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PubMed2001

Clinical and molecular characterization of vancomycin-resistant Enterococcus faecium strains during establishment of endemicity.

To characterize the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) in Tennessee, VREF isolates that were recovered from patients during a 3-year period at a tertiary care center and throughout the state were typed by means of pulsed-field gel electrophoresis. Clinical characteristics and outcomes of patients colonized or infected with different strain types were also examined. A total of 34 different strain types were identified. A single VREF strain (type O) predominated (63 [61%] of 103 single-patient isolates (i.e., 1 isolate per patient) obtained from 7 different health care institutions). There were no significant differences between patients harboring type O strains and those harboring non-type O strains (P> or =.05). The rate of recovery of type O subtypes and strains other than type O increased over the 3-year study period. Establishment of VREF endemicity was initially characterized by clonal spread of type O strains. Subsequently, polyclonal dissemination may have been due to microevolutionary changes among type O strains.

Anti-Bacterial Agents↗

PubMed2006

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping.

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.

Amino Acid Sequence↗

PubMed2006

Molecular characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for multiple human-animal interspecies transmissions.

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian-like VP7 and caprine-like VP4 genes, was isolated from a 2-year-old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000-2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4 gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4 and VP7 genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4 genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.

Animals↗

PubMed2002

Molecular characterization of prostate hyperplasia in prolactin-transgenic mice by using cDNA representational difference analysis.

BACKGROUND: Transgenic mice overexpressing the rat prolactin (PRL) gene develop a dramatic enlargement of the prostate gland. The objective of this study was to characterize the molecular mechanisms in the prostate of importance for the prostate hyperplasia seen in these transgenic mice. METHODS: cDNA representational difference analysis (cDNA RDA) was used to isolate differentially expressed transcripts in the prostate hyperplasia of the transgenic mice compared with wildtype littermates. Furthermore, cDNA microarray analysis was used to verify the RDA output. RESULTS: Here we report 10 transcripts, some of them described to be involved in proliferation and apoptosis, which are differentially expressed in the enlarged transgenic prostates compared with controls. CONCLUSION: The identified differentially expressed transcripts presented herein supports molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human BPH that may contribute to explain the molecular basis of prostate hyperplasia.

Animals↗

PubMed1997

Molecular characterization of a genetically unstable region containing the SMS critical area and a breakpoint cluster for human PNETs.

Recently we demonstrated the clustering of deletion breakpoints in the pericentromeric region of human chromosome 17p in human primitive neuroectodermal tumors (PNETs). Chromosomal disruption was shown to occur between the two markers D17S805 and D17S953, a region previously shown to be deleted in the Smith-Magenis syndrome. To characterize the molecular basis of this genomic instability, we established clone contigs covering this region. An initial physical map of chromosome 17p has been constructed with overlapping sets of YACs. YAC clones were transformed into five clone contigs according to their content of 30 previously known and 16 newly established sequence-tagged sites (STSs). To circumvent the complications inherent in YAC technologies, such as internal deletions, chimerism, and complex rearrangements, we then converted the YAC contigs to PAC and cosmid contigs. Thirty-nine individual PAC/cosmid clones were identified and were used to construct six different PAC/cosmid contigs ranging from 130 to 1200 kb in size and covering approximately 2.5 Mb of genomic DNA. The composite YAC/PAC/cosmid map covers a region of > 6 Mb of genomic DNA consisting of four different clone contigs of up to 2.9 Mb in size. We have demonstrated that three STSs (D17S58, PS1, and D17S842) are duplicated, suggesting the occurrence of low abundant repetitive sequences in this region. By integration of publicly available information we further mapped 10 genes and ESTs to their precise chromosomal positions and thus could exclude or identify them as candidate genes for PNET and/or the Smith-Magenis syndrome.

Base Sequence↗

PubMed1997

Molecular characterization of adenocarcinoma of the cervix.

In an attempt to characterize the molecular alterations of cervical adenocarcinoma, we analyzed 32 paraffin-embedded specimens for the presence of K-ras mutations, p53 overexpression, p16 and Rb protein expression, and the presence of HPV 16 and 18 DNA. Overall 25/32 (78%) of the tumors displayed an abnormality in at least one of these analyses. K-ras mutations were detected by PCR amplification and RFLP analysis in 3 tumors, including 2 at codon 12 and 1 at codon 61. p53 overexpression determined by immunohistochemistry was demonstrated with > 80% of tumor nuclei staining in 4 cases, 10-15% of nuclei staining in 3 cases, and < 1% of nuclei staining in 5 cases. The pattern of staining was diffuse in 6 cases, focal in 1 case, and scattered in 5 cases. Analysis of p16 protein expression in 23 specimens revealed 1 tumor with abnormal staining, while Rb protein expression was determined to be normal in all 25 tumors tested. HPV DNA, detected by PCR with type-specific primers, was found in 16 tumors (50%), including 7 (22%) with HPV 16 and 9 (28%) with HPV 18. There was no correlation among these abnormalities except that the presence of HPV and strong p53 overexpression (> 80% tumor nuclei staining) were mutually exclusive events. Clinical correlation demonstrated that p53 overexpression involving the majority of tumor cell nuclei is characteristic of advanced stage disease, while HPV positivity and activated ras genes are associated with early stage disease.

Adenocarcinoma↗

PubMed1995

Molecular characterization of plasminogen activators in human gingival crevicular fluid.

Plasminogen activators (PAs), a family of serine proteases, and their inhibitors (PAIs) are important in fibrinolysis, wound healing and tissue remodelling. Previous studies revealed differences in the localization of PA activity between healthy and diseased gingival tissues, suggesting that PAs and PAIs could play a part in periodontal homeostasis and disease. PAs and PAIs are synthesized by most of the cells types making up the periodontium and can be identified in gingival crevicular fluid (GCF). These studies sought to characterize the molecular species of PAs and their inhibitors in GCF collected from clinically healthy sites. PA enzymatic activity in GCF samples demonstrated by fibrin zymography revealed the presence of only tissue-type PA (tPA) activity. No urokinase-type PA (uPA) enzymatic activity was detected. tPA enzymatic activity appeared predominantly as an uncomplexed 70-kDa species, although some samples contained enzyme-inhibitor complexes. Quantitation of total tPA by enzyme immunoassay showed a mean concentration of 1.6 ng/microl. Analysis of GCF samples for uPA by immunoblotting and enzyme immunoassay disclosed the presence of small amounts of uPA (0.2 ng/microl), which were present predominantly in activator-inhibitor complexes. Immunoblotting showed specific PAI-2 immunoreactivity bands in high molecular-weight complexes and low molecular-weight degradation products, but less than nanogram amounts of free PAI-2 molecules. Enzyme immunoassay revealed that PAI-2 was present in an at least a seven times greater amount than PAI-1. These observations support the hypothesis that PA-generated proteolysis and its regulation by endogenous inhibitors has a role in the diverse biochemical mechanisms underlying periodontal physiology and pathology including host-microbial interaction, polymorphonuclear leucocyte infiltration, turnover and migration of epithelial cells, connective tissue degradation and remodelling, fibrinolysis and wound healing.

Adult↗

PubMed2006

Burkholderia respiratory tract infections in Italian patients with cystic fibrosis: molecular characterization.

OBJECTIVES: To characterize by molecular techniques Burkholderia strains responsible for respiratory tract infections in cystic fibrosis (CF) patients (children and adults), to assign the Burkholderia cepacia complex (Bcc) isolates to a genomovar and to assess the presence of cblA and esmR genes in bacteria. Unique or sequential Burkholderia isolates (n=48) that had been collected from eight CF children and 17 adults over several (4-6) years were investigated; moreover 11 reference strains were analyzed. METHODS: The microorganisms were identified by using biochemical methods, genotyped by pulse field gel electrophoresis (PFGE) and random-amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR), and assessed by PCR assays for the genomovar and cblA and esmR genes of Bcc. RESULTS: Among isolates 70.8% were identified as Bcc genomovar III-A; one child was infected by Burkholderia ambifaria and four adults were colonized with Burkholderia gladioli. The cblA gene was not detected in any of the isolates, while the esmR gene was detected in the 52.1% of the strains, all belonging to genomovar III-A. CONCLUSION: Molecular analysis of strains revealed in CF patients a colonization with a persistent Burkholderia flora with strains of one genotype. The prevalence of Bcc of genomovar III-A in the two categories of patients and of B. gladioli in four adults demonstrated that transmission may have occurred between subjects. Moreover the B. ambifaria infection demonstrated in a child may be environmentally derived.

Adult↗

PubMed2000

Molecular characterization of the specificity of interactions of various neurotoxins on two distinct nicotinic acetylcholine receptors.

Snake curaremimetic toxins are currently classified as short-chain and long-chain toxins according to their size and their number of disulfide bonds. All these toxins bind with high affinity to muscular-type nicotinic acetylcholine receptor, whereas only long toxins recognize the alpha7 receptor with high affinity. On the basis of binding experiments with Torpedo or neuronal alpha7 receptors using wild-type and mutated neurotoxins, we characterized the molecular determinants involved in these different recognition processes. The functional sites by which long and short toxins interact with the muscular-type receptor include a common core of highly conserved residues and residues that are specific to each of toxin families. Furthermore, the functional sites through which alpha-cobratoxin, a long-chain toxin, interacts with muscular and alpha7 receptors share similarities but also marked differences. Our results reveal that the three-finger fold toxins have evolved toward various specificities by displaying distinct functional sites.

Animals↗

PubMed1999

Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, the gene specific primers were designed and synthesized for 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fragments obtained by 3' and 5' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.

Amino Acid Sequence↗

PubMed2000

Molecular characterization of der(15)t(11;15) as a secondary cytogenetic abnormality in acute promyelocytic leukemia with cryptic PML-RAR alpha fusion on 17q.

A case of acute promyelocytic leukemia (APL) with cryptic PML-RAR alpha fusion on 17q and add(15p) as a secondary abnormality was characterized using molecular cytogenetic techniques. Spectral karyotyping (SKY) showed that chromosome 11 material was added to 15p, forming a der(15)t(11;15), which was refined to der(15)t(11;15)(q13.2;p13) with information obtained by comparative genomic hybridization (CGH). Interstitial insertion of chromosome 15 material into chromosome 17q was found by fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes. This study illustrates the necessity of a combination of molecular cytogenetics to decipher complex karyotypic abnormalities and cryptic translocations in leukemia.

Chromosomes, Human, Pair 11↗

PubMed1997

Molecular characterization of neurotrophin expression and the corresponding tropomyosin receptor kinases (trks) in epithelial and stromal cells of the human prostate.

The prostate is one of the most abundant sources of nerve growth factor (NGF) outside of the nervous system. NGF is a member of the neurotrophin family of growth factors which in mammals also includes brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These neurotrophins can bind with high affinity to a family of tropomyosin receptor kinases (trks). These receptors are trkA, which binds NGF; trkB, which binds both BDNF and NT-4/5; and trkC, which binds NT-3. In order to characterize the molecular expression of the neurotrophins and their corresponding trk receptors in the prostate we performed Northern blot analysis for the neurotrophins and reverse transcription-polymerase chain reaction (RT-PCR) coupled with Southern blot analysis for the trk family of receptors on smooth muscle stromal cells from the prostate, the androgen responsive LNCaP prostate tumor cell line and the androgen refractory TSU-pr1 prostate tumor cell line. The results show that smooth muscle stromal cells expressed NGF, BDNF and trkC, whereas both epithelial cell lines expressed trkA, trkB and trkC to various degrees. NT-3 was not detected in either the smooth muscle stromal cells or in both epithelial cell lines. This suggests that the stromal cell derived NGF and BDNF may interact via paracrine mechanisms with trkA and trkB receptors, respectively, on the adjacent epithelial cells. Interestingly, the androgen responsive LNCaP cell line did not express any of the neurotrophins, whereas the androgen refractory TSU-pr1 cell line expressed NGF, BDNF and NT-4/5. This suggests that the autocrine expression of NGF, BDNF and NT-4/5 is up-regulated in prostate epithelial cells following their transformation to an androgen refractory pathology. Hence, the malignant transformation of prostate epithelial tumor cells may facilitate their escape from a paracrine dependence on stromal cell derived neurotrophins by the acquisition of the autocrine expression of neurotrophins. Since the pathology of malignant cell migration within the prostate is predominantly by direct extension around prostatic nerves the upregulation of autocrine neurotrophin expression within prostate epithelial tumor cells may be concomitant with transformation to a malignant phenotype capable of invasion along the perineural space and extracapsular metastasis to distant sites of tumor formation.

Cell Line↗

PubMedDate not supplied

Molecular characterization of an n-alkane-degrading bacterial community and identification of a new species, Acinetobacter venetianus.

Twenty-five bacterial strains isolated from the Venice lagoon and implicated in the degradation of n-alkanes, n-alkanols, n-alkanals and n-alkanoates were characterized in molecular and physiological terms. The isolates were grouped by amplified ribosomal DNA restriction analysis (ARDRA) into seven clusters, corresponding to seven species, six of which were identified on the basis of 16S rDNA sequencing. Genetic variability among strains was shown by random amplified polymorphic DNA (RAPD). Only strains of the new species Acinetobacter venetianus grew with n-alkanes (C10, C14 and C20) and their respective oxidation products as sole carbon sources. Strains of the other three species identified thrived on n-alkane oxidation products (n-alkanols, n-alkanals, n-alkanoates). The other three species were not able to grow on any of the substrates tested. Analysis of plasmid content showed that only A. venetianus strains harboured plasmids. These plasmids contained sequences homologous to the Pseudomonas oleovorans alkBFGH genes.

Acinetobacter↗

PubMed1995

Molecular characterization and antibiotic susceptibility of Vibrio cholerae non-O1.

A collection of 64 clinical and environmental Vibrio cholerae non-O1 strains isolated in Asia and Peru were characterized by molecular methods and antibiotic susceptibility testing. All strains were resistant to at least 1 and 80% were resistant to two or more antibiotics. Several strains showed multiple antibiotic resistance (> or = three antibiotics). Plasmids most often of low molecular weight were found in 21/64 (33%) strains. The presence of plasmids did not correlate with antibiotic resistance or influence ribotype patterns. In colony hybridization studies 63/64 (98%) V. cholerae non-O1 strains were cholera toxin negative, whereas only strains recovered from patients were heat-stable enterotoxin positive. Forty-seven Bgl I ribotypes were observed. No correlation was shown between ribotype and toxin gene status. Ribotype similarity was compared by cluster analysis and two main groups of 13 and 34 ribotypes was found. Ribotyping is apparently a useful epidemiological tool in investigations of V. cholerae non-O1 infections.

Adult↗

PubMed1992

Molecular characterization of a low-molecular-mass matrix metalloproteinase secreted by glomerular mesangial cells as PUMP-1.

A polymerase chain reaction (PCR)-based homology cloning strategy was used to define the spectrum of stromelysin-like matrix metalloproteinases (MMPs) synthesized by cultured glomerular mesangial cells (MC). Using this technique, cDNAs encoding an unusual, truncated member of the MMP family, punctuated (putative) metalloproteinase (PUMP-1), were exclusively isolated. Incubation with the cytokines interleukin 1 and tumour necrosis factor increased the abundance of PUMP-1 mRNA in mesangial cells. The mesangial PUMP-1 mRNA is processed in a tissue-specific manner, yielding a transcript containing repeated 3'-untranslated region ATTTA motifs commonly found in cytokines with limited mRNA stability. Polyclonal antibodies prepared against the C-terminal region of the PUMP-1 protein documented release of this enzyme by cultures of cytokine-stimulated MC and permitted identification of PUMP-1-expressing mesangial cells within clinical biopsy specimens of acute glomerulonephritis. These findings represent new molecular and clinical evidence that non-malignant cells process and secrete this unusual member of the MMP family in a cytokine-mediated, tissue-specific manner. Mesangial synthesis of PUMP-1 may contribute to the progression of injury during glomerular inflammatory states.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of phenotypically CAMP-negative Streptococcus agalactiae isolated from bovine mastitis.

In the present study three phenotypically CAMP-negative Streptococcus agalactiae, isolated from three cows with mastitis, were characterized by molecular analysis. An identification of the S. agalactiae was performed by conventional methods and by PCR amplification of species specific parts of the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. In addition all three phenotypically CAMP-negative isolates harboured a normal sized CAMP-factor encoding cfb gene indicating a reduced expression of CAMP-factor or a gene defect elsewhere along the pathway of expression. The clonal identity of the three isolates could be demonstrated by macrorestriction analysis of their chromosomal DNA.

Animals↗

PubMed2002

Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.

Alleles↗

PubMed2002

Molecular characterization of ochratoxin A producing strains of the genus Penicillium.

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.

Base Sequence↗