Thr Substitution Research Articles

A transcription factor TaMYB5 modulates leaf rolling in wheat.

Leaf rolling is an important agronomic trait in wheat (Triticum aestivum L.). Moderate leaf rolling keeps leaves upright and maintains the relatively normal photosynthesis of plants under drought stress. However, the molecular mechanism of wheat leaf rolling remains unclear. Here, we identified a candidate gene TaMYB5-3A that regulates leaf rolling by using a genome-wide association study (GWAS) in a panel of 323 wheat accessions. Phenotype analysis indicated that the leaves of tamyb5 mutants were flatter than that of the wild type under drought condition. A nucleotide variation in the TaMYB5-3A coding region resulted in a substitution of Thr to Lys, which corresponds to two alleles SNP-3A-1 and SNP-3A-2. The leaf rolling index (LRI) of the SNP-3A-1 genotype was significantly lower than that of the SNP-3A-2 genotype. In addition, TaMYB5-3A alleles were associated with canopy temperature (CT) in multiple environments. The CT of the SNP-3A-1 genotype was lower than that of the SNP-3A-2 genotype. Gene expression analysis showed that TaMYB5-3A was mainly expressed in leaves and down-regulated by PEG and ABA treatment. TaMYB5 induces TaNRL1 gene expression through the direct binding to the AC cis-acting element of the promoter of the target gene, which was validated by EMSA (electrophoretic mobility shift assay). Our results revealed a crucial molecular mechanism in wheat leaf rolling and provided the theoretical basis and a gene resource for crop breeding.

Proteomic characterisation and phylogenetic derivation of ovine αS1-CN B and αS1-CN G genetic variants

A combination of chromatographic, immunoelectrophoretic and mass spectrometric methods was applied to healthy individual milk of ovine massese breed. Caseome of αS1-casein (αS1-CN) fraction was characterised in homozygous and heterozygous phenotypes for the novel B and G genetic variants. Using ovine αS1-CN C counterpart as reference, amino acid substitutions and phosphorylated components were determined. The replacement of p.Arg90>His and p.Ile194>Thr were found in αS1-CN B variant, while the single p.Ile194>Thr substitution occurred in αS1-CN G variant. The quali-quantitative composition of phosphorylated αS1-CN components in milk of animals bearing each variant was defined, highlighting possible genetic markers for assessment of dairy product quality. The heterogeneity of the electrophoretic profiles of the corresponding caseomes was due to the number of phosphorylated components and their net charge and pI values, and updated the ovine αS1-CN phylogenetic tree, suggesting αS1-CN G as the ancestral variant of αS1-CN B and other variants.

Probing the molecular basis for signal transduction through the Zinc-Activated Channel (ZAC)

The molecular basis for the signal transduction through the classical Cys-loop receptors (CLRs) has been delineated in great detail. The Zinc-Activated Channel (ZAC) constitutes a so far poorly elucidated fifth branch of the CLR superfamily, and in this study we explore the molecular mechanisms underlying ZAC signaling in Xenopus oocytes by two-electrode voltage clamp electrophysiology. In studies of chimeric receptors fusing either the extracellular domain (ECD) or the transmembrane/intracellular domain (TMD-ICD) of ZAC with the complementary domains of 5-HT3A serotonin or α1 glycine receptors, serotonin and Zn2+/H+ evoked robust concentration-dependent currents in 5-HT3A/ZAC- and ZAC/α1-Gly-expressing oocytes, respectively, suggesting that Zn2+ and protons activate ZAC predominantly through its ECD. The molecular basis for Zn2+-mediated ZAC signaling was probed further by introduction of mutations of His, Cys, Glu and Asp residues in this domain, but as none of the mutants tested displayed substantially impaired Zn2+ functionality compared to wild-type ZAC, the location of the putative Zn2+ binding site(s) in the ECD was not identified. Finally, the functional importance of Leu246 (Leu9′) in the transmembrane M2 α-helix of ZAC was investigated by Ala, Val, Ile and Thr substitutions. In concordance with findings for this highly conserved residue in classical CLRs, the ZACL9′X mutants exhibited left-shifted agonist concentration-response relationships, markedly higher degrees of spontaneous activity and slower desensitization kinetics compared to wild-type ZAC. In conclusion, while ZAC is an atypical CLR in terms of its (identified) agonists and channel characteristics, its signal transduction seems to undergo similar conformational transitions as those in the classical CLR.

Abstract MP240: Site-specific O-Glcnacylation Of Transient Receptor Potential Canonical Type 6 (trpc6) Channel Is Required For Normal Structure And Function

O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.

Effects of Serine or Threonine in the Active Site of Typical 2-Cys Prx on Hyperoxidation Susceptibility and on Chaperone Activity.

Typical 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous Cys-based peroxidases, which are stable as decamers in the reduced state, and may dissociate into dimers upon disulfide bond formation. A peroxidatic Cys (CP) takes part of a catalytic triad, together with a Thr/Ser and an Arg. Previously, we described that the presence of Ser (instead of Thr) in the active site stabilizes yeast 2-Cys Prx as decamers. Here, we compared the hyperoxidation susceptibilities of yeast 2-Cys Prx. Notably, 2-Cys Prx containing Ser (named here Ser-Prx) were more resistant to hyperoxidation than enzymes containing Thr (Thr-Prx). In silico analysis revealed that Thr-Prx are more frequent in all domains of life, while Ser-Prx are more abundant in bacteria. As yeast 2-Cys Prx, bacterial Ser-Prx are more stable as decamers than Thr-Prx. However, bacterial Ser-Prx were only slightly more resistant to hyperoxidation than Thr-Prx. Furthermore, in all cases, organic hydroperoxide inhibited more the peroxidase activities of 2-Cys Prx than hydrogen peroxide. Moreover, bacterial Ser-Prx displayed increased thermal resistance and chaperone activity, which may be related with its enhanced stability as decamers compared to Thr-Prx. Therefore, the single substitution of Thr by Ser in the catalytic triad results in profound biochemical and structural differences in 2-Cys Prx.

An ALA122 THR substitution in the AHAS/ALS gene confers imazamox-resistance in Aegilops cylindrica.

Wheat growers have limited herbicide options to manage Aegilops cylindrica Host (jointed goatgrass), with many relying on mesosulfuron or imazamox in Clearfield™ winter wheat. Both imazamox and mesosulfuron inhibit acetohydroxyacid synthase/acetolactate synthase (AHAS/ALS). In 2015, a suspected imazamox resistant biotype of Ae. cylindrica was found in eastern Washington. Imazamox and mesosulfuron were applied to the suspected resistant and susceptible Ae. cylindrica biotypes in increasing application rates to evaluate herbicide dose needed to cause 50% growth reduction (GR50 ). The imazamox resistant biotype had a GR50 of 308.5g ai ha-1 and was more than 5000 times more resistant to imazamox than a known susceptible biotype with a GR50 of 0.06 g ai ha-1 . The Ae. cylindrica resistant biotype was also resistant to mesosulfuron, with an GR50 of 46.82 g ai ha-1 , which was five times more than the susceptible GR50 of 8.6g ai ha-1 . Sequencing of the AHAS/ALS gene revealed an Ala122 Thr substitution in the herbicide binding region of the AHAS/ALS gene on the D genome of Ae. cylindrica. The resistance trait was inherited as a dominant trait, and the Ala122 Thr co-segregates with the resistance phenotype. An Ala122 Thr substitution in the AHAS/ALS gene on the D genome of Ae. cylindrica confers resistance to imazamox in Ae. cylindrica. © 2021 Society of Chemical Industry.

Mechanism of collagen folding propagation studied by Molecular Dynamics simulations.

Collagen forms a characteristic triple helical structure and plays a central role for stabilizing the extra-cellular matrix. After a C-terminal nucleus formation folding proceeds to form long triple-helical fibers. The molecular details of triple helix folding process is of central importance for an understanding of several human diseases associated with misfolded or unstable collagen fibrils. However, the folding propagation is too rapid to be studied by experimental high resolution techniques. We employed multiple Molecular Dynamics simulations starting from unfolded peptides with an already formed nucleus to successfully follow the folding propagation in atomic detail. The triple helix folding was found to propagate involving first two chains forming a short transient template. Secondly, three residues of the third chain fold on this template with an overall mean propagation of ~75 ns per unit. The formation of loops with multiples of the repeating unit was found as a characteristic misfolding event especially when starting from an unstable nucleus. Central Gly→Ala or Gly→Thr substitutions resulted in reduced stability and folding rates due to structural deformations interfering with folding propagation.

DGAT1 polymorphism in Egyptian Zaraibi goat breed and their association with milk yield and composition

BackgroundThe triacylglycerols in milk fat determine the physical and functional properties of dairy products rich in milk fat. Therefore, understanding the variability of genes related to fat synthesis is important for food production. We investigated the effect of diacylglycerol O-acyltransferase 1 (DGAT1) gene polymorphism on milk production parameters of the Zaraibi goat. Milk components were estimated by infrared spectroscopy. Moreover, Restriction Fragment Length Polymorphisms (RFLP) were used to detect genetic variants in DGAT1 genes. The amplified products were sequenced and aligned to the caprine reference sequence of this gene.ResultsTwo alleles (T and C) were identified in Zaraibi goats. The T allele resulted in one silent mutation while the C allele specified two-point mutations: one was located within a non-coding region (T703C) and the other (T713C) causing a Ile → Thr substitution in the deduced amino acid sequence. Moreover, the DGAT1 polymorphism significantly (p < 0.05) affected total solid content of milk, wherein harboring CC genotype had significantly higher amount of total solid than those with TC genotype. Milk contents and yields did not differ significantly between goats with CC and TC genotype.ConclusionThese results advance our understanding of the genetic architecture of Zaraibi milk composition and will help to improve the management and breeding program of the Egyptian dairy goat.Graphical

A novel wheat q’ allele identified by forward genetic in silico TILLING

The major wheat domestication allele Q (encoding an APETALA2 like transcription factor) is responsible for the free threshing and square-headed spikes of modern wheat. Wild type q and null q’ alleles cause a reversal to the speltoid phenotype. Q pleiotropically affects additional yield and quality traits so genetic variation in Q and its interaction partners remain important for crop improvement. Here we report the discovery of a novel q’ allele that contribute to the molecular characterization of Q. This induced mutation cause an Ala→Thr substitution at a highly conserved position in the first of two APETALA2 like domains of the Q protein. The mutation correlates with a speltoid phenotype as expected for a loss of Q function. Thus, it is demonstrated that the first APETALA2 like domain is essential for Q function. The phenotype was discovered in a mutant from an in silico TILLING population and the causative mutation could be identified in the existing sequence data. This report therefore provides an example of a forward genetic application of in silico TILLING.

Genotypic Characterization of katG, inhA, and ahpC in Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates in Shanghai, China

BackgroundMycobacterium tuberculosis is a pathogen that causes Tuberculosis and can invade various organs in infected patients. Its high morbidity and high mortality seriously threaten human health. In recent years, the continuous emergence of drug-resistant Tuberculosis bacteria has brought severe challenges to the prevention and control of Tuberculosis.ObjectivesThis study aimed to characterize the most frequent mutations of the katG, inhA, and ahpC genes in isoniazid (INH)-resistant M. tuberculosis clinical isolates in Shanghai Pulmonary Hospital, China, and investigate the relationship between gene mutations and the minimum inhibitory concentrations (MICs) of INH against M. tuberculosis.MethodsWe collected 92 INH-resistant and 30 INH-susceptible clinical isolates of M. tuberculosis. The drug resistance profiles of M. tuberculosis clinical isolates against common anti-tuberculosis drugs were determined and sequencing analysis was performed.ResultsOf 92 INH-resistant strains, mutations in the katG and inhA genes were observed in 64 (69.6%) isolates and five (5.4%) isolates, respectively, and only had one (1.1%) strain both katG and inhA mutations. Among them, 62 (67.4%) strains carried a single mutation at codon 315 of the katG gene and a new mutation site was found in the katG gene of two strains. We detected a single mutation site at codon 271 and three simultaneous mutation sites at codons 315, 431, and 439. Only one (3.3%) of the 30 isoniazid-sensitive strains had the katG mutation. The AhpC mutation was detected in no experimental strains. The KatG Ser 315 Thr (AGC315ACC) mutation occurred in 53 (68.8%) out of 77 strains with high MICs (≥ 1 g/mL) of isoniazid-resistant M. tuberculosis while five (33.3%) out of 15 strains with low MICs (less than 1 g/mL) had katG Ser 315 Thr (AGC315ACC) mutation.ConclusionsIsoniazid-resistant strains were dominated by Ser315 → Thr (AGC → ACC) substitution, which seems to be associated with multidrug resistance and high-level resistance to INH. Multisite mutations are related to multidrug-resistant M. tuberculosis and the discovery of new mutation sites provides a new basis for the detection of drug-resistant M. tuberculosis.

Evolution of metazoan oxygen-sensing involved a conserved divergence of VHL affinity for HIF1\u03b1 and HIF2\u03b1

Duplication of ancestral hypoxia-inducible factor (HIF)α coincided with the evolution of vertebrate species. Paralogs HIF1α and HIF2α are the most well-known factors for modulating the cellular transcriptional profile following hypoxia. However, how the processes of natural selection acted upon the coding region of these two genes to optimize the cellular response to hypoxia during evolution remains unclear. A key negative regulator of HIFα is von Hippel-Lindau (VHL) tumour suppressor protein. Here we show that evolutionarily-relevant substitutions can modulate a secondary contact between HIF1α Met561 and VHL Phe91. Notably, HIF1α binds more tightly than HIF2α to VHL due to a conserved Met to Thr substitution observed in the vertebrate lineage. Similarly, substitution of VHL Phe91 with Tyr, as seen in invertebrate species, decreases VHL affinity for both HIF1α and HIF2α. We propose that vertebrate evolution involved a more complex hypoxia response with fine-tuned divergence of VHL affinity for HIF1α and HIF2α.

Genetic Mutation Screening of DNA Polymerase in Human Lung Cancer

背景与目的DNA聚合酶β是参与DNA损伤修复的关键酶之一，国外有学者认为其编码基因Polb在30%的肿瘤中存在遗传突变，但受到所用标本量的限制，这一结论是否准确尚无定论。本研究基于基因测序技术，通过对69例肺癌患者组织标本的基因筛查，旨在明确DNA聚合酶β的基因突变在中国汉族人群肺癌患者中的发生频率。方法利用盐析法提取69例肺癌患者的癌及癌旁组织基因组DNA，并用于扩增Polb基因全部14个外显子区及启动子区。通过与NCBI数据库中野生型基因序列进行比对，系统分析肺癌组织中的Polb基因突变及其频率。结果相对于野生型，本研究共发现5种突变类型，其中3种（-196G > T, -188_-187insCGCCC, -168C > A）位于启动子区，2种（587C > G, 612A > T）位于外显子区，突变类型-188_-187insCGCCC和587C > G尚未见文献报道，后者可引起第196位氨基酸由苏氨酸突变为丝氨酸。另一方面，癌和癌旁组中均可检测到所有5种突变类型，提示这些突变并非肺癌组织所特有。结论肺癌组织中未发现特异性表达的Polb基因突变位点，Polb基因突变可能不是中国汉族肺癌患者的肿瘤标志物。

Moxifloxacin resistance and genotyping of Mycobacterium avium and Mycobacterium intracellulare isolates in Japan

BackgroundAlthough fluoroquinolones are considered as alternative therapies of pulmonary Mycobacterium avium complex (MAC) disease, the association between fluoroquinolone resistance and MAC genotypes in clinical isolates from individuals not previously treated for MAC infection is not fully clear. MethodsTotals of 154 M. avium isolates and 35 Mycobacterium intracellulare isolates were obtained from treatment-naïve patients with pulmonary MAC disease at the diagnosis of MAC infection at 8 hospitals in Japan. Their susceptibilities of moxifloxacin were determined by broth microdilution methods. Moxifloxacin-resistant isolates were examined for mutations of gyrA and gyrB. Variable numbers of tandem repeats (VNTR) assay was performed using 15 M. avium VNTR loci and 16 M. intracellulare VNTR loci. ResultsMoxifloxacin susceptibility was categorized as resistant and intermediate for 6.5% and 16.9%, respectively, of M. avium isolates and 8.6% and 17.1% of M. intracellulare isolates. Although the isolates of both species had amino acid substitutions of Thr 96 and Thr 522 at the sites corresponding to Ser 95 in the M. tuberculosis GyrA and Gly 520 in the M. tuberculosis GyrB, respectively, these substitutions were observed irrespective of susceptibility and did not confer resistance. The VNTR assays showed revealed three clusters among M. avium isolates and two clusters among M. intracellulare isolates. No significant differences in moxifloxacin resistance were observed among these clusters. ConclusionsAlthough resistance or intermediate resistance to moxifloxacin was observed in approximately one-fourth of M. avium and M. intracellulare isolates, this resistance was not associated with mutations in gyrA and gyrB or with VNTR genotypes.

Induced novel psbA mutation (Ala251 to Thr) in higher plants confers resistance to PSII inhibitor metribuzin in Lens culinaris.

Weed competition is a major limitation to worldwide lentil (Lens culinaris Medik.) production in part due to limited effective safe herbicide options. Metribuzin is a photosystem II inhibiting herbicide that provides broad spectrum weed control, however it causes excessive injury in lentil. Dose response analysis of photosystem II inhibiting herbicides and DNA sequencing of the psbA chloroplast gene occurred to quantify the spectrum and mechanism of herbicide resistance in two ethyl-methanesulfonate (EMS) induced mutant lentils. Compared to susceptible parent PBA Flash, the level of metribuzin resistance was 33-fold for mutant M043 and 10-fold for M009. No improvement in resistance occurred in either mutant to bromoxynil, diuron, bromacil and atrazine herbicides. Nucleotide sequencing of the psbA gene of both mutants identified a substitution at position 751 compared to PBA Flash. The resulting deduced amino acid sequence indicated an Ala251 Thr substitution as being most likely responsible for the high level of metribuzin resistance. The Ala251 Thr substitution discovered in this study is unique in mutagenized higher plants and the first report of an induced psbA target site mutation in higher plants. This target site metribuzin resistance is likely to have a significant impact on lentil production in Australia and worldwide. © 2019 Society of Chemical Industry.

Redox control of iodotyrosine deiodinase.

The redox chemistry of flavoproteins is often gated by substrate and iodotyrosine deiodinase (IYD) has the additional ability to switch between reaction modes based on the substrate. Association of fluorotyrosine (F-Tyr), an inert substrate analog, stabilizes single electron transfer reactions of IYD that are not observed in the absence of this ligand. The co-crystal of F-Tyr and a T239A variant of human IYD have now been characterized to provide a structural basis for control of its flavin reactivity. Coordination of F-Tyr in the active site of this IYD closely mimics that of iodotyrosine and only minor perturbations are observed after replacement of an active site Thr with Ala. However, loss of the side chain hydroxyl group removes a key hydrogen bond from flavin and suppresses the formation of its semiquinone intermediate. Even substitution of Thr with Ser decreases the midpoint potential of human IYD between its oxidized and semiquinone forms of flavin by almost 80 mV. This decrease does not adversely affect the kinetics of reductive dehalogenation although an analogous Ala variant exhibits a 6.7-fold decrease in its kcat /Km . Active site ligands lacking the zwitterion of halotyrosine are not able to induce closure of the active site lid that is necessary for promoting single electron transfer and dehalogenation. Under these conditions, a basal two-electron process dominates catalysis as indicated by preferential reduction of nitrophenol rather than deiodination of iodophenol.

Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific‑oxygenated signaling compounds.

243 - Tsa1 and Tsa2 Are Two Highly Similar 2-Cys Prx from Yeast That Display Remarkable Differences in Their Structural Switches

Typical 2-Cys Peroxiredoxins (2-Cys Prxs; AhpC-Prx1) are enzymes that reduce hydroperoxides with extraordinary rates and are involved in diseases such as cancer, neurodegeneration and Alzheimer. The active site is composed of a catalytic triad, containing the peroxidatic cysteine (C P ), an Arg, and a Thr (or Ser). After oxidation of C P , an intermolecular disulfide is formed between C P and a second cysteine residue (resolving cysteine – C R ). During catalysis, 2-Cys Prxs switch between dimers and decamers (pentamer of dimers) or even to higher molecular weight species, depending on redox state among other factors. Additionally, a conformational change in the active site loop (partial unfolding of the α helix C P ) is required for the generation of an intermolecular disulfide. Therefore, during catalytic cycle, 2-Cys Prx alternate between fully folded (FF - reduced enzyme), and locally unfolded (LU) states, when disulfide bond can be formed. The analysis of all available 2-Cys Prx structures in FF revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic Thr of a dimer and an aromatic residue of adjacent dimer. However, none of the structures with the natural substitution of Thr by Ser display this feature. Structural analysis of 2-Cys Prx containing Thr (but not Ser) in the active site indicated that this residue destabilizes the decamer in the oxidized state possibly due to steric hindrance. This hypothesis was experimentally confirmed using two homologs yeast 2-Cys Prx (Tsa1 and Tsa2). In spite of possessing a high degree of similarity (~86%), Tsa1 presents a Thr in the active site, while Tsa2 presents a Ser. Size exclusion chromatography demonstrated that Tsa1 decamer (but not Tsa2) was destabilized by the oxidation. Additionally, mutants of Tsa1 which were observed mainly as dimers also displayed a decreased catalytic activity. Taken all the data together, we believe that oligomeric state of these enzymes may be related to regulatory pathways.

The Thr to Met substitution of amino acid 118 in hepatitis B virus surface antigen escapes from immune-assay-based screening of blood donors.

Hepatitis B surface antigen (HBsAg) is the main diagnosis marker for hepatitis B virus (HBV) infection. In this study, a novel HBV mutant from an HBV-positive blood donor with false-negative results during HBsAg screening was identified. DNA sequencing discovered two mutations at nt 353 (A to T) and nt 349 (T to A), leading to Thr to Met and Ser to Thr substitutions at aa 118 and 117 of HBsAg, respectively. Further analysis showed that eight of ten HBsAg ELISA kits failed to detect this HBsAg mutant. A mutagenesis assay indicated that the Thr to Met substitution at aa 118 was the determinant for escape from HBsAg ELISA detection. A small-scale screening of blood donors identified two individuals infected by this unique HBV mutant, suggesting a certain level of prevalence among the general population. In conclusion, our study identified the aa 118 mutation in HBV surface antigen and provided information for improvement of HBV diagnosis products.

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

ABSTRACTVoltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.

A Computational Comparative Study of Α-Glucosidase Enzyme Divergence

α-glucosidases (α-Gls) catalyze the last step of carbohydrate digestion in mammals and release glucose in bloodstream, which results in the increased blood glucose level. The evolution and classification of this enzyme has been a matter of debate. In the present study the amino acid sequences of α-Gls from 12 species were aliened and the Phylogenetic trees were constructed. The data indicated that only the mammalian enzyme contained the glucoamylase region. Five amino acid regions were found to be the conservative blocks of mammalian α-Gls. These blocks were not fully conserved in plants, fungi and bacteria. The results were in favor of Chibas classification despite the Ile→Thr substitution in Aspergillus Niger and Ala→Pro substitution in chimpanzee and human α-Gls. The chimpanzee α-Gls showed the most similarity to human enzyme. Plants, fungi, bacteria, and the mammalian α-Gls seemed to separately create a specific class. Together, the data suggest that the eukaryotic enzyme had been diverged significantly from the prokaryotic α-Gls since the separation from the common ancestor.