Common polygenic variation and risk for childhood-onset schizophrenia.

Childhood-onset schizophrenia (COS) is a rare and severe form of the disorder, with more striking abnormalities with respect to prepsychotic developmental disorders and abnormities in the brain development compared with later-onset schizophrenia. We previously documented that COS patients, compared with their healthy siblings and with adult-onset patients (AOS), carry significantly more rare chromosomal copy number variations, spanning large genomic regions (>100 kb) (Ahn et al. 2014). Here, we interrogated the contribution of common polygenic variation to the genetic susceptibility for schizophrenia. We examined the association between a direct measure of genetic risk of schizophrenia in 130 COS probands and 103 healthy siblings. Using data from the schizophrenia and autism GWAS of the Psychiatric Genomic Consortia, we selected three risk-related sets of single nucleotide polymorphisms from which we conducted polygenic risk score comparisons for COS probands and their healthy siblings. COS probands had higher genetic risk scores of both schizophrenia and autism than their siblings (P<0.05). Given the small sample size, these findings suggest that COS patients have more salient genetic risk than do AOS.

[Association between long term systolic blood pressure variability index and cognitive function in middle-aged and elderly people].

OBJECTIVE: To investigate the association between different long term systolic blood pressure variability (SBPV) and cognitive function in middle-aged and elderly people. METHODS: A total of 101 510 employees from the Tangshan Kailuan Group participated in the 2006-2007 annual physical examination, 5 440 cases were selected by simple randomly sampling method. After excluding participants who did not underwent 2012-2013 examination and without complete blood pressure and mini-mental state examination (MMSE) score, 3 002 participants (1 627 males, (50.86±9.93) years old) with integrated data were included into the study. The long term SBPV was calculated by standard deviation(SD), maximum-minimum difference(MMD), average real variability (ARV) of mean systolic blood pressure measured in 2006-2007, 2008-2009, 2010-2011 and 2012-2013. Participants were grouped by the quartile of the different SBPV index. Multiple linear regressions analysis was used to analyze the correlation between the long term SBPV and cognitive function status. RESULTS: (1) The score of MMSE was 28.03±2.65. (2) The observation population was divided into four groups according to quartiles of different SBPV, respectively. The MMSE scores of SD<5.53 mmHg (1 mmHg=0.133 kPa)group, SD 5.53-8.90 mmHg group, SD 8.91-12.79 mmHg group and SD>12.79 mmHg group were 28.21±2.18, 28.26±3.09, 28.10±2.40 and 27.56±2.79, respectively(P<0.05). The MMSE scores of MMD<12.00 mmHg group, MMD 12.00-20.00 mmHg group, MMD 20.01-30.00 mmHg group and MMD>30.00 mmHg were 28.27±2.17, 28.25±3.09, 27.99±2.42 and 27.49±2.81, respectively(P<0.05). The MMSE scores of ARV<6.67 mmHg group, ARV 6.67-10.22 mmHg group, ARV 10.23-15.56 mmHg group and ARV>15.56 mmHg group were 28.27±2.20、28.28±3.20、28.00±2.42、27.57±2.65, respectively(P<0.05). (3) Adjusted for age, gender, baseline systolic blood pressure, body weight index, total cholesterol, fasting blood glucose, triglyceride, C reactive protein, smoke, drink, physical activity , the step-wise regressions analysis showed that SD(B=-0.129, P<0.05), MMD(B=-0.131, P<0.05), ARV(B=-0.125, P<0.05) had significant negative linear relationship with the MMSE score in the objects not taking the anti-hypertension drugs, and SD(B=-0.329, P<0.05), MMD(B=-0.314, P<0.05), but not ARV(B=-0.233, P>0.05), had significant negative linear relationship with the MMSE score in the objects taking the anti-hypertension drugs. CONCLUSION: The long term SBPV indexes (SD, MMD, ARV ) are negatively correlated with the MMSE score in middle-aged and elderly people not taking the anti-hypertension drugs, and SD, MMD are negatively correlated with the MMSE score in people taking the anti-hypertension drugs. Clinical Trail Registry: Chinese Clinical Trail Registry, ChiCTRTNC-11001489.

[Influencing factors of orthostatism brachial-ankle pulse wave velocity and ankle brachial index in the elderly].

OBJECTIVE: To investigate the distribution and influencing factors of orthostatism brachial-ankle pulse wave velocity(baPWV) and ankle brachial index(ABI) in the elderly. METHODS: Participants were selected with random sampling from ≥60 years old retired workers, who underwent 2010 to 2011 health check-up in the Tangshan Kailuan Hospital, Kailuan Linxi Hospital, Kailuan Zhaogezhuang Hospital. Multivariate linear regression analysis was used to analyze the influencing factors of orthostatism and supine baPWV and ABI in the elderly. RESULTS: (1) A total of 2 464 participants were included, and 1 601 participants (1 065 males(66.5%) and (67.5±6.1) years old) with integral data were analyzed. Orthostatism baPWV was (3 885.4±1 503.5)cm/s and Supine baPWV was (1 761.2±371.4)cm/s.Orthostatism ABI was 1.54±0.21 and supine ABI was 1.10±0.12. Orthostatism baPWV increased with increasing age, while orthostatism ABI decreased with aging(trend test, both P<0.01)in <65, 65-69, 70-74, and ≥75 years old participants.(2) Multiple linear regression analysis showed that the age(ß=0.19, P<0.01), lower limbs orthostatism systolic blood pressure(ß=0.18, P<0.01), lower limbs supine systolic blood pressure (ß=0.14, P<0.01), orthostatism heart rate (ß=0.30, P<0.01), supine heart rate (ß=0.23, P<0.01), body mass index (ß=-0.18, P<0.01) were associated with orthostatism baPWV, and female(ß=-0.055, P=0.01), upper limb orthostatism systolic blood pressure (ß=-0.834, P<0.01), lower limbs orthostatism systolic blood pressure (ß=0.708, P<0.01), lower limbs supine systolic blood pressure (ß=0.099, P<0.01) and fasting blood glucose(ß=-0.085, P<0.01) were associated with orthostatism ABI. CONCLUSIONS: Orthostatism baPWV and ABI were significantly higher than those of supine's. Age, lower limbs orthostatism and supine systolic blood pressure, orthostatism and supine heart rate, body mass index were associated with orthostatism baPWV. Female, upper limb orthostatism systolic blood pressure, lower limbs orthostatism, supine systolic blood pressure and fasting blood glucose were associated with orthostatism ABI in the elderly.

Primary irritation index and safety zone of cosmetics: retrospective analysis of skin patch tests in 7440 Korean women during 12 years.

BACKGROUND: Cosmetics are products used over long periods by the public, and their safety is very important. Several types of human tests are used widely for the evaluation of cosmetics including single patch tests, in-use tests, human repeated insult patch test (HRIPT). However, there is no clear and well-defined published objective and standardized criteria for primary skin irritation in regard to the large variety of cosmetic products. METHODS: This study analysed human patch tests conducted from May 2001 to December 2012 with 4606 materials of prototype or finished cosmetic products on 7440 normal Korean women aged 18-60 years. The tested products were patched under occlusion for 24 or 48 h, and skin tolerance was assessed twice at 30 min and 24 h after patch removal using a 5-step scale according to the CTFA guidelines. RESULTS: Human patch tests for cosmetics were performed of 4606 cases, and 30-33 subjects participated in each case. The response in each case was calculated based on total subject number, skin reaction intensity and the number of respondents. The calculated response was standardized using the z-score, and a safety zone was provided in terms of human primary irritation in accordance with the human skin reaction evaluation criteria and usage or formula of cosmetics. CONCLUSIONS: This study established the safety criteria for irritation in the cosmetics field.

Hypoxia-specific GM-CSF-overexpressing neural stem cells improve graft survival and functional recovery in spinal cord injury.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the differentiation and function of hematopoietic cells. GM-CSF has been implicated in nervous system function. The goal of the present study was to understand the effects of hypoxia-induced GM-CSF on neural stem cells (NSCs) in a model of spinal cord injury (SCI). GM-CSF-overexpressing NSCs were engineered utilizing a hypoxia-inducible gene expression plasmid, including an Epo enhancer ahead of an SV promoter (EpoSV-GM-CSF). Cells were then subjected to hypoxia (pO(2), 1%) or a hypoxia-mimicking reagent (CoCl(2)) in vitro. The progression of time of GM-CSF expression was tracked in EpoSV-GM-CSF-transfected NSCs. Overexpression of GM-CSF in undifferentiated and differentiated NSCs created resistance to H(2)O(2)-induced apoptosis in hypoxia. NSCs transfected with EpoSV-GM-CSF or SV-GM-CSF were transplanted into rats after SCI to assess the effect of GM-CSF on NSC survival and restoration of function. Moreover, a significantly higher amount of surviving NSCs and neuronal differentiation was observed in the EpoSV-GM-CSF-treated group. Significant improvement in locomotor function was also found in this group. Thus, GM-CSF overexpression by the Epo enhancer in hypoxia was beneficial to transplanted NSC survival and to behavioral improvement, pointing toward a possible role for GM-CSF in the treatment of SCI.

Differentiating blood samples from scrapie infected and non-infected hamsters by detecting disease-associated prion proteins using Multimer Detection System.

This communication describes the application of a modified sandwich enzyme-linked immunosorbent assay (ELISA), termed Multimer Detection System (MDS) for the detection of disease-associated multimeric forms of the prion protein (PrPd) in hamster blood. PrPd was detected in plasma of prion-affected hamsters while MDS revealed no PrPd in identically-treated plasma of healthy animals. This is the first report of a single ELISA- based immune detection of PrPd from blood samples.

A whole genome association study of neuroticism using DNA pooling.

We describe a multistage approach to identify single nucleotide polymorphisms (SNPs) associated with neuroticism, a personality trait that shares genetic determinants with major depression and anxiety disorders. Whole genome association with 452 574 SNPs was performed on DNA pools from approximately 2000 individuals selected on extremes of neuroticism scores from a cohort of 88 142 people from southwest England. The most significant SNPs were then genotyped on independent samples to replicate findings. We were able to replicate association of one SNP within the PDE4D gene in a second sample collected by our laboratory and in a family-based test in an independent sample; however, the SNP was not significantly associated with neuroticism in two other independent samples. We also observed an enrichment of low P-values in known regions of copy number variations. Simulation indicates that our study had approximately 80% power to identify neuroticism loci in the genome with odds ratio (OR)>2, and approximately 50% power to identify small effects (OR=1.5). Since we failed to find any loci accounting for more than 1% of the variance, the heritability of neuroticism probably arises from many loci each explaining much less than 1%. Our findings argue the need for much larger samples than anticipated in genetic association studies and that the biological basis of emotional disorders is extremely complex.

The perception threshold measurement can be a useful tool for evaluation of sensitive skin.

Sensitive skin is characterized by subjective symptoms that are hard to quantify. However, a neurobiological approach could improve our understanding of the nature of skin sensitivity. In this study, we measured the sensory perception of well-controlled electric currents on the skin that stimulated sensory nerve fibres such as the myelinated A fibre, A delta fibre and unmyelinated c-fibre. The sensory perception thresholds were obtained quantitatively from subjects with sensitive-prone skin and controls. Application of 0.075% capsaicin, known to stimulate the nociceptor c-fibre, was topically applied; then the sensory perception thresholds were measured to determine whether the exposure to nociceptive stimulation could affect the subsequent sensory perception. The results showed that the perception thresholds of skin sensitive-prone subjects were low for the c-fibre measurements at 5 Hz electric current stimulation. Furthermore, a wide variation in sensory perception was noted in the skin sensitive-prone subjects after topical application of capsaicin. In conclusion, the abnormal sensory perception in individuals with sensitive skin appears to be related to neurological instability, where c-fibre nociception plays a role. Thus, quantitative sensory perception threshold measurement was found to be a useful method for the identification of skin sensitive-prone subjects.

Association study between the serotonin 1A receptor (HTR1A) gene and neuroticism, major depression, and anxiety disorders.

The serotonin neurotransmitter system in general, and the serotonin 1A receptor in particular, has been broadly implicated in the pathophysiology of mood and anxiety disorders, although the results of genetic association studies have been mixed. In this study, we examined the serotonin 1A receptor gene, HTR1A, for its association with shared genetic risk across a range of anxiety and depression-related phenotypes. Using multivariate structural equation modeling, we selected twin pairs from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, and several anxiety disorders. One member from each selected pair was entered into a 2-stage, case-control association study for the HTR1A gene. In the resulting sample of 589 cases and 539 controls, four SNPs spanning the HTR1A locus, including the C(-1019)G functional promoter polymorphism (rs6295), were screened in stage 1, the positive results of which were tested for replication in stage 2. While one marker met threshold significance criteria in stage 1, this association was not replicated in stage 2. Post-hoc analyses did not reveal association to any of the specific psychiatric phenotypes. Our data suggests that the HTR1A gene may not play a major role in the genetic susceptibility underlying depressive and anxiety-related phenotypes.

LAMC2 enhances the metastatic potential of lung adenocarcinoma.

Lung cancer is the number one cancer killer, and metastasis is the main cause of high mortality in lung cancer patients. However, mechanisms underlying the development of lung cancer metastasis remain unknown. Using genome-wide transcriptional analysis in an experimental metastasis model, we identified laminin γ2 (LAMC2), an epithelial basement membrane protein, to be significantly upregulated in lung adenocarcinoma metastatic cells. Elevated LAMC2 increased traction force, migration, and invasion of lung adenocarcinoma cells accompanied by the induction of epithelial-mesenchymal transition (EMT). LAMC2 knockdown decreased traction force, migration, and invasion accompanied by EMT reduction in vitro, and attenuated metastasis in mice. LAMC2 promoted migration and invasion via EMT that was integrin ß1- and ZEB1-dependent. High LAMC2 was significantly correlated with the mesenchymal marker vimentin expression in lung adenocarcinomas, and with higher risk of recurrence or death in patients with lung adenocarcinoma. We suggest that LAMC2 promotes metastasis in lung adenocarcinoma via EMT and may be a potential therapeutic target.

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.

Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.

Purification and characterization of a manganese-containing superoxide dismutase from a carboxydobacterium, Pseudomonas carboxydohydrogena.

Superoxide dismutase from Pseudomonas carboxydohydrogena, a carboxydobacterium, grown on carbon monoxide was purified 37.6-fold in seven steps to homogeneity, with a yield of 1.4%. The final specific activity was 2,396 units per mg protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was determined to be 42,500. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 21,700. The optimal pH for enzyme activity was found to be 9.0. The enzyme was stable to heat treatment. The isoelectric point of the native enzyme was found to be 7.1. The enzyme showed an absorption peak at 280 nm with a shoulder at around 289 nm. Sodium azide, but not sodium cyanide and hydrogen peroxide, was found to inhibit the enzyme activity. One mol of native enzyme was found to contain 1.09 g-atom of manganese. Analysis of amino acid composition revealed that the enzyme contains cysteine. The superoxide dismutase of P. carboxydohydrogena was found to have antigenic sites identical to those of Oligotropha carboxydovorans. The enzyme showed partial identity to Hydrogenophaga pseudoflava and Escherichia coli enzymes, but no identity to Acinetobacter sp. strain JC1 enzyme.

Rapid genotyping of Plasmodium vivax Pvs25 and Pv38 genes by using mismatch specific endonuclease.

Mismatch specific endonuclease (MSE) method was used to detect natural polymorphisms in Pvs25 and Pv38 genes of Plasmodium vivax. Eighty seven patients with P. vivax were recruited in the Republic of Korea (ROK). Pvs25 and Pv38 genes were amplified by polymerase chain reaction (PCR), and the PCR amplicons were mixed with reference DNA sequences. Following the denaturation and gradual annealing, the product mixtures were cleaved by the MSE. Heteroduplex types were readily detected by gel electrophoresis, where extra bands with shorter sizes would appear from the cleavage. After MSE cleavage of 657- bp product from Pvs25 mixtures, three genotypes were detected, while Pv38 mixtures with 1220-bp products presented two genotypes in ROK isolates. After the MSE cleavage, the mismatched samples of Pvs25 and Pv38 were completely sequenced, and the results were in complete agreement with the MSE analyses. In conclusion, genotyping of Pvs25 and Pv38 with MSE cleavage could be a potential method for the high-throughput screening of the large field samples.

The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH.

BACKGROUND AND PURPOSE: Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown. EXPERIMENTAL APPROACH: Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown. KEY RESULTS: ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar 'dose'-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1. CONCLUSIONS AND IMPLICATIONS: These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Association between glutamic acid decarboxylase genes and anxiety disorders, major depression, and neuroticism.

Abnormalities in the gamma-aminobutyric acid (GABA) neurotransmitter system have been noted in subjects with mood and anxiety disorders. Glutamic acid decarboxylase (GAD) enzymes synthesize GABA from glutamate, and, thus, are reasonable candidate susceptibility genes for these conditions. In this study, we examined the GAD1 and GAD2 genes for their association with genetic risk across a range of internalizing disorders. We used multivariate structural equation modeling to identify common genetic risk factors for major depression, generalized anxiety disorder, panic disorder, agoraphobia, social phobia and neuroticism (N) in a sample of 9270 adult subjects from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. One member from each twin pair for whom DNA was available was selected as a case or control based on scoring at the extremes of the genetic factor extracted from the analysis. The resulting sample of 589 cases and 539 controls was entered into a two-stage association study in which candidate loci were screened in stage 1, the positive results of which were tested for replication in stage 2. Several of the six single-nucleotide polymorphisms tested in the GAD1 region demonstrated significant association in both stages, and a combined analysis in all 1128 subjects indicated that they formed a common high-risk haplotype that was significantly over-represented in cases (P=0.003) with effect size OR=1.23. Out of 14 GAD2 markers screened in stage 1, only one met the threshold criteria for follow-up in stage 2. This marker, plus three others that formed significant haplotype combinations in stage 1, did not replicate their association with the phenotype in stage 2. Subject to confirmation in an independent sample, our study suggests that variations in the GAD1 gene may contribute to individual differences in N and impact susceptibility across a range of anxiety disorders and major depression.

Mechanical strain modulates maximal phosphatidylinositol turnover in airway smooth muscle.

Mechanical strain regulates the maximal level of myosin light chain phosphorylation mediated by muscarinic activation in airway smooth muscle. Accordingly, we tested the hypothesis that mechanical strain regulates maximal phosphatidylinositol (PI) turnover (V(max)) coupled to muscarinic receptors in bovine tracheal smooth muscle. We found that PI turnover was not significantly length dependent in unstimulated tissues. However, carbachol-induced PI turnover was linearly dependent on muscle length at both 1 and 100 microM. The observed linear length dependence of PI turnover at maximal carbachol concentration (100 microM) suggests that mechanical strain regulates V(max). When carbachol concentration-PI turnover relationships were measured at optimal length and at 20% optimal length, the results could be explained by changes in V(max) alone. To determine whether the length-dependent step is upstream from heterotrimeric G proteins, we investigated the length dependence of fluoroaluminate-induced PI turnover. The results indicate that fluoroaluminate-induced PI turnover remained significantly length dependent at maximal concentration. These findings together suggest that regulating functional units of G proteins and/or phospholipase C enzymes may be the primary mechanism of mechanosensitive modulation in airway smooth muscle.

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle.

We tested the hypothesis that strain is the primary mechanical signal in the mechanosensitive modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle. We found that [Ca(2+)](i) was significantly correlated with muscle length during isotonic shortening against 20% isometric force (F(iso)). When the isotonic load was changed to 50% F(iso), data points from the 20 and 50% F(iso) experiments overlapped in the length-[Ca(2+)](i) relationship. Similarly, data points from the 80% F(iso) experiments clustered near those from the 50% F(iso) experiments. Therefore, despite 2.5- and 4-fold differences in external load, [Ca(2+)](i) did not deviate much from the length-[Ca(2+)](i) relation that fitted the 20% F(iso) data. Maximal inhibition of sarcoplasmic reticular (SR) Ca(2+) uptake by 10 microM cyclopiazonic acid (CPA) did not significantly change [Ca(2+)](i) in carbachol-induced isometric contractions and isotonic shortening. CPA also did not significantly change myosin light-chain phosphorylation or force redevelopment when carbachol-activated muscle strips were quickly released from optimal length (L(o)) to 0.5 L(o). These results are consistent with the hypothesis and suggest that SR Ca(2+) uptake is not the underlying mechanism.