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Study I represents a follow-up to a recently published study dealing with the initial development of the Sexual Abuse Questionnaire (SAQ). The SAQwas designed to be used to identify individuals experiencing psychological distress resulting from a history of childhood sexual abuse. Using a new data set (N = 2,806), this study utilized item analyses, exploratory factor analyses, and Receiver Operating Characteristics Curves to replicate and expand upon the initial psychometric properties of the SAQ, and to examine the sensitivity of the SAQ to other abuse categories. Findings from Study I indicated that the SAQ was sensitive to other abuse categories and in need of revision. Study II, using a new data set (N = 1,454), provided the initial psychometric evaluation of the newly constructed Binghamton Childhood Abuse Screen (BCAS). [ABSTRACT FROM AUTHOR]

Abstract: Stillbirth affects 1 in 175 pregnancies in the United States. There are significant racial and ethnic disparities in rates of stillbirth. Rates of stillbirth are highest among non-Hispanic Native Hawaiian or Other Pacific Islander and non-Hispanic Black women, more than twice the rate of non-Hispanic White women. Stillbirth is a public health crisis that warrants attention as it has significant physical, psychosocial, and economic effects on women and their family. Many stillbirths occur due to placental insufficiency, causing a lack of oxygenation of the fetus, which can result in decreased movement. Pregnant patients who experience stillbirth often observe decreased fetal movement days before birth. Daily fetal movement monitoring has the potential to identify pregnancies at risk so providers can intervene. Count the Kicks is a fetal movement monitoring program that provides standardized education and resources for expectant parents. Increased awareness of providers and childbearing families about the importance of fetal movement monitoring, standardized provision of education on fetal movement counting, and what to do if a baby's normal movement patterns change can be helpful in promoting healthy pregnancy outcomes., (Copyright © 2024 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.)

Competing Interests: The authors report no conflicts of interest.

Men who have sex with men (MSM), regardless of HIV infection status, have an intestinal microbiome that is compositionally distinct from men who have sex with women (MSW) and women. We recently showed HIV-negative MSM have elevated levels of intestinal CD4 + T cells expressing CCR5, a critical co-receptor for HIV. Whether elevated expression of CCR5 is driven by the altered gut microbiome composition in MSM has not been explored. Here we used in vitro stimulation of gut Lamina Propria Mononuclear Cells (LPMCs) with whole intact microbial cells isolated from stool to demonstrate that fecal bacterial communities (FBCs) from HIV-positive/negative MSM induced higher frequencies of CCR5 + CD4 + T cells compared to FBCs from HIV-negative MSW and women. To identify potential microbial drivers, we related the frequency of CCR5 + CD4 + T cells to the abundance of individual microbial taxa in rectal biopsy of HIV-positive/negative MSM and controls, and Holdemanella biformis was strongly associated with increased frequency of CCR5 + CD4 + T cells. We used in vitro stimulation of gut LPMCs with the type strain of H. biformis , a second strain of H. biformis and an isolate of the closely related Holdemanella porci , cultured from either a HIV-positive or a HIV-negative MSM stool. H. porci elevated the frequency of both CCR5 + CD4 + T cells and the ratio of TNF-α/IL-10 Genomic comparisons of the 3  Holdemanella isolates revealed unique cell wall and capsular components, which may be responsible for their differences in immunogenicity. These findings describe a novel mechanism potentially linking intestinal dysbiosis in MSM to HIV transmission and mucosal pathogenesis.

The results of a practice-based research project are described, in which parents of students with emotional and behavioral disorders (EBD) participated in a parent education presentation aimed at increasing parental self-efficacy. Results indicated that parents who participated did increase both their parental self-efficacy in regard to influencing their child's school-related performance and helping their child succeed in school. Qualitatively, parents also reported the parent education was helpful and that they had tried suggested interventions at home. Though a small-scale pilot project, results are promising and suggest parent education is a feasible technique school districts can use to reach parents of students with EBD. Limitations and future directions are discussed., (Published by Elsevier Ltd.)

Gaining a complete understanding of transmission risk factors will assist in efforts to reduce new HIV infections, especially within the disproportionally affected population of men who have sex with men (MSM). We recently reported that the fecal microbiota of MSM elevates immune activation in gnotobiotic mice and enhances HIV infection in vitro over that of fecal microbiota from men who have sex with women. We also demonstrated elevation of the gut homing marker CD103 (integrin αE) on CD4 + T cells by MSM-microbiota. Here we provide additional evidence that the gut microbiota is a risk factor for HIV transmission in MSM by showing elevated frequencies of the HIV co-receptor CCR5 on CD4 + T cells in human rectosigmoid colon biopsies. We discuss our interest in specific MSM-associated bacteria and propose the influx of CD103 + and CCR5 + CD4 + T cells into the colon as a potential link between the MSM microbiota and HIV transmission.

Rh alloimmunization remains a challenge for patients with sickle cell disease (SCD) despite transfusion of serologic Rh C, E, and K antigen-matched red cells. Inheritance of altered RH alleles contributes to the prevalence of Rh antibodies after blood transfusion in patients with SCD and explains approximately one-third of cases. The remainder seem to be stimulated by altered Rh proteins on African American donor red cells. Matching patients with donors on the basis of RH genotype may mitigate Rh alloimmunization, but the feasibility and resources required are not known. We compared RH allele frequencies between patients with SCD (n = 857) and African American donors (n = 587) and showed that RH allele frequencies are similar. Overall, 29% of RHD and 53% of RHCE alleles are altered in patients and African American donors. We modeled RH genotype matching compared with serologic Rh D, C, and E, along with K antigen matching, and found that approximately twice the number of African American donors would be required for RH genotype vs Rh serologic matching at our institution. We demonstrated that African American donor recruitment is necessary to maintain an adequate supply of C-, E-, and K-negative donor units to avoid depleting the Rh-negative (RhD - ) blood supply. Our results suggest that prophylactic RH genetic matching for patients with SCD is feasible with a donor pool comprised primarily of African-Americans and would optimize the use of our existing minority donor inventory. The current cost of RH genotyping all minority donors and management of the data remain limiting factors., (© 2018 by The American Society of Hematology.)

Allergic asthma is a chronic inflammatory lung disease characterized by sensitization of the airways, and the development of immunoglobulin E antibodies, to benign antigens. The established pathophysiology of asthma includes recurrent lung epithelial inflammation, excessive mucus production, bronchial smooth muscle hyperreactivity, and chronic lung tissue remodeling, resulting in reversible airflow restriction. Immune cells, including eosinophils and the recently characterized type 2 innate lymphoid cells, infiltrate into the lung tissue as part of the inflammatory response in allergic asthma. It is well established that a diet high in fruits and vegetables results in a reduction of the risk of developing inflammatory diseases. Secondary plant metabolites, such as proanthocyanidins which are found in apples, blackcurrants, boysenberries, cranberries, and grapes, have shown promising results in reducing or preventing allergic asthma airway inflammation. Recent evidence has also highlighted the importance of microbiome-mediated metabolism of plant polyphenols in modulating the immune system. In this review, we will discuss advances in our understanding of the pathophysiology of allergic asthma, including the role of the microbiome in lung immune function, and how proanthocyanidins modulate the airway inflammation. We will highlight the potential of dietary proanthocyanidins to impact on allergic asthma and the immune system.

Animal data shows that testosterone administration increases the volume of some parenchymal organs. However, the effects of exogenous testosterone on solid abdominal organs in humans remain unknown. The present study evaluated the effects of testosterone administration on the volume of liver, spleen and kidneys in a dose-response trial. Young healthy men aged 18-50 years participating in the 5α-Reductase (5aR) Trial. All participants received monthly injections of 7.5 mg leuprolide acetate to suppress endogenous testosterone secretion and weekly injections of 50, 125, 300 or 600 mg of testosterone enanthate, and were randomized to receive either 2.5 mg dutasteride (5 α-reductase inhibitor) or placebo daily for 20 weeks. Liver, spleen and kidney volumes were measured at baseline and the end of treatment using 1.5-Tesla magnetic resonance imaging. The dose-effect of testosterone on changes in the volume of parenchymal organs was evaluated by linear regression model. The association between changes in total testosterone (TT) levels and changes in organ volumes were assessed. Testosterone administration increased liver volume dose-dependently (17.4 cm 3 per 100 mg of weekly testosterone enanthate; p = 0.031); the increase in liver volume was positively associated with changes in TT levels (R 2  = 0.08, p = 0.024). A dose-dependent, but non-significant, increase in kidney volumes was also seen. Inclusion of dutasteride use into the models showed an independent association of randomization to dutasteride group with liver volume increase. In conclusion, Testosterone administration increased the liver volume in a dose-dependent manner. The potential changes in parenchymal organs should be considered when interpreting apparent changes in lean mass in response to anabolic interventions., (© 2017 American Society of Andrology and European Academy of Andrology.)

Allergic asthma is an inflammatory lung disease that is partly sustained by the chemokine eotaxin-3 (CCL26), which extends eosinophil migration into tissues long after allergen exposure. Modulation of CCL26 could represent a means to mitigate airway inflammation. Here we evaluated procyanidin A2 as a means of modulating CCL26 production and investigated interactions with the known inflammation modulator, Interferon γ (IFNγ). We used the human lung epithelial cell line A549 and optimized the conditions for inducing CCL26. Cells were exposed to a range of procyanidin A2 or IFNγ concentrations for varied lengths of time prior to an inflammatory insult of interleukin-4 (IL-4) for 24 h. An enzyme-linked immunosorbent assay was used to measure CCL26 production. Exposing cells to 5 μM procyanidin A2 (prior to IL-4) reduced CCL26 production by 35% compared with control. Greatest inhibition by procyanidin A2 was seen with a 2 h exposure prior to IL-4, whereas IFNγ inhibition was greatest at 24 h. Concomitant incubation of procyanidin A2 and IFNγ did not extend the inhibitory efficacy of procyanidin A2. These data provide evidence that procyanidin A2 can modulate IL-4-induced CCL26 production by A549 lung epithelial cells and that it does so in a manner that is different from IFNγ., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The purpose of this study was to determine the efficacy of CT to predict the development of bile leaks in hepatic trauma. This HIPAA-compliant retrospective study was IRB approved and consent was waived. All patients who sustained hepatic trauma between January 1, 2006, and January 31, 2012, and who underwent CT and hepatobiliary scans during the same hospital admission were included. One hundred and thirty-two patients met the inclusion criteria. Comparison between the presence of biliary injury relative to American Association for the Surgery of Trauma (AAST) hepatic injury grade and mean distance of the hepatic laceration to the inferior vena cava (IVC) was made. The ability of free fluid to predict bile injury was analyzed. Forty-one (31 %) of the 132 patients had positive hepatobiliary scans. Of these 41 patients, seven (17 %) sustained low-grade and 34 (83 %) sustained high-grade hepatic injury compared with the 37 (41 %) low-grade and 54 (59 %) high-grade hepatic injuries in the negative hepatobiliary scan group. The mean distance to the IVC was 2.4 cm (SD 2.9 cm) and 3.6 cm (SD 3.3 cm) in patients with and without bile leaks, respectively. A statistically significant difference in the proportion of high-grade injuries and the mean distance from the IVC between the two groups was identified. The presence of free fluid on CT is sensitive, but not specific, for detecting a bile leak. CT findings, including AAST liver injury grade and location of the liver laceration, are able to predict which patients are at risk for developing bile leaks as seen on hepatobiliary scintigraphy, whereas the presence of free fluid is not.

Reverse cholesterol transport (RCT), a process to deliver excess cholesterol from the periphery to the liver for excretion from body, is a major atheroprotective property of high-density lipoproteins. As major transporters for cholesterol efflux in macrophages, ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) are critical for RCT. We investigated mechanisms for the regulation of ABCA1 and ABCG1 expression by fatty acids (FA) in RAW264.7 macrophages. Cells were incubated with 100 μmol/L of palmitic, oleic, linoleic, linolenic or eicosapentaenoic acids in the absence or presence of T0901317, a liver X receptor (LXR) agonist. Unsaturated FA, but not saturated FA, significantly reduced ABCA1 and ABCG1 mRNA without the agonist. Trichostatin A (TSA), a histone deacetylase inhibitor, not only increased basal ABC transporter expression but abrogated the transcriptional repression by unsaturated FA. The increased basal ABCA1 and ABCG1 mRNA by TSA paralleled the increased peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator 1α expression, whereas LXRα and PGC-1β expression was significantly lowered. Although the repressive effect of ABCA1 and ABCG1 mRNA by unsaturated FA was abolished by T0901317, protein levels remained diminished. Chemical and genetic deficiency of protein kinase C δ did not abolish the repressive effect of linoleic acid on ABCA1 and ABCG1. In conclusion, unsaturated FA repressed ABCA1 and ABCG1 expression by two distinct mechanisms in RAW 264.7 macrophages: LXR-dependent transcriptional repression possibly by modulating histone acetylation state and LXR-independent posttranslational inhibition., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Adipocyte fatty acid binding protein (A-FABP) present in macrophages has been implicated in the integration of lipid metabolism and inflammatory response, contributing to development of insulin resistance and atherosclerosis., Aim of the Study: This study was conducted to test the hypothesis that the role of fatty acids in the inflammatory pathways is mediated through the modulation of A-FABP expression in macrophages., Methods: Murine RAW 264.7 macrophages were treated with inflammatory insults and fatty acids for quantitative real-time PCR and Western blot analysis. The cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor, for elucidating mechanisms for the regulation of A-FABP expression by fatty acids. RNA interference (RNAi) to knock down A-FABP was utilized to assess its role in inflammatory gene expression., Results: When RAW 264.7 were incubated with lipopolysaccharides (LPS; 100 ng/ml) or 2.5 ng/ml of tumor necrosis factor α for 18 h, A-FABP mRNA and protein levels were drastically increased. Unsaturated fatty acids (100 μmol/l in complexed with BSA) such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid, significantly repressed the basal as well as LPS-induced A-FABP expression, whereas palmitic acid did not elicit the same effect. TSA increased A-FABP mRNA levels and abolished the repressive effect of linoleic acid on A-FABP expression in unstimulated and LPS-stimulated macrophages. Depletion of A-FABP expression by 70-80% using RNAi markedly decreased cyclooxygenase 2 mRNA abundance and potentiated the repression by linoleic acid., Conclusion: Unsaturated fatty acids inhibited the basal as well as LPS-induced A-FABP expression. The mechanism may involve histone deacetylation and anti-inflammatory effect of unsaturated fatty acids may be at least in part attributed to their repression of A-FABP expression in RAW 264.7 macrophages.

In addition to the central role of compulsive behaviors in obsessive compulsive disorder (OCD), recent data have documented the presence of compulsive behaviors in individuals with generalized anxiety disorder (GAD). However, there is a lack of information about potential similarities and differences with regard to the quality, or content, of checking associated with worry and obsessions. The two studies presented herein are an initial step towards gathering this information. Findings of Study 1, from a large unselected undergraduate sample, showed that symptoms of OCD and GAD were both significantly associated with checking behaviors. However, while OCD symptoms were associated with checking related to both objects and interpersonal situations, GAD symptoms were only significantly associated with interpersonal checking. Findings of Study 2, using a separate sample, suggest links between interpersonal checking and features characteristic of GAD, namely emotion regulation difficulties, and between object checking and a cognitive feature of OCD, namely thought-action fusion. In summary, the current studies add to a growing body of literature suggesting that checking may be important in numerous forms of psychopathology, while also suggesting that the nature and function of checking may differ for various symptom profiles., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Anxiety disorders represent the single largest mental health problem in the United States [Greenberg et al., 1999. J Clin Psychiatry 60:427-435; Rice and Miller, 1998. Br J Psychiatry 173:4-9]. However most individuals with anxiety disorders never seek treatment [Henderson et al., 2002. Can J Psychiatry 47:819-824; Mojtabai et al., 2002. Arch Gen Psychiatry 59:77-84; Roness et al., 2005. Acta Psychiatr Scand 111:51-58]. Deficits in the ability to recognize anxiety disorders and beliefs about them, (i.e., "mental health literacy") may contribute to low levels of help seeking., Methods: Survey data assessing mental health literacy for multiple anxiety disorders and for depression were collected from 284 undergraduate students enrolled in psychology courses at a public university in the United States. Specifically, respondents were presented with vignettes portraying individuals experiencing various forms of mental illness and were asked to label the disorder, its cause and whether or not they would recommend treatment., Results: Findings showed that social phobia and obsessive compulsive disorder (OCD) were associated with recognition rates that were generally high and similar to depression (approximately 80%). In contrast, less than half of the respondents labeled panic disorder or generalized anxiety disorder (GAD) correctly. Symptoms of OCD were attributed to mental illness by approximately 50% of respondents, but such attributions were rare for the other anxiety disorders studied (<12%). Finally, data on help-seeking recommendations suggested that such recommendations are far from universal and varied between different anxiety disorders and according to perceptions of the causes of symptoms., Conclusions: Given that the current sample was well-educated young adults, mental health literacy of the general public may be even lower.

This article develops the cognitive-emotional forager (CEF) model, a novel application of a neural network to dynamical processes in foraging behavior. The CEF is based on a neural network known as the gated dipole, introduced by Grossberg, which is capable of representing short-term affective reactions in a manner similar to Solomon and Corbit's (1974) opponent process theory. The model incorporates a trade-off between approach toward food and avoidance of predation under varying levels of motivation induced by hunger. The results of simulations in a simple patch selection paradigm, using a lifetime fitness criterion for comparison, indicate that the CEF model is capable of nearly optimal foraging and outperforms a run-of-luck rule-of-thumb model. Models such as the one presented here can illuminate the underlying cognitive and motivational components of animal decision making.

A considerable proportion of heritable human phenotypic variation is thought to result from altered gene expression. Unfortunately, it is currently impossible to use bioinformatic analysis to discriminate between DNA sequence variants that are likely to influence gene expression and those that are not. In an attempt to define some of the characteristics of promoter polymorphisms with functional effects on gene expression, we examined 674 haplotypes representing 247 unique gene promoters using a standardized reporter gene assay system. Sequence variants that altered gene expression by 1.5-fold or more were strongly biased toward a location in the core and proximal promoter regions, 50% being within the first 100 bases 5' to the transcription start site. No bias was seen in the allele frequencies of functional and nonfunctional sequence variants. Only 33% of the functional variants were found in known consensus transcription factor binding sequences or motifs, which suggests that either there are many unknown transcription factor binding motifs or other, unknown mechanisms are involved. The genes with functional polymorphisms that are reported here for the first time include AGTRL2, CAT, CHRNA5, CTSG, CYP2D6, DLD, ERCC1, GABRA1, GABRP, HNRPH3, HIP1, IGKV1-9, KCNJ15, KCNK6, KLK1, MSMB, MYOC, NPY2R, NOTCH4, ORM2, PEDF, PTPRCAP, ST16 (IL24), SULT1A1, and TSHR.

Objective: Association has been reported between the C allele of a -759C/T polymorphism in the promoter of the 5-HT2C receptor gene (HTR2C) and antipsychotic-induced weight gain, suggesting that polymorphic HTR2C expression influences this phenotype. The authors tested this polymorphism, and other promoter variants, for effects on HTR2C transcription., Method: Six HTR2C promoter haplotypes constructed from four polymorphisms were cloned into a luciferase reporter gene plasmid. Their transcriptional activities were then compared in two human cell lines., Results: All haplotypes containing the -759C allele showed less transcriptional activity than haplotypes containing the -759T allele. The A allele of a -997G/A polymorphism was also associated with reduced expression., Conclusions: These findings suggest that the -759C allele is functional and results in relative underexpression of HTR2C. Reduced expression of HTR2C mRNA may underlie vulnerability to weight gain following antipsychotic treatment.

There is increasing interest in the possibility that polymorphisms affecting gene expression are responsible for a significant proportion of heritable human phenotypic variation, including human disease. We have sought to determine if polymorphisms in the promoters of brain expressed genes are commonly functional. We screened for polymorphism 56 genes previously reported to be differentially expressed in the brains of schizophrenics [Y. Hakak, J.R. Walker, C. Li, W.H. Wong, K.L. Davis, J.D. Buxbaum, V. Haroutunian, A.A. Fienberg, Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. 98 (2001) 4746-4751.]. We found 60 variants distributed across 31 of the genes. A total of 77 haplotypes representing 28 different putative promoters were analyzed in a reporter gene assay in two cell lines. Of a total of 54 sequence variants represented in the haplotypes, 12 (or around 22%) were functional according to a highly conservative definition. These were found in the promoters of eight genes: NPY, PCSK1, NEFL, KIAA0513, LMO4, HSPA1B, TF and MDH1. We therefore estimate that around 20-25% of promoter polymorphisms in brain expressed genes are functional, and this is likely to be an underestimate. Our data therefore provide for the first time empirical evidence that promoter element polymorphisms, at least in brain expressed genes, should be afforded a high priority for molecular genetic studies.

Segmental aneusomy, which includes chromosome 22 deletion syndrome (del(22)(q11.2q11.2)), has been associated with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face (CAF) syndrome, cat-eye syndrome (CES), der(22) syndrome, and duplication of the del(22)(q11.2q11.2) syndrome's typically deleted region. Adults with del(22)(q11.2q11.2) may develop psychiatric illnesses, including schizophrenia, schizoaffective disorder, and bipolar disorder, suggesting that lower gene dosage leads to a predisposition to these illnesses. In a bid to identify important regulatory polymorphisms (SNPs) that may emulate changes in gene dosage of the genes within the common deletion, we have analyzed the promoter region of 47 genes (44 of which encode a protein with known function) encoding proteins in and around 22q11 for sequence variants. A total of 33 of the promoters contained polymorphisms. Of those, 25 were cloned into a reporter gene vector, pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671), using a cotransfected CMV-SPAP plasmid as an internal control. Five genes (PRODH, DGCR14, GSTT2, SERPIND1, and a gene tentatively called DKFZP434P211) showed activity differences between haplotypes of greater than 1.5-fold. Of those, PRODH, which encodes proline dehydrogenase, has previously been highlighted in relation to schizophrenia, and the functional promoter polymorphism reported here may be involved in pathogenic mechanisms., (Copyright 2004 Wiley-Liss, Inc.)

Hyaluronan (HA) is a linear glycosaminoglycan of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2 and -3. The regulation of HA synthesis has been implicated in a variety of extracellular matrix-mediated and pathological processes, including renal fibrosis. We have recently described the genomic structures of each of the human HAS genes. In the present study, we analyzed the HAS2 promoter region. In 5'-rapid amplification of cDNA ends analysis of purified mRNA from human renal epithelial proximal tubular cells, we detected an extended sequence for HAS2 exon 1, relocating the transcription initiation site 130 nucleotides upstream of the reference HAS2 mRNA sequence, GenBank accession number NM&#95;005328. A luciferase reporter gene assay of nested fragments spanning the 5' terminus of NM&#95;005328 demonstrated the constitutive promoter activity of sequences directly upstream of the repositioned transcription initiation site but not of the newly designated exonic nucleotides. Using reverse transcription-PCR, expression of this extended HAS2 mRNA was demonstrated in a variety of human cell types, and orthologous sequences were detected in mouse and rat kidney. Alignment of human, murine, and equine genomic DNA sequences upstream of the repositioned HAS2 exon 1 provided evidence for the evolutionary conservation of specific transcription factor binding sites. The location of the HAS2 promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts as well as in developmental models in which HAS2 null animals have an embryonic lethal phenotype.

The abuse of gamma-hydroxybutyrate (GHB) and two of its precursors, gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are recognized as a public health concern. Here, we report dose-response and time-course analyses for effects of GBL and 1,4-BD on locomotor activity and body temperature in Swiss-Webster mice. Locomotor activity was measured for 2 h following a single injection of one of four doses of each agent plus a saline vehicle control. At 50 mg/kg, GBL produced an initial depression of locomotor activity which was followed by stimulation of locomotor activity. In contrast, 1,4-BD at 50 mg/kg stimulated locomotor activity without producing any depression of activity. At higher doses, GBL produced primarily a dose-dependent decrease in locomotor activity that returned to baseline within 50 min. In contrast, 1,4-BD produced an initial depression which was followed by stimulation of activity. Body temperature was measured rectally across a 2.5-h time course following injection with either agent. Both drugs produced hypothermia with peak effects occurring at 20 and 30 min for both drugs for the lower and higher dose, respectively. At 150 mg/kg, GBL produced a greater hypothermic response; however, no differences in hypothermic response were observed at 100 mg/kg. These studies demonstrate that the precursor drugs to GHB have some differential actions from each other.

We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes.

Objectives: The regulation of genes involved in glutamatergic function is thought to be a critical for many central nervous system processes including memory, learning, synaptic maintenance, and many pathological states. As part of a larger survey into the key regulatory elements in genes of neuro-psychiatric interest, we sought to identify the promoter regions of genes in this broad family, and to identify sequence variants that alter gene expression., Methods: Mutation analysis was carried out on the promoters of 20 genes encoding 13 glutamate receptor subunits, four transporters and three metabolizing enzymes using denaturing high performance liquid chromatography. Thirty-nine different promoter haplotypes were cloned into a luciferase reporter gene vector and tested for differences in their ability to drive transcription in both HEK293t and TE671 cell lines., Results: We have identified a total of 48 sequence variants in six glutamate receptor subunits, four glutamate transporters and two enzymes. Interestingly, seven promoter sequences gave three or more haplotypes from a single individual, indicating gene duplication. No differences in expression greater than 1.35-fold were found between haplotypes originating from the same or paralogous genes., Conclusion: The lack of common functional polymorphisms in any of these promoters indicates that expression of glutamate receptors and transporters is unusually tightly controlled, and suggests the possibility that non-coding polymorphisms in these genes are rare and may be unlikely to contribute in a major way to neuro-psychiatric phenotypes. This study represents the world's largest survey of the any group of promoters yet performed for any gene system.

The potential importance of gene regulation in disease susceptibility and other inherited phenotypes has been underlined by the observation that the human genome contains fewer protein coding genes than expected. Promoter sequences are potential sources of polymorphism affecting gene expression, although to date there are no large-scale systematic studies that have determined how frequently such variants occur. We have used denaturing high performance liquid chromatography to screen the first 500 bp of the 5' flanking region of 170 opportunistically selected genes identified from the Eukaryotic Promoter Database (EPD) for common polymorphisms. Using a screening set of 16 chromosomes, single-nucleotide polymorphisms were found in approximately 35% of genes. It was attempted to clone each of these promoters into a T-vector constructed from the reporter gene vector pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of three human cell lines (HEK293, JEG and TE671) using a co-transfected SEAP-CMV plasmid as a control. The findings suggest that around a third of promoter variants may alter gene expression to a functionally relevant extent.

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.

Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.

The ability to identify and examine promoter elements is important to researchers who wish to understand how gene expression is regulated in normal and pathological states. Unfortunately, the number of human promoters that have been directly experimentally defined is small. In order to determine if promoter sequences can be identified by simply aligning mRNA and genomic sequences, we have used a reporter gene assay to assess the promoter activity of the immediate 5' region flanking 38 mRNAs mapping to chromosome 21. For comparison, we have measured the activities of 19 sequences not thought to be promoters and 39 sequences taken from the Eukaryotic Promoter Database. Our results suggest that alignment of reference mRNAs to genomic sequence allows promoters to be identified for at least 75% of genes. These data provide the first empirical evidence that the current state of annotation of the genome is sufficient to allow molecular geneticists to correctly identify promoter sequences for most genes for which reference mRNA and genomic sequences are available.

We have developed a rapid method for identifying functional promoter-region polymorphisms. Using a modified pGL3 luciferase expression T-vector, we can amplify by PCR, clone, identify allelic pairs of a polymorphic gene promoter region, and prepare plasmids for cell culture 10 promoters (20 allele pairs) per week per researcher. By utilizing 96-well plate technology and an internal control plasmid expressing secreted alkaline phosphatase, each of these allele pairs can be tested for relative promoter activity in each of three cell lines (HEK293t, TE671, and JEG3) with similar resources.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

Three point mutations of the connexin26 (GJB2) gene associated with hereditary deafness were studied using in vitro expression systems. Mutation M34T results in an amino acid substitution in the first transmembrane domain of the connexin protein, W77R is located in the second transmembrane domain and W44C is in the first extracellular loop. Wild-type and mutated connexin vectors were constructed and transfected into communication-deficient HeLa cells to obtain transient expression of the connexin proteins. Intercellular coupling was subsequently assessed by examining transfer of Lucifer yellow between cells. All three mutations resulted in impaired intercellular coupling. The mechanistic reasons for the functional inadequacies of the mutated proteins were investigated. First, intracellular trafficking and targeting of the expressed connexins were determined by immunohistochemistry. Mutation W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma membrane. Oligomerization assays showed that connexins M34T and W77R failed to assemble efficiently into hexameric gap junction hemichannels, but the W44C mutation did so. A cell-free translation system showed that the mutated proteins were inserted into microsomal membranes but the mutations have different effects on the post-translational properties of the expressed proteins. The results point to the conclusion that mutations in the transmembrane domains of connexin proteins influence gap junction assembly.

Isoelectric focusing (IEF) in the pH range 2.5-5.0 has been used to compare the immunoreactive (ir) erythropoietin (Epo) in paired samples of serum and urine from three patients, two with idiopathic aplastic anaemia and one with paroxysmal nocturnal haemoglobinuria and also from three anaemic rats. Serum samples only were also examined from two further patients with aplastic anaemia and from three mice, made anaemic (like the rats) by irradiation and phenylhydrazine treatment. Most of the ir-Epo recovered after IEF was found in the pH range 2.5-3.9. For the sera, the proportion of more acidic ir-Epo with pI less than 3.0 recovered after IEF increased from human to rat to mouse. Human sera contained a greater proportion of ir-Epo with pI greater than 3.4 than rat or mouse sera. For the urines, the distribution of ir-Epo by IEF was similar between human and rat. For both species, the proportion of ir-Epo with pI less than 3.0 recovered after IEF was greater in urine than in the paired serum samples. The Second International Reference Preparation of Human Urinary Epo differed from the Epo in unextracted human urine in that there was a lower proportion of ir-Epo with pI less than 3.0. The differences observed between serum and urinary Epo are of particular interest because only the urinary form of native human Epo has ever been purified, and because this was used to compare native with rDNA-derived Epo.

A 71-year-old woman complained of "smoky" vision, which was found to be caused by a hyphema with blood dripping from a vascular tuft located in the 12 o'clock meridian of the iris. Fluorescein angiography delineated vascular tufts and argon laser photocoagulation eradicated one of the tufts that bled. Histopathologic studies of iris obtained at the time of cataract extraction showed an aggregate of small vessels at the pupillary margin. Most patients with vascular tufts of the pupillary margin have no systemic disease but they are also observed in diabetes mellitus and myotonic dystrophy.

A patient with recurrent intestinal obstruction and jejunocutaneous fistula was referred for treatment of the latter condition. Management with total parenteral nutrition was complicatied by bacteremia. Subsequently, a double-lumen tube was passed via an existing gastrostomy for purposes of aspirating above the level of the fistula and infusing appropriate nutrients and fluids distally. A period of marked clinical improvement was followed by increased fistula output and evidence of intestinal obstruction secondary to gaseous distention of a sealed latex terminal balloon which was retrieved only after percutaneous puncture. The unusual complication of prolonged intestinal intubation is discussed with special reference to this nonsurgical method of managing the impacted balloon and tube. Factors affecting balloon distention are discussed and the necessity of venting intestinal balloons reemphasized.