A linkage and cross-sectional study of hypertension and obesity using a poly(A) Alu-repeat polymorphism at the glucagon receptor gene locus (17q25)

1. Previous glucagon receptor gene (GCGR) studies have shown a Gly40Ser mutation to be more prevalent in essential hypertension and to affect glucagon binding affinity to its receptor. An Alu-repeat poly(A) polymorphism colocalized to GCGR was used in the present study to test for association and linkage in hypertension as well as association in obesity development. 2. Using a cross-sectional approach, 85 hypertensives and 95 normotensives were genotyped using polymerase chain reaction primers flanking the Alu-repeat. Both hypertensive and normotensive populations were subdivided into lean and obese categories based on body mass index (BMI) to determine involvement of this variant in obesity. For the linkage study, 89 Australian Caucasian hypertension affected sibships (174 sibpairs) were genotyped and the results were analysed using GENE-HUNTER, Mapmaker Sibs, ERPA and SPLINK (all freely available from http://linlkage.rockefeller. edu/soft/list.html). 3. Cross-sectional results for both hypertension and obesity were analysed using Chi-squared and Monte Carlo analyses. Results did not show an association of this variant with either hypertension (χ2 = 6.9, P = 0.14; Monte Carlo χ2 = 7.0, P = 0.11; n = 5000) or obesity (χ2 = 3.3, P = 0.35; Monte Carlo χ2 = 3.26, P = 0.34; n = 5000). In addition, results from the linkage study using hypertensive sib-pairs did not indicate linkage of the poly(A) repent with hypertension. Hence, results did not indicate a role far the Alu-repeat in either hypertension or obesity. However, as the heterozygosity of this poly(A) repeat is low (35%), a larger number of hypertensive sib-pairs may be required to draw definitive conclusions.

Sibpair studies implicate chromosome 18 in essential hypertension

Interest in chromosome 18 in essential hypertension comes from comparative mapping of rat blood pressure quantitative trait loci (QTL), familial orthostatic hypotensive syndrome studies, and essential hypertension pedigree linkage analyses indicating that a locus or loci on human chromosome 18 may play a role in hypertension development. To further investigate involvement of chromosome 18 in human essential hypertension, the present study utilized a linkage scan approach to genotype twelve microsatellite markers spanning human chromosome 18 in 177 Australian Caucasian hypertensive (HT) sibling pairs. Linkage analysis showed significant excess allele sharing of the D18S61 marker when analyzed with SPLINK (P=0.00012), ANALYZE (Sibpair) (P=0.0081), and also with MAPMAKER SIBS (P=0.0001). Similarly, the D18S59 marker also showed evidence for excess allele sharing when analyzed with SPLINK (P=0.016), ANALYZE (Sibpair) (P=0.0095), and with MAPMAKER SIBS (P = 0.014). The adenylate cyclase activating polypeptide 1 gene (ADCYAP1) is involved in vasodilation and has been co-localized to the D18S59 marker. Results testing a microsatellite marker in the 3′ untranslated region of ADCYAP1 in age and gender matched HT and normotensive (NT) individuals showed possible association with hypertension (P = 0.038; Monte Carlo P = 0.02), but not with obesity. The present study shows a chromosome 18 role in essential hypertension and indicates that the genomic region near the ADCYAP1 gene or perhaps the gene itself may be implicated. Further investigation is required to conclusively determine the extent to which ADCYAP1 polymorphisms are involved in essential hypertension. © 2003 Wiley-Liss, Inc.

Association of a RFLP for the insulin receptor gene, but not insulin, with essential hypertension

Insulin has cardiovascular actions and patients with essential hypertension display insulin resistance. A cross-sectional study of the R1 RFLP of the insulin receptor gene (INSR) was carried out in 67 hypertensive (HT) and 75 normotensive (NT) subjects whose parents had a similar blood pressure status at age ≥50. The frequency of the minor (+) allele was 0.31 in HTs and 0.44 in NTs, and the difference between observed alleles in all subjects in each group was significant (χ2 = 4.8, P<0.05). Allele frequencies of a BglI RFLP of the insulin gene, however, did not differ between the HT and NT groups. The data thus provide evidence in favour of an association of HT with a polymorphism at the INSR locus (19p 13.3-13.2), so implicating this locus, and possibly a genetic variant of the insulin receptor itself, in HT.

Non-linkage of insulin receptor locus with essential hypertension in an affected pedigree

A recent cross-sectional study has demonstrated a significant association of the R1 RsaI restriction fragment length polymorphism of the insulin receptor gene (INSR) with human essential hypertension. In the present study, an alternative approach, involving linkage analysis, was carried out using 8 hypertensive families with 5 or more affected members. Five of the families were found to be informative and in one of these pedigrees a conclusion of non-linkage of INSR and hypertension could be made on the basis of an obligate recombinant in one generation which yielded a Lod score of - ∞ at a recombination fraction (θ) of zero. In another family, the largest studied, a positive Lod score was obtained at θ = 0, but this was below the level required for a conclusion of linkage. Lod score at θ = 0 for a marker at the insulin locus in this family was negative. The present study has thus demonstrated one pedigree in which hypertension is not linked to the insulin receptor locus.

Molecular genetic analyses of RFLPs for PCR-amplified growth hormone gene, renal kallikrein gene and atrial natriuretic factor gene in essential hypertension

The present study examined polymorphisms of genes that might be involved in the onset of essential hypertension (HT). These included the (i) growth hormone gene (GH1), whose locus has recently been linked to elevated blood pressure (BP) in the stroke-prone SHR, although recent sib-pair analysis of a polymorphism near the human chorionic somatomammotropin gene (a member of the GH cluster) was unable to show linkage with HT; (ii) renal kallikrein gene (KLK1); and (iii) atrial natriuretic factor gene (ANF), where a primary defect in production or activity of kallikrein or ANF could cause NaCl retention and vasoconstriction. Association analyses were conducted to compare restriction fragment length polymorphisms (RFLPs) of each gene in 85 HT and 95 normotensive (NT) Caucasian subjects whose parents had a similar BP status at age ≥50 years. The frequency of the minor allele of (i) a RsaI RFLP in the promoter of GH1, amplified from leukocyte DNA by the polymerase chain reaction, was 0.15 in the HT group and 0.14 in the NT group (χ1=0.34, P=0.55); (ii) a TaqI RFLP for KLK1 was 0.035 in the HT group and 0.015 in the NT group (χ2=1.5, P=0.21); and (iii) a XhoI RFLP for ANF was 0.50 in HTs and 0.46 in NTs (χ2=0.20, P=0.65). Studies of HT pedigrees found one family in which the ANF locus and HT were not linked, owing to an obligate recombinant. The present data thus provide no evidence for involvement of the growth hormone, renal kallikrein, nor ANF gene in the causation of essential hypertension.

Association and linkage analyses of restriction fragment length polymorphisms for the human renin and antithrombin III genes in essential hypertension

Essential hypertension is a highly hereditable disorder in which genetic influences predominate over environmental factors. The molecular genetic profiles which predispose to essential hypertension are not known. In rats with genetic hypertension, there is some recent evidence pointing to linkage of renin gene alleles with blood pressure. The genes for renin and antithrombin III belong to a conserved synteny group which, in humans, spans the q21.3-32.3 region of chromosome I and, in rats, is linkage group X on chromosome 13. The present study examined the association of particular human renin gene (REN) and antithrombin III gene (AT3) polymorphisms with essential hypertension by comparing the frequency of specific alleles for each of these genes in 50 hypertensive offspring of hypertensive parents and 91 normotensive offspring of normotensive parents. In addition, linkage relationships were examined in hypertensive pedigrees with multiple affected individuals. Alleles of a REN HindIII restriction fragment length polymorphism (RFLP) were detected using a genomic clone, λHR5, to probe Southern blots of HindIII-cut leucocyte DNA, and those for an AT3 Pstl RFLP were detected by phATIII 113 complementary DNA probe. The frequencies of each REN allele in the hypertensive group were 0.76 and 0.24 compared with 0.74 and 0.26 in the normotensive group. For AT3, hypertensive allele frequencies were 0.49 and 0.51 compared with normotensive values of 0.54 and 0.46. These differences were not significant by χ2 analysis (P > 0.2). Linkage analysis of a family (data from 16 family members, 10 of whom were hypertensive), informative for both markers, without an age-of-onset correction, and assuming dominant inheritance of hypertension, complete penetrance and a disease frequency of 20%, did not indicate linkage of REN with hypertension, but gave a positive, although not significant, logarithm of the odds for linkage score of 0.784 at a recombination fraction of 0 for AT3 linkage to hypertension. In conclusion, the present study could find no evidence for an association of a REN HindIII RFLP with essential hypertension or for a linkage of the locus defined by this RFLP in a family segregating for hypertension. In the case of an AT3 Pstl RFLP, although association analysis was negative, linkage analysis suggested possible involvement (odds of 6:1 in favour) of a gene located near the 1q23 locus with hypertension in one informative family.

GEF-H1-RhoA signaling pathway mediates LPS-induced NF-κB transactivation and IL-8 synthesis in endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (Guanine-nucleotide exchange factor-H1)-RhoA signalling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signalling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.

Performance of a cognitive, but not visual, secondary task interacts with alcohol-induced balance impairment in novice and experienced motorcycle riders

The appropriateness of applying drink driving legislation to motorcycle riding has been questioned as there may be fundamental differences in the effects of alcohol on driving and motorcycling. It has been suggested that alcohol may redirect riders’ focus from higher-order cognitive skills such as cornering, judgement and hazard perception, to more physical skills such as maintaining balance. To test this hypothesis, the effects of low doses of alcohol on balance ability were investigated in a laboratory setting. The static balance of twenty experienced and twenty novice riders was measured while they performed either no secondary task, a visual (search) task, or a cognitive (arithmetic) task following the administration of alcohol (0%, 0.02%, and 0.05% BAC). Subjective ratings of intoxication and balance impairment increased in a dose-dependent manner in both novice and experienced motorcycle riders, while a BAC of 0.05%, but not 0.02%, was associated with impairments in static balance ability. This balance impairment was exacerbated when riders performed a cognitive, but not a visual, secondary task. Likewise, 0.05% BAC was associated with impairments in novice and experienced riders’ performance of a cognitive, but not a visual, secondary task, suggesting that interactive processes underlie balance and cognitive task performance. There were no observed differences between novice vs. experienced riders on static balance and secondary task performance, either alone or in combination. Implications for road safety and future ‘drink riding’ policy considerations are discussed.

Evaluating the dosimetric effect of treatment-induced changed in virally mediated head and neck cancer patients

Introduction Patients with virally mediated head and neck cancer (VMHNC) often present with advanced nodal disease that is highly radioresponsive as demonstrated by tumour and nodal regression during treatment. The resultant changes may impact on the planned dose distribution and so adversely affect the therapeutic ratio. The aim of this study was to evaluate the dosimetric effect of treatment-induced anatomical changes in VMHNC patients who had undergone a re-plan. Methods Thirteen patients with virally mediated oropharyngeal or nasopharyngeal cancer who presented for definitive radiotherapy between 2005 and 2010 and who had a re-plan generated were investigated. The dosimetric effect of anatomical changes, was quantified by comparing dose volume histograms (DVH) of primary and nodal gross target volumes and organs at risk (OAR), including spinal cord and parotid glands, from the original plan and a comparison plan. Results Eleven 3DCRT and 2 IMRT plans were evaluated. Dose to the spinal cord and brainstem increased by 4.1% and 2.6%, respectively. Mean dose to the parotid glands also increased by 3.5%. In contrast, the dose received by 98% of the primary and nodal gross tumour volumes decreased by 0.15% and 0.3%, respectively when comparing the initial treatment plan to the comparison plan. Conclusion In this study, treatment-induced anatomical changes had the greatest impact on OAR dose with negligible effect on the dose to nodal gross tumour volumes. In the era of intensity modulated radiotherapy (IMRT), accounting for treatment-induced anatomical changes is important as focus is placed on minimising the acute and long-term side effects of treatment.

Real - time prediction of rainfall induced instability of residual soil slopes associated with deep cracks

The early warning based on real-time prediction of rain-induced instability of natural residual slopes helps to minimise human casualties due to such slope failures. Slope instability prediction is complicated, as it is influenced by many factors, including soil properties, soil behaviour, slope geometry, and the location and size of deep cracks in the slope. These deep cracks can facilitate rainwater infiltration into the deep soil layers and reduce the unsaturated shear strength of residual soil. Subsequently, it can form a slip surface, triggering a landslide even in partially saturated soil slopes. Although past research has shown the effects of surface-cracks on soil stability, research examining the influence of deep-cracks on soil stability is very limited. This study aimed to develop methodologies for predicting the real-time rain-induced instability of natural residual soil slopes with deep cracks. The results can be used to warn against potential rain-induced slope failures. The literature review conducted on rain induced slope instability of unsaturated residual soil associated with soil crack, reveals that only limited studies have been done in the following areas related to this topic: - Methods for detecting deep cracks in residual soil slopes. - Practical application of unsaturated soil theory in slope stability analysis. - Mechanistic methods for real-time prediction of rain induced residual soil slope instability in critical slopes with deep cracks. Two natural residual soil slopes at Jombok Village, Ngantang City, Indonesia, which are located near a residential area, were investigated to obtain the parameters required for the stability analysis of the slope. A survey first identified all related field geometrical information including slope, roads, rivers, buildings, and boundaries of the slope. Second, the electrical resistivity tomography (ERT) method was used on the slope to identify the location and geometrical characteristics of deep cracks. The two ERT array models employed in this research are: Dipole-dipole and Azimuthal. Next, bore-hole tests were conducted at different locations in the slope to identify soil layers and to collect undisturbed soil samples for laboratory measurement of the soil parameters required for the stability analysis. At the same bore hole locations, Standard Penetration Test (SPT) was undertaken. Undisturbed soil samples taken from the bore-holes were tested in a laboratory to determine the variation of the following soil properties with the depth: - Classification and physical properties such as grain size distribution, atterberg limits, water content, dry density and specific gravity. - Saturated and unsaturated shear strength properties using direct shear apparatus. - Soil water characteristic curves (SWCC) using filter paper method. - Saturated hydraulic conductivity. The following three methods were used to detect and simulate the location and orientation of cracks in the investigated slope: (1) The electrical resistivity distribution of sub-soil obtained from ERT. (2) The profile of classification and physical properties of the soil, based on laboratory testing of soil samples collected from bore-holes and visual observations of the cracks on the slope surface. (3) The results of stress distribution obtained from 2D dynamic analysis of the slope using QUAKE/W software, together with the laboratory measured soil parameters and earthquake records of the area. It was assumed that the deep crack in the slope under investigation was generated by earthquakes. A good agreement was obtained when comparing the location and the orientation of the cracks detected by Method-1 and Method-2. However, the simulated cracks in Method-3 were not in good agreement with the output of Method-1 and Method-2. This may have been due to the material properties used and the assumptions made, for the analysis. From Method-1 and Method-2, it can be concluded that the ERT method can be used to detect the location and orientation of a crack in a soil slope, when the ERT is conducted in very dry or very wet soil conditions. In this study, the cracks detected by the ERT were used for stability analysis of the slope. The stability of the slope was determined using the factor of safety (FOS) of a critical slip surface obtained by SLOPE/W using the limit equilibrium method. Pore-water pressure values for the stability analysis were obtained by coupling the transient seepage analysis of the slope using finite element based software, called SEEP/W. A parametric study conducted on the stability of an investigated slope revealed that the existence of deep cracks and their location in the soil slope are critical for its stability. The following two steps are proposed to predict the rain-induced instability of a residual soil slope with cracks. (a) Step-1: The transient stability analysis of the slope is conducted from the date of the investigation (initial conditions are based on the investigation) to the preferred date (current date), using measured rainfall data. Then, the stability analyses are continued for the next 12 months using the predicted annual rainfall that will be based on the previous five years rainfall data for the area. (b) Step-2: The stability of the slope is calculated in real-time using real-time measured rainfall. In this calculation, rainfall is predicted for the next hour or 24 hours and the stability of the slope is calculated one hour or 24 hours in advance using real time rainfall data. If Step-1 analysis shows critical stability for the forthcoming year, it is recommended that Step-2 be used for more accurate warning against the future failure of the slope. In this research, the results of the application of the Step-1 on an investigated slope (Slope-1) showed that its stability was not approaching a critical value for year 2012 (until 31st December 2012) and therefore, the application of Step-2 was not necessary for the year 2012. A case study (Slope-2) was used to verify the applicability of the complete proposed predictive method. A landslide event at Slope-2 occurred on 31st October 2010. The transient seepage and stability analyses of the slope using data obtained from field tests such as Bore-hole, SPT, ERT and Laboratory tests, were conducted on 12th June 2010 following the Step-1 and found that the slope in critical condition on that current date. It was then showing that the application of the Step-2 could have predicted this failure by giving sufficient warning time.

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis- related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.

Synthesis and evaluation of resveratrol-based nitroxides as potential treatments for hypertension

The synthesis and evaluation of novel resveratrol-based nitroxides have been explored for the potential treatment of hypertension. New methodology for the direct aryl iodination of isoindoline and isoindoline nitroxide using periodic acid and potassium iodide in concentrated sulphuric acid was developed. Diiodinated tetramethyl and tetraethyl isoindolines and a tetramethyl isoindoline nitroxide were prepared in excellent yields (70 – 82%). A diiodinated tetraethyl isoindoline nitroxide was generated from the corresponding nitroxide in modest yield (37%) alongside iodinated nitrones. The mono-iodinated species were also generated in modest yields (34 – 48%). Incorporation of the nitroxide unit into the structure of resveratrol was achieved using palladium-catalysed Heck coupling. Use of the previously prepared iodo products 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloyl 18 and 5,6-diiodo-1,1,3,3-tetramethylisoindolin-2-yloyl 22 gave resveratrol nitroxides 12 and 13 in yields of 50% (optimized) and 1.6% respectively. Preliminary evaluation of the resveratrol analogue 12 as a treatment for hypertension was undertaken in the DOCA-salt rat model. A reduction in systolic blood pressure as well as alleviation of ventricular hypertrophy was observed. A larger study involving the DOCA salt rats is currently in progress.

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets × 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNF? protein expression, and IKKSer180/181 and p38MAPK Thr180/Tyr182 phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNF? and IKK Ser180/181. There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). AktSer473 and mTORSer2448 phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k Thr389 increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6Ser235/236 increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.