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PubMed1989

Molecular characterization of antithrombin Barcelona-2: 47 arginine to cysteine.

The molecular characterization of antithrombin Barcelona-2 is reported. The abnormal antithrombin was isolated from plasma by chromatography on heparin-Sepharose at pH 6.0, and ion exchange on DEAE-Sephadex at pH 8.6 and 6.0. The tryptic peptides were mapped by reverse-phase HPLC and amino acid sequencing and mass spectrometry showed arginine-47 to be replaced by cysteine. The affinity of Barcelona-2 for heparin is dramatically decreased. The new cysteine does not form a mixed disulphide with DTNB, implying it is present as a disulphide with some other available thiol molecule such as cysteine. This extra bulk at position 47 accounts for the low heparin affinity compared with two other mutations (Rouen-1 47 His; Rouen-2 47 Ser) at this residue. These results confirm the view that Arg-47 is an important residue in heparin binding. No dimers of Barcelona-2 were observed suggesting that steric hindrance of the new cysteine at residue 47 prevents dimerisation.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of the human airway smooth muscle Na+/Ca2+ exchanger.

The Na+/Ca2+ exchanger is an important plasma-membrane Ca2+ transport protein. Functional studies of the effects of removal of external Na+ on airway smooth muscle tone, and studies of Na+-dependent Ca2+ fluxes in membrane vesicles have provided indirect evidence of an important role for Na+/Ca2+ exchange in regulating airway smooth muscle tone. Recent molecular studies have identified seven isoforms of the Na+/Ca2+ exchanger (NCX1) gene in other tissues, but direct molecular characterization of the airway smooth muscle Na+/Ca2+ exchanger has not previously been performed. We have therefore used a reverse transcriptase-polymerase chain (RT-PCR) reaction method and DNA sequencing to study the Na+/Ca2+ exchanger isoform present in human airway smooth muscle. The dominant isoform was found to be virtually identical to the NACA3 isoform originally described in rabbit kidney cortex. The DNA sequence of the Na+/Ca2+ exchanger exon B that we obtained is previously unpublished for any human tissue. This study provides the first definitive molecular evidence of the existence of an Na+/Ca2+ exchanger in airway smooth muscle. The molecular characterization of the human airway smooth muscle Na+/Ca2+ exchanger will facilitate future electrophysiologic studies of its function.

Amino Acid Sequence↗
PubMed2001

Systematic search and molecular characterization of the antigenic targets of myeloma immunoglobulins: a monoclonal IgA from a female patient targeting sperm-specific cylicin II.

The identification of the antigenic stimuli of B-cell neoplasms might be of considerable importance since a causal relationship between these neoplasms and antigenic stimulation has been suggested. To date the identification of such antigens has been erratic and accidental. For a systematic search and molecular characterization of human proteins that are antigenic target structures of myeloma-associated immunoglobulins, we applied SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using a testis cDNA expression library and myeloma proteins from 42 patients. A monoclonal IgA from a female patient was shown to target sperm-specific cylicin II. The specificity of the reaction was confirmed by the characteristic staining of the equatorial belt of human sperm heads by the patient's myeloma protein. Serological analysis of recombinantly expressed cDNAs is a straightforward and high throughput approach for the molecular characterization of the targets of myeloma-associated immunoglobulins. The analysis of the antigenic spectrum of immunoglobulins associated with B-cell neoplasms will provide valuable information for the understanding of the pathogenesis of these diseases.

Aged↗
PubMed2005

Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization of 11 cases.

The microphthalmia with linear skin defects (MLS) syndrome (MIM 309801) is a severe and rare developmental disorder, which is inherited as an X-linked dominant trait with male lethality. In the vast majority of patients, this syndrome is associated with terminal deletion of the Xp22.3 region. Thirty-five cases have been described to date in the literature since the first description of the syndrome in the early 1990s. We now report on the clinical, cytogenetic, and molecular characterization of 11 patients, 7 of whom have not been described previously. Seven of these patients have chromosomal abnormalities of the short arm of the X-chromosome, which were characterized and defined by fluorescence in situ hybridization (FISH) analysis. Intriguingly, one of the patients displays an interstitial Xp22.3 deletion, which to the best of our knowledge is the first reported for this condition. Finally we report on the identification and molecular characterization of four cases with clinical features of MLS but apparently normal karyotypes, verified by FISH analysis using genomic clones spanning the MLS minimal critical region, and with genome-wide analysis using a 1 Mb resolution BAC microarray. These patients made it possible to undertake mutation screening of candidate genes and may prove critical for the identification of the gene responsible for this challenging and intriguing genetic disease.

Abnormalities, Multiple↗
PubMed2025

Isolation and Molecular Characterization of Three Staphylococcus pseudintermedius Strains from Dogs and Humans in Egypt.

Staphylococcus pseudintermedius is an opportunistic pathogen that is largely associated with canine hosts but is becoming more widely recognized as a zoonotic pathogen. Understanding its genetic and phenotypic properties, such as virulence factors and antimicrobial resistance (AMR) profiles, is critical for infection control and vaccine development. In this study, we isolated and molecularly characterized three S. pseudintermedius isolates from dogs (hereafter referred to as S. pseudintermedius D8) and humans (hereafter referred to as S. pseudintermedius H10 and S. pseudintermedius H11) in Egypt. All three isolates showed 100% sequence identity with the nuc gene of the S. pseudintermedius SP_11304-3A reference genome. Multilocus sequence typing (MLST) revealed novel sequence types (STs) in the three isolates. The AMR determinants varied substantially among the isolates. While the mecA gene was absent, blaZ was detected in the canine isolate, indicating beta-lactamase-mediated penicillin resistance. Additionally, tetK and tetM genes were found conferring tetracycline resistance in different isolates. Resistance genes for aminoglycosides, chloramphenicol, fusidic acid, macrolides, streptothricin, and trimethoprim were also identified. All isolates were positive for key virulence genes, including immune evasion (AdsA), coagulase (coa), immunoglobulin-binding protein (sbi/spsK), exfoliative toxin (speta), enterotoxins (se-int and siet), fibrinogen binding protein gene (fnbB), and two-component pore-forming leukocidin genes (lukF and lukS). The S. pseudintermedius H11 isolate uniquely harbored the neuraminidase gene (nanB), while none of the isolates contained the gene coding for immunoglobulin G binding protein (spsQ). These findings highlight the differences in virulence and antimicrobial resistance genes among these S. pseudintermedius isolates, underlining the need for global surveillance and molecular characterization of this pathogen.

Dogs↗
PubMed1993

Molecular characterization of Limulus polyphemus C-reactive protein. I. Subunit composition.

The molecular mass of whole native C-reactive protein (CRP) from Limulus polyphemus, the horseshoe crab, was precisely determined for the first time, by analytical ultracentrifugation after measurement of its absorption coefficient, A1cm, 1% at 280 nm (15.49) and its partial specific volume (0.72 +/- 0.003 ml/g). The apparent weight-average molecular mass, in the range over which it was independent of the concentration, was 300 kDa. Limulus CRP, isolated from an individual animal, was also analyzed by electrospray mass spectrometry after dissociation into its subunits, and nine components were detected in the mass range 24935-25617 Da. Four of these components corresponded precisely to one of the previously reported protein sequences of Limulus CRP subunits which is glycosylated by a single oligosaccharide chain of composition, Man2GlcNAc2, Man3GlcNAc2, Man4GlcNAc2 or Man5GlcNAc2, the structures of which are elucidated in the accompanying paper [Amatayakul-Chantler, S., Dwek, R. A., Tennent, G. A., Pepys, M. B. & Rademacher, T. W. Molecular characterization of Limulus polyphemus C-reactive protein. II. Asparagine-linked oligosaccharides (1993) Eur. J. Biochem. 214, 99-110]. All the remaining components, except one minor component, corresponded to the analogous glycoforms of peptides of molecular mass 24281 +/- 2 Da and 24523 +/- 1 Da, neither of which have previously been characterized. These results are consistent with the presence of 12 subunits in the Limulus CRP molecule, in contrast to the number of subunits in vertebrate members of the CRP family. The CRP family members in vertebrates are known as pentraxins because of the symmetrical pentameric arrangement of their subunits. The concentration of Limulus CRP in hemolymph from 20 individual animals, measured precisely by specific electroimmunoassay, covered the wide range 0.275-6.64 mg/ml, mean (SD) = 1.83 (2.06) mg/ml. It remains to be determined whether this protein is an acute-phase reactant, like its human and some other mammalian homologues.

Animals↗
PubMed1995

Chicken lecithin-cholesterol acyltransferase. Molecular characterization reveals unusual structure and expression pattern.

Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called "reverse cholesterol transport" (RCT) (Vieira, P.M., Vieira, A.V., Sanders, E.J., Steyrer, E., Nimpf, J., and Schneider, W.J. (1995) J. Lipid Res. 36, 601-610), pointing to the importance of this process in nonplacental reproduction. We have begun to delineate the details of this unique transport pathway branch by molecular characterization of the first nonmammalian lecithin-cholesterol acyltransferase (LCAT), the enzyme that catalyzes an early step in RCT. The biological significance of the enzyme is underscored by the high degree of protein sequence identity (73%) maintained from chicken to man. Interestingly, the conservation extends much less to the cysteine residues; in fact, two of the cysteines thought to be important in mammalian enzymes (residues 31 and 184 in man) are absent from the chicken enzyme, providing proof of their dispensability for enzymatic activity. Antibodies prepared against a chicken LCAT fusion protein cross-react with human LCAT and identify a 64-kDa protein present in enzymatically active fractions obtained by hydrophobic chromatography of chicken serum. The developmental and tissue distribution pattern of LCAT in females is striking; during embryogenesis and adolescence, LCAT expression is extremely high in liver but undetectable in brain. Upon onset of laying, however, brain LCAT mRNA increases suddenly and is maintained at levels 5 times higher than in liver, in stark contrast to most mammals. In adult roosters, the levels of LCAT transcripts in brain are lower than in liver. Together with the molecular characterization of chicken LCAT, these newly discovered developmental changes and gender differences in its expression establish the avian oocyte/liver system as a powerful model to delineate in vivo regulatory elements of RCT.

Amino Acid Sequence↗
PubMed2005

Molecular characterization of proteolytic processing of the Gag proteins of human spumaretrovirus.

Molecular characterization of proteolytic processing of the human spumaretrovirus (HSRV) Gag proteins and the precise determination of cleavage sites was performed. For in vitro processing of recombinant HSRV Gag proteins, a recombinant enzymatically active HSRV protease was employed. Recombinant Gag proteins and protease were cloned and expressed as hexa-histidine-tagged proteins in pET-32b and pET-22b vectors, respectively, in the E. coli BL21 expression strain. The recombinant proteins were purified by affinity chromatography on an immobilized metal ion matrix. To determine the precise processing sites, recombinant Gag proteins or synthetic peptides derived from Gag sequences were cleaved in vitro by the recombinant protease. Proteolytic processing reactions were carried out under optimal reaction conditions of HSRV protease in sodium phosphate buffer, pH 6.0, supplied with 2 M NaCl at 37 degrees C. The cleavage sites were determined by amino-terminal amino acid sequencing as well as by matrix-assisted laser desorption/ionization mass spectrometry analysis of the reaction products. Fluorescence spectrophotometry was used to determine cleavage kinetics of peptides mimicking different cleavage sites within the HSRV Gag proteins.

Blotting, Western↗
PubMed1999

Reverse transcriptase polymerase chain reaction-based diagnosis and molecular characterization of a new rat coronavirus strain.

BACKGROUND AND PURPOSE: Rat coronaviruses (RCV) are highly infectious and spread rapidly through laboratory rat colonies, causing sneezing, nasal and ocular discharges, photophobia, and cervical swelling. Current diagnostic methods include serologic testing and histologic examination. During a recent rat coronavirus outbreak, we tested a rapid, noninvasive method of RCV diagnosis that involved use of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to detect RCV RNA on cages housing infected rats. METHODS: The RT-PCR was used to detect RCV RNA in tissues from infected rats and on cages housing infected rats and to amplify portions of the RCV N, M, and S genes for molecular characterization. RESULTS: The RT-PCR detected RCV RNA on cages and in tissues from infected rats. The RCV-NJ N gene is most closely related to the MHV-Y N gene. The M proteins of RCV-NJ and RCV-SDA are 99% homologous, and the six RCV S protein fragments are 97 to 100% homologous. CONCLUSIONS: Use of RT-PCR with cage-swab specimens was capable of diagnosing RCV infection in and viral excretion from rats. Additionally, molecular characterization of the N, M, and S genes of RCV-NJ provided baseline information that can be used in performing further epidemiologic studies.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the creatine kinases and some historical perspectives.

Over the last 15 years, molecular characterization of the creatine kinase (CK) gene family has paralleled the molecular revolution of understanding gene structure, function, and regulation. In this review, we present a summary of advances in molecular analysis of the CK gene family with a few vignettes of historical interest. We describe how the muscle CK gene provided an essential model system to examine myogenic regulatory mechanisms, leading to the discovery of the binding site for the MyoD family of basic helix-loop-helix transcription factors essential in skeletal myogenesis and the characterization of the MEF2 family of factors with an A/T rich consensus binding site essential in skeletal myogenesis and cardiogenesis. Cloning and characterization of the four mRNAs and nuclear genes encoding the cytosolic CKs, muscle and brain CKs, and the mitochondrial (Mt) CKs, sarcomeric MtCK and ubiquitous MtCK, has allowed intriguing study of tissue-specific and cell-specific expression of the different CKs and analysis of structural, functional, regulatory, and evolutionary relationships among both the four CK proteins and genes. Current and future studies focus on understanding both cellular energetics facilitated by the CK enzymes, especially energy channelling from the site of production, the mitochondrial matrix and inner membrane, to various cytosolic foci of utilization, and regulation of MtCK gene expression at the cell and tissue-specific level as models of regulation of energy producing genes.

Amino Acid Sequence↗
PubMed2006

Molecular characterization of an 11q interstitial deletion in a patient with the clinical features of Jacobsen syndrome.

The 11q terminal deletion disorder or Jacobsen syndrome is a contiguous gene disorder. It is characterized by psychomotor retardation, cardiac defects, blood dyscrasias (Paris-Trousseau syndrome) and craniofacial anomalies. We report on a female patient with an approximately 10 Mb interstitial deletion with many of the features of Jacobsen syndrome: A congenital heart defect, dysmorphic features, developmental delay, and Paris-Trousseau syndrome. The karyotype of the patient is 46,XX,del(11)(q24.1q24.3). The interstitial deletion was confirmed using FISH probes for distal 11q, and the breakpoints were characterized by microarray analysis. This is the first molecularly characterized interstitial deletion in a patient with the clinical features of Jacobsen syndrome. The deletion includes FLI-1, but not JAM-3, which will help to determine the critical genes involved in this syndrome.

Abnormalities, Multiple↗
PubMed1997

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.

Acyltransferases↗
PubMed1990

Serologic and molecular characterization of a human monoclonal rheumatoid factor derived from rheumatoid synovial cells.

Molecular characterization of rheumatoid factors (RF) in rheumatoid arthritis (RA) has been hampered because of their polyclonality. To overcome this problem, we generated monoclonal RF-secreting hybridomas from rheumatoid synovial cells. Among the RF-secreting hybridomas, HAF10 secreted an IgM-RF that was monospecific for human IgG. It bound well to IgG1 and IgG2, but not to IgG3 and IgG4. Sequence analysis of its heavy and light chains showed that it contained a VH1 heavy chain and a V lambda light chain that did not belong to any known lambda light chain subgroup, and therefore, probably represented a new lambda subgroup. These results indicated that both the heavy and light chains of a monoclonal IgM-RF from rheumatoid synovial cells were quite different from the reported variable region sequences of several monoclonal RF derived mainly from patients with mixed cryoglobulinemia. Further studies of additional monoclonal RF from RA patients are warranted to define precisely their genetic basis and to further our understanding of the immunopathology of RA.

Amino Acid Sequence↗
PubMed2005

[Molecular characterization of invasive Haemophilus influenzae strains isolated in Tunisia].

We reported a molecular characterization of 25 Haemophilus influenzae strains derived from cases of meningitis and sepsis in children aged less than five years hospitalized in pediatric wards from three hospitals in the Sahel area (Tunisia) during the period 1997-2002. These strains were biotyped and subjected to a capsular typing by Slide agglutination serotyping and Polymerase Chain Reaction (PCR). The genetic polymorphism of these strains was also studied in Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) with two sets of primers: RAP IV and 217 delta(2) as in Pulsed Field Gel Electrophoresis after digestion of the total DNA with the restriction enzyme SmaI (PFGE SmaI). Nineteen strains among 25 (76%) were of biotype I. The bexA gene was highlighted in 13 strains (52%) and in all the cases it was of the type b. Twelve strains (48%) were shown to be unencapsulated by PCR. AP-PCR RAP IV (23 genotypes/25 with a discrimination index ID=0.993) had shown nearly the same discriminatory power than PFGE (20 genotypes/21 strains with a discrimination index ID=0.995). We thus note, how capsular typing by PCR is more sensitive than slide agglutination serotyping. We also note the genetic diversity of the invasive strains isolated with a remarkable presence of non typable strains. AP PCR seems to be an alternative of choice for the epidemiologic follow-up of the Haemophilus influenzae invasive infections.

Child, Preschool↗
PubMed1995

Molecular characterization of Charcot-Marie-Tooth patients in 15 pedigrees from France.

Molecular characterization of Charcot-Marie-Tooth patients in 15 pedigree from France: We collected 15 Charcot-Marie-Tooth (CMT) pedigrees from France. DNA polymorphisms analysis by Southern blotting with probes at the D17S122 locus demonstrated 17p duplication in three CMT1a families and in one sporadic case. Two families affected by CMT2 showed no evidence of the duplication.

Blotting, Southern↗
PubMed2006

Functional and molecular characterization for the damp-obstructed rat model in Chinese medicine.

Functional and molecular characterization was performed on the major organs of damp-obstructed rats by applying expression datasets of microarray experiments and real-time RT-PCR. Gene ontology repertoires, i.e. cellular component, molecular function, and biological process were used to classify differentially expressed genes in the major organs of rats upon treatment of dampness. As to the cellular component, over-expression of genes associated with the plasma membrane was observed in the stomach, spleen, kidney, heart, liver, and lung. Genes associated with translational machinery, endoplasmic reticulum membrane, Golgi apparatus, and nuclear envelope were down-regulated in the stomach. Concerning the molecular function, genes associated with oxidoreductase activity were up-regulated in the stomach, spleen, kidney, lung, and brain. Channel activity, membrane receptor, and electron transporter activity were up-regulated in stomach, kidney, and lung. Regarding the biological process, genes associated with signal transduction were up-regulated in the stomach, while genes associated with biosynthesis and ATP metabolism were down-regulated. In the spleen, melanin biosynthesis was up-regulated while hormone-related activities were down-regulated. In the kidney, genes associated with nucleotide biosynthesis and ATP metabolism were depressed. In the heart and liver, apoptosis was up-regulated while immune response and RAS signal transduction were down-regulated. Interestingly, genes associated with oncogenesis were up-regulated in the stomach and kidney. Functional fingerprints indicated that dampness weakened membrane structures, depressed metabolic activity (especially ATP metabolism), damaged matrix proteins, enhanced signal transduction, and revealed a positive association with oncogenesis. To quantify the functional impact at the molecular level, mRNA levels of key genes were determined by real-time RT-PCR. The results indicated that ATP storage in kidney, spleen, and stomach was depleted in damp-obstructed rats. We propose that oxidative stress, membrane integrity, melanin biosynthesis, ion channel activity, and ATP metabolism might be hallmarks for damp-obstructed rats. Our results also suggested dampness as a pathogenic factor in rats which is possibly associated with enhanced liabilities of cancer.

Animals↗
PubMedDate not supplied

Molecular characterization of G6PD Insuli--a novel 989 CGC --> CAC (330 Arg --> His) mutation in the Indian population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common red cell enzymopathy among humans. G6PD deficiency was reported in India more than 30 years ago and about 13 biochemically characterized variants have been reported. We now describe the molecular characterization of a distinct biochemical variant from India which was previously characterized by us as a unique Class IV variant with fast electrophoretic mobility. The sequence of the G6PD gene from this variant has revealed the presence of a novel 989 G --> A mutation in exon 9 with a predicted amino acid change of Arg330His.

Adolescent↗