Role of the Met(287)Thr polymorphism in the AS3MT gene on the metabolic arsenic profile

Chronic exposure to arsenic involves a biotransformation process leading to the excretion of methylated metabolites, such as monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), as well as the parental inorganic species (As(III) and As(V)). Inter-individual variations in arsenic biotransformation have been reported and polymorphisms affecting the genes involved in arsenic biotransformation have been considered as one of the plausible explanations for this variation. Coding and flanking regions of the human arsenic methyltransferase (AS3MT) gene have been analysed in 50 Chilean men exposed to arsenic. Nine polymorphisms were found, including one non-synonymous SNP at exon 9 (Met(287)Thr) with an allele frequency of 0.14. Other four changes occurred at potentially regulatory regions: a variable number of tandem repeats (VNTR) at the 5'-untranslated region (UTR5'), a G/C substitution at the promoter region, a GC/AT substitution inside the VNTR, and a G/A substitution at the 3'-untranslated region (UTR3'). The rest of polymorphisms were located in non-coding regions: a T/G substitution in intron 1, a CTC deletion in intron 2 and a TTT and ATT insertions in intron 5. In addition, the individual urinary arsenic profiles were analysed. Our results indicate that genetic polymorphisms in AS3MT contribute to inter-individual variation in arsenic biotransformation and, therefore, may contribute to inter-individual variations in risk of arsenic toxicity and arsenic carcinogenesis. Individuals with the Met(287)Thr polymorphism displayed increased arsenic methylation and might be at increased risk for toxic and genotoxic effects of arsenic exposure if, as the classical arsenic metabolic pathway indicates, methylation enhances toxicity.     

Asian specific low mutation frequencies of the M287T polymorphism in the human arsenic (+3 oxidation state) methyltransferase (AS3MT) gene

Groundwater pollution by arsenic is a serious worldwide problem, especially in Asian countries. Inter-individual variation in arsenic metabolism has been reported and recent studies demonstrate that 287T allele in human arsenic (+3 oxidation state) methyltransferase (AS3MT) increase the percentage of monomethylated arsenic in urine. The objectives of the present study was to evaluate the ethnic difference in M287T (T/C) polymorphism in AS3MT among Japanese (n=1074), Koreans (n=435), Chinese (n=154), Mongolians (n=246), Uygurs (n=56), Tibetans (n=180), Tamangs (n=53), Tamils (n=58), Sinhalese (n=54), Turks (n=243), Ovambos (n=185), Ghanaians (n=121), Xhosas (n=101), and other four populations from previous studies. Of 17 populations, Xhosas had the highest 287T frequency (0.233). Other African and Caucasian populations had similar287T frequencies above 0.100 with the exception of the Ghanaians (0.071). On the other hand, the Asian populations had relatively lower 287T allele frequencies ranging from 0.000 to 0.041 than the Africans and Caucasians. Our findings indicate that genetic susceptibility to arsenic toxicity in Asian is different from Africans and Caucasians.     

Thr164Ile polymorphism of the human beta2-adrenoceptor exhibits blunted desensitization of cardiac functional responses in vivo

In subjects heterozygous for Thr164Ile beta2-adrenoceptor (beta2AR) polymorphism, cardiac responses to beta2AR agonist stimulation are blunted. In this study, we investigated agonist-induced desensitization of Thr164Ile beta2ARs. For this purpose, we assessed in six subjects with heterozygous Thr164Ile beta2ARs and in 10 subjects with homozygous wild-type (WT) beta2ARs the effects of 2-wk oral treatment with 3 x 5 mg/day terbutaline on terbutaline infusion-induced increases in heart rate (HR) and contractility [measured as shortening of HR-corrected duration of electromechanical systole (QS2c)]. Compared with WT beta2AR subjects, Thr164Ile subjects exhibited a blunted terbutaline-induced maximum increase in HR (WT 32 +/- 4 beats/min, Thr164Ile 19 +/- 3 beats/min, P < 0.05) and contractility (WT -54 +/- 2 ms, Thr164Ile -37 +/- 6 ms, P < 0.05). Two-week oral terbutaline treatment desensitized cardiac beta2AR responses to terbutaline infusion (increase in HR: WT 10 +/- 2 beats/min, Thr164Ile 8 +/- 4 beats/min; increase in contractility: WT -22 +/- 5 ms Thr164Ile: -17 +/- 6 ms); however, the extent of desensitization was larger in WT than Thr164Ile beta2AR subjects. Thus, after 2-wk oral terbutaline treatment cardiac, beta2AR responses did not differ anymore between WT and Thr164Ile beta2AR subjects. We conclude that agonist-induced desensitization of cardiac beta2ARs is more pronounced in WT than Thr164Ile subjects. Thus cardiac Thr164Ile subjects appear to be somewhat protected against agonist-induced desensitization.     

Theoretical 3D model of histamine N-methyltransferase: insights into the effects of a genetic polymorphism on enzymatic activity and thermal stability

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou-Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105.     

Developmental Variation in Amylase Allozyme Activity Associated with Chromosome Inversions in DROSOPHILA PERSIMILIS

The amylase locus in Drosophila persimilis is polymorphic for allozymes, two of which show associations with naturally occurring chromosome 3 inversions. Amy1.09 occurs at high frequencies only in Whitney (WT), while the other common arrangements-Standard (ST), Klamath (KL) and Mendocino (MD)-are predominantly Amy 1.00. We have examined numerous strains, representing various electromorphs and inversions, for variation in cis-specific activity expression in both third-instar larvae and adults. Comparisons of these two life stages also allows the survey of developmental variation in amylase activities. The amount of activity variation exceeds electrophoretic variation at this locus. Moreover, this variation is largely nonrandom and reveals more genic divergence among inversions. The 1.00 allozyme of MD is more active than 1.00 KL in larvae and adults and shows a different developmental pattern. The activity of the 1.00 allozyme of KL is greater than 1.00 allozyme of ST in larvae and adults, but these two arrangements have similar developmental patterns. WT 1 with a 1.00 allele is dramatically different from the 1.00 allozymes of other arrangements in its developmental pattern. The 1.09 allozymes has high activity in WT and KL, but these arrangements differ in their developmental pattern of expression, WT being more active in adults. F2 segregational analyses are consistent with the variation being due to either structural enzyme variants or closely linked cis-acting regulatory elements. We argue that the suppression of recombination between arrangements has allowed the divergence in amylase activity among inversions.     

The Toll-Like Receptor 4 Polymorphism Asp299Gly but Not Thr399Ile Influences TLR4 Signaling and Function

The common, co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile, are associated with hyporesponsiveness to inhaled lipopolysaccharide (LPS) and increased susceptibility to Gram negative pathogens in humans. The purpose of this study was to identify the relative contributions of the Asp299Gly and the Thr399Ile variants in inhibiting the function of TLR4. 293/hMD2-CD14 cell line was transfected with lentiviral constructs containing human wild type (WT) TLR4-EGFP or TLR4-EGFP with Asp299Gly, Thr399Ile or Asp299Gly/Thr399Ile complementary DNA (cDNA). Multiple stable cell lines were established for each construct: three for WT TLR4, Asp299Gly, and Thr399Ile, and only two for Asp299Gly/Thr399Ile mutants and EGFP control. We did not observe a significant effect of polymorphisms on cell surface and intracellular TLR4 expression nor were there any significant differences in TLR4 and EGFP protein levels assessed by Western blotting and confocal microscopy among the multiple cell lines of each of the constructs. All cell lines had a dose-dependent responsiveness to LPS stimulation. However, compared to the WT TLR4, cells expressing TLR4 with Asp299Gly but not Thr399Ile polymorphism produced significantly less (P<0.05) IL-8 following LPS stimulation. Similarly, cells expressing TLR4 Asp299Gly but not Thr399Ile allele had significantly lower percentage of phosphorylated and total NF-κB P65 following LPS stimulation. While we could not do statistics on the Asp299Gly/Thr399Ile group, we observed a reduced responsiveness to LPS compared to WT TLR4. Taken together, we observed that the TLR4 Asp299Gly variant, but not the Thr399Ile variant, is responsible for impaired responsiveness of TLR4 to LPS and corresponding activation of NF-κB.     

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.     

Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches

The Thr29 residue in the hydrophobic core of goat alpha-lactalbumin (alpha-LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X-ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild-type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side-chain O-H group of Thr29 and the main-chain C=O group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main-chain C=O group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results.     

Residues in Human Arsenic (+3 Oxidation State) Methyltransferase Forming Potential Hydrogen Bond Network around S-adenosylmethionine

Residues Tyr59, Gly78, Ser79, Met103, Gln107, Ile136 and Glu137 in human arsenic (+3 oxidation state) methyltransferase (hAS3MT) were deduced to form a potential hydrogen bond network around S-adenosylmethionine (SAM) from the sequence alignment between Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) and hAS3MT. Herein, seven mutants Y59A, G78A, S79A, M103A, Q107A, I136A and E137A were obtained. Their catalytic activities and conformations were characterized and models were built. Y59A and G78A were completely inactive. Only 7.0%, 10.6% and 13.8% inorganic arsenic (iAs) was transformed to monomethylated arsenicals (MMA) when M103A, Q107A and I136A were used as the enzyme. The V_max (the maximal velocity of the reaction) values of M103A, Q107A, I136A and E137A were decreased to 8%, 22%, 15% and 50% of that of WT-hAS3MT, respectively. The K_M(SAM) (the Michaelis constant for SAM) values of mutants M103A, I136A and E137A were 15.7, 8.9 and 5.1 fold higher than that of WT-hAS3MT, respectively, indicating that their affinities for SAM were weakened. The altered microenvironment of SAM and the reduced capacity of binding arsenic deduced from K_M(As) (the Michaelis constant for iAs) value probably synergetically reduced the catalytic activity of Q107A. The catalytic activity of S79A was higher than that of WT despite of the higher K_M(SAM), suggesting that Ser79 did not impact the catalytic activity of hAS3MT. In short, residues Tyr59 and Gly78 significantly influenced the catalytic activity of hAS3MT as well as Met103, Ile136 and Glu137 because they were closely associated with SAM-binding, while residue Gln107 did not affect SAM-binding regardless of affecting the catalytic activity of hAS3MT. Modeling and our experimental results suggest that the adenine ring of SAM is sandwiched between Ile136 and Met103, the amide group of SAM is hydrogen bonded to Gly78 in hAS3MT and SAM is bonded to Tyr59 with van der Waals, cation-π and hydrogen bonding contacts.     

GSTO and AS3MT genetic polymorphisms and differences in urinary arsenic concentrations among residents in Bangladesh

We determined whether single nucleotide polymorphisms (SNPs) in the glutathione S-transferase omega (GSTO) and arsenic(III)methyltransferase (AS3MT) genes were associated with concentrations of urinary arsenic metabolites among 900 individuals without skin lesions in Bangladesh. Four SNPs were assessed in these genes. A pathway analysis evaluated the association between urinary arsenic metabolites and SNPs. GSTO1 rs4925 homozygous wild type was significantly associated with higher monomethylarsonic acid (MMA) and dimethylarsinic acid urinary concentrations, whereas wild-type AS3MT rs11191439 had significantly lower levels of As(III) and MMA. Genetic polymorphisms GSTO and As3MT modify arsenic metabolism as evidenced by altered urinary arsenic excretion.     

High-throughput screening-compatible assays of As(III) S-adenosylmethionine methyltransferase activity

Arsenic is a naturally existing toxin and carcinogen. As(III) S-adenosylmethionine methyltransferases (AS3MT in mammals and ArsM in microbes) methylate As(III) three times in consecutive steps and play a central role in arsenic metabolism from bacteria to humans. Current assays for arsenic methylation are slow, laborious, and expensive. Here we report the development of two in vitro assays for AS3MT activity that are rapid, sensitive, convenient, and relatively inexpensive and can be adapted for high-throughput assays. The first assay measures As(III) binding by the quenching of the protein fluorescence of a single-tryptophan derivative of an AS3MT ortholog. The second assay utilizes time-resolved fluorescence resonance energy transfer to directly measure the conversion of the AS3MT substrate, S-adenosylmethionine, to S-adenosylhomocysteine catalyzed by AS3MT. These two assays are complementary, one measuring substrate binding and the other catalysis, making them useful tools for functional studies and future development of drugs to prevent arsenic-related diseases.     

Threonine-74 Is a Key Site for the Activity of Clostridium perfringens Alpha-Toxin

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [⁶⁵Zn]²⁺ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.     

Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms in cancer: a meta-analysis

Toll-like receptor 4 (TLR4) is critical in the recognition of Gram-negative bacteria serving as a key immune system effector. Recently, a number of case-control studies were conducted to investigate the association between TLR4 gene polymorphism and cancer risk, especially Asp299Gly and Thr399Ile polymorphisms. However, published data were still conflicting. In this paper, we summarized 9463 cancer cases and 10,825 controls from 22 studies and attempted to assess the susceptibility of TLR4 gene polymorphism to cancers by a synthetical meta-analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the relationship. Our results suggested that Asp299Gly represented a risk factor on cancers in digestive system (G allele versus A allele, OR=1.64, 95% CI: 1.02-2.64; GA+GG versus AA, OR=1.64, 95% CI: 1.00-2.71) but tend to have a protective effect on prostate cancer (GG versus AA, OR=0.37, 95% CI: 0.14-0.98; GG versus GA+AA, OR=0.37, 95% CI: 0.14-0.98). Thr399Ile polymorphism was significantly associated with an elevated cancer risk in overall analysis (T allele versus C allele, OR=1.72, 95% CI: 1.27-2.33; TC versus CC, OR=1.63, 95% CI: 1.18-2.26; TT+TC versus CC, OR=1.70, 95% CI: 1.24-2.34) and especially in gastrointestinal subgroup (T allele versus C allele, OR=2.01, 95% CI: 1.40-2.89; TC versus CC, OR=1.86, 95% CI: 1.26-2.74; TT+TC versus CC, OR=1.97, 95% CI: 1.35-2.88). Further prospective researches with larger numbers of worldwide participants are warranted to draw comprehensive and true conclusions.     

Structural differences between wild-type and fish eye disease mutant of lecithin:cholesterol acyltransferase

Fluorescence spectroscopy has been used to investigate the conformational changes that occur upon binding of wild type (WT) and mutant (Thr123Ile) lecithin:cholesterol acyltransferase (LCAT) to the potential substrates (dioleoyl-phosphatidyl choline [DOPC] and high density lipoprotein [HDL]). For a detailed analysis of structural differences between WT and mutant LCAT, we performed decompositional analysis of a set of tryptophan fluorescence spectra, measured at increasing concentrations of external quenchers (acrylamide and KI). The data obtained show that Thr123Ile mutation in LCAT leads to a conformation that is likely to be more rigid (less mobile/flexible) than that of the WT protein with a redistribution of charged residues around exposed tryptophan fluorophores. We propose that the redistribution of charged residues in mutant LCAT may be a major factor responsible for the dramatically reduced activity of the enzyme with HDL and reconstituted high density lipoprotein (rHDL).     

Meta-analysis of the response of growing pigs to the isoleucine concentration in the diet

The efficiency of nitrogen utilization will be highest when the amino acid (AA) supply approaches the requirement of the animal. With the availability of different crystalline AA, it is theoretically possible to formulate low-protein diets for growing pigs in which seven AA are co-limiting for performance. In such a diet, the concentration of Lys, Met, Met + Cys, Thr, Trp and Val and a seventh AA would exactly match the requirement. To determine the extent to which low-protein diets can be used, it is important to have reliable information about the requirements for these AA. Isoleucine is often considered the seventh-limiting AA in diets for growing pigs; however, information about the Ile requirement is limited and sometimes conflicting. The purpose of this study was to carry out a meta-analysis of the available literature information to determine the Ile requirement in growing pigs. A total of 46 Ile dose–response experiments were identified that used at least four concentrations of Ile in the diet. Because of differences in experimental design, both the Ile concentration and the response criteria were standardized. In 13 dose–response experiments, there was no indication of a response to an increasing Ile concentration. For the other 33 experiments, a response to the increasing Ile concentration was observed and the Ile requirement estimates ranged from 53% to 114% of that of the National Research Council (1998). An Ile concentration below the requirement resulted in important reductions in both feed intake and growth. A 10% reduction in the Ile concentration (below the requirement) resulted in a 15% reduction in feed intake and a 21% reduction in daily gain. The use of blood products in the diet was the main factor determining whether a response to the Ile concentration was observed or not. Blood meal and blood cells are protein sources with a very low Ile concentration, but with high or very high concentrations of Leu, Val, Phe and His. Some of these AA compete with Ile for catabolic pathways or transport across the blood–brain barrier, thereby potentially increasing the requirement for Ile. In diets without blood products, the Ile requirement appears to be lower than the currently recommended requirement. On the basis of the outcome of this study, we recommend a Ile : Lys requirement ratio of at least 50% on a standardized ileal digestible basis.     

TLR4 and TLR9 polymorphism: Probable role in susceptibility among the population of Bihar for Indian visceral leishmaniasis

Genetic variations in the host TLRs genes play an important role in susceptibility and/or resistance to visceral leishmaniasis by altering the host-pathogen interaction. In this study, we investigated the association between polymorphisms of TLR4 (Asp299Gly, Thr399Ile) and TLR-9 (T-1237C), with susceptibility to visceral leishmaniasis. A bi-directional PCR amplification of specific alleles technique was used to characterize the distribution of TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) polymorphisms. A total of 60 samples were randomly selected from confirmed visceral leishmaniasis patients and 24 endemic healthy volunteers. The samples were genotyped and allele frequencies were determined. We observed that TLR4 Asp299Gly and Thr399Ile genotypes were more frequent in visceral leishmaniasis patients (10% and 15% respectively) compared to controls (4.2% and 8.3% respectively). However, the differences were not significant in TLR4 Asp299Gly and Thr399Ile alleles and genotypes. In the case of TLR9, we observed the frequency of T1237C genotype was higher in visceral leishmaniasis patients (43.3%) than in healthy controls (33.3%). Statistically significant differences were observed in TLR9 T1237C alleles and genotypes. We concluded that TLR9 T1237C, but not TLR4, gene polymorphisms can be regarded as contributors to visceral leishmaniasis susceptibility among the Indian population of Bihar state.     

Identification of structural determinants for G protein-independent activation of mitogen-activated protein kinases in the seventh transmembrane domain of the angiotensin II type 1 receptor

Although the intrareceptor mechanisms whereby the angiotensin II (AngII) type 1 receptor activates phospholipase C (PLC) have been extensively investigated, analogous studies of signaling through mitogen-activated protein kinases (MAPK) have been lacking. We investigated MAPK activation and traditional G(q)/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar(1),Ile(4),Ile(8)] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP(3)) production and thereby stabilizes an activated receptor state linked to G protein-independent MAPK signaling. Using receptor mutagenesis, we focused on the seventh transmembrane domain and identified three key residues-Tyr(292), Phe(293), and Thr(287). At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to G(q)/PLC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling. The mutant Y292F failed to exhibit AngII-induced IP(3) turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr(292) is a key epitope for activated states 1 and 3 but not required for activated state 2. Although the F293L mutant retained normal AngII responses, it also showed an IP(3) response to SII, indicating that Phe(293) may be involved in constraining the receptor to its inactive state. Mutations of Thr(287) abolished all SII-induced signaling without affecting any AngII responses. Thr(287) therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr(287)) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr(292) and Phe(293).     

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long-lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west-central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean H(E) = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (F(ST) = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (F(ST) = 0.018 and approximately 0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (F(ST) = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite F(ST) did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.     

Analysis of genotype polymorphism of tumor-related genes harbored in chromosome arm lp and 8p in hepatocellular carcinoma patients by cSNP chip

The majority of single nucleotide polymorphisms (SNPs) found in the coding region (cSNPs) are single base substitutions that may or may not lead to amino acid substitutions,most of which are related to diseases.Some cSNPs may prove useful for their potential links to functional cSNPs via linkage disequilibrium mapping.We have selected 48 cSNPs located in the coding regions of 25 genes to construct the cSNP chip.These genes are harbored in the high frequency loss regions of the chromosome 1p and 8p and related with apoptosis,cell cycles,signal transduction,oncogene,tumor suppressor genes and so on.All of the cSNPs can lead to amino acid substitutions except TP73 (rs1801174).The PCR products amplified from 31 hepatocellular carcinoma (HCC) specimens were labeled with Dig-dUTP and then hybridized with the cSNP chips.The results showed that there was no hybridization signal when there was more than one site of mutation in the amplification sequence,indicating that the cSNP chip had a high sensitivity.The statistic data of the SNP (MT,homozygous and HT,heterozygous) in the HCC patients with different phenotypes (HBV +/-,differentiation stage,family history positive or negative,tumor size) indicated that the number of MT was distinctly different between patients with positive HBV and negative HBV.The MT and HT numbers of all the 48 cSNPs were significantly different between low differentiation and high differentiation HCC patients.The numbers of MT and HT were not different between positived and negative family history groups and between tumor size＞3 cm and≤3 cm groups.The study results provided useful information for understanding the molecular mechanisms of HCC development.