An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus.

Management of viral diseases relies on definite and sensitive detection methods. Citrus yellow mosaic virus (CYMV), a double stranded DNA virus of the genus Badnavirus, causes yellow mosaic disease in citrus plants. CYMV is transmitted through budwood and requires a robust and simplified indexing protocol for budwood certification programme. The present study reports development and standardization of an isothermal based recombinase polymerase amplification (RPA) assay for a sensitive, rapid, easy, and cost-effective method for detection and diagnosis of CYMV. Two different oligonucleotide primer sets were designed from ORF III (coding for polyprotein) and ORF II (coding for virion associated protein) regions of CYMV to perform amplification assays. Comparative evaluation of RPA, PCR and immuno-capture recombinase polymerase amplification (IC-RPA) based assays were done using purified DNA and plant crude sap. CYMV infection was efficiently detected from the crude sap in RPA and IC-RPA assays. The primer set used in RPA was specific and did not show any cross-amplification with banana streak MY virus (BSMYV), another Badnavirus species. The results from the present study indicated that RPA assay can be used easily in routine indexing of citrus planting material. To the best of our knowledge, this is the first report on development of a rapid and simplified isothermal detection assay for CYMV and can be utilized as an effective technique in quarantine and budwood certification process.

Frequency of Defecation and Form of Stool among General Bangladeshi Population.

Data on stool form and defecation frequency which are a prerequisite for defining normal bowel habit are lacking in Bangladesh. This observational cross sectional study was designed to find out defecation frequency and stool form among general population in Bangladesh. This study was performed in the Department of Gastroenterology, Shaheed Suhrawardy Medical College Hospital, Dhaka, Bangladesh from July 2017 to June 2018. Apparently healthy 1090 respondents were evaluated for predominant stool form (Bristol chart) and frequency. Data on demographic and life-style were collected. The study population consisted of 1090 respondents, among them, 65.13% male and 34.87% female and mean age of them was 40.20±12.39 years. Most of the people 874(80.2%) passed stool between 12-14 times per week followed by 111(10.2%) less than 3 stools per week, 95(8.7%) passed more than 14 stools per week and 10(0.9%) between 3-12 stools per week, p<0.001. Most people passed predominantly Bristol type IV stool- 610(56.0%); followed by type III- 274(25.1%). Other stool forms were: type I- 52(4.8%), type II- 59(5.4%), type V- 31(2.8%), type VI- 33(3.0%), type VII- 31(2.8%), p<0.001. In regard to the physical activity, most of the respondents (70.0%) are physically active whereas about 13.0% are sedentary and about 17.0% are physically intermediate between the two, p<0.001. In the case of dietary habit, most of the participants are non-vegetarian (82.5%) and the remaining are vegetarian (11.1%) and occasional non vegetarian (6.4%), p<0.001. Median stool frequency in the studied population was 14 per week and predominant form was Bristol type IV. Older age was associated with lesser stool frequency, particularly among female subjects.

Serum transaminase level changes in dengue fever and its correlation with disease severity.

It was observed that liver enzymes are elevated in dengue fever. In this study our aims were to determine the changes in serum transaminases in dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) and to find out the relation of transaminase level changes with the disease severity. This cross sectional, prospective hospital based observational study was carried out in the department of Gastrointestinal Hepatobiliary and Pancreatic diseases and Internal Medicine department of BIRDEM Hospital, Dhaka. Patients are classified into 3 groups depending on clinical & laboratory findings: Group 1 dengue fever (DF), Group 2 dengue hemorrhagic fever & Group 3 dengue shock syndrome. A total of 240 cases were taken in this study who fulfilled the selection criteria. Out of whom 125 male and 115 female patients. DF was 157(65.4%) & DHF was 83(34.6%). Aminotransferases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] were significantly raised in DHF cases compared to those of classical dengue fever (AST 84.5±42.4 in DF vs. 507±106.8 IU/L in DHF and ALT 59.9±31.3 in DF vs. 234±30.6 IU/L in DHF). The rise of AST is far greater than ALT in both DF and DHF. Dengue fever is usually associated with mild to moderate elevations of aminotransferase levels. The increase in aminotransferases, mainly AST has been associated with disease severity and serves as an early indicator of dengue infection.

Structure of nitric oxide synthase oxygenase dimer with pterin and substrate.

Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate L-arginine (L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and L-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35 degrees helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and L-Arg suggest that pterin has electronic influences on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.

The structure of nitric oxide synthase oxygenase domain and inhibitor complexes.

The nitric oxide synthase oxygenase domain (NOSox) oxidizes arginine to synthesize the cellular signal and defensive cytotoxin nitric oxide (NO). Crystal structures determined for cytokine-inducible NOSox reveal an unusual fold and heme environment for stabilization of activated oxygen intermediates key for catalysis. A winged beta sheet engenders a curved alpha-beta domain resembling a baseball catcher's mitt with heme clasped in the palm. The location of exposed hydrophobic residues and the results of mutational analysis place the dimer interface adjacent to the heme-binding pocket. Juxtaposed hydrophobic O2- and polar L-arginine-binding sites occupied by imidazole and aminoguanidine, respectively, provide a template for designing dual-function inhibitors and imply substrate-assisted catalysis.

Estrogen increases precursor for pregnenolone synthesis with temperature-sensitive occupancy of P-450scc in mitochondria of rabbit corpus luteum.

To examine the mechanism of estrogen's direct stimulation of steroidogenesis in the rabbit corpus luteum, we tested the hypothesis that the effect of estrogen on progestin production occurs at the site of processing of the precursor for pregnenolone (i.e. cholesterol) in the mitochondrion. For this purpose, we manipulated a model of estrogen stimulation by 1) removing sc estradiol-filled polydimethylsiloxane capsules from superovulated rabbits on day 9 of pseudopregnancy or 2) leaving the capsules in place to preserve a chronic estrogen stimulus. In the estrogen-deprived rabbits, the serum progesterone level fell precipitously in vivo within 24 h, but in rabbits with chronic estrogen stimulation, serum progesterone levels remained high. Our results show that the loss in progestin production caused by estrogen deprivation could not be attributed to loss of the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc), a common rate-limiting step in progestin synthesis in many steroidogenic tissues. In addition, we confirmed that there was no loss in the catalytic activity of this enzyme. Treatment with aminoglutethimide in vivo followed by electron paramagnetic resonance spectroscopic analysis of mitochondria (prepared in aminoglutethimide-free buffers) showed that incubation of isolated mitochondria at 37 C and pH 6.2 caused an increased high spin state (g = 8.2 signal) and a concomitant decreased low spin state. This shift from low to high spin states, which is indicative of cholesterol-P-450scc complex formation, occurred in the luteal mitochondria from both estrogen-deprived and estrogen-stimulated rabbits. In further studies to localize estrogen's regulatory point, we determined that the initial (first minute) rate of production of pregnenolone (per mg protein or per U P-450scc) from endogenous precursor proceeded equally fast in mitochondria from estrogen-deprived and those from estrogen-stimulated rabbits. However, the rapid pregnenolone production in the estrogen-deprived group lasted for a shorter time and, after 30 min, yielded less pregnenolone per mg protein or per U P-450scc than did mitochondria from estrogen-stimulated rabbits. Addition of 25-hydroxycholesterol did not increase the initial rate of pregnenolone formation, indicating that precursor availability is not limiting during the initial period. In aggregate, these observations suggest that the effect of estrogen on progestin production in the rabbit corpus luteum is not regulation of the movement of cholesterol to the catalytic site on the inner mitochondrial membrane, even though this is a step in the regulation of protein hormone-stimulated steroidogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of progesterone synthesis and cytochrome P450 levels in rat luteal cells during human chorionic gonadotropin-induced desensitized state.

Down-regulation of hCG receptors and a steroidogenic lesion in progesterone production were observed in isolated luteal cells after 24-h treatment of pseudopregnant rats with 50 IU hCG. The luteal cells from hCG-desensitized rats exhibited a complete loss of steroidogenic response to lipoproteins in both the absence and presence of hCG. This loss of response was not overcome by the addition of 25-hydroxycholesterol. To examine if the loss of steroidogenic response is due to a transient loss of steroidogenic enzymes or inadequate delivery of substrate cholesterol, cholesterol side-chain cleavage enzyme (P450scc) activity was measured in the isolated mitochondria. No differences appeared in enzyme activity between control and hCG-treated groups. Furthermore, electron paramagnetic resonance spectra of mitochondrial cytochrome P450scc from control and hCG-treated groups exhibited no appreciable differences in low spin electron paramagnetic resonance signal (g = 1.9, 2.2, and 2.4) or high spin EPR signal (g = 8.2), suggesting that the binding of the substrate to P450scc was not impaired. Additionally, the iron sulfur protein signal (g = 1.95 and 2.03) of the reduced mitochondria remained unchanged in the desensitized group. These data were supported by immunoblotting analysis which revealed no difference in the relative amounts of iron sulfur protein or cytochrome P450scc between the two groups. Moreover, the inner mitochondrial membranes, the locus of cytochrome P450scc, showed an identical cholesterol content in control and desensitized mitochondria, indicating that the availability of cholesterol substrate is not impaired during desensitization. These results suggest that the intramitochondrial transfer of cholesterol and cholesterol side-chain cleavage enzyme activity are unaffected in the desensitized state, but an inhibitory substance might be responsible for the lack of steroidogenic response.

Regulation of cholesterol side-chain cleavage enzyme activity by gonadotropin in rat corpus luteum.

The locus of gonadotropin-induced acute stimulation of pregnenolone production by cholesterol side-chain cleavage (CSCC) enzyme containing cytochrome P450 (cytP450scc) was examined in rat corpus luteum. Mitochondria were isolated from pseudopregnant rat ovaries after treatment with different doses of human CG (hCG) (25-200 IU) for 30 min; Electron Paramagnetic Resonance (EPR) spectra of high spin cholesterol complex of cyt P450scc (type I high spin EPR signal) and the cyt P450scc activity were determined. hCG treatment increased the formation of type I EPR spectra compared to that obtained with saline-treated controls, and pretreatment with cycloheximide (30 mg/kg BW) before hCG abolished this increase. The magnitude of type I EPR signal diminished with increasing pH over the range of 6.2-7.3. The type I EPR signal increased with doses of hCG and correlated well with the pregnenolone production. Aminoglutethimide treatment (competitive inhibitor of CSCC) before hCG injection led to an increased accumulation of cholesterol in inner mitochondrial membranes with a corresponding decrease in the outer membrane cholesterol, and cycloheximide treatment inhibited this accumulation. This suggests that the transport of cholesterol to inner mitochondrial membranes from outer membranes is regulated by hCG. In addition, gonadotropin also regulates the redistribution of cholesterol within the inner mitochondrial membranes.

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins.

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.

Purification and partial characterization of two azoreductases from Shigella dysenteriae type 1.

Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.

Effect of bioamines on uptake of promastigotes of Leishmania donovani by hamster peritoneal macrophages.

Epinephrine and norepinephrine inhibit attachment of Leishmania donovani promastigotes to cultured hamster peritoneal macrophages. The inhibition was significant at catecholamine concentrations of 10(-4) and 10(-5) M and occurred when they were added to the cell mixtures, or after pre-treatment of either macrophages or parasites. Inhibition of attachment after pre-treatment was less marked than when the catecholamines were added to parasite-cell mixtures. Similar results were obtained with dibutyryl cyclic AMP, cholera toxin, theophylline, and cadaverine which raise intracellular cyclic AMP (cAMP). Pretreatment of parasites or macrophages with the bioamines elevated the intracellular cAMP concentration. It is suggested that the inhibitory effect on the host-parasite interaction is mediated through cAMP.

Effect of cold exposure on peroxidase and iodinase activities of rat submandibulary gland.

Exposure of rats to 6 +/- 2 degrees C for 3 h caused an increase in peroxidase activity (55%) and tyrosine iodinase activity (40%) of the submandibulary glands. Thiocyanate, hydrogen peroxide and formation of triiodide from iodide were also elevated under the same conditions. Administration of alpha-receptor blockers and indomethacin prevented the rise of peroxidase activity during cold stress whereas beta-receptor blockers were less effective.

An approach for immunodiagnosis of clinical cases of filariasis.

In this communication an immunodiagnostic approach has been adopted for detection of antigen and antibody in amicrofilaeamic Mf(-) patients by countercurrent immuno electrophoresis (CCIE) and immunodiffusion (ID). Using Setaria cervi and Immune Complex (IC) antigens, out of fifteen clinical cases the number of positive patients in CCIE were twelve and ten respectively. Sixty percent of the Mf(-) cases were positive in antigen detection against both the homologous and heterologous antibody. In ID nine Mf(-) cases gave precipitin bands against S. cervi antigen while with IC antigens ten patients were positive. In similar experiments, it was found that out of fifteen Mf(-) cases nine and eleven patients were positive in antigen detection against microfilaraemic Mf(+) sera and S. cervi antibody respectively. All the Mf(+) cases were positive in both antibody and antigen detection. From the standpoint of immunodiagnosis the data were analysed by two-way analysis of variance study and a newly developed system using Binomial distribution. The sera from the control group were negative in all the immunodiagnostic tests.

Effect of ureastibamine on Leishmania donovani amastigote.

Ureastibamine, a pentavalent antimonial, reduced the parasitic load in the 60-day model of infection of L. donovani in hamsters. It also inhibited the in vivo multiplication of I donovani amastigotes in hamster peritoneal macrophages. No inhibition in either promastigote multiplication or amastigotes transformation was noted with filtrate obtained after incubation of the drugs for 72 h in the macrophage culture. Incubation of macrophages with ureastibamine revealed an impairment in the uptake of deoxyglucose. The effect of ureastibamine was compared with that of another pentavalent antimonial, sodium stibogluconate. It is suggested that impairment of macrophage membrane may contribute towards the adverse effect of these drugs against the intracellular parasite.

Characterization of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India.

1. It is widely accepted that foul or polluted environments are the principal sources of potentially pathogenic species of free-living amoebae. The present paper is the first report of occurrence of potentially pathogenic free-living amoebae in sewage samples of Calcutta, India. 2. We describe the occurrence, isolation, specific identification and comparative mouse pathogenicity test of two pathogenic amoebae, viz., Naegleria fowleri (N. aerobia) Carter, 1970, causing human meningoencephalitis and Acanthamoeba castellanii Douglas, 1930, causing granulomatous amoebic encephalitis, and one non-pathogenic amoeba, viz., A. astronyxis Ray and Hayes, 1959, in sewage samples of Calcutta, India. 3. The existence of both pathogenic and non-pathogenic amoebae living side by side is of considerable epidemiological relevance.

Beryllium: an easily scavenged metal ion in the environment.

This study was specifically aimed to determine the levels of beryllium in environmental samples near the vicinity of the beryllium metal plant (BMP). Air particulate samples collected at the BMP site, in the non-monsoon and monsoon seasons, showed an average beryllium concentration of 0.3 and 0.1 ng m(-3) respectively, where as rain water samples showed the beryllium values in the range of 0.01-0.2 ng ml(-1). The suspended particulate matter (dust load) at the site studied was 570 and 250 microg m(-3) in the non-monsoon and monsoon seasons respectively. The results obtained show that, in the environment, 80% of the total beryllium present is removed by rain.

Mercury and organomercurial resistance in bacteria isolated from freshwater fish of wetland fisheries around Calcutta.

Mercury-resistant bacteria belonging to the genera Bacillus, Escherichia, Klebsiella, Micrococcus, Pseudomonas, Salmonella, Sarcina, Shigella, Staphylococcus and Streptococcus were isolated from gills and guts of fresh water fish collected from wetland fisheries around Calcutta, India, contaminated with mercury compounds. The total number of bacteria, as well as Hg-resistant bacteria, were always higher in guts than gills. Bottom-dwelling fish contained higher number of bacteria, including Hg-resistant bacteria, than surface and middle water dwelling fish. They belonged either to narrow-spectrum or to broad-spectrum Hg-resistant groups and they also possessed other heavy metal and antibiotic resistant properties. In the presence of toxic levels of HgCl(2), phenylmercuric acetate (PMA) and methylmercuric chloride (MMC), the lag in growth of the bacterial strains gradually increased with increasing concentration of Hg-compounds. Narrow-spectrum Hg-resistant bacterial strains volatilized only HgCl(2) from the liquid medium in the range of 64-89%, whereas the broad-spectrum group exhibited a high level of HgCl(2) (80-94%), PMA (72-84%) and MMC (64-80%) volatilizing capacity with inducible mercuric reductase and organomercurial lyase enzyme activities in their cell-free extracts. Cell-free extracts prepared from narrow-spectrum Hg-resistant bacterial strains induced by HgCl(2) exhibited Hg(+2)-dependent NADPH oxidation, indicating the presence of only mercuric reductase enzyme.

Lipid peroxidation of erythrocytes in visceral leishmaniasis.

Lipid peroxidation of erythrocytes was studied in kala-azar patients having a considerable degree of anemia. Enhanced formation of oxidative metabolic products was observed in the erythrocytes of these patients. Decreased activities of the protective enzymes suggest impairment of the defense mechanism against peroxidative threat. These may contribute to some extent to the shortened lifespan of red cells in visceral leishmaniasis.

Anemia in experimental visceral leishmaniasis in hamsters.

Experimental infection of hamsters with Leishmania donovani caused visceral leishmaniasis in which hematological changes occurred. The infected hamsters were anemic and reticulocyte counts were high. No significant change in the serum erythropoietin level was noted. Red cell membrane Na(+)-K(+)-ATPase and acetylcholinesterase activities increased. Osmotic fragility of the erythrocytes from infected animals increased. The level of 2,3-diphosphoglycerate of the red cells increased with the degree of anemia.

Kinetoplastid flagellates: surface-reactive carbohydrates detected by fluorescein-conjugated lectins.

Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL.