Genetic Covariation between Serum {gamma}-Glutamyltransferase Activity and Cardiovascular Risk Factors

Background: Several studies have shown that variation in serum gamma-glutamyltransferase (GGT) in the population is associated with risk of death or development of cardiovascular disease, type 2 diabetes, stroke, or hypertension. This association is only partly explained by associations between GGT and recognized risk factors. Our aim was to estimate the relative importance of genetic and environmental sources of variation in GGT as well as genetic and environmental sources of covariation between GGT and other liver enzymes and markers of cardiovascular risk in adult twin pairs. Methods: We recruited 1134 men and 2241 women through the Australian Twin Registry. Data were collected through mailed questionnaires, telephone interviews, and by analysis of blood samples. Sources of variation in GGT, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) and of covariation between GGT and cardiovascular risk factors were assessed by maximum-likelihood model-fitting. Results: Serum GGT, ALT, and AST were affected by additive genetic and nonshared environmental factors, with heritabilities estimated at 0.52, 0.48, and 0.32, respectively. One-half of the genetic variance in GGT was shared with ALT, AST, or both. There were highly significant correlations between GGT and body mass index; serum lipids, lipoproteins, glucose, and insulin; and blood pressure. These correlations were more attributable to genes that affect both GGT and known cardiovascular risk factors than to environmental factors. Conclusions: Variation in serum enzymes that reflect liver function showed significant genetic effects, and there was evidence that both genetic and environmental factors that affect these enzymes can also affect cardiovascular risk. (C) 2002 American Association for Clinical Chemistry.

Linkage of Paget disease of bone to a novel region on human chromosome 18q23

Paget disease of bone (PDB) is characterized by increased osteoclast activity and localized abnormal bone remodeling. PDB has a significant genetic component, with evidence of linkage to chromosomes 6p21.3 (PDB1) and 18q21-22 (PDB2) in some pedigrees. There is evidence of genetic heterogeneity, with other pedigrees showing negative linkage to these regions. TNFRSF11A, a gene that is essential for osteoclast formation and that encodes receptor activator of nuclear factor-kappa B (RANK), has been mapped to the PDB2 region. TNFRSF11A mutations that segregate in pedigrees with either familial expansile osteolysis or familial PDB have been identified; however, linkage studies and mutation screening have excluded the involvement of RANK in the majority of patients with PDB. We have excluded linkage, both to PDB1 and to PDB2, in a large multigenerational pedigree with multiple family members affected by PDB. We have conducted a genomewide scan of this pedigree, followed by fine mapping and multipoint analysis in regions of interest. The peak two-point LOD scores from the genomewide scan were 2.75, at D7S507, and 1.76, at D18S70. Multipoint and haplotype analysis of markers flanking D7S507 did not support linkage to this region. Haplotype analysis of markers flanking D18S70 demonstrated a haplotype segregating with PDB in a large subpedigree. This subpedigree had a significantly lower age at diagnosis than the rest of the pedigree (51.2 +/- 8.5 vs. 64.2 +/- 9.7 years; P = .0012). Linkage analysis of this subpedigree demonstrated a peak two-point LOD score of 4.23, at marker D18S1390 (theta = 0), and a peak multipoint LOD score of 4.71, at marker D18S70. Our data are consistent with genetic heterogeneity within the pedigree and indicate that 18q23 harbors a novel susceptibility gene for PDB.

Metal-insulator transition and charge ordering in the extended Hubbard model at one-quarter filling

We study, with exact diagonalization, the zero temperature properties of the quarter-filled extended Hubbard model on a square lattice. We find that increasing the ratio of the intersite Coulomb repulsion, V, to the bandwidth drives the system from a metal to a charge ordered insulator. The evolution of the optical conductivity spectrum with increasing V is in agreement with the observed optical conductivity of several layered molecular crystals with the theta and beta crystal structures.

Growth of subtropical ECM fungi with different nitrogen sources using a new floating culture technique

Eight species of ectomycorrhizal (ECM) fungi in the genera Amanita. Gymnoboletus, Lactarius, and Russula were isolated from subtropical plant communities in eastem Australia. Two species were isolated from each of rainforest, Nothofagus forest, Eucalyptus forest, and Eucalyptus dominated wallum (heath) forest. These communities differ strongly in their soluble soil nitrogen (N) composition. The ability of the fungi to use inorganic (nitrate, ammonium) and organic (amide, peptide, protein) nitrogen sources was determined. As the fungi did not grow in liquid culture, a 'floating culture' technique was devised that allows hyphal growth on a screen floating on liquid medium. With some exceptions, fungal biomass production in floating culture closely reflected fungal growth on solid media assessed by total colony glucosamine content. Most isolates grown in floating culture had similar glucosamine concentrations on all N sources, with isolate specific concentrations ranging from 6 to 12 mug glucosamine g(-1) DW. However, Russula spp. had up to 1.7-fold higher glucosamine concentrations when growing with glutamine or ammonium compared to nitrate, glutathione or protein. Floating cultures supplied with 0.5, 1.5. 4.5, or 10 mm N mostly produced greatest biomass with 4.5 mM N. In vitro nitrate reductase activity (NRA) ranged from very low (0.03 mumol NO2- g(-1) fw h(-1)) in Russula sp. (wallum) to high (2.16 mumol NO2- g(-1) fw h(-1)) in Gymnoboletus sp. (rainforest) and mirrored the fungi's ability to use nitrate as a N source. All Russula spp. (wallum, Nothofagus and Eucalyptus forests), Lactarills sp, (rainforest) and.4manita sp. (wallum) utilized ammonium and glutamine but had little ability to use other N sources. In contrast,Amanita species (Nothofagus and Eucalyptus forests) grew on all N sources but produced most biomass with ammonium and glutamine. Only Gymnoboletus sp. (rainforest) showed similar growth with nitrate and ammonium as N sources. Fungal N source use was not associated with taxonomic groups, but is discussed in the context of soil N sources in the different habitats.

Apoptosis and cell proliferation in the development of gastric carcinomas: Associations with c-myc and p53 protein expression

Background and Aim: Patients with gastric carcinomas have a poor prognosis and low survival rates. The aim of the present paper was to characterize cellular and molecular properties to provide insight into aspects of tumor progression in early compared with advanced gastric cancers. Methods: One hundred and nine graded gastric carcinomas (early or advanced stage, undifferentiated or differentiated type) with paired non-cancer tissue were studied to define the correlation between apoptosis (morphology, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling), cell proliferation (Ki-67 expression, morphology) and expression and localization of two proteins frequently having altered expression in cancers, namely p53 and c-myc. Results: Overall, apoptosis was lower in early stage, differentiated and undifferentiated gastric carcinomas compared with advanced-stage cancers. Cell proliferation was comparatively high in all stages. There was a high level of p53 positivity in all stages. Only the early- and advanced-stage undifferentiated cancers that were p53 positive had a significantly higher level of apoptosis (P< 0.05). Cell proliferation was significantly greater (P < 0.05) only in the early undifferentiated cancers that had either c-myc or p53-positivity. Conclusions: The results indicate that low apoptosis and high cell proliferation combine to drive gastric cancer development. The molecular controls for high cell proliferation of the early stage undifferentiated gastric cancers involve overexpression of both p53 and c-myc. Overexpression of p53 may also control cancer development in that its expression is associated with higher levels of apoptosis in early and late-stage undifferentiated, cancers. (C) 2002 Blackwell Publishing Asia Pty Ltd.

Shear stress and sediment transport calculations for sheet flow under waves

A simple method is provided for calculating transport rates of not too fine (d(50) greater than or equal to 0.20 mm) sand under sheet flow conditions. The method consists of a Meyer-Peter-type transport formula operating on a time-varying Shields parameter, which accounts for both acceleration-asymmetry and boundary layer streaming. While velocity moment formulae, e.g.., = Constant x calibrated against U-tube measurements, fail spectacularly under some real waves (Ribberink, J.S., Dohmen-Janssen, C.M., Hanes, D.M., McLean, S.R., Vincent, C., 2000. Near-bed sand transport mechanisms under waves. Proc. 27th Int. Conf. Coastal Engineering, Sydney, ASCE, New York, pp. 3263-3276, Fig. 12), the new method predicts the real wave observations equally well. The reason that the velocity moment formulae fail under these waves is partly the presence of boundary layer streaming and partly the saw-tooth asymmetry, i.e., the front of the waves being steeper than the back. Waves with saw-tooth asymmetry may generate a net landward sediment transport even if = 0, because of the more abrupt acceleration under the steep front. More abrupt accelerations are associated with thinner boundary layers and greater pressure gradients for a given velocity magnitude. The two real wave effects are incorporated in a model of the form Q(s)(t) = Q(s)[theta(t)] rather than Q(S)(t) = Q(S)[u(infinity)(t)], i.e., by expressing the transport rate in terms of an instantaneous Shields parameter rather than in terms of the free stream velocity, and accounting for both streaming and accelerations in the 0(t) calculations. The instantaneous friction velocities u(*)(t) and subsequently theta(t) are calculated as follows. Firstly, a linear filter incorporating the grain roughness friction factor f(2.5) and a phase angle phi(tau) is applied to u(infinity)(t). This delivers u(*)(t) which is used to calculate an instantaneous grain roughness Shields parameter theta(2.5)(t). Secondly, a constant bed shear stress is added which corresponds to the streaming related bed shear stress -rho ($) over bar((u) over tilde(w) over tilde)(infinity) . The method can be applied to any u(infinity)(t) time series, but further experimental validation is recommended before application to conditions that differ strongly from the ones considered below. The method is not recommended for rippled beds or for sheet flow with typical prototype wave periods and d(50) < 0.20 turn. In such scenarios, time lags related to vertical sediment movement become important, and these are not considered by the present model. (C) 2002 Elsevier Science B.V. All rights reserved.

Kainic acid induces 14-3-3 zeta expression in distinct regions of rat brain

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reactionbased differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex I h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid. (C) 2002 Elsevier Science B.V. All rights reserved.

CDT, GGT, and AST as markers of alcohol use: The WHO/ISBRA Collaborative Project

Background: Estimates of the performance of carbohydrate deficient transferrin (CDT) and gamma glutamyltransferase (GGT) as markers of alcohol consumption have varied widely. Studies have differed in design and subject characteristics. The WHO/ISBRA Collaborative Study allows assessment and comparison of CDT, GGT, and aspartate aminotransferase (AST) as markers of drinking in a large, well-characterized, multicenter sample. Methods: A total of 1863 subjects were recruited from five countries (Australia, Brazil, Canada, Finland, and Japan). Recruitment was stratified by alcohol use, age, and sex. Demographic characteristics, alcohol consumption, and presence of ICD-10 dependence were recorded using an interview schedule based on the AUDADIS, CDT was assayed using CDTect(TM) and GGT and AST by standard methods. Statistical techniques included receiver operating characteristic (ROC) analysis. Multiple regression was used to measure the impact of factors other than alcohol on test performance. Results: CDT and GGT had comparable performance on ROC analysis, with AST performing slightly less well. CDT was a slightly but significantly better marker of high-risk consumption in men. All were more effective for detection of high-risk rather than intermediate-risk drinking. CDT and GGT levels were influenced by body mass index, sex, age, and smoking status. Conclusions: CDT was little better than GGT in detecting high- or intermediate-risk alcohol consumption in this large, multicenter, predominantly community-based sample. As the two tests are relatively independent of each other, their combination is likely to provide better performance than either test alone, Test interpretation should take account sex, age. and body mass index.

Effects of vitamin E deficiency on fatigue and muscle contractile properties

Radical-mediated oxidative damage of skeletal muscle membranes has been implicated in the fatigue process. Vitamin E (VE) is a major chain breaking antioxidant that has been shown to reduce contraction-mediated oxidative damage. We hypothesized that VE deficiency would adversely affect Muscle contractile function, resulting in a more rapid development of muscular fatigue during exercise. To test this postulate, rats were fed either a VE-deficient (EDEF) diet or a control (CON) diet containing VE. Following a 12-week feeding period, animals were anesthetized and mechanically ventilated. Muscle endurance (fatigue) and contractile properties were evaluated using an in situ preparation of the tibialis anterior (TA) muscle. Contractile properties of the TA muscle were determined before and after a fatigue protocol. The muscle fatigue protocol consisted of 60 min of repetitive contractions (250 ms trains at 15 Hz; duty cycle = I I %) of the TA muscle. Prior to the fatigue protocol, no significant differences existed in the force-frequency curves between EDEF and CON animals. At the completion of the fatigue protocol, muscular force production was significantly (P

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2 mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2 mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24 h, The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity, Conversely, pro-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action. (C) 2002 Elsevier Science Ltd. All rights reserved.

Secretory pathway of trypanosomatid parasites

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Tryanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles it? the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory), transport and protein glycosylation that may be exploited in developing new antiparasite drugs.

Localization of a novel melanoma susceptibility locus to 1p22

Over the past 20 years, the incidence of cutaneous malignant melanoma (CMM) has increased dramatically worldwide. A positive family history of the disease is among the most established risk factors for CMM; it is estimated that 10% of CMM cases result from an inherited predisposition. Although mutations in two genes, CDKN2A and CDK4, have been shown to confer an increased risk of CMM, they account for only 20%-25% of families with multiple cases of CMM. Therefore, to localize additional loci involved in melanoma susceptibility, we have performed a genomewide scan for linkage in 49 Australian pedigrees containing at least three CMM cases, in which CDKN2A and CDK4 involvement has been excluded. The highest two-point parametric LOD score (1.82; recombination fraction [theta] 0.2) was obtained at D1S2726, which maps to the short arm of chromosome 1 (1p22). A parametric LOD score of 4.65 (theta = 0) and a nonparametric LOD score of 4.19 were found at D1S2779 in nine families selected for early age at onset. Additional typing yielded seven adjacent markers with LOD scores 13 in this subset, with the highest parametric LOD score, 4.95 (theta = 0) ( nonparametric LOD score 5.37), at D1S2776. Analysis of 33 additional multiplex families with CMM from several continents provided further evidence for linkage to the 1p22 region, again strongest in families with the earliest mean age at diagnosis. A nonparametric ordered sequential analysis was used, based on the average age at diagnosis in each family. The highest LOD score, 6.43, was obtained at D1S2779 and occurred when the 15 families with the earliest ages at onset were included. These data provide significant evidence of a novel susceptibility gene for CMM located within chromosome band 1p22.

Genetics of melanoma predisposition

Predisposition to melanoma is genetically heterogeneous. Two high penetrance susceptibility genes, CDKN2A and CDK4, have so far been identified and mapping is ongoing to localize and identify others. With the advent of a catalogue of millions of potential DNA polymorphisms, attention is now also being focused on identification of genes that confer a more modest contribution to melanoma risk, such as those encoding proteins involved in pigmentation, DNA repair, cell growth and differentiation or detoxification of metabolites. One such pigmentation gene, MC1R, has not only been found to be a low penetrance melanoma gene but has also been shown to act as a genetic modifier of melanoma risk in individuals carrying CDKN2A mutations. Most recently, an environmental agent, ultraviolet radiation, has also been established as a modifier of melanoma risk in CDKN2A mutation carriers. Hence, melanoma is turning out to be an excellent paradigm for studying gene-gene and gene-environment interactions.

Platelet inhibitory effects of the nitric oxide donor drug MAHMA NONOate in vivo in rats

The platelet inhibitory effects of the nitric oxide (NO) donor drug MAHMA NONOate ((Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino] diazen-1-ium-1,2-diolate) were examined in anaesthetised rats and compared with those of S-nitrosoglutathione (GSNO; an S-nitrosothiol). Bolus administration of the aggregating agent ADP dose-dependently reduced the number of circulating free platelets. Intravenous infusions of MAHMA NONOate (3-30 nmol/kg/min) dose-dependently inhibited the effect of 0.3 mumol/kg ADP. MAHMA NONOate was approximately 10-fold more potent than GSNO. MAHMA NONOate (0.3-10 nmol/kg/min) also reduced systemic artery pressure and was again 10-fold more potent than GSNO. Thus MAHMA NONOate has both platelet inhibitory and vasodepressor effects in vivo. The dose ranges for these two effects overlapped, although blood pressure was affected at slightly lower doses. The platelet inhibitory effects compared favourably with those of GSNO, even though NONOates generate free radical NO which, in theory, could have been scavenged by haemoglobin. Therefore platelet inhibition may be a useful therapeutic property of NONOates. (C) 2003 Elsevier B.V. All rights reserved.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.