Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean)

Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.     

Epidemiology of Tuberculosis in an Urban Slum of Dhaka City,Bangladesh

Background

The objectives of this study were to assess the tuberculosis (TB) burden and to provide an insight into the type of circulating M. tuberculosis species in urban slums of Bangladesh. We also aimed to test the feasibility of a larger transmission study in this setting.

Methods

This cross-sectional study was conducted in an urban slum of Dhaka city. The household members were actively screened to assess the presence of TB-related signs and symptoms; cough ≥3 weeks and body mass index (BMI) <17 kg/m². Sputum specimens from suspects were collected for acid fast bacilli (AFB) microscopy, culture and drug susceptibility testing. Genotyping of M. tuberculosis was done using spoligotyping and variable number tandem repeats of mycobacterial interspersed repetitive units typing.

Results

Among 9,877 adult screened for pulmonary TB (PTB), 25 were positive for AFB on microscopy and/or culture and the prevalence of new PTB cases was estimated to be 253/100,000. Only one child TB case was diagnosed among 5,147 child screened. Out of 26 cases, 21(81%) had cough for several duration and 5(19%) did not present with cough at the time of screening. One multidrug resistant case was found. Fifty two percent of all TB cases had BMI <17 kg/m2 (p = <0.001). Among the 20 analyzed isolates, 13 different spoligotype patterns were identified in which 5 clusters contained 12 strains and 8 strains had unique pattern.

Conclusions

The study revealed high prevalence of TB in urban slums. Screening using low BMI can be beneficial among risk group population. It is important to conduct larger study to validate clinical variables like cough <3 weeks and low BMI to define TB suspect and also to investigate the transmission of TB in slum settings.     

Length–weight relationships of 12 indigenous fish species in the Gajner Beel floodplain (NW Bangladesh)

The length–weight relationships (LWRs) were studied for 814 individuals covering eight families and 12 species of fish, e.g., Botia dario, Botia lohachata, Channa orientalis, Esomus danricus, Heteropneustes fossilis, Macrognathus pancalus, Nandus nandus, Ompok pabda, Pethia phutunio, Pethia ticto, Trichogaster fasciata and Trichogaster lalius captured from the Gajner beel floodplain, northwestern (NW) Bangladesh. Samples were collected occasionally from catches of fishermen from November 2014 to October 2015 using different types of traditional fishing gear, including gill net (mesh size ranges from 1.0 to 3.0 cm), cast net (mesh size ranges from 1.0 to 2.0 cm), square lift net (mesh size ~1.0 cm) and conical and box trap. The total length (TL) and body weight (BW) were measured for each individual. The allometric co‐efficient b values ranged between 2.64 (B. lohachata) to 3.27 (N. nandus) and were highly significant, with p < .001 for all species. Additionally, for the first time, the study provides LWR information for B. dario, B. lohachata, E. danricus, O. pabda, T. lalius and P. phutunio, beneficial for fishery biologists and to update the online database (FishBase). The study also recorded a maximum length for P. phutunio. The present observations are significant for the sustainable management and conservation of these fishes in the Gajner beel floodplain, NW Bangladesh and the surrounding ecosystems.     

Effect of pH,temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain

The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain fromAnanas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.     

Characterization of a proteinase inhibitor from Cajanus cajan (L.)

A protein proteinase inhibitor (PI) has been purified from pigeonpea Cajanus cajan (L.) PUSA 33 variety by acetic-acid precipitation, salt fractionation and chromatography on a DEAE-Cellulose column. The content of inhibitor was found to be 15 mg/20 g dry weight of pulse. The molecular weight of the inhibitor as determined by SDS-PAGE under reducing conditions was found to be about 14,000. It showed inhibitory activity toward proteolytic enzymes belonging to the serine protease group, namely trypsin and alpha-chymotrypsin. The inhibitory activity was stable over a wide range of pH and temperatures. Estimation of sulfhydryl groups yielded one free cysteine and at least two disulfide linkages. N-terminal sequence homology suggests that it belongs to the Kunitz inhibitor family. Structural analysis by circular dichroism shows that the inhibitor possesses a largely disordered structure.     

Imaging of the GI tract by QDs loaded heparin-deoxycholic acid (DOCA) nanoparticles

This study presents an approach to deliver non invasive, near-IR imaging agent using oral delivery system. Low molecular weight heparin (LMWH)-deoxycholic acid (DOCA)/(LHD) nanoparticles formed by a self-assembly method was prepared to evaluate their physicochemical properties and oral absorption in vitro and in vivo. Near-IR QDs were prepared and loaded into LHD nanoparticles for imaging of the gastro-intestinal (GI) tract absorption. Q-LHD nanoparticles were almost spherical in shape with diameters of 194-217nm. The size and fluorescent intensity of the Q-LHD nanoparticles were stable in 10% FBS solution and retained their fluorescent even after 5 days of incubation. Cell viability of Q-LHD nanoparticles maintained in the range of 80-95% for 24h incubation. No damage was found in tissues or organs during animal experiments. The in vivo oral absorption of Q-LHD was observed in SKH1 mice for 3h under different doses. From the results, we confirmed that Q-LHD was absorbed mostly into the ileum of small intestine containing intestinal bile acid transporter as observed in TEM and molecular imaging system. Our designed nanoparticles could be administered orally for bio-imaging and studying the bio-distribution of drug.     

Zinc supplementation in children with cholera in Bangladesh: randomised controlled trial

Objective To investigate the impact of zinc supplementation in children with cholera.Design Double blind, randomised, placebo controlled trial.Setting Dhaka Hospital, Bangladesh.Participants 179 children aged 3-14 years with watery diarrhoea and stool dark field examination positive for Vibrio cholerae and confirmed by stool culture.Intervention Children were randomised to receive 30 mg elemental zinc per day (n=90) or placebo (n=89) until recovery. All children received erythromycin suspension orally in a dose of 12.5 mg/kg every six hours for three days.Main outcome measures Duration of diarrhoea and stool output.Results 82 children in each group completed the study. More patients in the zinc group than in the control group recovered by two days (49% v 32%, P=0.032) and by three days (81% v 68%, P=0.03). Zinc supplemented patients had 12% shorter duration of diarrhoea than control patients (64.1 v 72.8 h, P=0.028) and 11% less stool output (1.6 v 1.8 kg/day, P=0.039).Conclusion Zinc supplementation significantly reduced the duration of diarrhoea and stool output in children with cholera. Children with cholera should be supplemented with zinc to reduce its duration and severity.Trial registration Clinical trials {"type":"clinical-trial","attrs":{"text":"NCT00226616","term_id":"NCT00226616"}}NCT00226616.     

The estimation of pollen viability in rice

Methods for testing pollen viability in rice were evaluatedby comparing staining with aniline blue in lactophenol, twotetrazolium salts and fluorescein diacetate with germinationin an in vitro culture medium. Staining with thiazolyl blue(MTT) was nicely correlated with germination. Under non-saline conditions, rice pollen was only viable fora short period of time, with an approximately 50% loss of viabilitywithin 20 min of shedding. When plants were salinized at eitherpanicle initiation or the booting stage, pollen viability wasreduced. Key words: Rice, Oryza sativa, pollen viability, salinity     

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Phosphorylation in the activation loop of the Raf-like kinase ARK is critical for SNF1-related protein kinase2 regulation during abscisic acid responses in the moss Physcomitrium (Physcomitrella) patens.