired the ability to undergo actin polymerization on stimulation with FMLP or phorbol myristate acetate. Kinetic experiments revealed that in the first 48 h of retinoic acid treatment there was no increase in the chemotactic peptide receptors on HL-60 cells, but the cells were capable of undergoing actin polymerization on stimulation with FMLP. Similarly, treatment with dbcAMP showed no increase in chemotactic peptide receptors until 24 h but stimulus-induced actin polymerization was demonstrable as early as 4 h after the treatment. In addition, with dbcAMP-treated cells the magnitude of stimulus-induced actin polymerization showed large variation depending on the duration of exposure to the drug. Dual-label studies using propidium iodide to measure DNA content and NBD-phallacidin to measure the F-actin content revealed that these variations were not related to the stages of cell cycle. Cells in all stages of the cell cycle responded to stimulus-induced actin polymerization, but the magnitude of the response appeared to be more in cells in G2/M phase. The observations reported here indicate that the small number of chemotactic peptide receptors present on HL-60 cells are adequate to mount an actin polymerization response, provided the required intracellular mechanisms exist. Differentiation-inducing agents, therefore, must cause changes within the cell, such as induction of actin-binding proteins, to cause actin polymerization following FMLP stimulation. The HL-60 system serves as a useful model for studying the molecular mechanisms regulating stimulus-induced actin polymerization in human neutrophils.

Phenylmethylsulfonyl fluoride inhibits chemotactic peptide-induced actin polymerization and oxidative burst activity in human neutrophils by an effect unrelated to its anti-proteinase activity.

Stimulation of polymorphonuclear leukocytes with the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) causes conversion of monomeric actin to polymeric actin. We studied the role of proteinase inhibitors phenylmethylsulfonyl fluoride PMSF) and diisopropyl fluorophosphate in fMet-Leu-Phe-induced actin polymerization in polymorphonuclear leukocytes. Pre-incubation of cells with PMSF (2 mM) for 1 min caused inhibition of fMet-Leu-Phe-induced actin polymerization, as studied by 7-nitrobenz-2-oxa-1,3-diazole (NBD) -phallacidin labeling and flow cytometry. PMSF also inhibited fMet-Leu-Phe-induced hydrogen peroxide release, superoxide anion generation and chemiluminescence. In contrast, diisopropyl fluorophosphate (5 mM) was unable to inhibit fMet-Leu-Phe-induced actin polymerization and superoxide generation, but was effective in inhibiting hydrogen peroxide production and chemiluminescence. PMSF did not cause any change in membrane potential by itself and failed to inhibit the membrane potential changes induced by fMet-Leu-Phe, indicating that PMSF does not affect the binding of fMet-Leu-Phe to the receptors. The high concentration of PMSF required coupled with the fact that diisopropyl fluorophosphate was unable to inhibit fMet-Leu-Phe-induced actin polymerization suggested that this activity of PMSF might be unrelated to proteinase inhibitory activity. Polymyxin B, a membrane-active antibiotic, had an effect similar to PMSF on fMet-Leu-Phe-induced actin polymerization. This suggests that PMSF may also be acting via its membrane effect rather than its anti-proteinase effect.

MAP kinase activation in macrophages.

Stimulation of macrophages by a variety of agents causes activation of mitogen-activated protein kinases (MAPKs). Activation of MAPKs by lipopolysaccharide involves CD14 and Toll receptors. Subsequent steps still remain to be explored. Tumor necrosis factor-alpha (TNF-alpha)-induced activation of MAPKs has been shown to involve the death domain proteins (TRADD, FADD, MADD) and TRAFs. Other molecules involved in this pathway include the protein kinases, ASK1, germinal center kinase (GCK), hematopoietic progenitor kinase 1 (HPK1), and GCK-related kinase (GCKR). Although, these pathways have been described in various cell types, their role in macrophages remains to be established. The availability of knockout mice and constitutively active and dominant-negative mutants of MAPKs should greatly enhance our understanding of this field. The activation of MAPKs seems to be different in cell lines compared with primary cells. Among the macrophages, cells from different compartments show different expression of receptors and signal transduction molecules. These differences may account for differences in MAPK activation and other phenotypic differences in macrophages from different compartments. Therefore, it is important to use primary cells for studying MAPK signal-transduction pathways, and the data from cell lines should not be extrapolated to primary cells.

Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation, and superoxide production.

The effects of pentoxifylline (Trental) on human neutrophil CR3 up-modulation, degranulation, and superoxide production were studied. We used the chemotactic peptide fMLP and the phorbol ester PMA as soluble stimuli, and beta-glucan particles as a CR3-specific solid phase stimulus of neutrophil superoxide production. Since neutrophils have adenosine A2 receptors, we compared effects of pentoxifylline to effects of adenosine, and we also looked at the effect of cytochalasin B, which breaks up actin filaments. Pentoxifylline inhibited both CR3 up-modulation and degranulation of myeloperoxidase and lysozyme. Pentoxifylline is a more potent inhibitor of fMLP- compared to PMA-induced degranulation, and is especially potent against superoxide production. While pentoxifylline is less potent than adenosine in its inhibition of fMLP-induced superoxide production, it is more potent in its inhibition of PMA- and beta-glucan particle-stimulated superoxide production. Cytochalasin B, which enhances degranulation and fMLP-stimulated superoxide production, was found to inhibit beta-glucan particle-stimulated superoxide production. These findings are consistent with the hypothesis that pentoxifylline can affect both the cytoskeletal architecture of unstimulated neutrophils and the activation and responses of neutrophils which involve actin polymerization and receptor-cytoskeletal interactions.

Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor.

Dichlorofluorescein (DCFH) oxidation assay measures hydrogen peroxide (H2O2), which is a derivative of superoxide anion. We found that a calmodulin antagonist, W-13, which is known to inhibit superoxide anion generation enhanced the capacity of human neutrophils to oxidize DCFH. To investigate this discrepancy we studied the role of nitric oxide (NO) in DCFH oxidation. Pure NO was capable of oxidizing DCFH, and the product formed had spectral properties identical to oxidized DCFH produced by H2O2. The arginine analog, NG-monomethyl-L-arginine (NMMA), which inhibits NO production, in combination with W-13 completely inhibited the stimulus-induced increase in DCFH oxidation. We conclude that the oxidation of DCFH in human neutrophils can occur by either H2O2 or NO.

Age-related increase in phorbol myristate acetate-induced lymphocyte adhesion to vascular endothelium.

Lymphocyte adhesion to vascular endothelium is an important part of immune function. This investigation sought to detect differences between the adhesion of lymphocytes from young and aged human donors to vascular endothelium with and without treatment of the lymphocytes with phorbol myristate acetate (PMA), an activator of protein kinase C (PKC) that stimulates this adhesive process. T-lymphocytes were isolated from young and aged donors and adhesion assays were conducted with human umbilical vein endothelial cells (HUVEC). In some cases the HUVEC were activated by pre-incubation with tumor necrosis factor, a cytokine that increases their adhesiveness, before the addition of lymphocytes in the presence or absence of PMA. The results show that, in the basal state, lymphocytes from young and aged donors had similar levels of adherence, while with PMA activation, lymphocytes from aged donors had a significantly higher level of adherence to both activated and non-activated HUVEC. No cytotoxic effect on the HUVEC was detected. These results suggest a role for lymphocytes in diseases that predominantly affect the elderly and that are thought to involve interaction between lymphocytes and endothelium. In addition, these results indicate that there may be a change in PKC function in lymphocytes with aging.

Age-related studies of SIg, Leu-4 and concanavalin A receptor densities and capping in human lymphocytes.

We compared the cell surface antigen density and capping of three antigens in lymphocytes obtained from healthy, young (mean age 27 years) and elderly (mean age 76), population. There were no differences in the expression of surface immunoglobulin (SIg), concanavalin A (con A) receptors and Leu-4 antigen between the two groups. Kinetic analysis of these molecules revealed a slight decrease in capping in the older population, but the differences were not statistically significant. In order to test the possibility that subjecting the cells to metabolic stress might bring out the differences, we performed a kinetic analysis of SIg and con A capping in the presence of various concentrations of the metabolic inhibitor sodium azide. Although the capping in cells from elderly subjects was slightly more sensitive to azide, no statistical difference was found. Analysis of con A capping by a flow cytometric method yielded similar results, confirming the data obtained by visual capping experiments. We conclude that although a trend toward decreased capping was observed, there is little alteration in the surface molecule capping phenomenon in the age-groups studied.

A simple method of raising antiserum to the third fraction of mouse serum complement.

A simple method of raising antiserum to the third fraction of mouse serum complement (C3) is described. C3 was precipitated with activated zymosan and was injected with complete Freund's adjuvant subcutaneously into rabbits. The antiserum raised is monospecific and found suitable for quantitation of C3 concentration in mouse serum by single radial diffusion method.

A simple bactericidal antibody test for sero-diagnosis of typhoid fever.

Serum samples were collected from 24 confirmed cases of typhoid fever, 15 clinically suspected cases and 23 normal healthy controls. The convalescent sera were obtained in 13 of the 24 confirmed typhoid cases. In all, 13 paired sera, 11 acute phase only, 15 clinically suspected and 23 normal serum samples were tested for eliciting bactericidal antibodies to Salmonella typhi. In addition, the Widal test was also performed for comparison. All the 24 acute phase sera as well as 13 convalescent sera were found to be positive by bactericidal antibody test (titre 1:80 or above). Of 15 clinically suspected cases, 5 were positive whereas one of the 23 normal controls sera gave a false positive reaction. In contrast, the Widal test could detect only one of the 24 cases in the acute stage, but all 13 cases showed antibodies at a diagnostic titre level during the convalescent stage. None of the 15 clinically suspected cases or 23 normal controls were positive by the Widal test. The feasibility of using a bactericidal antibody test in sero-diagnosis of typhoid fever is discussed.

Radial counter-immunoelectrophoresis for rapid sero-diagnosis of typhoid fever.

Serum samples were collected from 24 confirmed cases of typhoid fever and 23 normal healthy controls. Convalescent sera from the patients were obtained, wherever possible, one week after the first sample. In all, 13 paired sera, 11 acute phase only and 23 normal serum samples were tested for ability to elicit precipitins to Salmonella typhi by radial counter-immunoelectrophoresis using cellulose acetate membranes. In addition, conventional counter-immunoelectrophoresis (CIE) was performed using agar-gel layer for comparison. One of 24 acute phase sera gave positive results whereas all 13 convalescent sera were positive by both methods. The radial CIE test may be useful for rapid sero-diagnosis of typhoid fever as it takes only 4 min and can be used to screen large numbers of serum samples from patients suspected of typhoid fever.

Phorbol esters and retinoids induce actin polymerization in human leukocytes.

DNase I inhibition assay was used to determine the change in monomeric actin (G-actin) in human peripheral blood leukocytes following their treatment with phorbol esters and retinoids. Treatment of polymorphonuclear leukocytes (PMN) with 10(-6) M phorbol myristate acetate resulted in a decrease in G-actin content to 3.8 +/- 0.49 (microgram G-actin/100 micrograms total protein; mean +/- S.E.M.) from a control value of 5.6 +/- 0.51 (P less than 0.05). The effect of retinoic acid on mononuclear leukocytes varied depending on the concentration of the drug used. At 10(-5) M there is a slight increase in the amount of G-actin and maximal decrease in G-actin was noted at 10(-6) M. The decrease in G-actin can be prevented by pretreatment of cells with cytochalasin E (CE) indicating that the decrease is due to actin polymerization. The total actin, determined after guanidine hydrochloride (G X HCl) treatment, remained unchanged in drug treated cells. Only phorbol esters which are capable of inducing tumor promotion induce actin polymerization, suggesting that actin polymerization might have a role in tumor promotion. Actin polymerization might serve as a useful framework for further studies on delineating the mechanism of action of phorbol esters and retinoids.

Age-related differential effects of zinc on concanavalin A-induced capping of human lymphocytes.

Con A-induced capping of lymphocytes is altered by a number of cytoskeletal modulating agents. Zinc, in the presence of colchicine, demonstrated an age-dependent differential effect on con A-induced capping of human peripheral blood lymphocytes. In general, zinc enhanced capping of lymphocytes from young individuals and suppressed from those of old individuals. The effect of zinc on the cap formation adds further support to the evidence that zinc might be acting on the microfilaments. However, cytochalasin B, another microfilament modulating agent, does not show a differential effect similar to zinc. This suggests that zinc might modify microfilament formation or function in a manner different from cytochalasin B. The proposal that the effects of zinc might be due to its action on calmodulin suggests that calmodulin, the calcium regulating protein, may have a role in the physiological processes associated with aging. Further studies in this area might yield fruitful results in our understanding of the molecular mechanism of senescence.

Variability of plasma IL-6 and crosslinked fibrin dimers over time in community dwelling elderly subjects.

Because obtaining multiple blood samples from individuals involved in epidemiologic studies is difficult, conclusions must be drawn on the basis of one or two samples. In this study, the authors attempted to determine the variability of two plasma markers over time, namely IL-6 levels and crosslinked fibrin degradation products (D-Dimers), in 16 elderly community-dwelling individuals. The study group included both men and women and black and white subjects (four in each group). Eight blood samples were obtained from each subject over a period of 36 days. Blood was separated within 1 hour after collection and aliquots of plasma were stored at -70 degrees C until all samples were collected. All samples from an individual were analyzed at one time. IL-6 and D-Dimers were measured by commercially available ELISA kits. The variability in the levels of plasma IL-6 and D-Dimers was assessed by means of intra-class correlation coefficient (ICC). The estimates of the ICCs for one measurement of IL-6 and D-Dimers were .87 and .86, respectively. Reliability values of this magnitude indicate excellent reproducibility in the measurement. These values indicate that obtaining a single sample from a subject is fairly representative of that individual's IL-6 and D-Dimer levels over an extended period of time.