Carotid intima-media thickness, cardiovascular risk factors and albuminuria in a remote Australian Aboriginal community

Background: Rates of cardiovascular disease and renal disease in Australian Aboriginal communities are high, as is the prevalence of some 'traditional' cardiovascular (CV) risk factors, such as diabetes and cigarette smoking. Recent work has highlighted the importance of markers of inflammation, such as C-reactive protein (CRP), homocysteine and albuminuria as predictors of cardiovascular risk in urban westernised settings. It is not clear how these factors relate to outcome in the setting of these remote communities, but very high CRP concentrations have been shown in this and other Aboriginal communities. Methods and results: In a cross-sectional survey including 237 adults in a remote Aboriginal community in the Northern Territory of Australia, we measured carotid intima-media thickness (IMT), together with blood pressure, diabetes, lipid levels, smoking and albuminuria, CRP and fibrinogen, serum homocysteine concentration, and IgG titres for Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus. Median carotid IMT was 0.63 [interquartile range 0.54-0.71] mm. As a categorical outcome, the prevalence of the highest IMT quartile ('increased IMT', greater than or equal to0.72 mm) was compared with the lower three quartiles. Increased IMT was associated in univariate analyses with greater waist circumference, systolic BP, fibrinogen and serum albumin concentrations, urine albumin/creatinine ratio and older age as continuous variables. Associations of increased IMT with some continuous variables were not linear; univariate associations were seen with the highest quartile (versus all other quartiles) of CRP and homocysteine concentration and CMV IgG titre. In a multivariate model age, smoking, waist circumference and the highest quartile of CRP concentrations (greater than or equal to14 mg/l) remained significant predictors of IMT greater than or equal to0.72 mm. Conclusions: Measurement of carotid IMT was possible in this remote setting. Increased IMT (greater than or equal to0.72 mm) was associated with increased CRP concentrations over a range that suggests infection/inflammation may be important determinants of cardiovascular risk in this setting. The associations of IMT with markers of renal disease seen in univariate analyses were explained in this analysis by confounding due to the associations of urine ACR with other risk factors. (C) 2004 Published by Elsevier Ireland Ltd.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBIP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body. (C) 2004 Wiley-Liss, Inc.

The policy and ethical implications of genetic research on attention deficit hyperactivity disorder

Objective: To review the policy and ethical implications of recent research on the molecular genetics of attention deficit hyperactivity disorder (ADHD). Method: MEDLINE and psycINFO database searches were used to identify studies on the genetics of ADHD. The implications of replicated candidate genes are discussed. Results: The findings for most genes have been inconsistent but several studies have implicated the genes in the dopaminergic pathway in the aetiology of ADHD. Conclusions: The current evidence on the genetics of ADHD is insufficient to justify genetic screening tests but it will provide important clues as to the aetiology of ADHD. Genetic information on susceptibility to ADHD has the potential to be abused and to stigmatize individuals. Researchers and clinicians need to be mindful of these issues in interpreting and disseminating the results of genetic studies of ADHD.

Multicenter linkage study of schizophrenia loci on chromosome 22q

The hypothesis of the existence of one or more schizophrenia susceptibility loci on chromosome 22q is supported by reports of genetic linkage and association, meta-analyses of linkage, and the observation of elevated risk for psychosis in people with velocardiofacial syndrome, caused by 22q11 microdeletions. We tested this hypothesis by evaluating 10 microsatellite markers spanning 22q in a multicenter sample of 779 pedigrees. We also incorporated age at onset and sex into the analysis as covariates. No significant evidence for linkage to schizophrenia or for linkage associated with earlier age at onset, gender, or heterogeneity across sites was observed. We interpret these findings to mean that the population-wide effects of putative 22q schizophrenia susceptibility loci are too weak to detect with linkage analysis even in large samples.

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN > 44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

A rapid dietary assessment tool to determine intake of folate was developed and validated

Background and Objective: Estimates of dietary folate intake are currently of considerable interest, but no rapid tools are available to assess dietary intake of folate that are well suited to everyday health promotion activities, We developed and tested the reliability and validity of two prototypes of a rapid dietary assessment tool (a folate intake tool, FIT) to determine dietary intake of folate. Study Design and Setting: Five hundred and sixty eight men and women aged 33-93 years from Perth, Western Australia. Completed one of the two prototypes of the tool and gave a fasting blood sample for measurement of serum folate. A subset (n - 277) of participants completed the same tool on a second occasion 3-6 weeks later. Results: The Pearson correlations (r) between folate score from the tool and serum folate were moderately high for both prototypes (FIT-A r = 0.54-, FIT-B r = 0.49). The folate scores for the two prototypes were similar on repeat testing and correlated strongly (FIT-A r = 0.75; FIT-B r = 0.68). Conclusions: The rapid dietary assessment tool described here, FIT, provides a valid and reliable measurement of dietary intake of folate for both men and women. (c) 2005 Elsevier Inc. All rights reserved.

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide- linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design. Crown Copyright (c) 2005 Published by Elsevier B.V. All rights reserved.

The phasevarion: A genetic system controlling coordinated, random switching of expression of multiple genes

Several host-adapted bacterial pathogens contain methyltransferases associated with type III restriction-modification (R-M) systems that are subject to reversible, high-frequency on/off switching of expression (phase variation). To investigate the role of phase-variable expression of R-M systems, we made a mutant strain lacking the methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae and analyzed its phenotype. By microarray analysis, we identified a number of genes that were either up- or down-regulated in the mod mutant strain. This system reports the coordinated random switching of a set of genes in a bacterial pathogen and may represent a widely used mechanism.

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L(-/-) germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L(-/-) meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product fort-nation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation. (c) 2006 Elsevier Inc. All rights reserved.

Folate supplementation fails to affect vascular function and carotid artery intima media thickness in cyclosporin A-treated renal transplant recipients

Background: Cyclosporin A (CsA)-treated renal transplant recipients (RTR) exhibit relative hyperhomocystinemia and vascular dysfunction. Folate supplementation lowers homocysteine and has been shown to improve vascular function in healthy subjects and patients with coronary artery disease. The aim of this study was to assess the effects of 3 months of folate supplementation (5 mg/day) on vascular function and structure in RTR. Methods: A double-blind, placebo-controlled crossover study was conducted in 10 CsA-treated RTR. Vascular structure was measured as carotid artery intima media thickness (IMT) and function was assessed as changes in brachial artery diameter during reactive hyperemia (RE) and in response to glyceryl trinitrate (GTN). Function data were analyzed as absolute and percent change from baseline and area under the diameter/time curve. Blood samples were collected before and after supplementation and analyzed for total plasma homocysteine, folate, vitamin B-12 and asymmetric dimethyl arginine (ADMA) in addition to regular measures of hemoglobin, hematocrit, mean corpuscular volume (MCV) and serum creatinine. Results: Folate supplementation significantly increased plasma folate by 687% (p < 0.005) and decreased homocysteine by 37% (p < 0.05) with no changes (p > 0.05) in vitamin B 12 or ADMA. There were no significant (p > 0.05) changes in vascular structure or function during the placebo or the folate supplementation phases; IMT; placebo pre mean +/- SD, 0.52 +/- 0.12, post 0.50 +/- 0.11; folate pre 0.55 +/- 0.17, post 0.49 +/- 10.20 mm 5% change in brachial artery diameter (RH, placebo pre 10 +/- 8, post 6 +/- 5; folate pre 9 +/- 7, post 7 +/- 5; GTN, placebo pre 18 +/- 10, post 17 +/- 9, folate pre 16 +/- 9, post-supplementation 18 +/- 8). Conclusion: Three months of folate supplementation decreases plasma homocysteine but has no effect on endothelial function or carotid artery IMT in RTR.