Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.     

Prevalence of overweight and obesity among the affluent adolescent school children of Amritsar, Punjab

In the present study, an attempt has been made to assess the prevalence of overweight and obesity in adolescent children (between 10-15 years of age) of the affluent families of Amritsar district of Punjab, a state in rapid economic and epidemiological transition. A total of 640 children (323 boys and 317girls) were measured for height and weight. Overweight and obesity were assessed using age and sex-specific body mass index (BMI) cut-off points. 9.91% boys and 11.99% girls were overweight, and 4.95% boys and 6.31% girls were obese. The prevalence of overweight and obesity among the affluent children in Amritsar was as high or higher as in some industrialized countries.     

Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7^th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.     

The Effect of Intermittent Antenatal Iron Supplementation on Maternal and Infant Outcomes in Rural Viet Nam: A Cluster Randomised Trial

Background

Anemia affects over 500 million women, and in pregnancy is associated with impaired maternal and infant outcomes. Intermittent antenatal iron supplementation is an attractive alternative to daily dosing; however, the impact of this strategy on infant outcomes remains unclear. We compared the effect of intermittent antenatal iron supplementation with daily iron supplementation on maternal and infant outcomes in rural Viet Nam.

Methods and Findings

This cluster randomised trial was conducted in Ha Nam province, Viet Nam. 1,258 pregnant women (<16 wk gestation) in 104 communes were assigned to daily iron–folic acid (IFA), twice weekly IFA, or twice weekly multiple micronutrient (MMN) supplementation. Primary outcome was birth weight. Mean birth weight was 3,148 g (standard deviation 416). There was no difference in the birth weights of infants of women receiving twice weekly IFA compared to daily IFA (mean difference [MD] 28 g; 95% CI −22 to 78), or twice weekly MMN compared to daily IFA (MD −36.8 g; 95% CI −82 to 8.2). At 32 wk gestation, maternal ferritin was lower in women receiving twice weekly IFA compared to daily IFA (geometric mean ratio 0.73; 95% CI 0.67 to 0.80), and in women receiving twice weekly MMN compared to daily IFA (geometric mean ratio 0.62; 95% CI 0.57 to 0.68), but there was no difference in hemoglobin levels. Infants of mothers who received twice weekly IFA had higher cognitive scores at 6 mo of age compared to those who received daily IFA (MD 1.89; 95% CI 0.23 to 3.56).

Conclusions

Twice weekly antenatal IFA or MMN did not produce a clinically important difference in birth weight, when compared to daily IFA supplementation. The significant improvement in infant cognitive outcomes at 6 mo of age following twice weekly antenatal IFA requires further exploration, and provides additional support for the use of intermittent, rather than daily, antenatal IFA in populations with low rates of iron deficiency.

Trial registration

Australia New Zealand Clinical Trials Registry 12610000944033 Please see later in the article for the Editors'' Summary     

Transient gastric irritation in the neonatal rats leads to changes in hypothalamic CRF expression, depression- and anxiety-like behavior as adults

Aims

A disturbance of the brain-gut axis is a prominent feature in functional bowel disorders (such as irritable bowel syndrome and functional dyspepsia) and psychological abnormalities are often implicated in their pathogenesis. We hypothesized that psychological morbidity in these conditions may result from gastrointestinal problems, rather than causing them.

Methods

Functional dyspepsia was induced by neonatal gastric irritation in male rats. 10-day old male Sprague-Dawley rats received 0.1% iodoacetamide (IA) or vehicle by oral gavage for 6 days. At 8–10 weeks of age, rats were tested with sucrose preference and forced-swimming tests to examine depression-like behavior. Elevated plus maze, open field and light-dark box tests were used to test anxiety-like behaviors. ACTH and corticosterone responses to a minor stressor, saline injection, and hypothalamic CRF expression were also measured.

Results

Behavioral tests revealed changes of anxiety- and depression-like behaviors in IA-treated, but not control rats. As compared with controls, hypothalamic and amygdaloid CRF immunoreactivity, basal levels of plasma corticosterone and stress-induced ACTH were significantly higher in IA-treated rats. Gastric sensory ablation with resiniferatoxin had no effect on behaviors but treatment with CRF type 1 receptor antagonist, antalarmin, reversed the depression-like behavior in IA-treated rats

Conclusions

The present results suggest that transient gastric irritation in the neonatal period can induce a long lasting increase in depression- and anxiety-like behaviors, increased expression of CRF in the hypothalamus, and an increased sensitivity of HPA axis to stress. The depression-like behavior may be mediated by the CRF1 receptor. These findings have significant implications for the pathogenesis of psychological co-morbidity in patients with functional bowel disorders.     

Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.     

Design, synthesis and antitumor evaluation of phenyl N-mustard-quinazoline conjugates

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.     

Domain interaction in cyanobacterial phytochromes as a prerequisite for spectral integrity

Two phytochromes, CphA and CphB, from the cyanobacterium Calothrix PCC7601, with similar size (768 and 766 amino acids) and domain structure, were investigated for the essential length of their protein moiety required to maintain the spectral integrity. Both proteins fold into PAS-, GAF-, PHY-, and Histidine-kinase (HK) domains. CphA binds a phycocyanobilin (PCB) chromophore at a “canonical” cysteine within the GAF domain, identically as in plant phytochromes. CphB binds biliverdin IXα at cysteine24, positioned in the N-terminal PAS domain. The C-terminally located HK and PHY domains, present in both proteins, were removed subsequently by introducing stop-codons at the corresponding DNA positions. The spectral properties of the resulting proteins were investigated. The full-length proteins absorb at (CphA) 663 and 707 nm (red-, far red-absorbing P _r and P _fr forms of phytochromes) and at (CphB) 704 and 750 nm. Removal of the HK domains had no effect on the absorbance maxima of the resulting PAS–GAF–PHY constructs (CphA: 663/707 nm, CphB: 704/750 nm, P _r/P _fr, respectively). Further deletion of the “PHY” domains caused a blue-shift of the P _r and P _fr absorption of CphA (λ _max: 658/698 nm) and increased the amount of unproperly folded apoprotein, seen by a reduced capability to bind the chromophore in photoconvertible manner. In CphB, however, it practically impaired the formation of P _fr, i.e., showing a very low oscillator strength absorption band, whereas the P _r form remains unchanged (702 nm). This finding clearly indicates a different interaction between domains in the “typical”, PCB binding and in the biliverdin-binding phytochromes, and demonstrates a loss of oscillator strength for the latter, most probably due to a strong conformational distortion of the chromophore in the CphB P _fr form. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.