Donald R Dunbar

Abstract: Type 2 diabetes ultimately results from pancreatic β-cell failure. Abnormally elevated intracellular regeneration of glucocorticoids by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in fat or liver may underlie pathophysiological aspects of the metabolic syndrome. Elevated 11β-HSD1 is also found in pancreatic islets of obese/diabetic rodents and is hypothesized to suppress insulin secretion and promote diabetes. To define the direct impact of elevated pancreatic β-cell 11β-HSD1 on insulin secretion, we generated β-cell-specific, 11β-HSD1-overexpressing (MIP-HSD1) mice on a strain background prone to β-cell failure. Unexpectedly, MIP-HSD1(tg/+) mice exhibited a reversal of high fat-induced β-cell failure through augmentation of the number and intrinsic function of small islets in association with induction of heat shock, protein kinase A, and extracellular signal-related kinase and p21 signaling pathways. 11β-HSD1(-/-) mice showed mild β-cell impairment that was offset by improved glucose tolerance. The benefit of higher β-cell 11β-HSD1 exhibited a threshold because homozygous MIP-HSD1(tg/tg) mice and diabetic Lep(db/db) mice with markedly elevated β-cell 11β-HSD1 levels had impaired basal β-cell function. Optimal elevation of β-cell 11β-HSD1 represents a novel biological mechanism supporting compensatory insulin hypersecretion rather than exacerbating metabolic disease. These findings have immediate significance for current therapeutic strategies for type 2 diabetes.

Abstract: Rodent models exhibit only the earliest features of human diabetic nephropathy, which limits our ability to investigate new therapies. Hypertension is a prerequisite for advanced diabetic nephropathy in humans, so its rarity in typical rodent models may partly explain their resistance to nephropathy. Here, we used the Cyp1a1mRen2 rat, in which the murine renin-2 gene is incorporated under the Cytochrome P4501a1 promoter. In this transgenic strain, administration of low-dose dietary indole-3-carbinol induces moderate hypertension. In the absence of hypertension, streptozotocin-induced diabetes resulted in a 14-fold increase in albuminuria but only mild changes in histology and gene expression despite 28 weeks of marked hyperglycemia. In the presence of induced hypertension, hyperglycemia resulted in a 500-fold increase in albuminuria, marked glomerulosclerosis and tubulointerstitial fibrosis, and induction of many of the same pathways that are upregulated in the tubulointerstitium in human diabetic nephropathy. In conclusion, although induction of diabetes alone in rodents has limited utility to model human diabetic nephropathy, renin-dependent hypertension and hyperglycemia synergize to recapitulate many of the clinical, histological, and gene expression changes observed in humans.

Abstract: Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor-target of rapamycin (InR-TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR-TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demonstrates that Poly and InR interact and that poly mutants show an overall decrease in InR-TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR-TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling.

Abstract: Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl(4)-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B(hi) F4/80(int) Ly-6C(lo) macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter-diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C(hi) monocytes, a common origin with profibrotic Ly-6C(hi) macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C(lo) subset, compared with Ly-6C(hi) macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.

Abstract: AimsA pooled analysis of 14 genome-wide association studies revealed 23 susceptibility loci for coronary artery disease (CAD), thereby providing the most comprehensive genetic blueprint of CAD susceptibility. Here, we evaluated whether these 23 loci also predispose to recurrent myocardial infarction (MI) or cardiac death following an acute coronary syndrome (ACS).Methods and resultsA total of 2099 ACS patients enrolled in the Global Registry of Acute Coronary Events (GRACE) UK-Belgian study were prospectively followed for a median of 5 years (1668 days). C-allele carriers of the rs579459 variant, which is located upstream of the ABO gene and correlates with blood group A, were independently associated with recurrent MI [multivariable-adjusted hazard ratio (HR) 2.25, CI = 1.37-3.71; P = 0.001] and with recurrent MI or cardiac death [multivariable-adjusted (HR) 1.80, CI = 1.09-2.95; P = 0.021] within 5 years after an index ACS. The association of rs579459 was replicated in 1250 Polish patients with 6 months follow-up after an index ACS [multivariable-adjusted (HR) 2.70, CI = 1.26-5.82; P = 0.011 for recurrent MI]. Addition of rs579459 to a prediction model of 17 clinical risk factors improved risk classification for recurrent MI or cardiac death at 6 months as calculated by the integrated discrimination improvement method (P = 0.037), but not by C-statistics (P = 0.096).ConclusionIn this observational study, rs579459 was independently associated with adverse cardiac outcome after ACS. A weak improvement in clinical risk prediction was also observed, suggesting that rs579459 should be further tested as a potentially relevant contributor to risk prediction models for adverse outcome following ACS.

Abstract: OBJECTIVE The study objective was to determine the key early mechanisms underlying the beneficial redistribution, function, and inflammatory profile of adipose tissue in 11β-hydroxysteroid dehydrogenase type 1 knockout (11β-HSD1(-/-)) mice fed a high-fat (HF) diet. RESEARCH DESIGN AND METHODS By focusing on the earliest divergence in visceral adiposity, subcutaneous and visceral fat depots from 11β-HSD1(-/-) and C57Bl/6 J control mice fed an HF diet for 4 weeks were used for comparative microarray analysis of gene expression, and differences were validated with real-time PCR. Key changes in metabolic signaling pathways were confirmed using Western blotting/immunoprecipitation, and fat cell size was compared with the respective chow-fed control groups. Altered adipose inflammatory cell content and function after 4 weeks (early) and 18 weeks (chronic) of HF feeding was investigated using fluorescence (and magnetic)-activated cell sorting analysis, immunohistochemistry, and in situ hybridization. RESULTS In subcutaneous fat, HF-fed 11β-HSD1(-/-) mice showed evidence of enhanced insulin and β-adrenergic signaling associated with accretion of smaller metabolically active adipocytes. In contrast, reduced 11β-HSD1(-/-) visceral fat accumulation was characterized by maintained AMP kinase activation, not insulin sensitization, and higher adipocyte interleukin-6 release. Intracellular glucocorticoid deficiency was unexpectedly associated with suppressed inflammatory signaling and lower adipocyte monocyte chemoattractant protein-1 secretion with strikingly reduced cytotoxic T-cell and macrophage infiltration, predominantly in visceral fat. CONCLUSIONS Our data define for the first time the novel and distinct depot-specific mechanisms driving healthier fat patterning and function as a result of reduced intra-adipose glucocorticoid levels.

Abstract: Obesity and metabolic syndrome results from a complex interaction between genetic and environmental factors. In addition to brain-regulated processes, recent genome wide association studies have indicated that genes highly expressed in adipose tissue affect the distribution and function of fat and thus contribute to obesity. Using a stratified transcriptome gene enrichment approach we attempted to identify adipose tissue-specific obesity genes in the unique polygenic Fat (F) mouse strain generated by selective breeding over 60 generations for divergent adiposity from a comparator Lean (L) strain.

Abstract: We investigated the effects on urinary steroid and electrolyte excretion and renal gene expression of chronic infusions of ACTH in the mouse. ACTH caused a sustained increase in corticosteroid excretion; aldosterone excretion was only transiently elevated. There was an increase in the excretion of deoxycorticosterone, a weak mineralocorticoid, to levels of physiological significance. Nevertheless, we observed neither antinatriuresis nor kaliuresis in ACTH-treated mice, and plasma renin activity was not suppressed. We identified no changes in expression of mineralocorticoid target genes. Water turnover was increased in chronic ACTH-treated mice, as were hematocrit and hypertonicity: volume contraction is consistent with high levels of glucocorticoid. ACTH-treated mice exhibited other signs of glucocorticoid excess, such as enhanced weight gain and involution of the thymus. We identified novel ACTH-induced changes in 1) genes involved in vitamin D (Cyp27b1, Cyp24a1, Gc) and calcium (Sgk, Calb1, Trpv5) metabolism associated with calciuria and phosphaturia; 2) genes that would be predicted to desensitize the kidney to glucocorticoid action (Nr3c1, Hsd11b1, Fkbp5); and 3) genes encoding transporters of enzyme systems associated with xenobiotic metabolism and oxidative stress. Although there is evidence that ACTH-induced hypertension is a function of physiological cross talk between glucocorticoids and mineralocorticoids, the present study suggests that the major changes in electrolyte and fluid homeostasis and renal function are attributable to glucocorticoids. The calcium and organic anion metabolism pathways that are affected by ACTH may explain some of the known adverse effects associated with glucocorticoid excess.

Abstract: 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is a short-chain dehydrogenase/reductase (SDR) responsible for inactivating cortisol and preventing its binding to the mineralocorticoid receptor (MR). Non-functional mutations in HSD11B2, the gene encoding 11betaHSD2, cause the hypertensive syndrome of Apparent Mineralocorticoid Excess (AME). Like other such Mendelian disorders, AME is rare but has nevertheless helped to illuminate principles fundamental to the regulation of blood pressure. Furthermore, polymorphisms in HSD11B2 have been associated with salt-sensitivity, a major risk factor for cardiovascular mortality. It is therefore highly likely that sequence variation in HSD11B2, having subtle functional ramifications, will affect blood pressure in the wider population. In this study, a three-dimensional homology model of 11betaHSD2 was created and used to hypothesize the functional consequences in terms of protein structure of published mutations in HSD11B2. This approach underscored the strong genotype-phenotype correlation of AME: severe forms of the disease, associated with little in vivo enzyme activity, arise from mutations occurring in invariant alignment positions. These were predicted to exert gross structural changes in the protein. In contrast, those mutations causing a mild clinical phenotype were in less conserved regions of the protein that were predicted to be relatively more tolerant to substitution. Finally, a number of pathogenic mutations are shown to be associated with regions predicted to participate in dimer formation, and in protein stabilization, which may therefore suggest molecular mechanisms of disease.

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Abstract: Aim To define the long-term outcome of patients presenting with acute coronary syndrome [ST-segment elevation myocardial infarction (STEMI), and non-STEMI and unstable angina acute coronary syndrome (ACS) without biomarker elevation] and to test the hypothesis that the GRACE (Global Registry of Acute Coronary Events) risk score predicts mortality and death/MI at 5 years. Methods and results In the GRACE long-term study, UK and Belgian centres prospectively recruited and followed ACS patients for a median of 5 years (1797 days). Primary outcome events: deaths, cardiovascular deaths (CVDs) and MIs. Secondary events: stroke and re-hospitalization for ACS. There were 736 deaths, 19.8% (482 CVDs, 13%) and 347 (9.3%) MIs (>24 h), 261 strokes (7.7%), and 452 (17%) subsequent revascularizations. Rehospitalization was common: average 1.6 per patient; 31.2% had >1 admission, 9.2% had 5+ admissions. These events were despite high rates of guideline indicated therapies. The GRACE score was highly predictive of all-cause death, CVD, and CVD/MI at 5 years (death: chi(2) likelihood ratio 632; Wald 709.9, P< 0.0001, C-statistic 0.77; for CVD C-statistic 0.75, P< 0.0001; CVD/MI C-statistic 0.70, P< 0.0001). Compared with the low-risk GRACE stratum (ESC Guideline criteria), those with intermediate [hazard ratio (HR) 2.14, 95% CI 1.63, 2.81] and those with high-risk (HR 6.36, 95% CI 4.95, 8.16) had two- and six-fold higher risk of later death (Cox proportional hazard). A landmark analysis after 6 months confirmed that the GRACE score predicted long-term death (chi(2) likelihood ratio 265.4; Wald 289.5, P< 0.0001). Although in-hospital rates of death and MI are higher following STEMI, the cumulative rates of death (and CVD) were not different, by class of ACS, over the duration of follow-up (Wilcoxon = 1.5597, df = 1, P= 0.21). At 5 years after STEMI 269/1403 (19%) died; after non-STEMI 262/1170 (22%) after unstable angina (UA) 149/850 (17%). Two-thirds (68%) of STEMI deaths occurred after initial hospital discharge, but this was 86% for non-STEMI and 97% for UA. Conclusion The GRACE risk score predicts early and 5 year death and CVD/MI. Five year morbidity and mortality are as high in patients following non-ST MI and UA as seen following STEMI. Their morbidity burden is high (MI, stroke, readmissions) and the substantial late mortality in non-STE ACS is under-recognized. The findings highlight the importance of pursuing novel approaches to diminish long-term risk.

Abstract: Microarray analysis has become a core part of biomedical research and its value can be seen in thousands of research papers. A successful microarray experiment needs to be augmented by specialized data mining techniques if the data are to be fully exploited. Here, tools that concentrate on three areas - gene enrichment analysis, literature mining, and transcription factor binding site analysis - are described for the novice user of microarray technology. The focus of this chapter is on free, publicly available, web-based tools.

Abstract: Background Wiki technology has become a ubiquitous mechanism for dissemination of information, and places strong emphasis on collaboration. We aimed to leverage wiki technology to allow small groups of researchers to collaborate around a specific domain, for example a biological pathway. Automatically gathered seed data could be modified by the group and enriched with domain specific information. Results We describe a software system, BioKb, implemented as a plugin for the TWiki engine, and designed to facilitate construction of a field-specific wiki containing collaborative and automatically generated content. Features of this system include: query of publicly available resources such as KEGG, iHOP and MeSH, to generate 'seed' content for topics; simple definition of structure for topics of different types via an administration page; and interactive incorporation of relevant PubMed references. An exemplar is shown for the use of this system, in the the creation of the RAASWiki knowledgebase on the renin-angiotensin-aldosterone system (RAAS). RAASWiki has been seeded with data by use of BioKb, and will be the subject of ongoing development into an extensive knowledgebase on the RAAS. Conclusions The BioKb system is available from http://www.bioinf.mvm.ed.ac.uk/twiki/bin/view/TWiki/BioKbPlugin as a plugin for the TWiki engine.

Abstract: Severe forms of hypertension are characterized by high blood pressure combined with end-organ damage. Through the development and refinement of a transgenic rat model of malignant hypertension incorporating the mouse renin gene, we previously identified a quantitative trait locus on chromosome 10, which affects malignant hypertension severity and morbidity. We next generated an inducible malignant hypertensive model where the timing, severity and duration of hypertension was placed under the control of the researcher, allowing development of and recovery from end-organ damage to be investigated. We have now generated novel consomic Lewis and Fischer rat strains with inducible hypertension, and additional strains, which are reciprocally congenic for the refined chromosome 10 quantitative trait locus. We have captured a modifier of end-organ damage within the congenic region and, using a range of bioinformatic, biochemical and molecular biological techniques, have identified Angiotensin converting enzyme (Ace) as the modifier of hypertension-induced tissue microvascular injury. Reciprocal differences between Ace and the anti-inflammatory tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro in the kidney, a tissue susceptible to end-organ damage, suggests a mechanism for the amelioration of hypertension-dependent damage.

Abstract: Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-β1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-β1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-{alpha} and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-β1. These TGF-β1-producing DCs were poor at eliciting the activation of naive CD4+ T cells and sustaining their proliferation and differentiation into Th1 (IFN-{gamma}+) effectors. Instead, TGF-β1-producing DCs demonstrated an increased ability to generate CD4+CD25+Foxp3+ regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

Abstract: The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org.

Abstract: Understanding fundamental molecular mechanisms that govern the transmigration and interstitial migration of leukocytes to sites of tissue damage and infection is of potential significance in identifying novel therapeutic targets for the management of chronic inflammatory disorders. CD31 is a mammalian cell adhesion molecule that regulates the recruitment of leukocytes from the circulation. Our recent unpublished work has suggested that homophilic ligation of CD31 can negatively regulate the ether-à-go-go-related gene (ERG) current within leukocytes to regulate cell-cell adhesion. To validate and probe the functional significance of ERG in leukocytes, we developed an infected wound model of inflammation in zebrafish and assessed the efficacy of two ERG-specific inhibitors, dofetilide and E4031, as well as an ERG-specific antisense RNA morpholino on neutrophil recruitment. Our data confirm a hitherto undescribed role for ERG in leukocytes, where inhibition or translational knockdown of ERG resulted in significant attenuation of the inflammatory response to an infectious stimulus. Inhibition of ERG was verified independently by a decrease in the ventricular heart rate, where ERG also functions in the repolarization of the cardiac action potential. Our results suggest that ERG-specific Class III antiarrhythmic drugs can modulate inflammatory responses to infection.

Abstract: Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic loxP sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts.

Abstract: ABSTRACT: BACKGROUND: TGFbeta is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRB is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFbeta in disease. We asked how Rb-deficiency would affect responses to TGFbeta-induced cell cycle arrest. RESULTS: Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFbeta prevented cells to enter S phase via decreased cMYC activity, activation of P16 and P21 and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16 was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFbeta-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFbeta-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21 and P53 we investigated whether these proteins regulate pRB-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFbeta. Double deficiency had a similar impact showing that in cells containing functional pRB; P21 and P53 work through the same pathway to regulate G1/S in response to TGFbeta. In Rb-deficient cells however, p53 but not p21 deficiency had an additive effect highlighting a pRB-independent-P53-dependent effector pathway of inhibition of E2F activity. CONCLUSION: The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFbeta. As the function of these critical cellular proteins can be impaired by common causes of chronic liver disease and HCC, including viral hepatitis B and C proteins, we suggest that disruption of pRB function, and to a lesser extend P21Cip1 and P53 in hepatocytes may represent an additional new mechanism of escape from TGFbeta-growth-inhibition in the inflammatory milieu of chronic liver disease and contribute to cancer development.

Abstract: Salivary fluid, the collective product of numerous major and minor salivary glands, contains a range of secretory proteins that play key defensive, digestive, and gustatory roles in the oral cavity. To understand the distinct protein "signature" contributed by individual salivary glands to salivary secretions, we studied a family of proteins shown by in vitro mRNA translation to be abundantly expressed in mouse sublingual glands. Molecular cloning, Southern blotting, and restriction fragment length polymorphism analyses showed these to represent one known and two novel members of the common salivary protein (CSP-1)/Demilune cell and parotid protein (Dcpp) salivary protein family, the genes for which are closely linked in the T-complex region of mouse chromosome 17. Bioinformatic analysis identified a putative human CSP-1/Dcpp ortholog, HRPE773, expressed predominantly in human salivary tissue, that shows 31% amino acid identity and 45% amino acid similarity to the mouse Dcpp query sequence. The corresponding human gene displays a similar structure to the mouse Dcpp genes and is located on human chromosome 16 in a region known to be syntenic with the T-complex region of mouse chromosome 17. The predicted mouse and human proteins both display classical NH(2)-terminal signal sequences, putative jacalin-related lectin domains, and potential N-linked glycosylation sites, suggesting secretion via sublingual saliva into the oral cavity where they may display antimicrobial activity or provide a defensive coating to enamel. Identification of a human CSP-1/Dcpp ortholog therefore provides a key tool for investigation of salivary protein function in human oral health and disease.

Abstract: GPR50 is an orphan G protein-coupled receptor (GPCR) located on Xq28, a region previously implicated in multiple genetic studies of bipolar affective disorder (BPAD). Allele frequencies of three polymorphisms in GPR50 were compared in case-control studies between subjects with BPAD (264), major depressive disorder (MDD) (226), or schizophrenia (SCZ) (263) and ethnically matched controls (562). Significant associations were found between an insertion/deletion polymorphism in exon 2 and both BPAD (P=0.0070), and MDD (P=0.011) with increased risk associated with the deletion variant (GPR50(Delta502-505)). When the analysis was restricted to female subjects, the associations with BPAD and MDD increased in significance (P=0.00023 and P=0.0064, respectively). Two other single-nucleotide polymorphisms (SNPs) tested within this gene showed associations between: the female MDD group and an SNP in exon 2 (P=0.0096); and female SCZ and an intronic SNP (P=0.0014). No association was detected in males with either MDD, BPAD or SCZ. These results suggest that GPR50(Delta502-505), or a variant in tight linkage disequilibrium with this polymorphism, is a sex-specific risk factor for susceptibility to bipolar disorder, and that other variants in the gene may be sex-specific risk factors in the development of schizophrenia.

Abstract: Neurotransmitter transport systems are major targets for therapeutic alterations in synaptic function. We have cloned and sequenced a cDNA encoding the human type 2 glycine transporter GlyT2 from human brain and spinal cord. An open reading frame of 2391 nucleotides encodes a 797 amino acid protein that transports glycine in a Na+/Cl--dependent manner. When stably expressed in CHO cells, human GlyT2 displays a dose-dependent uptake of glycine with an apparent Km of 108 microM. This uptake is not affected by sarcosine at concentrations up to 1 mM. Radiation hybrid analysis mapped the GlyT2 gene to D11S1308 (LOD=8.988) on human chromosome 11p15.1-15.2.

Abstract: Mitochondrial mutations are associated with a wide spectrum of human diseases. A common class of point mutations affects tRNA genes, and mutations in the tRNA-leu(UUR) gene (MTTL1) are the most frequently detected. In earlier studies, we showed that lung carcinoma cybrid cells containing high levels (greater than 95%) of mutated mtDNA from a patient with the pathological nucleotide pair (np) 3243 tRNA-leu(UUR) mutation can remain genotypically stable over time, and exhibit severe defects in mitochondrial respiratory metabolism. From such a cybrid containing 99% mutated mtDNA, we have isolated a spontaneous derivative that retains mutant mtDNA at this level but which has nevertheless reverted to the wild-type phenotype, based on studies of respiration, growth in selective media, mitochondrial protein synthesis and biogenesis of mitochondrial membrane complexes. The cells are heteroplasmic for a novel anticodon mutation in tRNA-leu(CUN) at np 12300, predicted to generate a suppressor tRNA capable of decoding UUR leucine codons. The suppressor mutation represents approximately 10% of the total mtDNA, but was undetectable in a muscle biopsy sample taken from the original patient or in the parental cybrid. These results indicate that the primary biochemical defect in cells with high levels of np 3243 mutated mtDNA is the inability to translate UUR leucine codons.

Abstract: Using human and bovine short cDNA sequences as probes we screened human cosmid and P1 libraries for components of the complex I multi-subunit enzyme of oxidative phosphorylation. We isolated genomic recombinants encoding cI-B8 (gene NDUFA2), cI-B14 (gene NDUFA6), cI-B14.5a (gene NDUFA7), cI-ASHI (gene NDUFB8) and cI-23kD (gene NDUFS8). Genomic versions of these genes have not been previously cloned in the human although they are represented as anonymous entries in public cDNA databases. By using the derived genomic clones for in situ hybridisation studies we determined the following chromosome locations: NDUFA2, 5q31; NDUFA6, 21q22; NDUFA7, 20p13; NDUFB8, 12q21; NDUFS8, 3q28.

Abstract: We have studied the dynamics of mitochondrial DNA maintenance and segregation in human cells using serial cybrid transfer of partially duplicated mitochondrial DNA, from a mitochondrial myopathy patient, to two distinct recipient cell types. The results indicate two radically different outcomes dependent upon nuclear background. In one case (lung carcinoma) there is systematic loss of the partial duplication by an implied recombinational mechanism. In another nuclear background (osteosarcoma) the duplicated molecules can survive, having only a marginal effect on mitochondrial respiratory function. Moreover, in the osteosarcoma nuclear background further disturbances of mtDNA maintenance frequently follow from cybrid transfer. These are progressive, catastrophic loss of mtDNA and further rearrangement to generate partially triplicated molecules. The results imply differential expression of nuclear genes regulating mtDNA copy number, replication and recombination in different human cell types.

Abstract: 143B.206 rho degrees cells were repopulated with mitochondria from a MELAS patient harbouring a mixture of 3243G:C and 3243A:T mitochondrial DNA. A number of biochemical assays were performed on selected cybrids with various proportions of the two types of mitochondrial DNA. These assays revealed a marked decrease in oxygen consumption with pyruvate, a complex I substrate, in cybrids containing 60% to 90% 3243G:C mitochondrial DNA. Moreover, these cybrids showed decreased synthesis of a number of polypeptides in a mitochondrial in vitro translation assay. A cybrid line with a very high level of 3243G:C mitochondrial DNA (95%) had additional deficiencies in complexes III and IV and there was a marked generalised decrease in mitochondrial translation in this cybrid. The observation of complex I deficiency is consistent with previously reported enzymatic measurements of muscle homogenates from MELAS patients with the 3243G:C mutation.

Abstract: After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.

Abstract: Mitochondrial DNA from a 38 year old male with diabetes mellitus and features of mitochondrial dysfunction was analysed and shown to include a population with a partial duplication. The partially duplicated mitochondrial DNA molecules were evident in both muscle and blood. The region of mitochondrial DNA duplicated includes the origin of heavy strand replication, but not the light strand origin. This patient has features in common with other cases of partial direct tandem duplications and with a family which was reported to harbour a 10.4 kb mtDNA deletion. Initial restriction enzyme analysis of our case produced results consistent with a partial deletion of mitochondrial DNA. This leads us to propose that the rarity of reports of partial mitochondrial DNA duplications may stem in part from the classification of such mutants as partial deletions.

Abstract: Angiotensin (Ang) II plays an important role in the regulation of blood pressure, volume homeostasis, salt balance by triggering salt and water retention and vasoconstriction. The formation of Ang II results from the initial cleavage of angiotensinogen by renin. It has been established that Ang II is produced in tissues such as the vasculature, the brain or the heart [1,2]. Renin which is mainly produced in the kidney (as prorenin) is also found in tissues but it may come from de novo synthesis or uptake. von Lutterotti et al. however, concluded, that although controversial, renin synthesis does not occur in the heart [3]. Evidence of uptake of renin in the heart [4] is accumulating. The mechanisms of this uptake remain, however, unclear. Recently, a receptor specific to renin and prorenin was identified in human tissues [5]. Binding of this receptor to renin and prorenin resulted in an increased cleavage of angiotensinogen and the stimulation of a MAP kinase signaling pathway. We report here homology of the human renin receptor to a vacuolar proton-ATPase membrane sector-associated protein (M8-9) from a variety of species including mouse, chicken and C.elegans. In addition, we investigated the mouse homologue and found that at the cDNA and protein levels, the mouse and the human sequences are highly similar. The transcript was found (using RTPCR) in all adult tissues examined and in the kidney from E9.5dpc suggesting that the molecule is ubiquitously expressed in the mouse. Our findings indicate that the renin receptor and M8-9 may serve several functions and are encoded by the same gene.