Shveta Gupta

In vitro Neurogenesis from Neural Progenitor Cells Isolated from the Hippocampus Region of the Brain of Adult Rats Exposed to Ethanol during Early Development through Their Alcohol-Drinking Mothers

AIMS This study was aimed to determine whether ethanol exposure during early development altered neurogenesis in the brain of adult rats. METHODS Pregnant rats were given either ethanol-mixed or mannose-mixed (for control) rodent liquid diet ad libitum. Ethanol drinking continued during pregnancy and nursing. After weaning, the pups (AC(o): pups from control mothers, AE(o): pups from ethanol exposed mothers) received normal diet and water ad libitum for 11 weeks. Then the rats were anesthetized, their brains were collected and the hippocampal samples were processed for isolation of neural progenitor cells (NPCs). AC(o) NPCs and AE(o) NPCs were sequentially grown in media containing different growth factors that induced proliferation and differentiation. RESULTS AND CONCLUSIONS Neuronal maturation was significantly delayed in ethanol-exposed rats. AC(o) NPCs, up to day 7 of culture, exhibited high beta-catenin-probe binding, an increase in Ca(2+) when exposed to gamma-amino butyric acid (GABA) and lack of response to glutamate (Glu) exposure. beta-Catenin-probe binding and the stimulatory effects of GABA declined thereafter. AC(o) NPCs, at culture day 29, exhibited high beta-catenin-probe binding, lack of response to GABA and elevated Glu-induced increase in Ca(2+i). Cultures of AE(o) NPCs showed an amplified stimulatory effects of GABA, attenuated stimulatory effects of Glu and attenuated the delayed (culture day 29) increase in the expression of Wnt proteins and beta-catenin-probe binding. This suggests a significant alteration in neurogenesis and synapse formation in adult rats exposed to ethanol at early development through their alcohol-drinking mothers.

Effects of bacterial toxins on endothelial tight junction in vitro: a mechanism-based investigation.

ABSTRACT Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), principal cell wall components of Gram-negative and Gram-positive bacteria, respectively, play a central role in altering the blood-brain barrier and facilitate bacterial infection of the host brain. Despite the significance of bacterial toxins in disease pathogenesis, mechanisms by which toxins impair the barrier are not yet known. This study, using an in vitro cell culture model, showed that LPS and LTA interacted with the endothelial cells and disrupted the tight junction between the cells that increased the barriers permeability. Both toxins increased inducible nitric oxide synthase (iNOS) mRNA that is indicative of an increase in intracellular NO release, disrupted architecture of the tight junction proteins, suppressed zonula occludens-1 (ZO-1) and occludin (OCL) and junctional adhesive molecules (JAM) mRNA levels, and increased tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) mRNA levels. Anti-CD14 antibodies blocked the increase in TNFα and IL-1β mRNA levels but did not affect either changes in the tight junction or iNOS, ZO-1, OCL, and JAM mRNA levels in endothelial cells and astrocytes. Although both toxins did not cross the endothelial barrier, the abluminal neurons exhibited high inflammatory activity characterized by a sequential increase in TNFα, IL-1β, external receptor kinase (ERK), and RelA-p50 that induced inflammation, followed by an increase in anti-inflammatory/apoptotic factors including IL-10 and cysteine-aspartic acid protease-8 (CASPASE-8), which resolve inflammation and induce apoptosis. Anti-CD14 antibodies in luminal buffer blocked the pro- and anti-inflammatory effects of the toxins in neurons. Thus, the CD14-TLR cascade that participates in the inflammatory effects of toxins may not participate in the toxin-induced barrier disruption in vitro. Since the toxins did not cross the endothelial barrier, induction of inflammation in neurons was due to a release of proinflammatory cytokines in the abluminal fluid.

Anti-Inflammatory Potency of Nano-Formulated Puerarin and Curcumin in Rats Subjected to the Lipopolysaccharide-Induced Inflammation

Puerarin (PU) and curcumin (CU), used commonly in traditional Chinese medicine and Ayurveda, have been shown to possess potent anti-inflammatory, anti-oxidation, and neuro-protective properties. Despite the experimental success of CU and PU in in vitro and animal models, their effectiveness has not yet been demonstrated in clinical trials, possibly because of their poor bioavailability. We hypothesized that gold nanoparticle (AuNP)-formulated PU (PU-AuNP), CU (CU-AuNP), or a combination of PU and CU (PU-CU-AuNP) were a more effective and nontoxic alternative to their bulk (nonformulated) counterparts. To test the hypothesis, bioavailability, therapeutic potency, and toxicity of bulk CU and/or PU were compared with those of their nanotized counterparts in rats subjected to the lipopolysaccharide (LPS)-induced inflammation. This study showed that a 20-mg/kg dose of bulk PU or a mixture of PU and CU did not, while their nanotized counterparts, PU-AuNP, CU-AuNP, or PU-CU-AuNP, effectively suppressed the LPS-induced inflammation and cytotoxicity in rats. In addition, PU-CU-AuNP was more potent than PU-AuNP or CU-AuNP alone. The blank AuNP (bAuNP) at ≤40 mg/kg dose did not cause any adverse effects (blood and brain lactic acid concentrations, kidney function, and neuronal apoptosis were measured) in animals. Therefore, the present observations suggest that a bi-functional AuNP loaded with CU and PU may effectively suppress the LPS-induced inflammation and cytotoxicity provided the following conditions are met: (1) The AuNP dose is at or below the no-effect dose; (2) the nanoparticles release a therapeutic dose of CU and PU in vivo; and (3) the active ingredients are released into the intracellular component of the brain.

Quantitative analysis of conjugated and free estrogens in swine manure: solutions to overcome analytical problems due to matrix effects.

Although animal manure is an important source for environmental estrogens, quantitative analysis of estrogens in manure is complicated due to matrix interference. In the present study, chromatographic methods have been developed for quantification of conjugated and free estrogens in manure samples collected from pig farms. The whole manure samples, immediately after collection, were stored at 4°C, acidified (pH≈2.0) and spiked with (i) (13)C-labeled internal standards to account for possible storage related degradation and (ii) deuterium labeled internal standards for calibration and quantitative analysis. The liquid samples were extracted with ethyl acetate for separating conjugated and free estrogens. The solid samples were eluted with water for desorbing conjugated hormones followed by methanol for desorbing free hormones. The water and extracts were further purified using hydrophilic-lipophilic balance and/or aminopropyl cartridges. The conjugated estrogens were analyzed using high-performance liquid chromatograph-mass spectrometer, while the free estrogens were analyzed using gas chromatograph-mass spectrometer. The extraction and calibration methods used in the present study yielded excellent sensitivity, reproducibility and >85% recovery of both free and conjugated estrogens that was independent of the manure matrix. In general, the total estrogen loads in liquid and solid samples were 5.1mg/l and 4.93mg/kg, respectively. This may represent the hormonal load of approximately 2.3tons estrogen per day.

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Darbepoietin (DAR) and recombinant human erythropoietin (rhEPO) stimulate erythropoiesis, leading to an increase in red blood cells. Along with their legitimate clinical use, rhEPO and DAR are also misused in racing horses for performance enhancement. To control the illegal use of DAR and rhEPO, it is important to develop analytical methods for the detection and confirmation of these proteins in plasma. Analysis of rhEPO and DAR in plasma is challenging due to the presence of a number of high abundance proteins including albumin that interferes with their extraction. The present study showed that the extraction of rhEPO or DAR from plasma using anti‐EPO–antibody coupled immunoaffinity (IA) extraction yielded low (25–40%) recovery. Albumin‐depletion using antialbumin–antibody coupled IA columns also depleted the target proteins and further reduced their recovery. Pre‐extraction of spiked plasma using hydroxyapatite (HTP)‐ProGel or ConA columns followed by the IA column yielded 65 to 70% recovery. The extracted samples were (i) analyzed directly with or without SDS‐PAGE for intact proteins and (ii) analyzed after trypsin hydrolysis, with or without SDS‐PAGE, for peptide fingerprinting using MALDI‐TOF. Trypsin and enolase were used as internal calibrators for intact protein analysis and a peptide EYEATLEECCAK was used as internal calibrator for fragment analysis. Analysis of extracted sample without SDS‐PAGE yielded, along with the target proteins (rhEPO and DAR), albumin and other related proteins. SDS‐PAGE separated the target proteins with albumin and yielded clean samples. Inclusion of internal calibrators resulted in a linear dose–response relationship for both intact protein and digested fragments and allowed quantification of the target peptides. Thus, extraction of plasma using a combination of ConA and IA extractions yielded approximately 70% recovery of target proteins with a small amount of albumin and other proteins. SDS‐PAGE improved the quality of the MALDI‐TOF results. Minimum detection limits for digested fragments were lower than those for intact proteins.