Purification and Partial Characterization of Low Molecular Weight Vicilin-Like Glycoprotein from the Seeds of <Emphasis Type="Italic">Citrullus lanatus</Emphasis>

The watermelon (Citrullus lanatus) seeds are highly nutritive and contain large amount of proteins and many beneficial minerals such as magnesium, calcium, potassium, iron, phosphorous, zinc etc. In various parts of the world, C. lanatus seed extracts are used to cure cancer, cardiovascular diseases, hypertension, and blood pressure. C. lanatus seed extracts are also used as home remedy for edema and urinary tract problems. In this study, we isolated protein fraction of C. lanatus seeds using various protein separation methods. We successfully purified a low molecular weight vicilin-like glycoprotein using chromatographic methods followed by SDS-PAGE and MALDI-TOF/MS identification. This is the first report of purification of a vicilin like polypeptide from C. lanatus seeds. In next step, we extracted mRNA from immature seeds and reverse transcribed it using suitable forward and reverse primers for purified glycoprotein. The PCR product was analysed on 1% agarose gel and was subsequently sequenced by Dideoxy DNA sequencing method. An amino acid translation of the gene is in agreement with amino acid sequences of the identified peptides.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in saliva

A high performance liquid chromatographic method for determination of moxifloxacin in human saliva was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.25 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from saliva was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Humanizing π-class glutathione S-transferase regulation in a mouse model alters liver toxicity in response to acetaminophen overdose

Background

Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of π-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. π-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/2−/− mice escape liver injury.

Methodology/Principal Findings

To more faithfully model the contribution of π-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/2−/− strain, π-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/2−/− mice, when hGSTP1+mGstp1/2−/− mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that π-class GSTs may be critical determinants of toxin-induced hepatocyte injury even when not expressed by hepatocytes.

Conclusions

By recapitulating human π-class GST expression, hGSTP1+mGstp1/2−/− mice may better model human drug and xenobiotic toxicology.     

Male use of female sex work in India: a nationally representative behavioural survey

Heterosexual transmission of HIV in India is driven by the male use of female sex workers (FSW), but few studies have examined the factors associated with using FSW. This nationally representative study examined the prevalence and correlates of FSW use among 31,040 men aged 15–49 years in India in 2006. Nationally, about 4% of men used FSW in the previous year, representing about 8.5 million FSW clients. Unmarried men were far more likely than married men to use FSW overall (PR = 8.0), but less likely than married men to use FSW among those reporting at least one non-regular partner (PR = 0.8). More than half of all FSW clients were married. FSW use was higher among men in the high-HIV states than in the low-HIV states (PR = 2.7), and half of all FSW clients lived in the high-HIV states. The risk of FSW use rose sharply with increasing number of non-regular partners in the past year. Given the large number of men using FSW, interventions for the much smaller number of FSW remains the most efficient strategy for curbing heterosexual HIV transmission in India.     

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer''s disease, and head injury.Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.Click here to view the related article Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.Download video file.^{(53M, mov)}     

The vividness of happiness in dynamic facial displays of emotion

Rapid identification of facial expressions can profoundly affect social interactions, yet most research to date has focused on static rather than dynamic expressions. In four experiments, we show that when a non-expressive face becomes expressive, happiness is detected more rapidly anger. When the change occurs peripheral to the focus of attention, however, dynamic anger is better detected when it appears in the left visual field (LVF), whereas dynamic happiness is better detected in the right visual field (RVF), consistent with hemispheric differences in the processing of approach- and avoidance-relevant stimuli. The central advantage for happiness is nevertheless the more robust effect, persisting even when information of either high or low spatial frequency is eliminated. Indeed, a survey of past research on the visual search for emotional expressions finds better support for a happiness detection advantage, and the explanation may lie in the coevolution of the signal and the receiver.     

Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation

Inflammation has classically been defined histopathologically, especially by the presence of immune cell infiltrates. However, more recent studies suggest a role for "low-grade" inflammation in a variety of disorders ranging from metabolic syndrome to cancer, which is defined by modest elevations in pro-inflammatory gene expression. Consequently, there is a need for cost-effective, non-invasive biomarkers that, ideally, would have the sensitivity to detect low-grade inflammation and have a dynamic range broad enough to reflect classic robust intestinal inflammation. Herein, we report that, for assessment of intestinal inflammation, fecal lipocalin 2 (Lcn-2), measured by ELISA, serves this purpose. Specifically, using a well-characterized mouse model of DSS colitis, we observed that fecal Lcn-2 and intestinal expression of pro-inflammatory cytokines (IL-1β, CXCL1, TNFα) are modestly but significantly induced by very low concentrations of DSS (0.25 and 0.5%), and become markedly elevated at higher concentrations of DSS (1.0 and 4.0%). As expected, careful histopathologic analysis noted only modest immune infiltrates at low DSS concentration and robust colitis at higher DSS concentrations. In accordance, increased levels of the neutrophil product myeloperoxidase (MPO) was only detected in mice given 1.0 and 4.0% DSS. In addition, fecal Lcn-2 marks the severity of spontaneous colitis development in IL-10 deficient mice. Unlike histopathology, MPO, and q-RT-PCR, the assay of fecal Lcn-2 requires only a stool sample, permits measurement over time, and can detect inflammation as early as 1 day following DSS administration. Thus, assay of fecal Lcn-2 by ELISA can function as a non-invasive, sensitive, dynamic, stable and cost-effective means to monitor intestinal inflammation in mice.