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At least 163 records · Page 9Linked to original sources
PubMed1998

Identification of a new heterozygous point mutation in the COL1A2 gene leading to skipping of exon 9 in a patient with joint laxity, hyperextensibility of skin and blue sclerae. Mutations in brief no. 166. Online.

A heterozygous deletion of exon 9 in the COL1A2-mRNA of a patient with symptoms of both the Ehlers-Danlos-Syndrome and the Osteogensis Imperfecta is described. In the genomic DNA of the patient, exon 9 is homozygously present. We identified a novel heterozygous point mutation in the splice donor site of intron 9, leading to a G-->A substitution in position +5. This mutation leads to heterozygous skipping of exon 9 in the COL1A2-mRNA of this patient. The deletion results in a shortened (by 18 amino acids) but in frame 12(1) chain, which probably leads to the formation of abberantly processed triple helices.

Alternative Splicing↗
PubMed1999

Spectrum of CFTR mutations in the Middle North of Spain and identification of a novel mutation (1341G-->A). Mutation in brief no. 252. Online.

We have analyzed 39 unrelated cystic fibrosis (CF) families by denaturing gradient gel electrophoresis (DGGE) and direct sequencing in order to determine the spectrum of CF mutations in our population. This approach has allowed us to detect 72 out of the 78 CF chromosomes (92.3%). The DF508 mutation was found to be present in 51/78 (65.4%) CF chromosomes, in accordance with the predicted Northwest-Southeast gradient within the European population. Another 14 known mutations, and the novel 1341G-->A mutation were identified. Nine out of fifteen non DF508 mutations were present in a single chromosome. The 1341G-->A mutation, found in 2 unrelated patients, is a new mutation associated to severe phenotype, causing pancreatic insufficiency and chronic lung infections. Our data suggest a different distribution of non-DF508 mutations in our population when compared with previous studies carried out in Spanish CF families. Six out of the 14 non-F508 in our study were not present in a recent study carried out in 640 Spanish families with CE These six mutations account for 29.6% non DF508 chromosomes in our sample.

Cystic Fibrosis↗
PubMed1999

Identification of 9 novel FBN1 mutations in German patients with Marfan syndrome.

We report 9 new mutations in German patients presenting with classical Marfan syndrome. All mutations occur in exons with calcium-binding (cb) epidermal growth factor-like (EGF) domains. Five mutations are missense involving exons 12, 27, 30, 44, and 52 with the resultant substitution of cysteine by phenylalanine (C504F), cysteine by tyrosine (C1129Y), tyrosine by cysteine (Y1261C), cysteine by serine (C1833S), and cysteine by tyrosine (C2142Y), respectively. The other four mutations are single base deletions in exons 39, 43, 48, and 58, at nucleotide A4826, C5311, T6018, and A7291, respectively, each resulting in frameshift with premature termination. Four mutations were detected in sporadic cases and are likely to be de novo.

Adult↗
PubMed1995

One-dimensional genome scanning: identification of the basis of a mouse mutation and identification of genomic changes in ovarian carcinoma.

We have developed a simple one-dimensional electrophoretic method, genome scanning, that can be used to identify large-scale genomic differences between two or more DNA samples. Genome scanning is especially useful in the detection of genetic amplifications, deletions, and rearrangements. The assay is essentially a high-resolution Southern analysis, comparing equivalent amounts of genomic DNA samples that are variant for a given trait. The Southern blots are hybridized to a probe sequence derived from a medium copy number repetitive element (1000-2000 copies per haploid genome) naturally dispersed throughout the genome. The hybridization pattern that results is complex and consists of hundreds of bands. If the DNA samples are otherwise equivalent, a net difference in hybridization intensity between homologous bands of different samples indicates a genetic change. In this report, we discuss the origin of the method, its premise, and review its application to mouse mutational analysis and to human cancer research (a more detailed discussion of the theory is presented elsewhere in this issue; Y. Gondo and M. H. Brilliant, Electrophoresis 1995, 16, 174-178).

Animals↗
PubMed1995

Identification of an endotoxin and IFN-inducible cDNA: possible identification of a novel protein family.

The response of macrophages to agents such as lipopolysaccharide (LPS) and interferon (IFN) includes the transcriptional activation of numerous genes. We have used the method of differential screening of a RAW 264.7 macrophage cell line cDNA library to isolate and characterize LPS-induced messages. One such message, LRG-47, is induced by LPS, IFN-gamma, and IFN-alpha/beta, but not by a panel of other cytokines or pharmacological activating agents. LRG-47 is homologous to two other IFN-gamma-induced genes, IRG-47 and Mg21. The LRG-47 sequence is approximately 33% identical and 52% similar to both these putative protein products. All three putative proteins, particularly Mg21, bear homology to a T cell product, Tgtp, induced by T cell receptor cross-linking. The three macrophage-derived proteins share areas of homology with GTP-binding proteins, are approximately 415 amino acids in length, and have similar kinetics of induction by IFN-gamma. This suggests that these genes may be members of a new family of IFN-inducible proteins.

Amino Acid Sequence↗
PubMed1998

Identification of genetic variation in the human hormone-sensitive lipase gene and 5' sequences: homology of 5' sequences with mouse promoter and identification of potential regulatory elements.

Hormone-sensitive lipase (HSL) plays a crucial role in triglyceride hydrolysis in adipose tissue and exhibits cholesterol hydrolase activity in steroidogenic tissue and macrophages. Thus, common genetic variation in the HSL gene could affect both energy metabolism and atherogenesis. Using overlapping single-strand conformational polymorphism analysis (SSCP), a common single base change (T/C) in intron 4 [allele frequency 0.012 (95% CI 0.007-0. 018)] and a variable CA repeat in intron 6 with 9 alleles (heterozygosity index = 0.66) were identified. Sequence of 1.123 kb upstream from the reported 5'UTR (1) which includes intron B, exon B, and 159 bp of the promoter (2) was obtained and a single common nucleotide change, -60C/G [allele frequency 0.052 (95% CI 0.039-0. 064)], identified. Preliminary in vitro studies show that the -60G construct has 38.5% lower luciferase activity compared to the -60C construct (P = 0.035), suggesting a functional change affecting HSL gene expression. The 5' sequence shows 57-59% homology with the mouse promoter with higher homology at potential regulatory motifs. Thus, the 1.7 kb of 5' sequences is well conserved and may play a part in the regulation of HSL gene expression.

Adipocytes↗
PubMed1996

Characterization of Bacterial Communities from Activated Sludge: Culture-Dependent Numerical Identification Versus In Situ Identification Using Group- and Genus-Specific rRNA-Targeted Oligonucleotide Probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7-8.3% on R2A agar; 45.0-63.7% on TS agar), Acinetobacter spp. (5.4-9.0% on R2A agar; 5.0-9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp.. The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.

Journal Article↗
PubMed1996

mtDNA as a tool for identification of human remains. Identification using mtDNA.

Two cases are presented in order to emphasize the importance of mitochondrial DNA in forensic medicine. The first case involved a charred body which could not be identified by morphological means because of severe destruction of all tissues. The parallel use of PCR methods using genomic DNA and sequencing of the mitochondrial d-loop region produced unequivocal and reproducible results. In the second case, various parts of a highly decomposed body were investigated. The application of standard PCR methods for genomic DNA proved unsuitable to answer the question whether the body parts belonged to the same body. However, when sequencing of mitochondrial DNA segments amplified from tissue and bone samples was performed, clearly interpretable results were obtained.

Adult↗
PubMed1991

Metabolism of metandienone in man: identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites.

After oral administration of metandienone (17 alpha-methyl-androsta-1,4-dien-17 beta-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl-N-trimethyl-silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17 alpha-methyl-5 beta-androst-1-en-17 beta-ol-3-one (II), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (III), 17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (IV) and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites--6 beta,12-dihydroxy-metandienone (IX), 6 beta,16 beta-dihydroxy-metandienone (X), 6 beta,16 alpha-dihydroxy-metandienone (XI) and 6 beta,16 beta-dihydroxy-17-epimetandienone (XII)--were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6 beta-hydroxy-metandienone (VII) and 6 beta-hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.

Administration, Oral↗
PubMed2005

The identification of osteoporosis in patients presenting with low-impact fracture: a pilot study of a method for identification of these patients.

OBJECTIVES: To assess the use of a portable ultrasound unit in screening for osteoporosis in older patients presenting to the Emergency Department (ED) with low-impact fractures. METHOD: All patients over 65 years with a possible low-impact fracture were to be identified and screened. Full ethical approval was obtained. An initial pilot study of "non-osteoporotic" ED staff was carried out before the full study was to occur. RESULTS: The pilot study showed portable ultrasound measuring proximal phalanx bone mineral density lacked both precision and accuracy. CONCLUSIONS: The aims of the study were worthwhile. However, the initial pilot study showed that the tools were inadequate for a full study. The full study was abandoned and new ways of identifying these patients are being looked at.

Adult↗
PubMed2006

Multidimensional chromatographic approach applied to the identification of novel aroma compounds in wine. Identification of ethyl cyclohexanoate, ethyl 2-hydroxy-3-methylbutyrate and ethyl 2-hydroxy-4-methylpentanoate.

A multidimensional chromatographic strategy has been developed and optimized with the purpose of identifying different odorants potentially relevant to the aroma and flavor of aged wines from Madeira or Sherry. Different techniques of extraction and fractionation were studied in order to get clear olfactometric and spectrometric signals from the target odorants. The best results were obtained with a dynamic headspace extraction followed by a fractionation on a normal phase medium pressure liquid chromatography on a silicagel column. Large volumes (50 microl) of the concentrated fractions were further analyzed in a dual gas chromatography-mass spectrometric system (GC-MS) equipped with two olfactometric ports. The strategy made it possible to identify in wine by first time the presence of the powerful strawberry-smelling compound, ethyl cyclohexanoate, and of two other novel fruity esters, ethyl 2-hydroxy-3-methylbutyrate and ethyl 2-hydroxy-4-methylpentanoate. Some other unidentified odorants could be isolated and their mass spectra are given.

Butyrates↗
PubMed2006

Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast.

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.

Amino Acid Sequence↗
PubMed2006

Identification and biochemical characterization of a eukaryotic-type serine/threonine kinase and its cognate phosphatase in Streptococcus pyogenes: their biological functions and substrate identification.

A eukaryotic-type signaling system in group A Streptococcus (GAS) was identified and characterized. This system comprises primarily the products of two co-transcribed genes, a eukaryotic-type Ser/Thr kinase (SP-STK) and phosphatase (SP-STP) and their endogenous substrate histone-like protein (SP-HLP). Enzyme activities of SP-STK and SP-STP primarily depended on Mn(2+). The site on the substrate for reversible phosphorylation by these enzymes was found to be only the threonine residue. Using specific antibodies generated against these proteins, SP-STK was found to be membrane-associated with its N-terminal kinase domain facing the cytoplasm and its C-terminal repeat domain outside the membrane and cell-wall associated. Further, SP-STP, primarily a cytoplasmic protein, was found to be a major secretory protein of GAS and essential for bacterial survival. Three isogenic mutants, lacking either the entire SP-STK, or one of its two domains, were found displaying distinct pleiotropic effects on growth, colony morphology, cell division/septation, surface protein/virulence factor expression, bacterial ability to adhere to and invade human pharyngeal cells, and resist phagocytosis by human neutrophils. In addition to these properties, the ability of these three proteins to modulate the expression of the major virulence factors, the M protein and the capsule, indicates that these proteins are structurally and functionally distinct from the kinases and phosphatases described in other microorganisms and play a key role in GAS pathogenesis.

Amino Acid Sequence↗