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At least 433 records · Page 24Linked to original sources
PubMed2005

New concepts in the chromatography of peptides and proteins.

Although chromatography using a variety of novel bed configurations (e.g. fluidized beds, expanded beds, simulated moving beds, annular rotating beds, etc.) has been of recent interest, the majority of practical applications of analytical and preparative chromatography employ a stationary adsorbent bed into which a feed slug is charged periodically, similar to the technique first described by Mikhail Tswett over 100 years ago. However, new concepts in both the practice and theory of fixed-bed chromatography are continuing to expand the available range of applications for separating peptides and proteins.

Algorithms↗
PubMed2006

Prevalence and spectrum of mutations in the sarcomeric troponin T and I genes in a cohort of Spanish cardiac hypertrophy patients.

We sequenced the coding exons of the cardiac troponins T (TNNT2) and I (TNNI3) genes in 115 Spanish HCM-patients (32% with a family history of the disease). Only two (2%) had mutations in the TNNT2 (Arg278>Cys and Arg92>Lys). These mutations were associated with variable clinical outcomes. No patient had TNNI3-mutation. We also genotyped these patients and 320 healthy controls for a 5 bp insertion/deletion (I/D) polymorphism in intron 3 of TNNT2. DD-homozygotes for the 5 bp I/D polymorphism were significantly more frequent among the patients (OR=1.83, 95% CI=2.10-5.16).

Adolescent↗
PubMed2006

Chemical ligation--an unusual paradigm in protease inhibition.

Some protease inhibitors use uncommon mechanisms to restrain the activity of their target enzymes. A recent paper in Chemistry and Biology (Lu et al., 2006) demonstrates a curious mechanism of inhibition of a caspase, relying on principles of native peptide ligation.

Apoptosis↗
PubMed2006

Mutation analysis of DARPP-32 as a candidate gene for schizophrenia.

Dopamine- and cAMP-regulated phosphoprotein of relative molecular mass 32kDa (DARPP-32) plays a pivotal role in the signal transduction of several neurotransmitters and neuromodulators that are implicated in the pathophysiology of a variety of neuropsychiatric disorders. A postmortem study reported a significantly reduced DARPP-32 expression in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia, suggesting possible involvement of DARPP-32 in the pathophysiology of schizophrenia. Hence, DARPP-32 was considered as a candidate gene for schizophrenia in this study. We first systemically searched for mutations in the DARPP-32 gene in 50 Han Chinese patients with schizophrenia from Taiwan. Five molecular variants were identified, including a C-to-G substitution (g.-2036C>G) in the putative core promoter that obliterated a predictive AP-2 transcription factor binding site, a G deletion in the untranslated exon 2 (g.1238delG), a G-to-A and an A-to-G substitutions in intron 2 (IVS2+31G>A) and intron 6 (IVS6+32A>G), respectively, and a three-base pair deletion of AGA in exon 6 that resulted in deletion of a glutamate at codon 135 (E135del). Further SNP- and haplotype-based association study in 249 patients and 273 control subjects, however, did not detect association of these markers with schizophrenia. Hence, our results suggest that the reduced DARPP-32 protein in patients with schizophrenia is unlikely caused by mutations in the DARPP-32 gene itself and the DARPP-32 gene is also unlikely a major susceptibility gene for schizophrenia. Nevertheless, the identification of these molecular variants should help the study of gene regulation and structure-function relationship of DARPP-32, and the association study of DARPP-32 gene with other neuropsychiatric disorders.

Alleles↗
PubMed2006

Post-translational enzyme activation in an animal via optimized conditional protein splicing.

Control over the timing, location and level of protein activity in vivo is crucial to understanding biological function. Living systems are able to respond to external and internal stimuli rapidly and in a graded fashion by maintaining a pool of proteins whose activities are altered through post-translational modifications. Here we show that the process of protein trans-splicing can be used to modulate enzymatic activity both in cultured cells and in Drosophila melanogaster. We used an optimized conditional protein splicing system to rapidly trigger the in vivo ligation of two inactive fragments of firefly luciferase in a tunable manner. This technique provides a means of controlling enzymatic function with greater speed and precision than with standard genetic techniques and is a useful tool for probing biological processes.

Animals↗
PubMed2006

Determinants of RING-E2 fidelity for Hrd1p, a membrane-anchored ubiquitin ligase.

A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function.

Amino Acid Motifs↗
PubMed2006

Chemical synthesis and biosynthesis of the cyclotide family of circular proteins.

Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed.

Amino Acid Sequence↗
PubMed2006

A mutation in At-nMat1a, which encodes a nuclear gene having high similarity to group II intron maturase, causes impaired splicing of mitochondrial NAD4 transcript and altered carbon metabolism in Arabidopsis thaliana.

To elucidate the mechanism of cellulose synthesis, we isolated a mutant of Arabidopsis (changed sensitivity to cellulose synthesis inhibitors 1, css1) that showed changed sensitivity to cellulose biosynthesis inhibitor. The analysis of phenotypes indicated that the css1 mutation influenced various fundamental metabolic pathways including amino acid metabolism, triacylglycerol degradation and polysaccharide synthesis (cellulose and starch) during the early stage of plant growth. Unexpectedly, the map-based cloning of the gene responsible for the css1 mutation identified a protein (At-nMat1a) that was assumed to be a splicing factor of the mitochondrial group II intron. In accordance with this result, this mutant exhibited improper splicing of the mitochondrial NAD4 transcript. We noticed that the phenotypes of the css1 mutant are similar to the responses to anoxia that hinders mitochondrial aerobic respiration. It seems that the defect in the function of mitochondria influences various aspects of fundamental cellular metabolism including cellulose synthesis. Our results suggested that sucrose synthase (SuSy), an enzyme involved in the biosynthesis of cellulose, plays key roles in the connection between mitochondria and cellulose synthesis. The isolation of the css1 mutant also provides a useful resource in the study of post-transcriptional gene regulation in mitochondria.

Amino Acids↗
PubMed2006

Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain.

The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR-ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62-kDa EGFR-ICD, suggesting an increase in the mobility of the C-terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c-Src and the phosphorylated EGFR C-terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C-terminal domain possessed a significant level of secondary structure in the form of alpha-helices and beta-sheets, with a marginal change in beta-sheet content occurring upon phosphorylation.

Circular Dichroism↗
PubMed2006

Genetic polymorphisms in KCNQ1, HERG, KCNE1 and KCNE2 genes in the Chinese, Malay and Indian populations of Singapore.

AIMS: To determine the genetic variability of long QT syndrome (LQTS)-associated genes (KCNQ1, HERG, KCNE1 and KCNE2) among three distinct ethnic groups in the Singapore population. METHODS: Genomic DNA samples from up to 265 normal healthy Chinese, 118 Malay and 139 Indian volunteer subjects were screened for genetic variations in the coding region of the LQTS-associated genes using denaturing high-performance liquid chromatography and sequencing analyses. RESULTS: In total, 37 single nucleotide polymorphisms (SNPs) were identified in the coding exons of the LQTS-associated potassium ion channel genes, seven of which were novel nonsynonymous polymorphisms. SNPs 356G-->A (exon 1 of KCNQ1), 2624C-->T and 2893G-->A (exon 11 of HERG), 3164G-->A, 3322C-->G and 3460G-->A (exon 14 of HERG), and 79C-->T (exon 3 of KCNE2) resulted in Gly119Asp, Thr875Met, Gly965Arg, Arg1055Gln, Leu1108Val, Gly1154Ser and Arg27Cys amino acid substitutions, respectively. In addition, 16 intronic variants were detected. The functional consequence of these variants has not been studied and their association with risk of LQTS is unclear. CONCLUSIONS: There exist multiple genetic polymorphisms of the LQTS-associated genes in the three distinct Asian populations. Though the functional significance of many of these SNPs is unknown, this interindividual and interethnic genetic variability may underlie the different susceptibilities of individuals to developing LQTS.

Adolescent↗
PubMed2005

Mutation analysis in 51 patients with haemophilia A: report of 10 novel mutations and correlations between genotype and clinical phenotype.

We report the results of genetic analysis on a series of 51 patients attending this Haemophilia Comprehensive Care Centre. The most common cause of severe haemophilia A--the factor VIII intron 22 inversion was detected in eight families and the factor VIII intron 1 inversion in three families. Mutation analysis was carried out on the remaining patients by nucleotide sequencing of genomic DNA after screening with conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC). A total of 27 different FVIII non-inversion mutations were detected. Severe haemophilia was associated with 12 null mutations (six nonsense, six frameshift) and four missense mutations. A further 11 different missense mutations were associated with moderate or mild disease. To our knowledge, six null mutations [1950del 4(tttg), 3270-75insA, 4416del 10, 6735-38delA, W1029X, Y1792X] and four missense mutations (E1682K, M1947V, P2048L, P2143L) have not been previously published. Each novel missense mutation occurred at a highly conserved residue, no other candidate mutation was detected on screening the entire coding region of the FVIII gene and they were not detected in a screen of individuals without haemophilia A. The genotype-phenotype correlations of the FVIII mutations detected will be discussed.

Base Sequence↗
PubMed2005

Utility of a (GT) dinucleotide repeat in intron 1 of the factor 8 gene for haemophilia A carrier diagnosis.

We report the usefulness of a dinucleotide GT repeat in intron 1 of the factor 8 gene in carrier diagnosis of haemophilia A (HA). We analyzed 47 women from HA families in which the mutation was not identified in the index case and in which the common intragenic polymorphic markers were uninformative. The intron 1 GT repeat was useful to identify the X chromosome with the mutation in 19 of them. The analysis of this easily detectable marker in conjunction with other current markers may facilitate X chromosome identification in a large proportion of HA families.

Base Sequence↗