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Rationale: Early-life stress (ELS) can increase anxiety, reduce prosocial behaviors, and impair brain regions that facilitate emotional and social development. This knowledge greatly stems from assessing disrupted mother-child relationships, while studies investigating the long-term effects of father-child relationships on behavioral development in children are scarce. However, available evidence suggests that fathers may uniquely influence a child's behavioral development in a sex-specific manner. Rodent models examining mother-offspring interaction demonstrate relationships among ELS, neuroinflammatory mediators, and behavioral development; yet, the role paternal care may play in neuroimmune functioning remains unreported., Objectives: Using the biparental California mouse (Peromyscus californicus), we examined to what extent paternal deprivation impairs social and anxiety-like behaviors, augments peripheral corticosterone (CORT) response, and alters central proinflammatory cytokine production following an acute stressor in adulthood., Methods: Biparentally reared and paternally deprived (permanent removal of the sire 24 h post-birth) adult mice were assessed for sociability, preference for social novelty, social vigilance, and social avoidance behaviors, followed by novelty-suppressed feeding (NSF) testing for general anxiety-like behavior. Following an acute stressor, circulating CORT concentrations and region-specific proinflammatory cytokine concentrations were determined via radioimmunoassay and Luminex multianalyte analysis, respectively., Results: In response to a novel same-sex conspecific, social vigilance behavior was associated with reduced sociability and increased avoidance in paternally deprived mice-an effect not observed in biparentally reared counterparts. Yet, in response to a familiar same-sex conspecific, social vigilance persisted but only in paternally deprived females. The latency to consume during NSF testing was not significantly altered by paternal deprivation. In response to an acute physical stressor, lower circulating CORT concentrations were observed in paternally deprived females. Compared to control-reared males, paternal deprivation increased hypothalamic interleukin-1β, but decreased hippocampal IL-6 protein concentration., Conclusion: Greater social vigilance behavior was demonstrated in paternally deprived mice while they avoided social interaction with a novel same-sex conspecific; however, in response to a familiar same-sex conspecific, paternal deprivation increased social vigilance behavior but only in females. It is possible that different neurobiological mechanisms underlie these observed behavioral outcomes as sex-specific central proinflammatory cytokine and stress responsivity were observed in paternally deprived offspring., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The aim of this review is to highlight our knowledge of the various drugs of abuse that can prove potential teratogens affecting the brain and cognitive development in an individual exposed to maternal consumption of such agents. Among several drugs of abuse in women, we specifically highlighted the commonly used alcohol, nicotine, opioids, cannabis, cocaine and marijuana. These drugs can affect the fetal development and slow the cognitive maturation apart from physical disabilities. However, no known therapy exists to counter the toxic potential of these drugs. Several researchers used animal models of drug abuse to understand the underlying mechanisms affecting brain development and the relevant neurotransmitter system. Identifying such targets can potentially help in drug discovery research. We reported in depth analysis of such mechanisms and discussed the potential targets for drug development research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gaucher disease (GD) is the most common lysosomal storage disease caused by deficiency of beta-glucocerebrosidase (GCase) resulting in lysosomal accumulation of its glycolipid substrate glucosylceramide. The activity of GCase depends on many factors such as proper folding and lysosomal localization, which are influenced by mutations in GCase encoding gene, and regulated by various GCase-binding partners including Saposin C, progranulin and heat shock proteins. In addition, proinflammatory molecules also contribute to pathogenicity of GD. In this review, we summarize the molecules that are known to be important for the pathogenesis of GD, particularly those modulating GCase lysosomal appearance and activity. In addition, small molecules that inhibit inflammatory mediators, calcium ion channels and other factors associated with GD are also described. Discovery and characterization of novel molecules that impact GD are not only important for deciphering the pathogenic mechanisms of the disease, but they also provide new targets for drug development to treat the disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) increases the risk of various liver injuries, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and ultimately hepatocellular carcinoma (HCC). Ample evidence has suggested that aberrant expression of microRNAs (miRNAs) is functionally involved in the activation of cellular stress, inflammation and fibrogenesis in hepatic cells, including hepatocytes, Kupffer and hepatic stellate cells (HSCs), at different pathological stages of NAFLD and liver fibrosis. Here, we overview recent findings on the potential role of miRNAs in the pathogenesis of NAFLD, including lipotoxicity, oxidative stress, metabolic inflammation and fibrogenesis. We critically assess the literatures on both human subjects and animal models of NAFLD and liver fibrosis with miRNA dysregulation and their mechanisms of actions in liver damage. We further highlight the potential use of miRNA mimics or antimiRNAs as therapeutic approaches for the prevention and treatment of NAFLD and liver fibrosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fructose is a highly lipogenic sugar that can alter energy metabolism and trigger metabolic disorders. In the current study, microRNAs (miRNAs) altered by a high-fructose diet were comprehensively explored to elucidate their significance in the pathogenesis of chronic metabolic disorders. miRNA expression profiling using small noncoding RNA sequencing revealed that 19 miRNAs were significantly upregulated and 26 were downregulated in the livers of high-fructose-fed mice compared to chow-fed mice. Computational prediction and functional analysis identified 10 miRNAs, miR-19b-3p, miR-101a-3p, miR-30a-5p, miR-223-3p, miR-378a-3p, miR-33-5p, miR-145a-3p, miR-128-3p, miR-125b-5p and miR-582-3p, assembled as a regulatory network to potentially target key genes in lipid and lipoprotein metabolism and insulin signaling at multiple levels. qRT-PCR analysis of their potential target genes [IRS-1, FOXO1, SREBP-1c/2, ChREBP, insulin-induced gene-2 (Insig-2), microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (apoB)] demonstrated that fructose-induced alterations of miRNAs were also reflected in mRNA expression profiles of their target genes. Moreover, the miRNA profile induced by high-fructose diet differed from that induced by high-fat diet, indicating that miRNAs mediate distinct pathogenic mechanisms in dietary-induced metabolic disorders. This study presents a comprehensive analysis of a new set of hepatic miRNAs, which were altered by high-fructose diet and provides novel insights into the interaction between miRNAs and their target genes in the development of metabolic syndrome., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Microglia are the immune effector cells that are activated in response to pathological changes in the central nervous system. Microglial activation is accompanied by the alteration of integrin expression on the microglia surface. However, changes of integrin expression upon chemoattractant (ADP) stimulation still remain unknown. In this study, we investigated whether ADP induces the alteration of integrin species on the cell surface, leading to changes in chemotactic ability on different extracellular matrix proteins. Flow cytometry scans and on-cell Western assays showed that ADP stimulation induced a significant increase of α6 integrin-GFP, but not α5, on the surface of microglia cells. Microglia also showed a greater motility increase on laminin than fibronectin after ADP stimulation. Time lapse microscopy and integrin endocytosis assay revealed the essential role of calcium-independent phospholipase A 2 activity for the recycling of α6 integrin-GFP from the endosomal recycling complex to the plasma membrane. Lack of calcium-independent phospholipase A 2 activity caused a reduced rate of focal adhesion formation on laminin at the leading edge. Our results suggest that the alteration of integrin-mediated adhesion may regulate the extent of microglial infiltration into the site of damage by controlling their chemotactic ability., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Insulin induced gene-2 (Insig-2) is an ER-resident protein that inhibits the activation of sterol regulatory element-binding proteins (SREBPs). However, cellular factors that regulate Insig-2 expression have not yet been identified. Here we reported that cyclic AMP-responsive element-binding protein H (CREBH) positively regulates mRNA and protein expression of a liver specific isoform of Insig-2, Insig-2a, which in turn hinders SREBP-1c activation and inhibits hepatic de novo lipogenesis. CREBH binds to the evolutionally conserved CRE-BP binding elements located in the enhancer region of Insig-2a and upregulates its mRNA and protein expression. Metabolic hormone glucagon and nutritional fasting activated CREBH, which upregulated expression of Insig-2a in hepatocytes and inhibited SREBP-1c activation. In contrast, genetic depletion of CREBH decreased Insig-2a expression, leading to the activation of SREBP-1c and its downstream lipogenic target enzymes. Compromising CREBH-Insig-2 signaling by siRNA interference against Insig-2 also disrupted the inhibitory effect of this signaling pathway on hepatic de novo triglyceride synthesis. These actions resulted in the accumulation of lipid droplets in hepatocytes and systemic hyperlipidemia. Our study identified CREBH as the first cellular protein that regulates Insig-2a expression. Glucagon activated the CREBH-Insig-2a signaling pathway to inhibit hepatic de novo lipogenesis and prevent the onset of hepatic steatosis and hypertriglyceridemia.

Objective: Akkermansia muciniphila (A muciniphila) is a mucin-degrading bacterium that resides in the mucus layer whose abundance inversely correlates with body weight and the development of diabetes mellitus in mice and humans. The objective of this study was to explore the regulatory effect of A muciniphila on host lipoprotein metabolism, insulin sensitivity, and hepatic metabolic inflammation., Approach and Results: By establishing a novel mouse model that colonized the A muciniphila in the gastrointestinal tract of the cAMP-responsive binding protein H (CREBH)-deficient mouse and in vivo chylomicron assay, we found that increased colonization of A muciniphila in the gastrointestinal tract of wild-type mice protected mice from an acute fat load-induced hyperlipidemia compared with vehicle-treated mice. A muciniphila administration also significantly ameliorated chronic hypertriglyceridemia, improved insulin sensitivity, and prevented overproduction of postprandial chylomicrons in CREBH-null mice. Mechanistic studies revealed that increased A muciniphila colonization induced expression of low-density lipoprotein receptors and apolipoprotein E in the hepatocytes of CREBH-null mice, which facilitated the uptake of intermediate-density lipoprotein via the mediation of apolipoprotein B100 and apolipoprotein E, leading to the increased clearance of triglyceride-rich lipoprotein remnants, chylomicron remnants, and intermediate-density lipoproteins, from the circulation. Treatment with A muciniphila further improved hepatic endoplasmic reticulum stress and metabolic inflammation in CREBH-null mice., Conclusions: Increased colonization of the disease-protective gut bacteria A muciniphila protected the host from acute and chronic hyperlipidemia by enhancing the low-density lipoprotein receptor expression and alleviating hepatic endoplasmic reticulum stress and the inflammatory response in CREBH-null mice., (© 2016 American Heart Association, Inc.)

Background: Periodontal disease is a significant public health issue worldwide. Motivational techniques in combination with financial incentives are shown to lead to effective behavior change. The current study sought to examine whether a brief oral health promotion program (self-management cues that were based on self-efficacy and self-regulatory skills) in combination with an incentive (free dental treatment) would make a difference in the adoption of regular dental flossing in a population of Indian periodontal disease outpatients., Methods: One hundred and twelve participants (n = 55 oral health promotion intervention group; n = 57 control group) were assigned to the intervention (self-management cues + incentive) or control groups, and follow-up assessments were performed three weeks later. Flossing frequency, behavioral intentions, and perceived self-efficacy served as dependent variables. Data were analyzed with mixed models, ANCOVAs, and path analyses., Results: The intervention yielded effects on flossing frequency (p < 0.01) and flossing intentions (p < 0.01) at follow-up. Women developed stronger intentions than men. Moreover, by path analysis a sequential mediation chain was found that demonstrated an indirect effect of the intervention on flossing via self-efficacy and intentions: the intervention predicted changes in self-efficacy which, in turn, were associated with changes in intentions, predicting flossing frequency at follow up, while controlling for baseline behavior, gender, and age., Conclusions: Combining incentives with minimal self-management cues has been found effective in improving interdental cleaning intentions and habits in periodontal disease patients, and the facilitating role of dental self-efficacy has been demonstrated.

Background: The double stranded RNA (dsRNA)-activated protein kinase PKR is a well-established protein kinase that is activated by dsRNA during viral infection, and it inhibits global protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor 2α (eIF2α). Recent studies have greatly broadened the recognized physiological activities of PKR by demonstrating its fundamental role in inflammatory signaling, particularly in chronic, low-grade inflammation induced by metabolic disorders, known as metaflammation. Metaflammation is initiated by the activation of the NOD-like receptor (NLR), leucine-rich repeat, pyrin domaincontaining 3 (NLRP3) gene by mitochondrial reactive oxygen species (ROS). A protein complex defined as the metaflammasome is assembled in the course of metaflammation. This complex integrates nutritional signaling with cellular stress, inflammatory components, and insulin action and is essential in maintaining metabolic homeostasis. PKR is a key constituent of the metaflammasome and interacts directly with several inflammatory kinases, such as inhibitor κB (IκB) kinase (IKK) and c-Jun N-terminal kinase (JNK), insulin receptor substrate 1 (IRS1), and component of the translational machinery such as eIF2α., Conclusion: This review highlights recent findings in PKR-mediated metaflammation and its association with the onset of metabolic syndrome in both human and animal models, with a focus on the molecular and biochemical pathways that underlie the progression of obesity, insulin resistance, and type-2 diabetes.

Noncoding RNAs and microRNAs (miRNAs) represent an important class of regulatory molecules that modulate gene expression. The role of miRNAs in diverse cellular processes such as cancer, apoptosis, cell differentiation, cardiac remodeling, and inflammation has been intensively explored. Recent studies further demonstrated the important roles of miRNAs and noncoding RNAs in modulating a broad spectrum of genes involved in lipid synthesis and metabolic pathways. This overview focuses on the role of miRNAs in hepatic lipid and lipoprotein metabolism and their potential as therapeutic targets for metabolic syndrome. This includes recent advances made in the understanding of their target pathways and the clinical development of miRNAs in lipid metabolic disorders., (© 2015 Wiley Periodicals, Inc.)

Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERalpha) within the plasma membrane of endothelial cells (EC). ERalpha is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERalpha to the plasma membrane. However, the role played by caveolin 1 in regulating 17beta-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17beta-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERalpha localization to the plasma membrane in EC. Further, before estrogen stimulation ERalpha associates with caveolin 1, whereas stimulation promotes a pp60(Src)-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERalpha-PI3 kinase interactions and disrupting caveolin 1-ERalpha interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERalpha/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62-73 of caveolin 1 as the ERalpha-binding site. Delivery of a synthetic peptide based on this sequence decreased ERalpha plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17beta-estradiol-induced NO production by promoting ERalpha to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation.

Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of Ser1177. However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear-mediated increases in ROS play a role in stimulating Ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC, there was an increase in H2O2 levels. The increases in H2O2 were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at Ser1177 phosphorylation and a decrease in phosphorylation at Thr495. We also found that the overexpression of catalase significantly attenuated the shear-mediated increases in H2O2, phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKCdelta activity in response to shear stress, and the overexpression of PKCdelta attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKCdelta overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H2O2. Together, our data indicate that shear stress decreases PKCdelta activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H2O2 signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation.

Previous studies demonstrate impaired nitric oxide (NO) signaling in children and animal models with congenital heart defects and increased pulmonary blood flow. However, the molecular mechanisms underlying these alterations remain incompletely understood. The purpose of this study was to determine if early changes in arginine metabolic pathways could play a role in the reduced NO signaling demonstrated in our lamb model of congenital heart disease with increased pulmonary blood flow (Shunt lambs). The activities of the arginine recycling enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) were both decreased in lung tissues of Shunt lambs while arginase activity was increased. Associated with these alterations, lung L-arginine levels were decreased. These changes correlated with an increase in NO synthase-derived reactive oxygen species (ROS) generation. This study provides further insights into the molecular mechanisms leading to decreased NO signaling in Shunt lambs and suggests that altered arginine metabolism may play a role in the development of the endothelial dysfunction associated with pulmonary hypertension secondary to increased pulmonary blood flow.

In an ovine model of persistent pulmonary hypertension of the newborn (PPHN), endothelin-1 (ET-1) expression is increased, while endothelial nitric oxide synthase (eNOS) expression is decreased. However, the molecular mechanisms by which ET-1 attenuates eNOS expression in endothelial cells are not completely understood. Thus, the goal of this study was to determine if the overexpression of ET-1 decreases eNOS expression in pulmonary arterial endothelial cells isolated from fetal lambs. To increase the ET-1 expression, cells were transfected with a plasmid coding for Prepro-ET-1, a precursor of ET-1. After overexpression of Prepro-ET-1, ET-1 levels in the culture medium were significantly increased (control = 805.3 +/- 69.8; Prepro-ET-1 overexpression = 1351 +/- 127.9). eNOS promoter activity, protein levels, and NO generation were all significantly decreased by the overexpression of Prepro-ET-1. The decrease in transcription correlated with increased activity of protein kinase Cdelta (PKCdelta) and STAT3. Further, DNA binding activity of STAT3 was also increased by Prepro-ET-1 overexpression. The increase in STAT3 activity and decrease in eNOS promoter activity were inhibited by the overexpression of dominant negative mutants of PKCdelta or STAT3. Further, a 2 bp mutation in the STAT3 binding site in the eNOS promoter inhibited STAT3 binding and led to enhanced promoter activity in the presence of Prepro-ET-1 overexpression. In conclusion, ET-1 secretion is increased by Prepro-ET-1 overexpression. This results in activation of PKCdelta, which phosphorylates STAT3, increasing its binding to the eNOS promoter. This in turn decreases eNOS promoter activity, protein levels, and NO production. Thus, ET-1 can reduce eNOS expression and NO generation in fetal pulmonary artery endothelial cells through PKCdelta-mediated activation of STAT3.

We have previously shown that the regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells isolated from fetal lamb under static conditions is positively regulated by PKCdelta. In this study, we explore the role of PKCdelta in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKCdelta activation. Modulation of PKCdelta before shear with a dominant negative mutant of PKCdelta (DN PKCdelta) or bryostatin (a known PKCdelta activator) demonstrated that PKCdelta inhibition potentiates the shear-mediated increases in eNOS expression and activity, while PKCdelta activation inhibited these events. To gain insight into the mechanism by which PKCdelta inhibits shear-induced eNOS expression, we examined activation of STAT3, a known target for PKCdelta phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKCdelta reduced, while PKCdelta activation enhanced, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and nitric oxide production in response to shear. Finally, we found that mutating the STAT3 binding site sequence within the eNOS promoter increased promoter activity in response to shear and that this was no longer inhibited by bryostatin. In conclusion, shear decreases PKCdelta activity and, subsequently, reduces STAT3 binding to the eNOS promoter. This signaling pathway plays a previously unidentified role in the regulation of eNOS expression by shear stress.

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.

Previously, we have shown that pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs produce significant levels of nitric oxide (NO) but minimal superoxide upon stimulation, whereas PAECs isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAECs during postnatal development. In this study, we sought to extend these studies by investigating the potential role of heat shock protein 90 (HSP90) in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAECs isolated from fetal compared with 4-wk-old lambs, whereas the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAECs isolated from 4-wk-old lambs, we utilized an adenovirus to overexpress HSP90. We found that overexpression of HSP90 significantly increased the shear-stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS-dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor radicicol led to significant decreases in eNOS-HSP90 interactions, decreased shear-stimulated NO generation, and increased NOS-dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90-eNOS interactions were decreased in shunt lambs and that this was associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together, our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.

The bioactive phospholipid lysophosphatidic acid (LPA) promotes cell proliferation, survival, and migration by acting on cognate G protein-coupled receptors named LPA(1), LPA(2), and LPA(3). We profiled gene expression of LPA receptors in androgen-dependent and androgen-insensitive prostate cancer cells and found that LPA(1) gene is differentially expressed in androgen-insensitive and LPA-responsive but not androgen-dependent and LPA-resistant cells. In human prostate specimens, expression of LPA(1) gene was significantly higher in the cancer compared with the benign tissues. The androgen-dependent LNCaP cells do not express LPA(1) and do not proliferate in response to LPA stimulation, implying LPA(1) transduces cell growth signals. Accordingly, stable expression of LPA(1) in LNCaP cells rendered them responsive to LPA-induced cell proliferation and decreased their doubling time in serum. Implantation of LNCaP-LPA(1) cells resulted in increased rate of tumor growth in animals compared with those tumors that developed from the wild-type cells. Growth of LNCaP cells depends on androgen receptor activation, and we show that LPA(1) transduces Galphai-dependent signals to promote nuclear localization of androgen receptor and cell proliferation. In addition, treatment with bicalutamide inhibited LPA-induced cell cycle progression and proliferation of LNCaP-LPA(1) cells. These results suggest the possible utility of LPA(1) as a drug target to interfere with progression of prostate cancer.

Herein, we describe the identification of GPCR56, an orphan G-protein coupled receptor, to be differentially expressed in esophageal squamous cell carcinoma. Although, GPCRs have been demonstrated to be altered in various human cancers, much is still unknown about GPCR56 expression in tumors. To evaluate the expression of these genes in esophageal tissues, we performed semi-quantitative Reverse-Transcription Polymerase Chain Reaction in ESCCs, dysplasia and matched normal esophageal epithelium. Increased transcript levels of GPCR56 were detected in 48% of ESCCs, while the adjacent non-malignant esophageal tissue did not show the expression of this transcript. Interestingly, most of the dysplastic tissues analyzed also exhibited increased expression of GPCR56 suggesting that alteration in GPCR56 expression is an early event in esophageal tumorigenesis. In depth analysis of GPCR56 in different stages of development and progression of esophageal tumorigenesis is warranted to explore its utility as potential early diagnostic marker and its function in esophageal cancer.

Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978bp open reading frame interrupted by two introns. The first intron is located in the 5'-untranslated region while the second one is positioned 174bp downstream to the ATG codon. The encoded approximately 40kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model.

Objectives: Early detection of esophageal cancer is hampered by paucity of molecular markers for diagnosis of this aggressive gastrointestinal malignancy in early stages. We recently identified TC21/R-Ras2, a small GTP-binding protein (SMG) in esophageal squamous cell carcinomas (ESCCs) by differential display. This study was designed to test the hypothesis that differential expression of TC21 in normal, dysplastic and malignant esophageal tissues may be of clinical relevance in esophageal tumorigenesis., Methods: Immunohistochemical analysis of TC21 was carried out in 83 ESCCs, 37 dysplasias and 29 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. The cellular localization of TC21 was determined by confocal microscopy., Results: Expression of TC21 protein was observed in 60/83 (73%) ESCCs predominantly localized in tumor nuclei. Intriguingly, intense TC21 immunoreactivity was observed in all endoscopic biopsies with histological evidence of dysplasia (16 cases) as well as in dysplastic areas distant to ESCCs (21 cases), while matched distant histologically normal epithelia did not show detectable TC21 expression. Immunoblotting and semi-quantitative RT-PCR confirmed TC21 expression in dysplastias and ESCCs. Confocal microscopy showed nuclear as well as cytoplasmic TC21 expression in ESCCs and TE13 cells., Conclusions: To our knowledge, this is the first report demonstrating differential expression of TC21 in normal, dysplastic and ESCC tissues, suggesting that TC21 expression is associated with early stages of esophageal tumorigenesis. Nuclear localization of TC21 makes it the third of over 100 small SMGs identified to be localized in the nucleus.

HAART therapy has led to a significant reduction of general and neurological morbidity, and mortality among HIV-1 infected patients. It can also decrease HIV-1 RNA titres in plasma and CSF towards undetectable level. However, the initial hope of achieving total eradication of the virus from the body has vanished. Even in patients who do not develop viral resistance or treatment intolerance, two kinds of viral persistence have been demonstrated both in lymphoid and central nervous system. The first one is a smoldering infection that persists, despite prolonged and apparently efficient HAART, in monocytes, tissue macrophages and most probably microglia. The second one is an integration of proviral DNA in the genome of subpopulations of CD4 lymphocytes of patients receiving efficient HAART. A similar viral integration in astrocytes and less likely in resting microglia is suggested by several studies, although it has yet to be demonstrated conclusively.

Histological and histochemical changes (lipids, phospholipids, neutral polysaccharides, acid mucopolysaccharides and sialic acid) were studied in the rat at pre- and postpubertal stages. At 10 days lipid and phospholipid staining was not observed both in the testis and epididymis though neutral and acid mucopolysaccharides and sialic acid were demonstrable. By 21 days, lipid and phospholipid staining was present in moderate amounts both in testis and epididymis. There was also a slight increase in other parameters studied. Maximum histochemical staining for all the parameters was seen at 60 days when the testicular and further components were well organized and functional. These findings reveal that both the testis and epididymis follow a similar pattern of development and are possibly governed by a common controlling factor--the androgens.