Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1ß and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome.

Keutel syndrome (KS, MIM 245150) is an autosomal recessive disorder characterized by abnormal cartilage calcification, peripheral pulmonary stenosis and midfacial hypoplasia. A genome search using homozygosity mapping provided evidence of linkage to chromosome 12p12.3-13.1 (maximum multipoint lod score, 4.06). MGP was a candidate on the basis of its localization to this chromosomal region and the known function of its protein. MGP maps to chromosome 12p near D12S363. Human MGP is a 10-kD skeletal extracellular matrix (ECM) protein that consists of an 84-aa mature protein and a 19-aa transmembrane signal peptide. It is a member of the Gla protein family, which includes osteocalcin, another skeletal ECM protein, and a number of coagulation factors (factors II, VII, IX, X and proteins S and C). All members of this family have glutamic acid residues modified to gamma-carboxyglutamic acids (Gla) by a specific gamma-carboxylase using vitamin K as a cofactor. The modified glutamic acid residues of Gla proteins confer a high affinity for mineral ions such as calcium, phosphate and hydroxyapatite crystals, the mineral components of the skeletal ECM. The pattern and tissue distribution of Mgp expression in mice suggest a role for Mgp in regulating ECM calcification. Mglap-deficient mice (Mglap-/-) have been reported to have inappropriate calcification of cartilage. Mutational analysis of MGP in three unrelated probands identified three different mutations: c.69delG, IVS1-2A-->G and c.113T-->A. All three mutations predict a non-functional MGP. Our data indicate that mutations in MGP are responsible for KS and confirm its role in the regulation of extracellular matrix calcification.

Analytical electron microscopy of black carbon and microaggregated mineral matter in Amazonian dark Earth.

Black carbon (BC) is one of the most stable forms of soil organic matter. Its surface functional groups and structure have been well characterized by a range of analytical methods. However, little is known about the mechanisms of interactions between the BC particles and the surrounding mineral matter. In this paper a range of microscopy techniques, such as transmission electron microscopy and scanning transmission electron microscopy, were used to investigate the possible reactions of BC particles within microaggregates (<2 mm) found in Amazonian dark Earth. Attention is given to the interactions that occur at the interfacial regions between the organic and inorganic phases. Examination of Amazonian dark Earth showed that the carbon-rich phase detected within the BC particles has a significant calcium concentration and a high density of micropores was found at the BC-mineral interface. These observations provide evidence to support suggested mechanisms of interaction between these phases.

First genome-wide association study investigating blood pressure and renal traits in domestic cats.

Hypertension (HTN) and chronic kidney disease (CKD) are common in ageing cats. In humans, blood pressure (BP) and renal function are complex heritable traits. We performed the first feline genome-wide association study (GWAS) of quantitative traits systolic BP and creatinine and binary outcomes HTN and CKD, testing 1022 domestic cats with a discovery, replication and meta-analysis design. No variants reached experimental significance level in the discovery stage for any phenotype. Follow up of the top 9 variants for creatinine and 5 for systolic BP, one SNP reached experimental-wide significance for association with creatinine in the combined meta-analysis (chrD1.10258177; P = 1.34 × 10-6). Exploratory genetic risk score (GRS) analyses were performed. Within the discovery sample, GRS of top SNPs from the BP and creatinine GWAS show strong association with HTN and CKD but did not validate in independent replication samples. A GRS including SNPs corresponding to human CKD genes was not significant in an independent subset of cats. Gene-set enrichment and pathway-based analysis (GSEA) was performed for both quantitative phenotypes, with 30 enriched pathways with creatinine. Our results support the utility of GWASs and GSEA for genetic discovery of complex traits in cats, with the caveat of our findings requiring validation.

Genetics of hypertension.

In the past year, substantial progress has been made in both mapping and fine mapping the genes involved in blood pressure regulation. Genome scans have been carried out in humans and mice and these reveal many new potential chromosomal locations for blood pressure susceptibility loci. The chromosomal regions containing blood pressure genes for many of the inbred hypertensive rat models have been refined using new congenic strains. Further genetic studies support a role for antiotensinogen, aldosterone synthase and a region close to the epithelial sodium channel in blood pressure regulation. Finally, comprehensive single-nucleotide polymorphism analysis of cardiovascular genes has been undertaken using chip technology.

Phenotypic consequences of variation across the aldosterone synthase and 11-beta hydroxylase locus in a hypertensive cohort: data from the MRC BRIGHT Study.

BACKGROUND: Aldosterone is an important cardiovascular hormone; 15% of hypertensive subjects have alteration in aldosterone regulation, defined by a raised ratio of aldosterone to renin (ARR). Studies of the aldosterone synthase gene (CYP11B2) have focused on a single nucleotide polymorphism in the 5'promoter region (-344 C/T). In normotensive subjects, the T allele associates with raised levels of the 11-deoxysteroids, deoxycorticosterone and 11-deoxycortisol which are substrates for 11beta-hydroxylase, encoded by the adjacent and homologous gene, CYP11B1. We have speculated that this altered 11beta-hydroxylase efficiency leads to increased ACTH drive to the adrenal gland to maintain cortisol production and reported herein the association between the -344 C/T single nucleotide polymorphism (SNP) and adrenal steroid production in subjects with essential hypertension. METHODS: The CYP11B2-344 C/T polymorphism was genotyped and urinary excretion of adrenal steroid metabolites was measured (by GCMS) in 511 unrelated hypertensives from the Medical Research Council (MRC) British Genetics of Hypertension (BRIGHT) study. RESULTS: Thirty-five per cent of subjects were homozygous for the -344T allele whilst 16% were CC homozygotes. There was no difference in cortisol excretion rate between the two genotype groups but the index of adrenal 11beta-hydroxylation (ratio of tetrahydrodeoxycortisol/total cortisol) was significantly higher in the TT group (P < 0.005) than in the CC group. Excretion rates of the major urinary metabolite of aldosterone (tetrahydroaldosterone) correlated strongly with the ACTH-regulated steroids, cortisol (r = 0.437, P < 0.0001) and total androgen metabolites (r = 0.4, P < 0.0001) in TT but not CC subjects. CONCLUSIONS: Hypertensives homozygous for the -344 T allele of CYP11B2 demonstrate altered 11beta-hydroxylase efficiency (CYP11B1); this is consistent with the hypothesis of a genetically determined increase in adrenal ACTH drive in these subjects. The correlation between excretion of aldosterone and cortisol metabolites and suggests that, in TT subjects, ACTH exerts an important common regulatory influence on adrenal corticosteroid production in subjects with hypertension.

Imaging and analysis of 3-D structure using a dual beam FIB.

The application of focused ion beam instrumentation in the generation of three-dimensional microstructural data is described. The methodologies used to acquire and manipulate this data are explained, and the technique is illustrated by a number of examples from the material sciences. The limitations of this method, and practical pointers to the generation of meaningful data, are also discussed.

Uromodulin gene variants and their association with renal function and blood pressure in cats: a pilot study.

OBJECTIVES: In humans, genome-wide association studies have identified variants in the uromodulin gene (UMOD) associated with blood pressure and renal function. This study aimed to evaluate the association of single nucleotide polymorphisms at the UMOD locus with renal function and blood pressure in cats. METHODS: We retrospectively identified cats aged 14 years that had participated in a geriatric monitoring program, and from which stored DNA samples were available, from a computerised database. We then measured the association of specific single nucleotide polymorphisms in the feline UMOD gene with renal function and systolic blood pressure as continuous variables and, also, the dichotomous outcome of azotaemic chronic kidney disease and systemic hypertension. RESULTS: Eight intronic single nucleotide polymorphisms, one 1372 base pairs upstream from UMOD and two exonic single nucleotide polymorphisms were evaluated in 227 cats with renal and blood pressure data. An analysis of 188 cats found four single nucleotide polymorphisms to be significantly associated (P<0·01) with systolic blood pressure although all were in linkage disequilibrium. No significant associations were identified between single nucleotide polymorphisms and renal function or chronic kidney disease. CLINICAL SIGNIFICANCE: Results of this pilot study suggest that genetic variation in UMOD might influence blood pressure in cats, similar to findings in humans. Validation of these results is required.

Lowering N2O emissions from soils using eucalypt biochar: the importance of redox reactions.

Agricultural soils are the primary anthropogenic source of atmospheric nitrous oxide (N2O), contributing to global warming and depletion of stratospheric ozone. Biochar addition has shown potential to lower soil N2O emission, with the mechanisms remaining unclear. We incubated eucalypt biochar (550 °C)--0, 1 and 5% (w/w) in Ferralsol at 3 water regimes (12, 39 and 54% WFPS)--in a soil column, following gamma irradiation. After N2O was injected at the base of the soil column, in the 0% biochar control 100% of expected injected N2O was released into headspace, declining to 67% in the 5% amendment. In a 100% biochar column at 6% WFPS, only 16% of the expected N2O was observed. X-ray photoelectron spectroscopy identified changes in surface functional groups suggesting interactions between N2O and the biochar surfaces. We have shown increases in -O-C = N /pyridine pyrrole/NH3, suggesting reactions between N2O and the carbon (C) matrix upon exposure to N2O. With increasing rates of biochar application, higher pH adjusted redox potentials were observed at the lower water contents. Evidence suggests that biochar has taken part in redox reactions reducing N2O to dinitrogen (N2), in addition to adsorption of N2O.

A model for Batten disease protein CLN3: functional implications from homology and mutations.

In an attempt to understand the molecular nature of Batten disease, we have examined the amino acid sequence of the affected CLN3 gene product (The International Batten Disease Consortium (1995) Cell 82, 949-957) and the site-specific mutations which give rise to the biological defect. Homology searches and molecular modeling have led to the development of a model for the folding and disposition of the protein, possibly within a mitochondrial membrane. High homology with a yeast protein of unknown function suggests a strong evolutionary conservation of function. We speculate that a possible role for the protein may be in chaperoning the folding/unfolding or assembly/ disassembly of other proteins, specifically subunit c of the mitochondrial ATP synthase complex.

Delayed classic and protracted phenotypes of compound heterozygous juvenile neuronal ceroid lipofuscinosis.

OBJECTIVE: To correlate the phenotypes with the genotypes of 10 Finnish juvenile neuronal ceroid lipofuscinosis (JNCL; late-onset Batten disease) patients who all are compound heterozygotes for the major 1.02-kb deletion in the CLN3 gene. METHODS: The mutations on the non-1.02-kb deletion chromosomes were screened in 6 patients; in the other 4 patients the mutations were known (one affecting a splice site, two missense mutations, and one deletion of exons 10 through 13). Clinical features were examined, and MRI, MRS, somatosensory evoked magnetic field (SEF), and overnight polysomnography (PSG) studies were performed. RESULTS: A novel deletion of exons 10 through 13 was found in 6 patients belonging to three families. In the patients carrying the deletions of exons 10 through 13 the clinical course of the disease was fairly similar. Variation was greatest in the time course to blindness. In these patients the mental and motor decline was slower than in classic JNCL, but more severe than in the two patients with missense mutations in exons 11 and 13. MRI showed brain atrophy in 4 patients. One patient had hyperintense periventricular white matter, otherwise brain signal intensities were normal. SEFs were enhanced in patients older than 14 years, whereas in PSG all but the youngest 6-year-old patient showed epileptiform activity in slow-wave sleep. CONCLUSIONS: JNCL can manifest as at least three different phenotypes: classic, delayed classic, and protracted JNCL with predominantly ocular symptoms. Finnish compound heterozygotes have the delayed classic or the protracted form of JNCL.

Genes and hypertension.

The combination of investigation of rare Mendelian forms of hypertension, candidate gene studies, comparative mapping and genome-wide screening in both animal models and man has led to significant progress in determining new mechanisms of blood pressure control. In this review, the newly discovered blood pressure/cardiovascular genes, WNK kinases and angiotensin converting enzyme 2 and the development of a new anti-hypertensive agent PST2238 are discussed. Major genes causing essential hypertension have yet to be discovered, however, there are now over 20 published genome-wide screens for blood pressure controlling genes. Several regions demonstrate suggestive linkage to the trait and there is some overlap of regions between the different studies. It is hoped that new blood pressure genes will ultimately be discovered using this method. Pharmacogenetic studies in hypertension have only been initiated recently, some are described in this paper. Small studies upon single candidate genes, suggest that the contribution of genetics to the inter-individual variation in blood pressure response to anti-hypertensive therapy, is small, approximately 3-5%. Recently micro-arrays with multiple polymorphisms in multiple genes have been used. After accounting for the additive affects of multiple blood pressure loci, an individual's genetic profile appeared to explain up to 50% of the variation in blood pressure response to therapy. Knowledge of the genetic variants that cause hypertension and influence response to anti-hypertensive therapy will ultimately provide a greater understanding of the molecular mechanisms underlying blood pressure control.

Analysis of Batten disease candidate genes STP and STM.

We have sequenced a large proportion of the open reading frames (ORFs) of two phenol sulphotransferase gene transcripts (STP and STM) from three patients with Batten disease. This was done using reverse transcription and PCR amplification of total RNA followed by direct sequencing of the PCR products. No mutations or changes have been observed in either gene after sequencing 93% of the STP ORF and 72% of the STM ORF. Work is in progress to finish sequencing both genes which will allow the confirmation or exclusion of these phenol sulphotransferases having a role in the development of Batten disease.

Absence of linkage of the epithelial sodium channel to hypertension in black Caribbeans.

Hypertensives of African origin have low-renin, sodium-sensitive blood pressure and respond poorly to treatment with angiotensin converting enzyme inhibitors. The epithelial sodium channel may be important in the pathogenesis of essential hypertension in this population. This is supported by the identification of mutations within this channel, which lead to excess sodium reabsorption and hypertension in Liddle's syndrome. In this study we tested whether there was linkage of the genes encoding the three subunits of the epithelial sodium channel to essential hypertension in 63 affected sibling pairs of West African origin from St. Vincent and the Grenadines. We found no support for linkage of the epithelial sodium channel to essential hypertension in this population. However, further studies will be needed in larger populations of African ancestry to exclude a contribution of the genes encoding the epithelial sodium channel to hypertension.

Progress in determining the genes for hypertension, insulin resistance, and dyslipidemia.

For the first time, we have techniques available that may enable us to determine cause and effect in common cardiovascular diseases. The observations presented herein require cautious interpretation until replicated. There are currently several large programs under way that seek to establish substantial family resources for investigation of the genetic basis of hypertension. When interpreting the results of genome screens, it will be necessary to consider that we may link genetic loci for coexistent features such as dyslipidemia and insulin resistance to hypertension. Therefore, careful phenotypic data will be necessary to dissect out the causes of hypertension.

Microstructural analysis of a FeAl/quasicrystal-based composite prepared using a focused ion beam miller.

A composite consisting of a brittle multiphase matrix containing both an Al-based quasicrystalline phase (psi) and an ordered body centred cubic phase (beta) and a relatively ductile ordered body centred cubic intermetallic FeAl phase has been developed as an abrasive wear-resistant coating material. It is applied as a 500 µm thick layer onto stainless steel substrates through plasma spray processing. The microstructure of such materials can be readily examined by optical and scanning electron microscopy, but the inherent difficulty of preparing transmission electron microscope (TEM) samples has inhibited higher resolution studies. However, the relatively recent development of the focused ion beam (FIB) miller as a tool in materials science provides a method ideal for the preparation of TEM specimens of these materials. In this study a coating consisting of a mixture of an Al-Cu-Fe based quasicrystal and FeAl+Cr was deposited on to a 304 stainless steel substrate. TEM specimens were prepared using a FIB and subjected to detailed microstructural characterization. The structure consisted of elongated bands of a FeAl phase about 100 nm in width and several micrometres in length, which enclosed more equiaxed regions about 1 µm in diameter that consisted of fine mixtures of quasicrystal and two Al-Fe-Cu phases isostructurally related to FeAl.

Full-field ERG in patients with Batten/Spielmeyer-Vogt disease caused by mutations in the CLN3 gene.

PURPOSE: To investigate, using full-field ERG, the retinal function in patients with Batten/Spielmeyer-Vogt disease caused by mutations in the CLN(3) gene. METHODS: Batten disease status of five patients was confirmed by the presence of vacuolated lymphocytes in peripheral blood and the identification of mutations in the Batten disease gene (CLN(3)). Visual acuity, fundus appearance, and full-field ERG were examined in all patients (age 4-19 years). The examination was repeated in one patient after 16 months. RESULTS: Three unrelated patients were homozygous for the most common mutation in CLN(3), the 1.02 kb deletion; two patients (sisters) were heterozygous for the 1.02 kb deletion and an as yet unidentified mutation in the CLN(3) gene. Full-field ERG recordings in all five patients demonstrated no rod responses and only small remaining cone responses, which could be detected with 30 Hz-flicker stimulation. Re-examination of a six-year-old girl after 16 months revealed a fast progression of the retinal degeneration. CONCLUSION: Full-field ERG recordings in Batten disease patients, both homozygous and heterozygous for the 1.02 kb deletion in the CLN( 3) gene, confirm retinal degeneration to be severe, widespread, and with a rapid progression early in the disease course. The onset of visual failure may be delayed when compared to the classic disease course, particularly in patients who are not homozygous for the most common CLN(3) mutation, a 1.02 kb deletion. In that case, the disease progression in terms of other symptoms may also be further delayed.

Absence of genetic linkage between polymorphisms of the insulin receptor gene and essential hypertension.

This study evaluated whether hypertensive siblings had excess sharing of RsaI and SstI alleles of the insulin receptor gene compared with a random population. Thirty families consisting of 60 affected individuals with established hypertension were genotyped for the RsaI and SstI restriction fragment length polymorphisms and the resulting genotype data was analysed using the affected pedigree member method of linkage analysis. The hypertensive siblings were found to have increased sharing of INSR alleles; however, this linkage could not be confirmed using a maximum LOD score method. Thus, the results from this study do not support a role for the INSR gene in the genesis of essential hypertension in the population studied.

The atrial natriuretic peptide gene and essential hypertension in African-Caribbeans from St Vincent and the Grenadines.

Atrial natriuretic peptide (ANP) which alters sodium balance, blood volume and vascular tone represents an important candidate for investigating the genetic basis of essential hypertension (EH). Accordingly, we have studied Bgl1 and Xho1 restriction fragment length polymorphisms (RFLPs) of the ANP gene in 147 hypertensive, 141 normotensive and 67 population-based control subjects from a homogenous population of West African origin from St Vincent and the Grenadines. We found no association of either Bgl1 and Xho1 RFLPs with EH. This study suggests that the ANP locus may not exert a major gene effect on EH amongst the black people of St Vincent and the Grenadines.

Analysis of CLN3-protein interactions using the yeast two-hybrid system.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene. The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive. In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins. Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions. CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain. Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested. No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions. Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested. No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3. We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system.