Top PDF Amino acid properties conserved in molecular evolution.

Amino acid properties conserved in molecular evolution.

Amino acid properties conserved in molecular evolution.

The connection between amino acid properties and molecular evolution was proposed very soon after the discovery of the latter [1–4]. It has been shown that the most frequently occurring single nucleotide mutations of DNA lead to amino acid changes that conserve certain amino acid properties [3,5]. It has also been suggested that this property of the genetic code was acquired in the process of evolution – the code itself evolved to minimize changes in important properties of amino acids upon mutation [5–10]. Those mutations which conserve important residue properties are much more likely to preserve the structure and function of protein, than those which dramatically change these properties. Therefore, the importance of the amino acid properties should be reflected in the data on the actual mutations of proteins. Such data can be collected in the laboratory by studying the effects of mutations on protein activity in numerous mutants of various proteins. These mutants can be obtained via site-directed mutagenesis, and one can measure directly how mutations affect protein activity [11,12]. However, the amount of work necessary to perform such analysis limits practical application of this approach, since only a small subset of all possible combinations of mutations can be studied in this way. One can also study the mutational experiments performed by Nature, using proteins from all living organisms as experimental material. The evolving proteins retain their structure and function, so by studying the mutations occurring in natural sequences, and observing which properties are conserved in these
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Evolutionary pressure on mitochondrial cytochrome b is consistent with a role of CytbI7T affecting longevity during caloric restriction.

Evolutionary pressure on mitochondrial cytochrome b is consistent with a role of CytbI7T affecting longevity during caloric restriction.

We used TreeSAAP to gain a historical context of the amino acid property changes at site 7 in cytb. Specifically, the property changes that TreeSAAP detected in step 1 of the analysis were correlated with the same properties TreeSAAP detected to be under natural selection throughout mammalian evolution. The overall purpose of this second step is to establish context for properly interpreting the results of step one. By estimating the naturally occurring pattern of adaptation, the effects of SNPs may be compared to the location and effects of extant genetic variation and historical adaptations within the broader taxonomic group. When a SNP fails to share characteristics with the historical adaptations of the group, that SNP is more likely have a detrimental effect. Conversely, if a SNP has much in common with the adaptations of the group, that SNP may have a similar adaptive effect [26].
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Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324.

Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324.

Reports on the crystal structures of AOx enzymes from fungal sources are limited with PDB id 1VAO and 3FIM from P. simplicissimum [29] and P. eryngii [44], respectively are the most prominent submissions. Both these crystal structures revealed significant dissimilarities between their 3D structures as well as in their amino acid sequence content. Multiple sequence alignment of A.terreus rAOx with the above aryl AOxs from these lignin degrading strains revealed significant sequence diversity. The cDNA sequence of the aryl AOx from A.terreus deduced by us consisted of 666 amino acids, whereas, the widely studied aryl AOxs from P. pulmonarius, P. eryngii and P. simplicissimum consisted of 594, 593 and 560 amino acid residues, respectively [43,44,2]. The amino acid sequence identity (using NCBI BLAST) of A.terreus AOx with other aryl AOxs from P. pulmonarius, P. eryngii and P. simplicissimum showed 27%, 25% and 37%, respectively. Even with the prevailing sequence variation, the predicted model of A.terreus rAOx showed significant structural homology with chain B of aryl AOx from P. eryngii (PDB id: 3FIM) [9] and its function was that of an aromatic AOx. The Ramachandran plot predicted our modeled protein to be stereo-chemically significant, thus increas- ing the authenticity of the ab-initio based 3D model. Further validations of our modeled 3D structure were proven through docking simulation studies with its co-factor FAD, which precisely predicted the conserved N-terminal binding region (Rossmann fold; GXGXXG motif, X = any amino acid residue) in our model. The docking also confirmed the b-a-b fold essential for non- covalent interaction with FAD. Function as predicted by I- TASSER was further validated through docking simulations carried out with our modeled rAOx along with four aromatic alcohols used in our kinetic studies. Based on MolDock scoring function the substrates were evaluated on the basis of its binding energies and was found to be consistent with the kinetics studies. The docking studies clearly demonstrated the close proximity of the active substrate binding site and the co-factor FAD binding site separated by a narrow funnel shaped cavity connecting the both (data not shown). It also confirmed that the active site lies in close vicinity of the FAD isoalloxazine ring. This kind of topology is conserved across all members of Glucose-Methanol-Choline (GMC) oxidoreductase family of proteins and is well reported [10,16]. Presence of few conserved aromatic amino acid residues (Phe 98 and Tyr 55) near the FAD isoalloxazine ring and substrate binding site could be involved in p-p stacking interaction with FAD isoalloxazine ring, thus stabilizing the co-factor. Molecular modeling and docking results gave a visual insight into better
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Amino acid and soluble nitrogen evolution throughout ripening of Serra da Estrela cheese

Amino acid and soluble nitrogen evolution throughout ripening of Serra da Estrela cheese

The water-soluble fraction is very heterogenous in terms of composition, and includes whey proteins, and high-, medium- and low-molecular weight peptides, as well as FAA. The set of reactions normally designated as secondary proteolysis result from the action of residual rennet and indigenous milk proteinases, in addition to adventitious microflora. Similar trends have been reported by Vioque et al. (2000) in a ewe’s milk cheese coagulated with C. cardunculus, and by Macedo and Malcata (1997) for Serra da Estrela cheese. The latter authors reported 34.6% WSN, 5.8% TCASN and 1.2% PTASN for 35 d-old cheeses, similar to our average values pertaining to the four dairies (i.e. 23.8%, 5.43% and 1.3%, respectively). Fern !andez- Salguero and Sanju!an (1999) reported values of 33.4% for WSN in ewe’s milk cheese after 60 d of ripening that had been coagulated with aqueous extracts of C. cardunculus. In La Serena cheese by the same time of ripening, WSN was 38.8%, whereas non-protein nitro- gen leveled off at 14.5% (Fern!andez del Pozo, Gaya, Medina, Rodr !ıguez-Mar!ın, & N!un˜ez, 1988; Fern!andez- Salguero, Matos, & Marsilla, 1978); other researchers (N !un˜ez, Fern!andez del Pozo, Rodr!ıguez-Mar!ın, Gaya, & Medina, 1991) reported even higher values for WSN and non-protein nitrogen, thus confirming that cheeses coagulated with plant rennets undergo extensive proteolysis.
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Partial Molar Volumes of Amino Acids in Aqueous MgSO4

Partial Molar Volumes of Amino Acids in Aqueous MgSO4

In this work, four amino acids (glycine, L-alanine, DL-2-aminobutyric acid and L- valine) were selected as model compounds, as they are the fundamental structural units of proteins, and magnesium sulphate was chosen as the electrolyte since it provokes dramatic and, sometimes, opposite effects on amino acid solubility’s. The volumetric properties of these solutions will provide important information about solute-solute and solute-solvent interactions that govern protein hydration, denaturation, and aggregation. The results, combined with information gathered from the literature review, aim to con- tribute to a better understanding of the forces that manage important biological struc- tures, in other words, for a better molecular interpretation of the interactions between the ions (Mg 2+ and SO
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Physico-chemical properties and amino acid profiles of sap from Tunisian date palm

Physico-chemical properties and amino acid profiles of sap from Tunisian date palm

which could be involved in various biological functions, including redox regulation, stress responses, defence reactions, signal and transport pathways, and carbohy- drate metabolism. All proteins identified with reference to yeast Saccharomyces cerevisiae, which belongs to the natural microflora of date palm sap, showed a molecular weight in excess of 45 kDa. In fact, as reported by Ben Thabet et al. (2010), all the glycolytic enzymes which form part of the fermentation processes that occur in Saccharomyces cerevisiae to produce alcohol were iden- tified by MALDI-TOF analyses and showed a molecu- lar weight varying from 44.7 to 62.1 kDa. In addition, proteins that this yeast involves against stress such as ATPase of Hsp90 chaperone complex, present a molec- ular weight nearly 77.6 kDa. The metabolism proteins produced by Saccharomyces cerevisiae, like Acetohy- droxyacid reductoisomerase, were also identified with a molecular weight of about 45 kDa (Ben Thabet et al., 2010b).
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Molecular evolution of the ent -kaurenoic acid oxidase gene in Oryzeae

Molecular evolution of the ent -kaurenoic acid oxidase gene in Oryzeae

We surveyed the substitution patterns in the ent-kaurenoic acid oxidase (KAO) gene in 11 species of Oryzeae with an outgroup in the Ehrhartoidaea. The synonymous and non-synonymous substitution rates showed a high positive correlation with each other, but were negatively correlated with codon usage bias and GC content at third codon posi- tions. The substitution rate was heterogenous among lineages. Likelihood-ratio tests showed that the non- synonymous/synonymous rate ratio changed significantly among lineages. Site-specific models provided no evi- dence for positive selection of particular amino acid sites in any codon of the KAO gene. This finding suggested that the significant rate heterogeneity among some lineages may have been caused by variability in the relaxation of the selective constraint among lineages or by neutral processes.
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Characterization of the deleted in autism 1 protein family: implications for studying cognitive disorders.

Characterization of the deleted in autism 1 protein family: implications for studying cognitive disorders.

Autism spectrum disorders (ASDs) are a group of commonly occurring, highly-heritable developmental disabilities. Human genes c3orf58 or Deleted In Autism-1 (DIA1) and cXorf36 or Deleted in Autism-1 Related (DIA1R) are implicated in ASD and mental retardation. Both gene products encode signal peptides for targeting to the secretory pathway. As evolutionary medicine has emerged as a key tool for understanding increasing numbers of human diseases, we have used an evolutionary approach to study DIA1 and DIA1R. We found DIA1 conserved from cnidarians to humans, indicating DIA1 evolution coincided with the development of the first primitive synapses. Nematodes lack a DIA1 homologue, indicating Caenorhabditis elegans is not suitable for studying all aspects of ASD etiology, while zebrafish encode two DIA1 paralogues. By contrast to DIA1, DIA1R was found exclusively in vertebrates, with an origin coinciding with the whole-genome duplication events occurring early in the vertebrate lineage, and the evolution of the more complex vertebrate nervous system. Strikingly, DIA1R was present in schooling fish but absent in fish that have adopted a more solitary lifestyle. An additional DIA1-related gene we named DIA1-Like (DIA1L), lacks a signal peptide and is restricted to the genomes of the echinoderm Strongylocentrotus purpuratus and cephalochordate Branchiostoma floridae. Evidence for remarkable DIA1L gene expansion was found in B. floridae. Amino acid alignments of DIA1 family gene products revealed a potential Golgi- retention motif and a number of conserved motifs with unknown function. Furthermore, a glycine and three cysteine residues were absolutely conserved in all DIA1-family proteins, indicating a critical role in protein structure and/or function. We have therefore identified a new metazoan protein family, the DIA1-family, and understanding the biological roles of DIA1-family members will have implications for our understanding of autism and mental retardation.
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Key words: Agar; Aeromonas sp.; -agarase; Pseudoalteromonas sp.; GHF-16; Neoagaro-

Key words: Agar; Aeromonas sp.; -agarase; Pseudoalteromonas sp.; GHF-16; Neoagaro-

belong to the GHF-16 family showed overall sequence identities ranging from 24-97%. Some of the -agarases encode for a modular protein consisting of a signal peptide, catalytic module and C-terminal domain of unknown function or a carbohydrate binding module (1, 35), and this may be the reason for having low identity, even within the same family. It has been reported that homologous regions in most of the reported GHF-16 agarases were present in catalytic module of the -agarases (1). This further confirms the heterogeneity of the amino acid sequences in length, catalytic properties and substrate specificities in agarases. Furthermore, the results of the phylogenetic analysis support the idea that many -agarases may have evolved from a common ancestral form, and, that domain shuffling may contribute significantly to the diversity of the agarases (7,10). Even though the protein is divergent from the primary sequences, AgaA features strictly conserved catalytic residues, which show conservation among the GHF- 16 family members. Glutamic and aspartic acid are the highly conserved active site residues, which are responsible for catalytic activity in the GHF (30). According to previous reports (1, 31), the conserved Glu 148 and Glu 153 are responsible
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Novel nucleotide and amino acid covariation between the 5'UTR and the NS2/NS3 proteins of hepatitis C virus: bioinformatic and functional analyses.

Novel nucleotide and amino acid covariation between the 5'UTR and the NS2/NS3 proteins of hepatitis C virus: bioinformatic and functional analyses.

The highly variable HCV genome has been classified into 6 genotypes [16] which affect pathogenesis and therapeutic outcome [17]. Covarying amino acid residues are believed to evolve for persistent viral replication or egression, to be functionally conserved and constrained within certain viral components. Certain compensatory mutations at the protein level have been reported recently [18-22]. Coordinated substitutions in NS3 and NS4A affect HCV replication via modulation of NS5A phosphor- ylation [19]. Compensatory mutations in p7 and NS2 restore assembly-defective core protein mutants, whereas chimeric HCV with coordinated mutations in envelope 1, p7, NS2, and NS3 increase the intergenotypic compatibilities for virus assembly and release [20,22]. More importantly, amino acid covariance networks have been identified to predict the response in HCV patients receiving anti-viral therapy [18,21]. Such studies underscore the significance of the functional linkage of certain proteins and their covariant amino acid residues for HCV persistency, raising the possibility that molecular covariation can be computationally predicted during persistent infection for diagnosis, prognosis and optimal drug selection.
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Analysis of Genes Encoding Penicillin-Binding Proteins in Clinical Isolates of Acinetobacter baumannii

Analysis of Genes Encoding Penicillin-Binding Proteins in Clinical Isolates of Acinetobacter baumannii

There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acineto- bacter baumannii to ␤-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to ␤-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.
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Molecular Cloning and Sequencing of Alkalophilic Cellulosimicrobium cellulans CKMX1 Xylanase Gene Isolated from Mushroom Compost and Characterization of the Gene Product

Molecular Cloning and Sequencing of Alkalophilic Cellulosimicrobium cellulans CKMX1 Xylanase Gene Isolated from Mushroom Compost and Characterization of the Gene Product

gene (Fig. 1). The nucleotide sequence of 1671 bp amplicon shared 98% homology to xylanase gene of Cellulosimicrobium sp. HY-13 endo-beta-1,4- xylanase (xylK-1) gene, (Accession no. FJ859907), thus confirming the isolation of xylanase gene from C. cellulans CKMX1 (Fig. 1). A positive clone, indicating that the clone contained an insert of 1671 bp in size with a 58% G+C content, in conformity with the high G+C levels was found in nucleotide sequences from other Cellulosimicrobium sp., e.g., endo-beta-1, 4- xylanase gene from Cellulosimicrobium sp. HY-13 (Kim et al. 2009). The conserved domain search of the xylanases gene across the Cellulosimicrobium sp. and other bacterial species indicates the significance of enzyme in xylan hydrolysis. The mature protein consisted of amino acids 556 with calculated molecular weight of 58 KDa. To-date, many xylanases genes have been cloned from different microorganisms, including Actiomadura sp. S14 (Thayat et al. 2011), Paenibacillus sp. 12- 11 (Zhao et al. 2011) and Streptomyces sp. S27 (Li et al. 2009). However, this study was to clone a xylanase gene from C. cellulans CKMX1 and a very little information was available regarding cellulase-free xylanases from C. cellulans CKMX1. Further, the work regarding hyperxylanolytic production could be done in vivo. Amino acid composition determines the fundamental properties of the enzyme (Arora et al. 2009). The amino acid composition of xylanase sequences is represented in Table 1. The pI values of all protein sequences were in the range of 4.32- 9.57, indicating that all considered xylanase sequences were acidic except C. flavigena XIB (9.57), C. flavigena DSM 20109 (8.94), Cellvibrio gilvus ATCC 13127 (8.70), B. subtilis (9.18) and Pedobacter saltans DSM 12145 (9.16). The xylanase enzyme of C. cellulans CKMX1 had the pI value of 4.66 which showed that the xylanase sequence was acidic (Arora et al. 2009). The calculated isoelectric point (pI) will be useful because at pI, solubility is least and mobility in an electro focusing system is zero. The instability index, which gives clue about the stability of a protein in vitro, can be calculated using Expasy’s ProtParam server (Geourjon and Deleage 1995). All the considered sequences were classified as stable with values ranging from 16.14 to 38.99, except Granulicella mallensis MP5ACTX8 (42.29). The xylanase sequence of C. cellulans CKMX1 (22.89) indicated a stable protein (Table 2).
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A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase.

A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase.

The study was supported by grants from International Health Cooperation Research (27-S- 1102 and 26-A-103) and Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development, AMED. BML Inc. provided support in the form of salaries for the author [MS], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manu- script. The specific roles of the author are articulated in the ‘author contributions’ section.

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Identification of a type I nitroreductase gene in non-virulent Trypanosoma rangeli

Identification of a type I nitroreductase gene in non-virulent Trypanosoma rangeli

Trypanosomatid type I nitroreductases (NTRs), i.e., mitochondrial enzymes that metabolise nitroaromatic pro- drugs, are essential for parasite growth, infection, and survival. Here, a type I NTR of non-virulent protozoan Try- panosoma rangeli is described and compared to those of other trypanosomatids. The NTR gene was isolated from KP1(+) and KP1(-) strains, and its corresponding transcript and 5’ untranslated region (5’UTR) were determined. Bioinformatics analyses and nitro-drug activation assays were also performed. The results indicated that the type I NTR gene is present in both KP1(-) and KP1(+) strains, with 98% identity. However, the predicted subcellular locali- sation of the protein differed among the strains (predicted as mitochondrial in the KP1(+) strain). Comparisons of the domains and 3D structures of the NTRs with those of orthologs demonstrated that the nitroreductase domain of T. rangeli NTR is conserved across all the strains, including the residues involved in the interaction with the FMN cofactor and in the tertiary structure characteristics of this oxidoreductase protein family. mRNA processing and expression were also observed. In addition, T. rangeli was shown to be sensitive to benznidazole and nifurtimox in a concentration-dependent manner. In summary, T. rangeli appears to have a newly discovered functional type I NTR.
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Three hundred and twenty Hy-Line W36

Three hundred and twenty Hy-Line W36

Three hundred and tw enty Hy-Line W36  commercial laying hens, 39 w eeks of age, w ere used to determine the sequence and the length of time needed for hens to recover performance characteristics after an eight-w eek period under graded levels of threonine deficiency. Eight experimental diets w ith Thr levels ranging from 0.35 to 0.53% w ere randomly fed w ith eight replicates of five hens each. After the previous experiment, the hens w ere fed a control diet (0.53% Thr) for a four- w eek period. Feed consumption (FC), energy intake (EI), egg production (EP), egg w eight (EW), egg mass (EM ), and body w eight (BW) w ere evaluated. All performance characteristics w ere impaired on Thr deficient diets. The recovery sequence order w as FC and EI; EP, EW and EM , and f inally BW, w it h t he lengt h of t ime of t w o, t hree, and f our w eeks, respectively. The data indicated that an amino acid deficiency does not cause permanent damage to the reproductive system of the hens.
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Arq. Gastroenterol.  vol.53 número3

Arq. Gastroenterol. vol.53 número3

Until recently, little structure information about HCV protein and thyroid self-protein human were available and molecular models has been built on partial structures, with as- sembly guided by biochemical data. The purpose of this study is to explore the possible sequence similarity between the AA sequences of thyroid self-protein, which are potential B- and T-cell epitopes of these antigens and proteins of HCV, using da- tabanks of proteins and immunogenic peptides to explain ATD.

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Does low-protein diet improve broiler performance under heat stress conditions?

Does low-protein diet improve broiler performance under heat stress conditions?

a commercial diet, with or without essential amino acid supplementation (10% over commercial levels), in broiler chicken raised during the summer and concluded that amino acid supplementation had a minimum effect on heat production, with more energy utilization for fat deposition than to protein synthesis. Temim et al. (2000b) evaluated protein turnover in Pectoralis major (breast muscle), Sartorius and Gastrocnemius (leg muscles) of broiler raised at 22 or 32ºC and fed diets with 20 or 25% of crude protein. These authors found that high-protein diet did not change muscle protein turnover. Considering that heat production is positively correlated with body protein synthesis instead of protein intake, and there is no evidence indicating an increase of heat production due to amino acid excess (Macleod, 1997), we should speculate that utilization of high-protein diets for broiler chickens reared under heat stress is not able to change heat production in the birds. Beside, at high temperature, diets with high protein might help muscle protein synthesis due to proteolysis reduction (Temim et al., 2000b). However, high-protein diets increase nitrogen excretion (Aletor et al., 2000), which has a negative environmental impact. Thus, basically three items need to be considered for defining the better diet protein level for broiler chickens: 1) productivity; 2) final product quality (low protein diets increase fat deposition) and, 3) environmental impact (nitrogen excretion).
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Predictive properties of plasma amino acid profile for cardiovascular disease in patients with type 2 diabetes.

Predictive properties of plasma amino acid profile for cardiovascular disease in patients with type 2 diabetes.

Prevention of cardiovascular disease (CVD) is an important therapeutic object of diabetes care. This study assessed whether an index based on plasma free amino acid (PFAA) profiles could predict the onset of CVD in diabetic patients. The baseline concentrations of 31 PFAAs were measured with high-performance liquid chromatography-electrospray ionization-mass spectrometry in 385 Japanese patients with type 2 diabetes registered in 2001 for our prospective observational follow-up study. During 10 years of follow-up, 63 patients developed cardiovascular composite endpoints (myocardial infarction, angina pectoris, worsening of heart failure and stroke). Using the PFAA profiles and clinical information, an index (CVD-AI) consisting of six amino acids to predict the onset of any endpoints was retrospectively constructed. CVD-AI levels were significantly higher in patients who did than did not develop CVD. The area under the receiver-operator characteristic curve of CVD-AI (0.72 [95% confidence interval (CI): 0.64–0.79]) showed equal or slightly better discriminatory capacity than urinary albumin excretion rate (0.69 [95% CI: 0.62–0.77]) on predicting endpoints. A multivariate Cox proportional hazards regression analysis showed that the high level of CVD-AI was identified as an independent risk factor for CVD (adjusted hazard ratio: 2.86 [95% CI: 1.57–5.19]). This predictive effect of CVD-AI was observed even in patients with normoalbuminuria, as well as those with albuminuria. In conclusion, these results suggest that CVD-AI based on PFAA profiles is useful for identifying diabetic patients at risk for CVD regardless of the degree of albuminuria, or for improving the discriminative capability by combining it with albuminuria.
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Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipaseA

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipaseA

Reverse phase chromatography (RP-HPLC) on C18 column Fraction D3 obtained in the chromatographic step on DEAE Sepharose was subjected to a C18 reverse phase column (4.6 mm ID × 25 cm, CLC-ODS, Shimadzu, Japan) using ÄKTA™ purifier system (GE Healthcare, USA). The column had been previously equilibrated with a solution of 0.1 % trifluoroacetic acid (TFA) (solvent A), and about 10 mg of fraction D3 was diluted in the same solvent and applied to the system using a 500 μL loop. Elution was performed at a flow rate of 0.5 mL/minute with a linear concentration gradient solution containing 70 % aceto- nitrile and 0.1 % TFA (Solvent B): 0-100 % solvent B in ten column volumes. All eluted fractions were assessed for their phospholipase activity and on SDS-PAGE, as described below. The fraction that showed phospholipase activity was pooled, lyophilized and rechromatographed in the same column, this time using a segmented concentra- tion gradient of 0-60 % solvent B in three column volumes, 60-80 % in five column volumes, and 80-100 % in one column volume.
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Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features.

Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features.

Recently, we have physically mapped the large Z chromosome of the codling moth using fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) probes, the so-called BAC-FISH, and showed that it is in fact a neo-Z chromosome that has arisen by fusion between an ancestral Z chromosome and an autosome corresponding to chromosome 15 in the Bombyx mori reference genome. Further experiments, performed by quantita- tive PCR (qPCR) showed a Z-linkage of selected orthologs of B. mori chromosome 15 genes in two other tortricids, Lobesia botrana and Eupoecilia ambiguella (see below). The results suggest that the Z chromosome-autosome fusion originated in a common ancestor of the main tortricid subfamilies, Olethreutinae and Tortricinae [41]. In this study, we examined karyotype features in four other species of tortricids by standard cytogenetic techniques and by mapping multigene families (major rRNA genes and histone genes) using fluorescence in situ hybridization (FISH) with 18S rDNA and H3 histone probes. We also used comparative genomic hybrid- ization (CGH) to determine the level of molecular differentiation of the W and Z sex chromosomes. Cytogenetic characteristics were compared with those of the codling moth [32] with the aim to understand karyotype and sex chromosome evolution in the family Tortricidae. Such complex comparisons have never been done across any lepidopteran family, except for the W chromosome divergence in the family Pyralidae [37]. For our research we chose two pests of pome and stone fruits, the Oriental fruit moth, Grapholita molesta (Busck) and the plum fruit moth, Grapholita funebrana (Treitschke), both close relatives of the codling moth (Olethreutinae: Grapholitini), and two pests of cultivated grapes, the European grapevine moth, Lobesia botrana (Denis & Schiffer- mu¨ller) from the tribe Olethreutini and the vine moth, Eupoecilia ambiguella (Hu¨bner) representing the tribe Cochylini of Tortricinae. Our choice was also motivated by the fact that G. molesta and possibly the two grape pests are candidate species for their control by sterile insect technique (SIT), which is currently used against the codling moth [42], and the acquired cytogenetic knowledge may facilitate transfer of the technology to these and other tortricid pests.
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