Glucocorticoid receptor polymorphisms and post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is reported in some studies to be associated with increased glucocorticoid (GC) sensitivity. Two common glucocorticoid receptor (GR) potymorphisms (N363S and 8cll) appear to contribute to the population variance in GC sensitivity. There is some evidence that there may be a genetic predisposition to PTSD. Hence we studied 118 Vietnam war veterans with PTSD for (i) GR polymorphisms, particularly the N363S and the Bcll polymorphisms which are thought to be GC sensitising, and (ii) two measures of GC sensitivity, the tow-dose 0.25 mg dexamethasone suppression test (LD-DST) and the dermal vasoconstrictor assay (DVVA). The DST and GR polymorphisms were also performed in 42 combat exposed Vietnam war veterans without PTSD. Basal plasma cortisol levels were not significantly different in PTSD (399.5 +/- 19.2 nmol/L, N=75) and controls (348.6 +/- 23.0 nmol/L, N = 33) and the LD-DST resulted in similar cortisol suppression in both groups (45.6 +/- 3.2 vs. 40.8 +/- 4.1%). The cortisol suppression in PTSD patients does not correlate with Clinician Administered PTSD Scores (CAPS), however there was a significant association between the Bcll GG genotype and low basal cortisol levels in PTSD (P=0.048). The response to the DVVA was similar to controls (945 +/- 122, N = 106 vs. 730 +/- 236, N = 28, P = 0.42). PTSD patients with the GG genotype, however, tended to be more responsive to DVVA and in this group the DVVA correlated with higher CAPS scores. The only exon 2 GR polymorphisms detected were the R23K and N363S. Heterozygosity for the N363S variant in PTSD, at 5.1% was not more prevalent than in other population studies of the N363S polymorphism in Caucasians (6.0-14.8%). The GG genotype of the Bcll polymorphism found to be associated with increased GC sensitivity in many studies showed a tendency towards increased response with DVVA and correlated with higher CAPS scores. In conclusion, the N363S and Bcll GR polymorphisms were not more frequent in PTSD patients than controls and reported population frequencies. Our PTSD group did not display GC hypersensitivity, as measured by the LD-DST and DVVA. In a subset of PTSD patients with the Bcll GG genotype, CAPS scores and basal cortisol Levels were negatively correlated. (C) 2004 Elsevier Ltd. All rights reserved.

Medial prefrontal cortex suppression of the hypothalamic–pituitary–adrenal axis response to a physical stressor, systemic delivery of interleukin-1β

Previous studies have shown that the medial prefrontal cortex can suppress the hypothalamic-pituitary-adrenal axis response to stress. However, this effect appears to vary with the type of stressor. Furthermore, the absence of direct projections between the medial prefrontal cortex and corticotropin-releasing factor cells at the apex of the hypothalamic-pituitary-adrenal axis suggest that other brain regions must act as a relay when this inhibitory mechanism is activated. In the present study, we first established that electrolytic lesions involving the prelimbic and infralimbic medial prefrontal cortex increased plasma adrenocorticotropic hormone levels seen in response to a physical stressor, the systemic delivery of interleukin-1beta. However, medial prefrontal cortex lesions did not alter plasma adrenocorticotropic hormone levels seen in response to a psychological stressor, noise. To identify brain regions that might mediate the effect of medial prefrontal cortex lesions on hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1beta, we next mapped the effects of similar lesions on interleukin-1beta-induced Fos expression in regions previously shown to regulate the hypothalamic-pituitary-adrenal axis response to this stressor. It was found that medial prefrontal cortex lesions reduced the number of Fos-positive cells in the ventral aspect of the bed nucleus of the stria terminalis. However, the final experiment, which involved combining retrograde tracing with Fos immunolabelling, revealed that bed nucleus of the stria terminalis-projecting medial prefrontal cortex neurons were largely separate from medial prefrontal cortex neurons recruited by systemic interleukin-1beta, an outcome that is difficult to reconcile with a simple medial prefrontal cortex-bed nucleus of the stria terminalis-corticotropin-releasing factor cell control circuit.

Suppression of spurious intermediate frequency modes in under-integrated elements by combined stiffness/viscous stabilization

Solutions employing perturbation stiffness or viscous hourglass control with one-point quadrature finite elements often exhibit spurious modes in the intermediate frequency range. These spurious frequencies are demonstrated in several examples and their origin is explained. Then it is shown that by critically damping the hourglass modes, these spurious mid-range frequency modes can be suppressed. Estimates of the hourglass frequency and damping coefficients are provided for the plane 4-node quadrilateral and a 4-node shell element. Results are presented that show almost complete annihilation of spurious intermediate frequency modes for both linear and non-linear problems. Copyright (c) 2005 John Wiley & Sons, Ltd.

E2F suppression and Sp1 overexpression are sufficient to induce the differentiation-specific marker, transglutaminase type 1, in a squamous cell carcinoma cell line

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.

Copper sensitivity of cueO mutants of Escherichia coli K-12 and the biochemical suppression of this phenotype

The cueO gene of Escherichia coli encodes a multi-copper oxidase, which contributes to copper tolerance in this bacterium. It was observed that a cueO mutant was highly sensitive to killing by copper ions when cells were grown on defined minimal media. Copper sensitivity was correlated with accumulation of copper in the mutant strain. Growth of the cueO mutant in the presence of copper could be restored by addition of divalent zinc and manganese ions or ferrous iron but not by other first row transition metal ions or magnesium ions. Copper toxicity towards a cueO mutant Could also be suppressed by addition of the superoxide quencher 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron), suggesting that a primary cause of copper toxicity is the copper-catalyzed production of superoxide anions in the cytoplasm. (C) 2005 Elsevier Inc. All rights reserved.

Physiological evidence for reproductive suppression in the introduced population of brown tree snakes (Boiga irregularis) on Guam

Introduced species are an increasingly pervasive problem. While studies on the ecology and behavior of these pests are numerous, there is relatively little known of their physiology, specifically their reproductive and stress physiology. One of the best documented introduced pest species is the brown tree snake, Boiga irregularis, which was introduced onto the Pacific island of Guam sometime around World War II. The snake is responsible for severely reducing Guam's native vertebrates. We captured free-living individuals throughout the year and measured plasma levels of stress and sex hormones in an effort to determine when they were breeding. These data were compared to reproductive cycles from a captive population originally collected from Guam. Free-living individuals had chronically elevated plasma levels of the stress hormone corticosterone and basal levels of sex steroids and a remarkably low proportion were reproductively active. These data coincide with evidence that the wild population may be in decline. Captive snakes, had low plasma levels of corticosterone with males displaying a peak in plasma testosterone levels during breeding. Furthermore, we compared body condition between the free-living and captive snakes from Guam and free-living individuals captured from their native range in Australia. Male and female free-living snakes from Guam exhibited significantly reduced body condition compared to free-living individuals from Australia. We suggest that during the study period, free-living brown tree snakes on Guam were living under stressful conditions, possibly due to overcrowding and overexploitation. of food resources, resulting in decreased body condition and suppressed reproduction. (C) 2004 Elsevier Ltd. All rights reserved.

S100A8 chemotactic protein is abundantly increased, but only a minor contributor to LPS-induced, steroid resistant neutrophilic lung inflammation in vivo

Neutrophilic lung inflammation is an essential component of host defense against diverse eukaryotic and prokaryotic pathogens, but in chronic inflammatory lung diseases, such as chronic obstructive lung disease (COPD), severe asthma, cystic fibrosis, and bronchiolitis, it may damage the host. Glucocorticosteroids are widely used in these conditions and in their infectious exacerbations; however, the clinical efficacy of steroids is disputed. In this study, we used a proteomic approach to identify molecules contributing to neutrophilic inflammation induced by transnasal administration of lipopolysaccharide (LPS) that were also resistant to the potent glucocorticosteroid dexamethasone (Dex). We confirmed that Dex was biologically active at both the transcript (suppression of GM-CSF and TNFalpha transcripts) and protein levels (induction of lipocortin) and used 2D-PAGE/MALDI-TOF to generate global expression profiles, identifying six LPS-induced proteins that were Dex resistant. Of these, S100A8, a candidate neutrophil chemotactic factor, was profiled in detail. Steroid refractory S100A8 expression was highly abundant, transcriptionally regulated, secreted into lung lavage fluid and immunohistochemically localized to tissue infiltrating neutrophils. However, in marked contrast to other vascular beds, neutralizing antibodies to S100A8 had only a weak anti-neutrophil recruitment effect and antibodies against the related S100A9 were ineffective. These data highlight the need for extensive in vivo profiling of proteomically identified candidate molecules and demonstrates that S100A8, despite its abundance, resistance to steroids and known chemotactic activity, is unlikely to be an important determinant of LPS-induced neutrophilic lung inflammation in vivo.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Fate of hairpin transcript components during RNA silencing and its suppression in transgenic virus-resistant tobacco

Transgenic tobacco plants, carrying a Potato virus Y (PVY)-NIa hairpin sequence separated by a unique unrelated spacer sequence were specifically silenced and highly resistant to PVY infection. In such plants neither PVY-NIa nor spacer transgene transcripts were detectable by specific quantitative real time reverse transcriptase PCR (RT-qPCR) assays of similar relative efficiencies developed for direct comparative analysis. However, small interfering RNAs (siRNAs) specific for the PVY sequence of the transgene and none specific for the LNYV spacer sequence were detected. Following infection with Cucumber mosaic virus (CMV), which suppresses dsRNA-induced RNA silencing, transcript levels of PVY-NIa as well as spacer sequence increased manifold with the same time course. The cellular abundance of the single-stranded (ss) spacer sequence was consistently higher than that of PVY dsRNA in all cases. The results show that during RNA silencing and its suppression of a hairpin transcript in transgenic tobacco, the ssRNA spacer sequence is affected differently than the dsRNA. In PVY-silenced plants. the spacer is efficiently degraded by a mechanism not involving the accumulation of siRNAs, while following suppression of RNA silencing by CMV, the spacer appears protected from degradation. Crown Copyright (c) 2006 Published by Elsevier B.V. All rights reserved.

Arthritic disease suppression and cartilage protection with glycosaminoglycan polypeptide complexes (Peptacans) derived from the cartilage extracellular matrix: A novel approach to therapy

Molecular fragments of cartilage are antigenic and can stimulate an autoimmune response. Oral administration of type II collagen prevents disease onset in animal models of arthritis but the effects of other matrix components have not been reported. We evaluated glycosaminoglycan polypeptides (GAG-P) and matrix proteins (CaP) from cartilage for a) mitigating disease activity in rats with collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA) and b) stimulating proteoglycan (PG) synthesis by chondrocytes in-vitro. CIA and AIA were established in Wistar rats using standard methods. Agents were administered orally (10–200 mg/kg), either for seven days prior to disease induction (toleragenic protocol), or continuously for 15 days after injecting the arthritigen (prophylactic protocol). Joint swelling and arthritis scores were determined on day 15. Histological sections of joint tissues were assessed post-necropsy. In chondrocyte cultures, CaP + / − interleukin-1 stimulated PG biosynthesis. CaP was also active in preventing arthritis onset at 3.3, 10 or 20 mg/kg in the rat CIA model using the toleragenic protocol. It was only active at 20 and 200 mg/kg in the CIA prophylactic protocol. GAG-P was active in the CIA toleragenic protocol at 20 mg/kg but chondroitin sulfate and glucosamine hydrochloride or glucosamine sulfate were all inactive. The efficacy of CaP in the rat AIA model was less than in the CIA model. These findings lead us to suggest that oral CaP could be used as a disease-modifying anti-arthritic drug.

Vitamin D action and regulation of bone remodeling: Suppression of osteoclastogenesis by the mature osteoblast

Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone. This study indicates that vitamin D-mediated osteoclastogenesis is regulated locally by OPG production in the mature osteoblast.

Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8(+) T-cell function in Hodgkin lymphoma patients

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg (HRS) cells constitute only 0.5% of 10% of the diseased tissue. The surrounding cellular infiltrate is enriched with T cells that are hypothesized to modulate antitumor immunity. We show that a marker of regulatory T cells, LAG-3, is strongly expressed on infiltrating lymphocytes present in proximity to HRS cells. Circulating regulatory T cells (CD4(+) CD25(hi) CD45 ROhi, CD4(+) CTLA4(hi), and CD4(+) LAG-3(hi)) were elevated in HL patients with active disease when compared with remission. Longitudinal profiling of EBV-specific CD8(+) T-cell responses in 94 HL patients revealed a selective loss of interferon-gamma expression by CD8(+) T cells specific for latent membrane proteins 1 and 2 (LMP1/2), irrespective of EBV tissue status. Intratumoral LAG-3 expression was associated with EBV tissue positivity, whereas FOXP3 was linked with neither LAG-3 nor EBV tissue status. The level of LAG-3 and FOXP3 expression on the tumor-infiltrating lymphocytes was coincident with impairment of LMP1/2-specific T-cell function. In vitro pre-exposure of peripheral blood mono-nuclear cells to HRS cell line supernatant significantly increased the expansion of regulatory T cells and suppressed LMP-specific T-cell responses. Deletion of CD4(+) LAG-3(+) T cells enhanced LMP-specific reactivity. These findings indicate a pivotal role for regulatory T cells and LAG-3 in the suppression of EBV-specific cell-mediated immunity in HL.