Expression of a linear Sry transcript in the mouse genital ridge.

Sry is the Y-chromosomal gene that is pivotal in the determination of sex in mammals, however the structure of the Sry transcript produced in the embryo has not been determined. We show here that the transcript expressed in the developing mouse gonad at the sex determining stage of development is linear, polyadenylated and encoded by a single exon, in contrast to the circular, apparently untranslated transcript produced in adult testes. The linear transcript was not detected in any other fetal tissue nor in any adult tissue tested, and was expressed only in the genital ridge portion of the urogenital ridge. The spatial and temporal profile of Sry expression suggests that its role in the mouse fetus is limited to initiating Sertoli cell development during testis determination.

An H-YDb epitope is encoded by a novel mouse Y chromosome gene.

Rejection of male tissue grafts by genotypically identical female mice has been explained by the existence of a male-specific transplantation antigen, H-Y (ref. 1), but the molecular nature of H-Y antigen has remained obscure. Hya, the murine locus controlling H-Y expression, has been localized to delta Sxrb, a deletion interval of the short arm of the Y chromosome. In mice, H-Y antigen comprises at least four distinct epitopes, each recognized by a specific T lymphocyte clone. It has recently been shown that one of these epitopes, H-YKk, is a peptide encoded by the Y-linked Smcy gene, presented at the cell surface with the H-2Kk major histocompatibility complex (MHC) molecule. However, deletion mapping and the analysis of variable inactivation of H-Y epitopes has suggested that the Hya locus may be genetically complex. Here we describe a novel mouse Y chromosome gene which we call Uty (ubiquitously transcribed tetratricopeptide repeat gene on the Y chromosome). We identify the peptide WMHHNMDLI derived from the UTY protein as an H-Y epitope, H-YDb. Our data formally demonstrate that H-Y antigen is the product of more than one gene on the Y chromosome.

The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos.

Mutations in the human SRY-related gene, SOX9, located on chromosome 17, have recently been associated with the sex reversal and skeletal dysmorphology syndrome, campomelic dysplasia. In order to clarify the role of this gene in skeletal development, we have studied the expression of mouse Sox9 during embryogenesis. Sox9 is expressed predominantly in mesenchymal condensations throughout the embryo before and during the deposition of cartilage, consistent with a primary role in skeletal formation. Interspecific backcross mapping has localized mouse Sox9 to distal chromosome 11. The expression pattern and chromosomal location of Sox9 suggest that it may be the gene defective in the mouse skeletal mutant Tail-short, a potential animal model for campomelic dysplasia.

Molecular cloning and functional expression of glutamate receptor subunit genes.

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.

Cloning of a novel glutamate receptor subunit, GluR5: expression in the nervous system during development.

We have isolated cDNAs encoding a glutamate receptor subunit, designated GluR5, displaying 40%-41% amino acid identity with the kainate/AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4. This level of sequence similarity is significantly below the approximately 70% intersubunit identity characteristic of kainate/AMPA receptors. The GluR5 protein forms homomeric ion channels in Xenopus oocytes that are weakly responsive to L-glutamate. The GluR5 gene is expressed in subsets of neurons throughout the developing and adult central and peripheral nervous systems. During embryogenesis, GluR5 transcripts are detected in areas of neuronal differentiation and synapse formation.

Optimization of normal-phase chromatographic separation of compounds with primary, secondary and tertiary amino groups.

The retention behavior of primary, secondary and tertiary amines was studied using normal-phase-HPLC on silica, diol, and cyano stationary phases. Several classes of amines, including benzylamines, anilines, ephedrines, tryptamines, and azatryptamines were chromatographed using mixtures of hexane and ethoxynonafluorobutane with methylene chloride and methanol. Peak tailing, diminished selectivity and low plate count were minimized by the addition of volatile amines to the mobile phase. The optimal additive was n-propylamine at 0.1% concentration. On diol columns, the elution order of free primary, N-N-methyl, and N,N-dimethylamines was predictable, while the elution order of primary and secondary amines on cyano columns varied depending on the alcohol modifier concentration. The feasibility of preparative normal-phase chromatography was demonstrated by the separation of a mixture of primary, secondary and tertiary amines obtained by direct methylation of norephedrine. The procedures described may provide a practical alternative to traditional methods of analysis and purification of potential drug candidates.

GENETICS IN ENDOCRINOLOGY: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 'DSDnet'

The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 'DSDnet' was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV) or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case.

Association between Carotid Plaque Features on CTA and Cerebrovascular Ischemia: A Systematic Review and Meta-Analysis.

BACKGROUND: CTA is a widely available imaging examination that may allow the evaluation of high-risk carotid plaque features. PURPOSE: Our aim was to evaluate the association between specific carotid plaque features on CTA and ipsilateral cerebrovascular ischemia. DATA SOURCES: We performed a systematic review of Ovid MEDLINE, Ovid Embase, Scopus, and the Cochrane Library from inception to March 2016 for articles that evaluated the relationship between CTA-detected carotid plaque features and ischemic events, defined as ipsilateral ischemic stroke or transient ischemic attack. STUDY SELECTION: Sixteen studies were ultimately included after screening 12,557. DATA ANALYSIS: Two readers recorded data from each study and assessed the study quality with all disagreements resolved by a third reader. A random-effects OR was used to evaluate the association between cerebrovascular ischemia and each of the evaluated plaque features. DATA SYNTHESIS: We found significant positive relationships with cerebrovascular ischemia for the presence of soft plaque (OR, 2.9; 95% CI, 1.4-6.0), plaque ulceration (OR, 2.2; 95% CI, 1.4-3.4), and increased common carotid artery wall thickness (OR, 6.2; 95% CI, 2.5-15.6). We found a significant negative relationship between calcified plaque and ipsilateral ischemia (OR, 0.5; 95% CI, 0.4-0.7). LIMITATIONS: We found heterogeneity in the existing literature secondary to lack of standardized plaque features and clinical definitions. CONCLUSIONS: Soft plaque, plaque ulceration, and increased common carotid artery wall thickness on CTA are associated with ipsilateral cerebrovascular ischemia, while calcified plaque is negatively associated with downstream ischemic events.

Normal-phase chiral liquid chromatography-mass spectrometry of non-UV-active compounds: applications for pharmaceutically relevant racemates.

Mixtures of hexane-like ethoxynonafluorobutane with alcohols were used as MS-friendly mobile phases for separation and efficient detection of non-UV-active enantiomers and diastereomers using normal-phase HPLC-APCI-MS. Racemic muscone, camphorsulfonamide, camphorsultam, BOC-protected 1-(3-aminopropyl)-2-pipecoline and diastereomeric 2-methylhexanoyl camphorsultams were resolved on Chiralpak AS and AD and achiral Luna CN columns. The responses of UV and APCI-MS detectors were compared under separation conditions studied, with MS detection achieving lowest detectable quantity in the range of 0.5-2 ng per chromatographic peak. The absolute configuration of crystalline derivatives of racemic 2-methylhexanoic acid with (S)-(-)-2,10-camphorsultam was determined by X-ray analysis after their automatic purification by preparative LC-MS. The technique described can be used to purify and determine the absolute stereochemistry of compounds of unknown structure which contain free carboxy group and lack sufficient UV absorbance.

Factors Associated With Nutritional Risk Among Homebound Older Adults With Depressive Symptoms.

OBJECTIVES: This study used the Evans model of public health determinants to identify factors associated with nutritional risk in older adults. DESIGN: The Evans model domains (physical and mental well-being, social/environmental statuses, individual choice, and economic security) were measured in a sample of homebound older adults. Regularized logistic regression analysis with LASSO penalty function was used to determine the strongest domain of the Evans model. Using traditional logistic regression, individual variables across all domains were compared to identify the significant predictors. SETTING: Older adults receiving home meal services were referred to the study by community program staff. PARTICIPANTS: Participants included 164 homebound older adults (age > 60) who endorsed at least one gateway symptom of depression. MEASUREMENTS: Measurements: Nutritional risk was determined using the Mini Nutritional Assessment. Domains of the Evans model were measured using the MAI Medical Condition Checklist, items from the IADL scale, the Structured Clinical Interview for DSM-IV Axis I Disorders, the Duke Social Support Index, living arrangements, marital status, the Alcohol Use Disorders Identification Test, items from the SCID Screening Module, and a self-report of perceived financial security. RESULTS: Poor mental well-being, defined by a diagnosis of major depressive disorder, was identified as the strongest Evans model domain in the prediction of nutritional risk. When each variable was independently evaluated across domains, instrumental support (Wald's Z=-2.24, p=0.03) and a history of drug use (Wald's Z=-2.40, p=0.02) were significant predictors. CONCLUSIONS: The Evans model is a useful conceptual framework for understanding nutritional health, with the mental domain found to be the strongest domain predictor of nutritional risk. Among individual variables across domains, having someone to help with shopping and food preparation and a history of drug use were associated with lower nutritional risk. These analyses highlight potential targets of intervention for nutritional risk among older adults.

Over-expression of p55Cdc inhibits granulocyte differentiation and accelerates apoptosis in myeloid cells.

p55Cdc is a protein identified in cycling mammalian cells. It is highly expressed in proliferating but not in differentiated or growth-arrested cells. Structurally, p55Cdc is similar to the Cdc4 and Cdc20 proteins, which have been proposed to regulate DNA synthesis and mitosis in Saccharomyces cerevisiae. To define the role of p55Cdc during myelopoiesis, we studied the expression and regulation of this protein in response to the hematopoietic growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF). We analysed the time course of expression of p55Cdc in response to GM-CSF and G-CSF stimulation in the murine factor-dependent myeloid leukemic cell line, 32Dc13, and demonstrated differential regulation of p55Cdc in response to these two growth factors. Over-expression of p55Cdc resulted in acceleration of apoptosis in growth factor- and serum-free conditions, although no difference was observed in the rate of cell proliferation. Decreases in p55Cdc protein levels correlated with cells undergoing apoptosis. p55Cdc over-expression also inhibited granulocyte differentiation of 32Dc13 cells treated with G-CSF. Our studies suggest that p55Cdc regulation is critical for normal cell cycle control during myeloid cell proliferation and differentiation.

Expression of a novel mammalian epidermal growth factor-related gene during mouse neural development.

We have recently reported the preliminary characterisation of a novel EGF-related gene, Scube1 (signal peptide-CUB domain-EGF-related, gene 1), that is expressed prominently in the developing gonad, nervous system, somites, surface ectoderm and limb buds of the mouse. Here we describe the expression pattern of a closely related gene, Scube2 (also known as Cegp1), which maps to the distal region of mouse chromosome 7. Scube2 transcription is restricted to the embryonic neurectoderm but is also detectable in the adult heart, lung and testis.

Screening for novel ENU-induced rhythm, entrainment and activity mutants.

Chemical mutagenesis has provided an opportunity to develop and expand the repertoire of behavioural mutants for gene function studies. With this in mind, we have established a screen in mice for mutations affecting circadian rhythms, entrainment to light and other wheel-running parameters. The screen consists of an assessment of mouse wheel-running activity in a 12:12 h light/dark cycle for 7-10 days followed by assessment in constant darkness for up to 20 days. Responses to light are assessed using two protocols; a 15 minute light pulse given at circadian time 16 on the tenth day in constant darkness and an additional 12 h of light upon transition from light/dark conditions to constant darkness. To date, approximately 1300 progeny of chemically mutagenised mice have been screened. Computer-aided assessment of wheel-running parameters has helped in identifying abnormal phenotypes in approximately 5% of all animals screened. Inheritance testing of mice with abnormal phenotypes has confirmed the number of robustly inherited mutant phenotypes to be 1% of the total screened. Confirmed mutants including those affecting free-running period, light-responsiveness and wheel-running endurance have been identified. Thus far, low-resolution map positions have been established for four mutants by completing genome scans in backcross progeny. Mutant loci do not correspond with those previously associated with wheel-running behaviour. This result confirms that phenotype-driven approaches such as this should continue to provide material for mammalian gene function studies.

A model to describe the size distribution of mammalian genomic fragments recovered by microcloning.

Experiments involving the use of microdissection and microcloning of mammalian chromosomes to obtain genomic clones from individual chromosome regions have demonstrated an aberrant clone recovery. The number average microclone size is well below the empirically observed number average size of genomic restriction fragments. A model is proposed that describes the distribution of microclone sizes by the use of two parameters: (1) the frequency of restriction-enzyme cleavage per bp and (2) the frequency of a second DNA event per bp. The model assumes that any DNA fragment subject to this second event is unclonable. The model shows good fit to the observed microclone size distribution from various experiments when the frequency of the second event is of the order of 0.01. The nature of the second event is unknown but it likely represents a hydrolytic event on the DNA caused by acid fixation of metaphase chromosomes prior to microdissection and microcloning.

Characterisation of phosphate binding to mitochondrial and bacterial membrane-bound ATP synthase by studies of inhibition with 4-chloro-7-nitrobenzofurazan.

The effect of phosphate on the inhibition by 4-chloro-7-nitrobenzofurazan of the ATPase activity of the proton-translocating ATP synthase in heart submitochondrial particles was investigated. Binding of phosphate protected strongly against the inhibition. A dissociation constant of 0.2 mM was determined for the enzyme X Pi complex and shown to be independent of pH in the range 7.0-8.0. The protective effect of phosphate was mimicked by arsenate but not by sulphate or malonate. Similar results were obtained for the enzyme from Paracoccus denitrificans. 2,4-Dinitrophenol enhanced phosphate binding to the mitochondrial enzyme since the protective effect of phosphate was increased. The data are compatible with protection arising from binding of phosphate to a catalytic site.

Molecular phenotyping of the mouse ky mutant reveals UCP1 upregulation at the neuromuscular junctions of dystrophic soleus muscle.

The ky mutant mouse displays a muscular dystrophy that affects almost exclusively slow type muscles in which persistent muscle regeneration, neuromuscular junction instability and an absence of the hypertrophic response are prominent features. In order to gain insights into the pathogenesis of this muscular dystrophy we have undertaken RNA profiling of the extensor digitorum longus, a fast unaffected muscle, and the highly pathological soleus slow muscle, followed by further expression studies to validate the results. In dystrophic soleus, there is a coordinated change in the expression level of genes encoding energy transducing mitochondrial proteins and an increase in the expression of stretch response genes. Upregulation of uncoupling proteins 1 and 2 is a unique molecular signature of the ky muscular dystrophy and was further characterised at the protein level. Our results show a spatial and temporal association between disorganisation of acetylcholine receptor clusters and upregulation of uncoupling protein 1. There is also evidence of a breakdown of neuromuscular junction muscle-specific kinase-dependent signalling in adult mutant soleus. Sarcolemma-associated proteins implicated in muscular dystrophies revealed no differences on microarrays and were confirmed as normally distributed by immunofluorescence. Altogether, the data presented suggest that the ky muscular dystrophy develops by a distinctive pathogenic mechanism.

Is the transjugular intrahepatic portocaval shunt procedure beneficial for liver transplant recipients?

Recent reports document the efficacy of transjugular intrahepatic portocaval shunts (TIPS) for the prevention of portal hypertensive bleeding and have advocated its use as a bridge to liver transplantation. There are no reports, however, analyzing liver transplant results for patients with indwelling TIPS. We reviewed the records of all adult primary recipients with a history of portal hypertensive bleeding or unmanageable ascites transplanted since the TIPS procedure became available in our institution in July 1991. Seven of 20 recipients underwent TIPS before transplant. There were no significant differences between patients with or without TIPS in age, United Network for Organ Sharing status, Child-Pugh score, preoperative prothrombin time, operative time, operative blood product requirement, overall length of stay, and 6-month patient survival after transplant. We noted a trend toward less operative red cell (26.0 +/- 26.2 vs. 31.8 +/- 38.1 U, mean +/- SD) and autologous blood (4,762 +/- 3,335 vs. 13,355 [corrected] +/- 20,460 ml) transfusion and improved patient survival for those with a TIPS. Patients with a TIPS in place waited significantly longer for their transplant (282 +/- 113 vs. 149 +/- 113 days, P = 0.014). There were 2 technical complications related to the TIPS, 1 in a patient who died after rupture of the suprahepatic vena caval anastomosis where the device had traversed the caval/hepatic vein junction and weakened the tissues, and the other in a survivor in whom the device extended into the right atrium and was extracted during the transplant procedure. Three patients with TIPS in place died of sepsis while waiting for a donor organ. We conclude that while the TIPS offers benefits for the liver transplant recipient, placement of the device in small shrunken cirrhotic livers must be precise. Immediate benefits for the transplant candidate may be offset by increased waiting time and technical complications at the transplant operation.

Estrogen induces phosphorylation of cyclic AMP response element binding (pCREB) in primary hippocampal cells in a time-dependent manner.

Using hippocampal primary cell cultures at 14 days in vitro (div), we have investigated actions of 17-beta estradiol (E; 10 nM) on the phosphorylation of CREB and on signaling pathways that regulate CREB phosphorylation. After demonstrating that 14 div is optimal for these studies, we examined the time course of E induction of CREB phosphorylation (pCREB) at serine residue 133. The induction of pCREB occurs as early as 1 h following E treatment, presumably via a mechanism involving an E-stimulated signal transduction system, which is sustained for at least 24 h but inhibited by 48 h. The early activity may represent an initial signal required for events leading to phosphorylation of CREB while the sustained signal may lead to CREB-mediated gene expression for cell survival and synapse formation. Furthermore, we examined the pathways for E action preceding pCREB induction by blocking three major kinases (protein kinase; mitogen activated protein kinase, MAPK; and calcium-calmodulin kinase II, CaMKII) upstream of pCREB. We found that E stimulates each pathway at 24 h and that phosphorylation of CREB is dependent on both MAPK and CaMK activities, but less dependent on the Akt pathway. Because CREB has been linked to E induction of excitatory spine synapses, we used a spine marker, spinophilin, to establish E effects on spine formation. Spinophilin expression was up-regulated in response to E and this effect was blocked by an inhibitor of (CaMKII). These studies demonstrate the central role played by CaMKII pathway in the actions of E on both transcriptional regulation and structural reorganization in neurons.

Evaluation of gastrointestinal bleeding by red blood cells labeled in vivo with technetium-99m.

To determine the effectiveness of abdominal imaging with RBCs labeled in vivo with Tc-99m, for the detection of gastrointestinal (GI) bleeding, 28 control subjects and ten patients with suspected bleeding underwent scintigraphy at 0-24 hr after tracer injection. Colonic activity was noted in one of the controls within 3 hr of injection, and in five of ten controls at 24 hr, all of whom had initial gastric activity. Of the ten patients with suspected GI bleeding, eight had documented active bleeding; seven of these had positive scintigrams. Nasogastric (NG) suction markedly decreased the presence of initial gastric activity in the patients with active bleeding. With this blood-pool radiopharmaceutical, frequent imaging of the abdomen over 24 hr can be done to test active bleeding. Continuous NG suction is recommended to reduce accumulation of gastric activity. These results suggest that red blood cells labeled in vivo with Tc-99m provide a sensitive method of detecting active GI bleeding.

Intraoperative localization of small bowel bleeding sites with combined use of angiographic methods and methylene blue injection.

Superselective catheter placement with angiographic techniques and methylene blue injection at laparotomy through a prepositioned angiographic catheter have helped to localize small bowel bleeding lesions. The technique has been applied successfully in two patients with arteriovenous malformations and one patient with bleeding mucosal ulcerations of the small bowel.