Recent Research Articles from UNTHSC

Astrocyte elevated gene-1 regulates astrocyte responses to neural injury: implications for reactive astrogliosis and neurodegeneration.

J Neuroinflammation. 2012;9:195

Authors: Vartak-Sharma N, Ghorpade A

Abstract
BACKGROUND: Reactive astrogliosis is a ubiquitous but poorly understood hallmark of central nervous system pathologies such as trauma and neurodegenerative diseases. In vitro and in vivo studies have identified proinflammatory cytokines and chemokines as mediators of astrogliosis during injury and disease; however, the molecular mechanism remains unclear. In this study, we identify astrocyte elevated gene-1 (AEG-1), a human immunodeficiency virus 1 or tumor necrosis factor α-inducible oncogene, as a novel modulator of reactive astrogliosis. AEG-1 has engendered tremendous interest in the field of cancer research as a therapeutic target for aggressive tumors. However, little is known of its role in astrocytes and astrocyte-mediated diseases. Based on its oncogenic role in several cancers, here we investigate the AEG-1-mediated regulation of astrocyte migration and proliferation during reactive astrogliosis.
METHODS: An in vivo brain injury mouse model was utilized to show AEG-1 induction following reactive astrogliosis. In vitro wound healing and cell migration assays following AEG-1 knockdown were performed to analyze the role of AEG-1 in astrocyte migration. AEG-1-mediated regulation of astrocyte proliferation was assayed by quantifying the levels of cell proliferation markers, Ki67 and proliferation cell nuclear antigen, using immunocytochemistry. Confocal microscopy was used to evaluate nucleolar localization of AEG-1 in cultured astrocytes following injury.
RESULTS: The in vivo mouse model for brain injury showed reactive astrocytes with increased glial fibrillary acidic protein and AEG-1 colocalization at the wound site. AEG-1 knockdown in cultured human astrocytes significantly reduced astrocyte migration into the wound site and cell proliferation. Confocal analysis showed colocalization of AEG-1 to the nucleolus of injured cultured human astrocytes.
CONCLUSIONS: The present findings report for the first time the novel role of AEG-1 in mediating reactive astrogliosis and in regulating astrocyte responses to injury. We also report the nucleolar localization of AEG-1 in human astrocytes in response to injury. Future studies may be directed towards elucidating the molecular mechanism of AEG-1 action in astrocytes during reactive astrogliosis.

PMID: 22884085 [PubMed - indexed for MEDLINE]

R- and S-equol have equivalent cytoprotective effects in Friedreich's ataxia.

BMC Pharmacol Toxicol. 2012;13:12

Authors: Richardson TE, Simpkins JW

Abstract
BACKGROUND: Estradiol (E2) is a very potent cytoprotectant against a wide variety of cellular insults in numerous different cell models, including a Friedreich's ataxia (FRDA) model. Previously, we demonstrated that estrogen-like compounds are able to prevent cell death in an FRDA model independent of any known estrogen receptor (ER) by reducing reactive oxygen species (ROS) and the detrimental downstream effects of ROS buildup including oxidative damage to proteins and lipids and impaired mitochondrial function.
RESULTS: We have previously demonstrated by western blot that our cell model lacks ERα and expresses only very low levels of ERβ. Using L-buthionine (S,R)-sulfoximine (BSO) to induce oxidative stress in human FRDA fibroblasts, we determine the potency and efficacy of the soy-derived ERβ agonist S-equol and its ERα-preferring enantiomer, R-equol in vitro on cell viability and ROS accumulation. Here we demonstrate that these equol biphenolic compounds, while significantly less potent and efficacious than E2, provide statistically similar attenuation of ROS and cytoprotection against a BSO-induced oxidative insult.
CONCLUSIONS: These preliminary data demonstrate that estrogen and soy-derived equols could have a beneficial effect in delaying the onset and decreasing the severity of symptoms in FRDA patients by an antioxidant mechanism. In addition, these data confirm that the protection seen previously with E2 was indeed unrelated to ER binding.

PMID: 23088310 [PubMed - indexed for MEDLINE]

Therapeutic Strategies in Friedreich's Ataxia.

Brain Res. 2013 Apr 12;

Authors: Richardson TE, Kelly HN, Yu AE, Simpkins JW

Abstract
First established as a diagnosis by Nikolaus Friedreich in 1863, Friedreich's ataxia (FA) is an autosomal recessive progressive neurodegenerative disorder cause by a trinucleotide repeat expansion. FA begins with the functional absence of the FXN gene product frataxin, a protein whose exact function still remains unknown. This absence results in impaired intracellular antioxidant defenses, dysregulation of iron-sulfur cluster proteins, depression of aerobic electron transport chain respiration, massive mitochondrial dysfunction, and ultimately cell death in the brain, spinal cord and heart. Herein, we review the molecular and cellular pathogenesis leading to widespread organ system dysfunction, as well as current therapeutic research aimed at preventing the debilitating effects of frataxin loss and preventing the signs and symptoms associated of FA. We also discuss the ongoing treatment strategies employed by our laboratory to prevent mitochondrial damage using synergistic effects of 17β-estradiol and methylene blue, previously shown by our group and others to have protective effects in human FA fibroblasts.

PMID: 23587934 [PubMed - as supplied by publisher]

An evaluation of the transfer of saliva-derived DNA.

Int J Legal Med. 2012 Nov;126(6):851-61

Authors: Warshauer DH, Marshall P, Kelley S, King J, Budowle B

Abstract
Studies of DNA transfer have focused largely on the transfer of sloughed off epithelial cells from individuals' hands. This research examines primary, secondary, and tertiary transfer events involving DNA originating from saliva, a commonly encountered body fluid. More routine human behaviors were simulated to evaluate transfer, and the effects of drying time, moisture, and surface composition were investigated. The results agree with previous findings which indicate that the presence of moisture, as well as a smooth nonporous surface as the primary substrate, increases the efficiency of transfer. Previous transfer studies have found that the last individual to come into contact with an item is usually the major contributor to the resulting DNA mixture, unless conditions are simulated in which a "good shedder" serves as the primary depositor and a poor shedder serves as the secondary depositor. The results of this study indicate that when saliva is the source of the transferred DNA, the primary depositor is often the major contributor. These findings suggest that shedder status is less relevant with regard to touch DNA samples in a forensic setting and emphasize the need for caution when analyzing such samples.

PMID: 22797945 [PubMed - indexed for MEDLINE]

Neuroprotective actions of methylene blue and its derivatives.

PLoS One. 2012;7(10):e48279

Authors: Poteet E, Winters A, Yan LJ, Shufelt K, Green KN, Simpkins JW, Wen Y, Yang SH

Abstract
Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection in vitro and in rodent models of Parkinson's disease and stroke. In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB's 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.

PMID: 23118969 [PubMed - indexed for MEDLINE]

Inhibition of HIV-1 transcription and replication by a newly identified cyclin T1 splice variant.

J Biol Chem. 2013 Apr 8;

Authors: Gao G, Wu X, Zhou J, He M, He JJ, Guo D

Abstract
A variety of cellular factors participate in the HIV-1 life cycle and transcription. Among them is the well-characterized cyclin T1 (CycT1). CycT1 binds to cyclin-dependent kinase 9 (CDK9) and forms the positive transcription elongation factor-b (P-TEFb), which is then recruited by HIV-1 Tat to the HIV-1 long terminal repeat (LTR) promoter to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAP II), leading to enhanced processivity of RNAP II and transcription of a full-length HIV-1 RNA. In this study, we report the identification of a new CycT1 splice variant, designated as CycT1b and accordingly renamed CycT1 as CycT1a. CycT1b was detected in several cell lines, including primary human CD4 T cells, and its expression was subject to cell cycle regulation. Similar to CycT1a, CycT1b was primarily localized in the nucleus. CycT1b expression was found to be inversely correlated with HIV-1 gene expression and replication. This inverse correlation appeared to involve Tat transactivation, CDK9 binding, and subsequent recruitment of p-TEFb to the HIV-1 LTR promoter and RNAP II CTD phosphorylation. In agreement with these findings, CycT1b expression led to direct inhibition of Tat-transactivated transcription of the HIV-1 LTR promoter. Taken together, these results show that the newly identified CycT1b splice variant inhibits HIV-1 transcription and may provide new clues for the development of anti-HIV strategies.

PMID: 23569210 [PubMed - as supplied by publisher]

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).

Anal Bioanal Chem. 2013 Apr 6;

Authors: Rich RM, Mummert M, Gryczynski Z, Borejdo J, Sørensen TJ, Laursen BW, Foldes-Papp Z, Gryczynski I, Fudala R

Abstract
Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

PMID: 23564284 [PubMed - as supplied by publisher]

Upregulation of PKCη by PKCε and PDK1 involves two distinct mechanisms and promotes breast cancer cell survival.

Biochim Biophys Acta. 2013 Apr 4;

Authors: Pal D, Outram SP, Basu A

Abstract
BACKGROUND: Protein kinase C (PKC) serves as the receptor for tumor-promoting phorbol esters, which are potent activators of conventional (c) and novel (n) PKCs. We recently showed that these activators induced selective upregulation of PKCη in breast cancer cells. The objective of this study is to understand unique regulation of PKCη and its importance in breast cancer. METHODS: The levels of PKC isozymes were monitored in breast cancer cells following treatment with inhibitors of kinases, proteasome and proteases by Western blotting. PKCε was introduced by adenoviral delivery. PKCη and PDK1 were depleted by siRNA silencing. Cell growth was determined by the MTT or clonal assay. RESULTS: The general PKC inhibitors Gö 6983 and bisindolylmaleimide but not cPKC inhibitor Gö 6976 led to substantial PKCη downregulation, which was partly rescued by the introduction of nPKCε. Inhibition of phosphoinositide-dependent kinase-1 (PDK1) by Ly294002 or knockdown of PDK1 also led to downregulation of basal PKCη but had no effect on PKC activator-induced upregulation of PKCη. Proteasome inhibitors blocked PKCη downregulation triggered by PDK1 inhibition/depletion but not by Gö 6983. PKCη level increased in malignant but not in non-tumorigenic or pre-malignant cells in the progressive MCF-10A series associated with activated PDK1, and knockdown of PKCη inhibited breast cancer cell growth and clonogenic survival. CONCLUSION: Upregulation of PKCη contributes to breast cancer cell growth and targeting either PKCε or PDK1 triggers PKCη downregulation but involves two distinct mechanisms. GENERAL SIGNIFICANCE: The status of PKCη may serve as a potential biomarker for breast cancer malignancy.

PMID: 23562764 [PubMed - as supplied by publisher]

Rural-Urban Differences in Health Services Utilization in the US-Mexico Border Region.

J Rural Health. 2013 Mar;29(2):215-223

Authors: Su D, Pratt W, Salinas J, Wong R, Pagán JA

Abstract
Purpose: Evaluate the association between driving distance to the US-Mexico border and rural-urban differences in the use of health services in Mexico by US border residents from Texas. Methods: Data for this study come from the Cross-Border Utilization of Health Care Survey, a population-based telephone survey conducted in the Texas border region in spring 2008. Driving distances to the border were estimated from the nearest border crossing station using Google Maps. Outcome measures included medication purchases, physician visits, dentist visits, and inpatient care in Mexico during the 12 months prior to the survey. A series of adjusted logit models were estimated after controlling for relevant confounding factors. Findings: The average driving distance to the nearest border crossing station among rural respondents was 4 times that of urban respondents (42.0 miles vs 10.3 miles [P < .001]). Rural respondents were more likely to be dissatisfied than urban respondents with the health care provided on the US side of the border, yet they were less likely to use health services in Mexico. Driving distance to the border largely explained the observed rural-urban differences in medication purchases from Mexico. In the case of inpatient care, however, rural respondents reported a higher utilization rate than urban respondents and this rural-urban difference became more pronounced after adjusting for the effect of driving distance to the border. Conclusions: Dissatisfaction with US health care services in rural communities in the US-Mexico border region seems to be compounded by the lack of access to health care services in Mexico due to travel distance constraints.

PMID: 23551652 [PubMed - as supplied by publisher]

Effects of Thailanstatins on Glucocorticoid Response in Trabecular Meshwork and Steroid-Induced Glaucoma.

Invest Ophthalmol Vis Sci. 2013 Apr 2;

Authors: Jain A, Liu X, Wordinger RJ, Yorio T, Cheng YQ, Clark AF

Abstract
PURPOSE: Elevated intraocular pressure (IOP) is a major risk factor in glaucoma. Various changes in the trabecular meshwork (TM) are responsible for elevated IOP. Glucocorticoids (GCs) increase IOP and mediate biochemical changes in the TM, similar to those associated with primary open angle glaucoma (POAG). There are differences in steroid responsiveness among the population. Approximately 40% of individuals significantly elevate IOP (i.e. responders) upon GC administration, while others do not (i.e. nonresponders). The responders are at higher risk of developing POAG compared to the nonresponders. In addition, almost all POAG patients are steroid responders. GC responsiveness is regulated by the relative levels of the active GC receptor alpha (GRα) and the alternatively spliced dominant negative regulator isoform GRβ. Glaucomatous TM cell strains have lower a GRβ/GRα ratio compared to normal TM cells, making them more sensitive to GCs. Our purpose was to investigate the role of a special class of natural products called thailanstatins (TSTs), in GR alternative splicing and GC response in cultured human TM cells. METHODS: QRT-PCR and western immunoblotting were used to study the effect of TSTs on GRβ/GRα ratios in human TM cell strains. Effects of TSTs on dexamethasone (DEX) responsiveness were assessed by GRE-luciferase reporter activity and fibronectin induction in TM cells. RESULTS: TSTs increased the GRβ/GRα ratio in TM cells. Increased GRβ/GRα ratios were associated with decreased DEX-mediated fibronectin induction and GRE-luciferase activity. CONCLUSIONS: TSTs modulate the GR splicing process to enhance GRβ levels and thereby decrease the GC response in cultured human TM cells. These TSTs, or similar compounds, may potentially be new glaucoma therapeutic agents.

PMID: 23548621 [PubMed - as supplied by publisher]

Factors associated with attention deficit/hyperactivity disorder among US children: results from a national survey.

BMC Pediatr. 2012;12:50

Authors: Lingineni RK, Biswas S, Ahmad N, Jackson BE, Bae S, Singh KP

Abstract
BACKGROUND: The purpose of this study was to investigate the association between Attention Deficit/Hyperactivity Disorder (ADHD) and various factors using a representative sample of US children in a comprehensive manner. This includes variables that have not been previously studied such as watching TV/playing video games, computer usage, family member's smoking, and participation in sports.
METHODS: This was a cross-sectional study of 68,634 children, 5-17 years old, from the National Survey of Children's Health (NSCH, 2007-2008). We performed bivariate and multivariate logistic regression analyses with ADHD classification as the response variable and the following explanatory variables: sex, race, depression, anxiety, body mass index, healthcare coverage, family structure, socio-economic status, family members' smoking status, education, computer usage, watching television (TV)/playing video games, participation in sports, and participation in clubs/organizations.
RESULTS: Approximately 10% of the sample was classified as having ADHD. We found depression, anxiety, healthcare coverage, and male sex of child to have increased odds of being diagnosed with ADHD. One of the salient features of this study was observing a significant association between ADHD and variables such as TV usage, participation in sports, two-parent family structure, and family members' smoking status. Obesity was not found to be significantly associated with ADHD, contrary to some previous studies.
CONCLUSIONS: The current study uncovered several factors associated with ADHD at the national level, including some that have not been studied earlier in such a setting. However, we caution that due to the cross-sectional and observational nature of the data, a cause and effect relationship between ADHD and the associated factors can not be deduced from this study. Future research on ADHD should take into consideration these factors, preferably through a longitudinal study design.

PMID: 22583686 [PubMed - indexed for MEDLINE]

Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and p38 regulate the expression of chemokine CXCL8 in human astrocytes.

PLoS One. 2012;7(9):e45596

Authors: Mamik MK, Ghorpade A

Abstract
CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.

PMID: 23029125 [PubMed - indexed for MEDLINE]

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.

Exp Eye Res. 2013 Feb;107:21-31

Authors: Mueller BH, Park Y, Daudt DR, Ma HY, Akopova I, Stankowska DL, Clark AF, Yorio T

Abstract
Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

PMID: 23183135 [PubMed - indexed for MEDLINE]

Novel Interaction between Proliferating Cell Nuclear Antigen and HLA I on the Surface of Tumor Cells Inhibits NK Cell Function through NKp44.

PLoS One. 2013;8(3):e59552

Authors: Horton NC, Mathew SO, Mathew PA

Abstract
NK cell function is closely regulated by numerous inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface PCNA. Other reports have pointed to potential associations between NKp44 and HLA I molecules, as well as HLA I and Damage Associated Molecular Pattern molecules (DAMPs) on the surface of tumor cells. In this report, we have identified novel interaction between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK cell cytotoxicity. We further postulate NCR ligands are composed of DAMP molecules localized to the cell surface, colocalizing with HLA I, and potentially heparin sulfate proteoglycans.

PMID: 23527218 [PubMed - in process]

Nuclear factor-[kappa]B mediates suppression of canonical transient receptor potential 6 expression by reactive oxygen species and protein kinase C in kidney cells.

J Biol Chem. 2013 Mar 22;

Authors: Wang Y, Ding M, Chaudhari S, Ding Y, Yuan J, Stankowska D, He S, Krishnamoorthy R, Cunningham JT, Ma R

Abstract
Abstract The present study was carried out to explore the molecular mechanism for downregulation of canonical transient receptor potential 6 (TRPC6) expression in the reactive oxygen species (ROS)/protein kinase C (PKC) signaling in kidney cells. In cultured human mesangial cells, H2O2 and TNF-α inhibited TRPC6 mRNA expression in a time dependent manner. Inhibition of NF-κB reversed both H2O2- and phorbol 12-myristate 13-acetate (PMA)-induced decrease in TRPC6 protein expression. Activation of NF-κB by knocking down IκB-α using siRNA could mimic the suppressive effect of ROS/PKC on TRPC6. Ca2+ imaging study showed that activation and inhibition of NF-κB significantly decreased and increased the TRPC6-mediated Ca2+ entry, respectively. Further experiments showed that PMA, but not its inactive analog 4-αPDD, caused phosphorylation of IκBα and stimulated the nuclear translocation of NF-κB p50 and p65 subunits. The PMA-dependent IκBα phosphorylation was significantly inhibited by Go6976. Electrophoretic mobility shift assay revealed that PMA stimulated DNA binding activity of NF-κB. Furthermore, specific knockdown of p65, but not p50, prevented H2O2 inhibitory effect on TRPC6 protein expression, suggesting p65 being a predominant NF-κB subunit repressing TRPC6. In agreement with a major role of p65, chromatin immunoprecipitation assays showed that PMA treatment induced p65 binding to the TRPC6 promoter. Moreover, PMA treatment increased associations of p65 with histone deacetylase (HDAC) and decreased histone acetylation at the TRPC6 promoter. Consistently, knockdown of HDAC2 by siRNA or inhibition of HDAC with trichostatin A prevented H2O2-induced decrease in TRPC6 mRNA and protein expressions, respectively. Taken together, our findings imply an important role of NF-κB in a negative regulation of TRPC6 expression at the gene transcription level in kidney cells.

PMID: 23525112 [PubMed - as supplied by publisher]

The Androgen Metabolite, 5α-Androstane-3β,17β-Diol (3β-Diol), Activates the Oxytocin Promoter Through an Estrogen Receptor-β Pathway.

Endocrinology. 2013 Mar 20;

Authors: Hiroi R, Lacagnina AF, Hinds LR, Carbone DG, Uht RM, Handa RJ

Abstract
Testosterone has been shown to suppress the acute stress-induced activation of the hypothalamic-pituitary-adrenal axis; however, the mechanisms underlying this response remain unclear. The hypothalamic-pituitary-adrenal axis is regulated by a neuroendocrine subpopulation of medial parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN). These neurons are devoid of androgen receptors (ARs). Therefore, a possibility is that the PVN target neurons respond to a metabolite in the testosterone catabolic pathway via an AR-independent mechanism. The dihydrotestosterone metabolite, 5α-androstane-3β,17β-diol (3β-diol), binds and activates estrogen receptor-β (ER-β), the predominant ER in the PVN. In the PVN, ER-β is coexpressed with oxytocin (OT). Therefore, we tested the hypothesis that 3β-diol regulates OT expression through ER-β activation. Treatment of ovariectomized rats with estradiol benzoate or 3β-diol for 4 days increased OT mRNA selectively in the midcaudal, but not rostral PVN compared with vehicle-treated controls. 3β-Diol treatment also increased OT mRNA in the hypothalamic N38 cell line in vitro. The functional interactions between 3β-diol and ER-β with the human OT promoter were examined using an OT promoter-luciferase reporter construct (OT-luc). In a dose-dependent manner, 3β-diol treatment increased OT-luc activity when cells were cotransfected with ER-β, but not ER-α. The 3β-diol-induced OT-luc activity was reduced by deletion of the promoter region containing the composite hormone response element (cHRE). Point mutations of the cHRE also prevented OT-luc activation by 3β-diol. These results indicate that 3β-diol induces OT promoter activity via ER-β-cHRE interactions.

PMID: 23515287 [PubMed - as supplied by publisher]

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays.

Anal Bioanal Chem. 2012 Nov;404(8):2223-31

Authors: Rich R, Li J, Fudala R, Gryczynski Z, Gryczynski I, Mandecki W

Abstract
Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments, we found the depth of the polymer matrix to be 1-2 μm. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2 μm. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform.

PMID: 22960796 [PubMed - indexed for MEDLINE]

Osteopathic Manual Treatment and Ultrasound Therapy for Chronic Low Back Pain: A Randomized Controlled Trial.

Ann Fam Med. 2013 Mar;11(2):122-129

Authors: Licciardone JC, Minotti DE, Gatchel RJ, Kearns CM, Singh KP

Abstract
PURPOSE We studied the efficacy of osteopathic manual treatment (OMT) and ultrasound therapy (UST) for chronic low back pain. METHODS A randomized, double-blind, sham-controlled, 2 × 2 factorial design was used to study OMT and UST for short-term relief of nonspecific chronic low back pain. The 455 patients were randomized to OMT (n = 230) or sham OMT (n = 225) main effects groups, and to UST (n = 233) or sham UST (n = 222) main effects groups. Six treatment sessions were provided over 8 weeks. Intention-to-treat analysis was performed to measure moderate and substantial improvements in low back pain at week 12 (30% or greater and 50% or greater pain reductions from baseline, respectively). Five secondary outcomes, safety, and treatment adherence were also assessed. RESULTS There was no statistical interaction between OMT and UST. Patients receiving OMT were more likely than patients receiving sham OMT to achieve moderate (response ratio [RR] = 1.38; 95% CI, 1.16-1.64; P <.001) and substantial (RR = 1.41, 95% CI, 1.13-1.76; P = .002) improvements in low back pain at week 12. These improvements met the Cochrane Back Review Group criterion for a medium effect size. Back-specific functioning, general health, work disability specific to low back pain, safety outcomes, and treatment adherence did not differ between patients receiving OMT and sham OMT. Nevertheless, patients in the OMT group were more likely to be very satisfied with their back care throughout the study (P <.001). Patients receiving OMT used prescription drugs for low back pain less frequently during the 12 weeks than did patients in the sham OMT group (use ratio = 0.66, 95% CI, 0.43-1.00; P = .048). Ultrasound therapy was not efficacious. CONCLUSIONS The OMT regimen met or exceeded the Cochrane Back Review Group criterion for a medium effect size in relieving chronic low back pain. It was safe, parsimonious, and well accepted by patients.

PMID: 23508598 [PubMed - as supplied by publisher]

Allopatric tuberculosis host-pathogen relationships are associated with greater pulmonary impairment.

Infect Genet Evol. 2013 Mar 14;

Authors: Pasipanodya JG, Moonan PK, Vecino E, Miller T, Fernandez M, Slocum P, Drewyer G, Weis SE

Abstract
BACKGROUND: Host pathogen relationships can be classified as allopatric, when the pathogens originated from separate, non-overlapping geographic areas from the host; or sympatric, when host and pathogen shared a common ancestral geographic location. It remains unclear if host-pathogen relationships, as defined by phylogenetic lineage, influence clinical outcome. We sought to examine the association between allopatric and sympatric phylogenetic M. tuberculosis lineages and pulmonary impairment after tuberculosis (PIAT). METHODS: Pulmonary function tests were performed on patients 16 years of age and older who had received ⩾20 weeks of treatment for culture-confirmed M. tuberculosis complex. Forced Expiratory Volume in 1 min (FEV1) ⩾80%, Forced Vital Capacity (FVC) ⩾80% and FEV1/FVC > 70% of predicted were considered normal. Other results defined pulmonary impairment. Spoligotype and 12-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) were used to assign phylogenetic lineage. PIAT severity was compared between host-pathogen relationships which were defined by geography and ethnic population. We used multivariate logistic regression modeling to calculate adjusted odds ratios (aOR) to assess the association between phylogenetic lineage and PIAT. RESULTS: Self-reported continental ancestry was correlated with M. tuberculosis lineage (p<0.001). In multivariate analyses adjusting for phylogenetic lineage, age and smoking, the overall aOR for subjects with allopatric host-pathogen relationships and PIAT was 1.8 (95% confidence interval [CI]: 1.1, 2.9) compared to sympatric relationships. Smoking >30 pack-years was also associated with PIAT (aOR: 3.2; 95% CI: 1.5, 7.2) relative to smoking <1 pack-years. CONCLUSIONS: PIAT frequency and severity varies byhost-pathogen relationship and heavy cigarette consumption, but not phylogenetic lineage alone. Patients who had disease resulting from allopatric-host-pathogen relationship were more likely to have PIAT than patients with disease from sympatric-host-pathogen relationship infection. Further study of this association may identify ways that treatment and preventive efforts can be tailored to specific lineages and racial/ethnic populations.

PMID: 23501297 [PubMed - as supplied by publisher]

Estrogen Receptors and Ischemic Neuroprotection: Who, What, Where and When?

Brain Res. 2013 Mar 8;

Authors: Schreihofer DA, Ma Y

Abstract
Estrogens, particularly 17β-estradiol (E2), are powerful neuroprotective agents in animal models of cerebral ischemia. Loss of endogenous E2 in women at menopause or after surgical oopherectomy leads to an increase risk of stroke, neurodegenerative disease, and cognitive decline. However, several clinical trials found detrimental effects of E2 therapy after menopause, including increased stroke risk and dementia. Recent animal and human studies now support the "critical period" hypothesis for E2 neuroprotection whereby E2 therapy must begin soon after the loss of endogenous E2 production to have a beneficial effect. Although a wide array of mechanisms has been proposed for estradiol (E2)-dependent neuroprotection in cerebral ischemia and neurodegenerative disease, most of these mechanisms involve interactions of E2 with one of its cognate receptors, estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), or the G protein-coupled estrogen receptor (GPER). However, these receptors are not uniformly distributed throughout the brain, across different cell types, and within cellular compartments. Such differences likely play a role in the ability of E2 and ER selective ligands to protect the brain from ischemia. This review examines the changes in ER expression and location that may underlie the loss of E2 neuroprotection seen with aging and long-term estrogen deprivation (LTED). Recent results suggest that the loss of ERα that accompanies aging and LTED plays an important role in the loss of E2-dependent neuroprotection.

PMID: 23500634 [PubMed - as supplied by publisher]