Dominance of the α1B-Adrenergic Receptor and its Subcellular Localization in Human and TRAMP Prostate Cancer Cell Lines

The function and distribution of α₁-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the α₁-AR subtype and suggested that the α_{1 A}-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of α₁-ARs was determined by saturation binding and the distribution of the different α₁-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major α₁-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the α_1B-AR. DU145 cells contained 100% of the α_1B-AR subtype, whereas PC3 cells were composed of 21% α_{1 A}-AR and 79% α_1B-AR. TRAMP cell lines contained between 66% and 79% of the α_1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing α _1B-AR and increasing α_{1 A}- and α_1D-AR densities. Transfection with EGFP-tagged α_1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of α ₁-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that α_1B-ARs are the major α₁-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

Ross operation in children and young adults: the Alder Hey case series

Background

Plasma levels of tumor necrosis factor (TNF)-α and of C-reactive protein (CRP) are elevated in smokers. Previous studies failed to show an association between the G-308A polymorphism in the promoter region of the TNF-α gene and coronary artery disease (CAD). We investigated whether smoking would interact with the TNF-α G-308A polymorphism in determining plasma levels of TNF-α and CRP.

Methods

Study participants with a complete data set in terms of smoking and the TNF-α G-308A polymorphism were 300 middle-aged male and female industrial employees. After excluding 24 irregular smokers, analyses were performed on 198 "non-smokers" (life-long non-smokers or subjects who quit smoking >6 months ago) as compared to 78 "regular smokers" (subjects currently smoking >3 cigarettes/day). All subjects had a fasting morning blood draw to measure plasma levels of TNF-α and CRP by high-sensitive enzyme-linked immunosorbent assays.

Results

The cardiovascular risk factor adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a significant main effect for smoking status (F_1,250 = 5.67, p = .018) but not for genotype (F_1,250 = 0.33, p = .57). The interaction-term between genotype and smoking status was not significant (F_1,250 = 0.09, p = .76). The fully adjusted model with plasma TNF-α failed to show significant main effects for smoking and genotype, as well as for the smoking-status-genotype interaction.

Conclusions

The findings suggest that the TNF-α G-308A polymorphism does not mediate the effect of smoking on plasma CRP levels. It remains to be seen whether other genetic polymorphisms along the inflammatory pathway may modulate vascular risk in smokers.     

Clustering heart rate dynamics is associated with β-adrenergic receptor polymorphisms: analysis by information-based similarity index

Background

Genetic polymorphisms in the gene encoding the β-adrenergic receptors (β-AR) have a pivotal role in the functions of the autonomic nervous system. Using heart rate variability (HRV) as an indicator of autonomic function, we present a bottom-up genotype–phenotype analysis to investigate the association between β-AR gene polymorphisms and heart rate dynamics.

Methods

A total of 221 healthy Han Chinese adults (59 males and 162 females, aged 33.6±10.8 years, range 19 to 63 years) were recruited and genotyped for three common β-AR polymorphisms: β₁-AR Ser49Gly, β₂-AR Arg16Gly and β₂-AR Gln27Glu. Each subject underwent two hours of electrocardiogram monitoring at rest. We applied an information-based similarity (IBS) index to measure the pairwise dissimilarity of heart rate dynamics among study subjects.

Results

With the aid of agglomerative hierarchical cluster analysis, we categorized subjects into major clusters, which were found to have significantly different distributions of β₂-AR Arg16Gly genotype. Furthermore, the non-randomness index, a nonlinear HRV measure derived from the IBS method, was significantly lower in Arg16 homozygotes than in Gly16 carriers. The non-randomness index was negatively correlated with parasympathetic-related HRV variables and positively correlated with those HRV indices reflecting a sympathovagal shift toward sympathetic activity.

Conclusions

We demonstrate a bottom-up categorization approach combining the IBS method and hierarchical cluster analysis to detect subgroups of subjects with HRV phenotypes associated with β-AR polymorphisms. Our results provide evidence that β₂-AR polymorphisms are significantly associated with the acceleration/deceleration pattern of heart rate oscillation, reflecting the underlying mode of autonomic nervous system control.     

Pro-inflammatory genetic profile and familiarity of acute myocardial infarction

Background

Acute myocardial infarction (AMI) is a multifactorial disease with a complex pathogenesis where lifestyle, individual genetic background and environmental risk factors are involved. Altered inflammatory responses are implicated in the pathogenesis of atherosclerosis and a premature AMI of parents is associated with an increased risk of the disease in their offspring (Offs). However, the genetic background of familiarity for AMI is still largely unknown. To understand which genes may predispose to increased risk of cardiovascular disease gene polymorphism of immune regulatory genes, and clinical events from the Offs of parents with an early AMI were investigated. Genetics data from Offs were compared with those obtained from healthy subjects and an independent cohort of patients with clinical sporadic AMI. Rates of clinical events during a 24?years follow up from Offs and from an independent Italian population survey were also evaluated.

Results

This study showed that a genetic signature consisting of the concomitant presence of the CC genotype of VEGF, the A allele of IL-10 and the A allele of IFN-?? was indeed present in the Offs population. In fact, the above genetic markers were more frequent in unaffected Offs (46.4%) and patients with sporadic AMI (31.8%) than in the CTR (17.3%) and the differences were highly statistically significant (Offs vs CTR: p?=?0.0001, OR?=?4.129; AMI vs CTR: p?=?0.0001, OR?=?2.224). During the 24-year follow-up, Offs with a positive familiarity in spite of a relatively young age showed an increased prevalence of diabetes, ischemic heart disease and stroke. These findings reinforce the notion that subjects with a familial history of AMI are at risk of an accelerated aging of cardiovascular system resulting in cardiovascular events.

Conclusion

Our data suggest that selected genes with immune regulatory functions are part of the complex genetic background contributing to familiarity for cardiovascular diseases. This inflammatory genetic profile, along with classical cardiovascular risk factors, may be used for better defining individual risk of AMI in unaffected subjects.     

A Deletion in the α2B‐Adrenergic Receptor Gene and Autonomic Nervous Function in Central Obesity

Objective: We investigated the impact of a three‐amino acid deletion (12Glu9) polymorphism in the α_2B‐adrenergic receptor gene on autonomic nervous function. The short form (Glu⁹/Glu⁹) of the polymorphism has previously been associated with a reduced basal metabolic rate in obese subjects. Because autonomic nervous function participates in the regulation of energy metabolism, there could be a link between this polymorphism and autonomic nervous function. Research Methods and Procedures: Data of a 10‐year follow‐up study with 126 nondiabetic control subjects and 84 type 2 diabetic patients were used to determine the effects of the 12Glu9 polymorphism on autonomic nervous function. A deep breathing test and an orthostatic test were used to investigate parasympathetic and sympathetic autonomic nervous function. In addition, cardiovascular autonomic function was studied using power spectral analysis of heart rate variability. Results: No significant differences were found in the frequency of the 12Glu9 deletion polymorphism between nondiabetic and diabetic subjects. The nondiabetic men with the Glu⁹/Glu⁹ genotype, especially those with abdominal obesity, had significantly lower total and low‐frequency power values in the power spectral analysis when compared with other men. Furthermore, in a longitudinal analysis of 10 years, the decrease in parasympathetic function was greater in nondiabetic men with the Glu⁹/Glu⁹ genotype than in the men with the Glu⁹/Glu¹² or Glu¹²/Glu¹² genotypes. Discussion: The results of the present study suggest that the 12Glu9 polymorphism of the α_2B‐adrenergic receptor gene modulates autonomic nervous function in Finnish nondiabetic men. In the nondiabetic men with the Glu⁹/Glu⁹ genotype, the general autonomic tone is depressed, and vagal activity especially becomes impaired with time. Furthermore, this association is accentuated by central obesity.     

Intracellularly Truncated Human α2B-Adrenoceptors: Stable and Functional GPCRs for Structural Studies

All three α₂-adrenoceptor subtypes have a long third intracellular loop (3i), which is conserved by overall size and charge-hydrophobic properties but not by amino acid sequence similarity. These properties must be relevant for function and structure, because they have been preserved during hundreds of millions of years of evolution. The contribution of different loop portions to agonist/antagonist binding properties and G protein coupling of the human α_2B-adrenoceptor (α_2B-AR) was investigated with a series of 3i truncated constructs (Δ 3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated α_2B-AR Δ3i with various agonists and measured [³⁵S]GTPγS binding in isolated cell membranes with or without antagonist inhibition. We also evaluated the ability of oligopeptides, analogous to the amino and carboxyl terminal parts of 3i, to promote G protein activation, monitored with the [³⁵S]GTPγS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for G_i/G_o protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some α_2B-AR agonists, indicating that agonist binding is dependent on the conformation of the 3i domain, possibly through the involvement of G protein interactions. The truncated receptor constructs may be more stable on purification and thus be useful for structural characterization of α_2B-AR.     

Human α1-adrenoceptor subtype selectivity of substituted homobivalent 4-aminoquinolines

A series of ring-substituted ethyl- and heptyl-linked 4-aminoquinoline dimers were synthesized and evaluated for their affinities at the 3 human α₁-adrenoceptor (α₁-AR) subtypes and the human serotonin 5-HT_1A-receptor (5-HT_1A-R). We find that the structure-specificity profiles are different for the two series at the α₁-AR subtypes, which suggests that homobivalent 4-aminoquinolines can be developed with α₁-AR subtype selectivity. The 8-methyl (8-Me) ethyl-linked analogue has the highest affinity for the α_1A-AR, 7 nM, and the greatest capacity for discriminating between α_1A-AR and α_1B-AR (6-fold), α_1D-AR (68-fold), and the 5-HT_1A-R (168-fold). α_1B-AR selectivity was observed with the 6-methyl (6-Me) derivative of the ethyl- and heptyl-linked 4-aminoquinoline dimers and the 7-methoxy (7-OMe) derivative of the heptyl-linked analogue. These substitutions result in 4- to 80-fold selectivity for α_1B-AR over α_1A-AR, α_1D-AR, and 5-HT_1A-R. In contrast, 4-aminoquinoline dimers with selectivity for α_1D-AR are more elusive, since none studied to date has greater affinity for the α_1D-AR over the other two α₁-ARs. The selectivity of the 8-Me ethyl-linked 4-aminoquinoline dimer for the α_1A-AR, and 6-Me ethyl-linked, and the 6-Me and 7-OMe heptyl-linked 4-aminoquinoline dimers for the α_1B-AR, makes them promising leads for drug development of α_1A-AR or α_1B-AR subtype selective ligands with reduced 5-HT_1A-R affinity.     

Functional Promoter -94 ins/del ATTG Polymorphism in NFKB1 Gene Is Associated with Bladder Cancer Risk in a Chinese Population

Background

A functional -94 insertion/deletion polymorphism (rs28362491) in the promoter of the NFKB1 gene was reported to influence NFKB1 expression and confer susceptibility to different types of cancer. This study aims to determine whether the polymorphism is associated with risk of bladder cancer.

Materials and methods

TaqMan assay was used to determine genotype among 609 cases and 640 controls in a Chinese population. Logistic regression was used to assess the association between the polymorphism and bladder cancer risk, and quantitative real-time polymerase chain reaction was used to determine NFKB1 mRNA expression.

Results

Compared with the ins/ins/ins/del genotypes, the del/del genotype was associated with a significantly increased risk of bladder cancer [adjusted odd ratio (OR)  = 1.92, 95% confidence interval (CI)  = 1.42–2.59]. The increased risk was more prominent among subjects over 65 years old (OR  = 2.37, 95% CI  = 1.52–3.70), male subjects (OR  = 1.97, 95% CI = 1.40–2.79) and subjects with self-reported family history of cancer (OR  = 3.59, 95% CI  = 1.19–10.9). Furthermore, the polymorphism was associated with a higher risk of developing non-muscle invasive bladder cancer (OR  = 2.07, 95% CI  = 1.51–2.85), grade 1 bladder cancer (OR  = 2.40, 95% CI  = 1.68–3.43), single tumor bladder cancer (OR  = 2.04, 95% CI  = 1.48–2.82) and smaller tumor size bladder cancer (OR  = 2.10, 95% CI  = 1.51–2.92). The expression of NFKB1 mRNA in bladder cancer tissues with homozygous insertion genotype was higher than that with deletion allele.

Conclusions

In conclusion, the -94 ins/del ATTG polymorphism in NFKB1 promoter may contribute to the etiology of bladder cancer in the Chinese population.     

Functional studies with membrane-bound and detergent-solubilized α2-adrenergic receptors expressed in Sf9 cells

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.We have expressed the three subtypes of α₂-adrenergic receptor (α₂-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α_2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K_d values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α₂-AR is active even in a lipid-free environment. The K_d values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α₂-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.     

Angiotensin-converting enzyme genotype and late respiratory complications of mustard gas exposure

Background

Exposure to mustard gas frequently results in long-term respiratory complications. However the factors which drive the development and progression of these complications remain unclear. The Renin Angiotensin System (RAS) has been implicated in lung inflammatory and fibrotic responses. Genetic variation within the gene coding for the Angiotensin Converting Enzyme (ACE), specifically the Insertion/Deletion polymorphism (I/D), is associated with variable levels of ACE and with the severity of several acute and chronic respiratory diseases. We hypothesized that the ACE genotype might influence the severity of late respiratory complications of mustard gas exposure.

Methods

208 Kurdish patients who had suffered high exposure to mustard gas, as defined by cutaneous lesions at initial assessment, in Sardasht, Iran on June 29 1987, underwent clinical examination, spirometric evaluation and ACE Insertion/Deletion genotyping in September 2005.

Results

ACE genotype was determined in 207 subjects. As a continuous variable, FEV₁ % predicted tended to be higher in association with the D allele 68.03 ± 20.5%, 69.4 ± 21.4% and 74.8 ± 20.1% for II, ID and DD genotypes respectively. Median FEV₁ % predicted was 73 and this was taken as a cut off between groups defined as having better or worse lung function. The ACE DD genotype was overrepresented in the better spirometry group (Chi² 4.9 p = 0.03). Increasing age at the time of exposure was associated with reduced FEV₁ %predicted (p = 0.001), whereas gender was not (p = 0.43).

Conclusion

The ACE D allele is associated with higher FEV₁ % predicted when assessed 18 years after high exposure to mustard gas.     

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α₁-adrenoceptor (α₁-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α_1A- and α_1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α_1A-AR, but not α_1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α_1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α_1D antagonist BMY7378. Following activation by phenylephrine, α_1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α_1D-AR subtype was not observed via CypHer5 technology. The three α₁-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α_1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.     

Association of a deletion of GSTT2B with an altered risk of oesophageal squamous cell carcinoma in a South African population: a case-control study

Background

Polymorphisms in the Glutathione S-transferase genes are associated with altered risks in many cancers, but their role in oesophageal cancer is unclear. Recently a 37-kb deletion polymorphism of GSTT2B that reduces expression of GSTT2 has been described. We evaluated the influence of the GSTT1 and GSTT2B deletion polymorphisms, and the GSTP1 Ile105Val polymorphism (rs1695) on susceptibility to oesophageal squamous cell carcinoma (OSCC) in the Black and Mixed Ancestry populations of South Africa.

Methods and Results

The GSTT1, GSTT2B and GSTP1 variants were genotyped in 562 OSCC cases and 907 controls, and tested for association with OSCC and for interaction with smoking and alcohol consumption. Linkage disequilibrium (LD) between the deletions at GSTT1 and GSTT2B was determined, and the haplotypes tested for association with OSCC. Neither the GSTT1 deletion nor the GSTP1 Ile105Val polymorphism was associated with OSCC risk in the Black or Mixed Ancestry populations. The GSTT2B deletion was not associated with OSCC risk in the Black population, but was associated with reduced risk of OSCC in the Mixed Ancestry population (OR = 0.71; 95% CI 0.57–0.90, p = 0.004). Case-only analysis showed no interaction between the GST polymorphisms and smoking or alcohol consumption. LD between the neighboring GSTT1 and GSTT2B deletions was low in both populations (r² _Black = 0.04; r² _MxA = 0.07), thus these deletions should be assessed independently for effects on disease risk.

Conclusions

Although there was no association between the GSTT1 deletion polymorphism or the GSTP1 Ile105Val polymorphism with OSCC, our results suggest that the presence of the recently described GSTT2B deletion may have a protective effect on the risk of OSCC in the Mixed Ancestry South African population. This is the first report of the contribution of the GSTT2B deletion to cancer risk.     

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake

While α₁-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α₁-AR stimulation protected against increased lactate dehydrogenase release or Annexin V⁺ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α₁-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α₁-AR stimulation increased ³H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α₁-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α₁-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α₁-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α_1A-AR but not α_1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α_1A-AR but not α_1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α₁-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α_1A-AR subtype.     

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires

Background

Plasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine.

Methods

Isolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp -1 block 2, and msp -2 block 3, to analyze both parasite population polymorphism and clone fluctuations.

Results

Twenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence.

Conclusion

P. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity.