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At least 649 records · Page 36Linked to original sources
PubMed2001

B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection.

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS. Cryosections from an AS-derived SM were stained for B and T lymphocytes. B cells were isolated from different areas of a focus. The rearranged VH genes were amplified by semi-nested polymerase chain reaction (PCR) using oligonucleotides specific for the six different VH families and heavy chain joining segments (JHs). PCR products were cloned and sequenced.Fifty-nine of 70 different heavy chain gene rearrangements were potentially functional. Most of the rearranged genes were mutated (range, 1-15%). Thirty of 70 products had a mutational pattern typical for antigen selection. Most of the rearranged VH genes belonged to the VH3 family (54%), consistent with data from healthy donors and patients with RA, while VH4 genes, in contrast to RA, were identified less frequently (10%) and VH5 genes were over-represented (11%). In contrast to RA, neither VH6 genes nor the autoimmunity-prone VH4-34 were seen, whereas another autoimmunity-prone gene, V3-23, was predominantly used (11%). One VH1-derived and one VH3-derived B cell clone were expanded. CDR3 were shorter and more variable in length than in RA. Comparable with RA and reactive arthritis, there is a biased repertoire of selected VH genes, whereas the panel of represented genes is different and less clonal expansion was observed.

Amino Acid Sequence↗
PubMed1995

Evaluation and comparison of the Amplicor HIV-1 PCR kit with an 'in-house' nested PCR.

BACKGROUND: The polymerase chain reaction (PCR), a powerful gene amplification technique, is moving rapidly from the research laboratory into routine clinical use. OBJECTIVES: To evaluate the specificity and sensitivity of a commercially available PCR kit, the Amplicor HIV-1 PCR kit (AMP) and to compare it with an in-house nested PCR, which amplified part of the POL gene (POL(n)). STUDY DESIGN: A total of 517 samples were tested by AMP, including 159 fresh whole blood specimens from HIV-1 antibody positive Europeans and 358 archival samples (338 seropositive and 20 seronegative individuals) originating from 35 different countries in Africa and Europe. We compared the performance of AMP on the archival samples with POL(n). RESULTS: The overall sensitivity and specificity as compared to HIV-1 serology were 93% and 100%, and 96% and 100%, for AMP and the in-house PCR, respectively. Repeat testing on co-cultured lymphocytes increased the sensitivity of AMP to 95%. CONCLUSIONS: AMP is a rapid, and easy to use commercially available PCR kit, since only one amplification of the test sample is required. Moreover, the use of radioactivity is omitted, and reading of the test can be done with a spectrophotometer. The use of at least one additional primer pair may increase confidence in distinguishing a positive and negative sample by both PCR techniques.

Journal Article↗
PubMed1998

Detection of Bacteroides fragilis enterotoxin gene by PCR.

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.

Animals↗
PubMed2003

Mapping of the bovine herpesvirus 1 glycoprotein C promoter region and its specific transactivation by the viral BICP27 gene product.

We investigated the cis-acting sequences of the promoter regulating the gene encoding the glycoprotein C (gC) of bovine herpesvirus 1 (BoHV-1), a gene of the gamma1 class. S1 nuclease protection assays revealed that gC transcription initiated predominantly at position C18281 of the viral genome, corresponding to 21 and 56 bases downstream from putative TATA-like and CAAT boxes, respectively. To map the gC promoter (pgC), we measured the ability of a series of nested deletions of the region +71 to -1155 with respect to the major gC transcriptional start site, to drive expression of the firefly luciferase (Luc) reporter gene following transient transfection of a cell host, in the absence as well as in the presence of viral factors expressed in trans. We show that the minimal pgC sequences required to drive maximal BoHV-1 independent expression was within the region +71 to -76 which harbours the putative TATA and CAAT boxes, the mRNA start site, and the complete 5' transcript leader sequence. This small pgC fragment was highly trans-activated by the co-expression of the BoHV-1-encoded BICP27 protein, but not BICP0 nor BTIF. In contrast, the pgC fragment spanning the region +71 to -1155 was only minimally trans-activated by BICP27, but substantially stimulated by BICP0. These findings thus suggest that gC gene regulation may involve the combined action of several viral transactivators.

Animals↗
PubMed2005

[Hypermethylation of CpG island of p16 gene and arsenic trioxide induced p16 gene demethylation in multiple myeloma].

OBJECTIVE: To investigate the role of hypermethylation of p16 gene in the pathogenesis of multiple myeloma (MM) and the effect of arsenic trioxide (As2O3) induced p16 gene demethylation. METHODS: Methylation status of p16 gene in MM and U266 cell line exposed to As2O3 were detected the nested-methylation specific PCR. The expression of p16 gene mRNA was determined with RT-PCR. The induced growth inhibition of U266 cell by growth curve and MTT and the DNA content of U266 cell were analyzed with flow cytometry after exposure to As2O3. RESULTS: Hypermethylation of CpG island of p16 gene was observed in 54.8% of the MM patients in our group. p16 gene fail to express in U266 cell line after methylation. As compared with beta-actin, the expression of p16 gene mRNA in U266 cell was increased to 0.22 +/- 0.10, 0.59 +/- 0.11, 0.68 +/- 0.09 after exposure to 0.5 micromol/L, 1.0 micromol/L and 2.0 micromol/L As2O3 for 72 h. CONCLUSIONS: These results indicate that methylation of p16 gene is essential important in the pathogenesis of MM and may provide a new diagnostic technique and drug target for the treatment of MM. As2O3 may activate the expression of p16 gene by demethylation.

Aged↗
PubMed2004

Metabolic primers for detection of (Per)chlorate-reducing bacteria in the environment and phylogenetic analysis of cld gene sequences.

Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (cld) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were cld gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the cld and 16S ribosomal DNA (rDNA) gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.

Amino Acid Sequence↗
PubMed1999

Development of a PCR system for porcine cytomegalovirus detection and determination of the putative partial sequence of its DNA polymerase gene.

After PCR amplification with conservative cytomegalovirus primers, a 520 nucleotide putative partial sequence of the DNA polymerase gene of porcine cytomegalovirus (PCMV) was determined. Sequence comparison revealed homology to DNA polymerase genes from various beta herpes viruses and a dendrogram was constructed depicting the relationship of PCMV to other members of the Herpesviridae family. The dendrogram indicates that PCMV is indeed a beta herpes virus that is more closely related to human herpes virus types 6 and 7 than to type 5. To address the difficulties encountered during conventional PCMV detection and characterization a set of nested PCR primers were constructed which generated DNA fragments of 415 and 257 bp from the DNA polymerase gene. The nested PCR system proved specific for PCMV and provided a novel means for the detection of this poorly characterized herpes virus in pig populations, vaccines and in organs used in xenotransplantation.

Animals↗
PubMed1995

Gene transcripts of eleven proteins with specific functions are all detected in human normal cells and tumor cell lines: a possible DNA-->RNA basal constant flow.

Existance of messenger ribonucleic acids of eleven functionally differentiated proteins in normal human cells and tumor cell lines was investigated using reverse transcription--nested polymerase chain reaction method. Examined gene transcripts were those of progesterone receptor, estrogen receptor, interleukin 2, CD8, parathyroid hormone, cholecystokinin/pancreozymin, glucagon, insulin, adrenocorticotropic hormone, enkephalin and thyroid stimulating hormone. In RT-PCR almost all primers were originally designed and sequences of PCR products were confirmed by the Sanger's method. Investigated cells were normal human peripheral mononuclear cells and their subsets, lymphokine-activated killer cells, gastric mucosal cells, sperm and fifteen established tumor cell lines. All of the examined mRNAs were detected in all of the above cell groups. Therefore, cells of independent tissues or tumors share same kinds of mRNA such as steroid hormone receptors, cytokine, lymphocyte surface molecule and parathyroid, digestive and cerebral hormones. These findings strongly suggest that every cell can express every mRNA. Beneath the cell differentiation there may exist a DNA-->RNA basal constant flow, the arrow of time captured in a cell.

Base Sequence↗
PubMed1995

Novel methods for cloning and engineering genes using the polymerase chain reaction.

Use of the polymerase chain reaction (PCR) has become increasingly widespread in virtually all aspects of molecular biology. Recently, novel ligation-independent methods have been developed for the cloning of DNA fragments amplified using PCR. Ligation-independent cloning utilizing the enzyme uracil DNA glycosylase (termed UDG cloning) provides an efficient method for gene cloning and recombinant PCR. This technology is now being applied to site-directed mutagenesis, the generation of nested deletions, and the engineering of novel gene constructs. The ease and flexibility of this methodology, combined with PCR amplification, simplify gene cloning and engineering techniques.

Animals↗
PubMed2002

[Detection and analysis of bcl-1/IgH rearrangement in mantle cell lymphoma]

OBJECTIVE: To investigate the specificity and sensitivity of the PCR technique in the identification of bcl-1/IgH gene rearrangement in mantle cell lymphoma (MCL) and to characterize the bcl-1/IgH junction DNA sequences. METHODS: A semi-nested PCR method to amplify bcl-1/IgH gene rearrangement in DNA from fresh frozen lymphonode specimen was established. Twenty-eight cases of mantle cell lymphoma were analyzed for the presence of bcl-1/IgH gene rearrangement. The rearrangement products was cloned and sequenced to recognize the junction sequences, the breakpoints in the bcl-1 region, and J(H) gene involved in the rearrangement. RESULTS: A bcl-1/IgH gene rearrangement was detected in 17 out of 28 cases of MCL, while only 9 cases was detected with single step PCR method (X(2)=4.59, P<0.05). The rearrangement product varied in size between 74 to 162 base pairs, and the length of the junction sequences ranged from 6 to 24 base pairs. Ten different bcl-1 breakpoints were clustered within 65 base-pair spans, among which, 5 breakpoints (located at 430, 440, 451, 486 and 492) were never reported by other authors. The most common J(H) gene segments utilized in the translocation were J(H) 4 (8/18), then J(H)5 (3/18), J(H)6 (2/18), J(H)4/5 (2/18). J(H)1 2/18, and J(H)3 (1/18). CONCLUSION: These results indicate that the semi-nested PCR is a specific and sensitive method for the detection of bcl-1/IgH gene rearrangement in mantle cell lymphoma, which has implications for both the diagnosis and clinical management of mantle cell lymphoma. The recognization the potential mechanism of bcl-1/IgH gene rearrangement will help us to know the exact pathogenic machanisms of MCL.

Journal Article↗
PubMed1996

Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay.

A simple approach for the determination of drug susceptibilities by using human immunodeficiency virus type 1 (HIV-1) RNA from the sera of patients is described. HIV-1 RNA was extracted from patient sera, and the 5' part of the reverse transcriptase (RT) gene was transcribed into DNA and amplified in a nested PCR. The amplified fragment covers the 3' part of the protease gene and amino acids 1 to 304 of the RT gene. This fragment can be introduced through homologous recombination, as described previously, into a novel HIV-1 reference strain (pHXB2 delta 2-261RT) from which amino acids 2 to 261 of RT have been deleted. The resulting recombinant virus expresses all properties of the HXB2 reference strain except for those encoded by the introduced part of the patient RT gene. Recombinant viruses were subsequently tested for drug susceptibility in a microtiter format killing assay [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay] as well as in the standard HeLa CD4+ plaque reduction assay. Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from patients receiving alternating nevirapine and zidovudine treatment or lamivudine-zidovudine combination therapy. In conclusion, this approach enables high-through-put determination of the drug susceptibilities of serum RNA-derived RT genes, independent of the patient's viral background, and generates the possibility of relating changes in susceptibility to changes in viral genotypes.

Anti-HIV Agents↗
PubMed2000

Detection of circulating cancer cells with von hippel-lindau gene mutation in peripheral blood of patients with renal cell carcinoma.

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene have been detected in up to 60% of sporadic clear cell renal carcinomas (RCCs). Even patients with RCCs believed to be curable with radical nephrectomy sometimes develop distant metastasis 5-10 years after surgery, suggesting hematogenous circulation of cancer cells. Useful tumor markers have not yet been established for RCC. To detect patients at high risk of metastasis after surgery, we developed a highly sensitive and specific nested reverse transcription-PCR method using VHL gene mutation to detect circulating cancer cells. We screened 29 sporadic clear cell RCCs from patients for mutations of the VHL gene by direct sequencing. We next examined blood samples from patients with the VHL gene mutation using mutation-specific nested reverse transcription-PCR. Somatic mutations were detected in 20 of 29 (69.0%) sporadic clear cell RCCs. The VHL gene mutations were detected in peripheral and/or renal venous blood from 15 of 20 (75%) patients. The mutations were detected in the peripheral blood in 2 of 17 (11.8%) patients before surgery, 6 of 16 (37.5%) patients within 24 h after surgery, 3 of 16 (18.8%) patients on day 7 after surgery, and 2 of 11 (18.2%) patients on day 30 after surgery. In seven of nine (77.8%) patients, mutations were detected in renal venous blood during surgery. These findings indicate the presence of circulating cancer cells with VHL gene mutation. Although much larger studies are needed to determine the clinical significance, our study shows that this technique is feasible for detecting circulating RCC cells.

Adult↗
PubMed2002

Deletion of the FHIT gene in human colorectal cancer is independent of high-risk HPV infection.

BACKGROUND AND AIMS: The fragile histidine triad (FHIT) gene, which is frequently lost in many cancers, has been identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. Human papillomaviruses (HPVs) are the major cause of cervical carcinoma and have been found to be able to integrate its genes into the chromosome 3 fragile site of cultured cells, deleting a piece of DNA which includes the FHIT gene. MATERIALS AND METHODS: We used nested reverse transcriptase PCR and DNA sequencing to evaluate 32 cases of colorectal adenocarcinoma and matched nearby normal tissues for aberrant transcripts of FHIT and infection of high-risk HPVs. RESULTS: Aberrant transcripts of the FHIT gene were observed in 34.4% of colorectal adenocarcinoma and in 6.3% of matched nearby normal tissues. The HPV(16) DNA was detected in 21.9% of colorectal adenocarcinomas and 3.1% of matched nearby normal tissues using PCR. Only two cases of colorectal adenocarcinoma contained both aberrant transcripts of FHIT and HPV(16) infection. No HPV(18) infection was detected in the present study. CONCLUSION: Our results indicate that alteration of the FHIT gene and HPV(16) infection are important genetic events associated with colorectal adenocarcinoma. However, there is no correlation between FHIT abnormalities, clinicopathological features, and HPV(16) infection in colorectal adenocarcinoma.

Acid Anhydride Hydrolases↗
PubMed1995

Detection of bcl-1 gene rearrangement and B-cell clonality in mantle cell lymphoma using formalin-fixed, paraffin-embedded tissues.

Mantle cell lymphoma (MCL) has recently emerged as a distinct clinicopathologic entity with characteristic molecular genetic features. Specifically, MCL are clonal B-cell neoplasms and often harbor bcl-1 gene rearrangements. Although this genetic profile is well documented, scant or no data are available on the molecular assessment of MCL using formalin-fixed, paraffin-embedded tissue as a sample source. The polymerase chain reaction (PCR) was employed to study bcl-1 and immunoglobulin heavy chain (IgH) gene rearrangements (B-cell clonality) using formalin-fixed tissue from 12 cases of MCL. In addition, 12 cases of low grade B-cell lymphoma and 5 cases of reactive lymphocytic hyperplasia were studied as comparison controls. A hemi-nested PCR assay was developed to identify major translocation cluster (MTC) bcl-1 gene rearrangements, whereas IgH gene rearrangements were evaluated by both a single-step and hemi-nested approach. Bcl-1 gene rearrangements were amplified in 4 of 12 (33%) MCL, but in none of the controls. With the hemi-nested approach, B-cell monoclonality was demonstrated in 11 of 12 (92%) MCL; 6 of 6 (100%) small lymphocytic lymphomas; 1 of 2 marginal zone lymphomas; 1 of 4 follicular lymphomas; and 0 of 5 reactive lymphocytic hyperplasias. When one-step PCR was used for B-cell clonality assessment, the overall detection rate was lower, specifically: 8 of 12 (67%) MCL; 4 of 6 (67%) small lymphocytic lymphomas; 1 of 2 marginal zone lymphomas; 0 of 4 follicular lymphomas; and 0 of 5 reactive lymphocytic hyperplasias were identified as monoclonal. We have demonstrated that MTC bcl-1 gene rearrangements can be amplified from formalin-fixed tissue. In addition, monoclonal B-cell populations from MCL are better amplified with a hemi-nested approach rather than a single-step PCR assay. With specialized nucleic acid isolation techniques and appropriate PCR protocol design, formalin-fixed, paraffin-embedded tissue is an adequate source of DNA for assessing MTC bcl-1 and IgH gene rearrangements.

B-Lymphocytes↗
PubMed2001

Sensitive detection of Borrelia burgdorferi sensu lato DNA and differentiation of Borrelia species by LightCycler PCR.

In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.

Bacterial Proteins↗
PubMed1992

csgA expression entrains Myxococcus xanthus development.

The development cycle of the myxobacterium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring approximately 20% maximum csgA expression, fruiting body formation requiring approximately 30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending approximately 930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.

Base Sequence↗
PubMed2003

Bovine herpesvirus 5 (BoHV-5) in bull semen: amplification and sequence analysis of the US4 gene.

Bovine herpesvirus type 5 (BoHV-5), which is potentially neuropathogenic, was detected in clinical samples of bovine semen, both directly and after isolation in cell culture, using a nested PCR system for amplifying the US4 gene. Nucleotide sequences generated from the amplicons were analysed and deposited at GenBank (NCBI, Bethesda, MD, USA) under the accession numbers AF298174 and AF330157. Alignment of these sequences and previously deposited sequences of BoHV-1 and BoHV-5 showed 82% and 98% similarity, respectively. The bulls, which were maintained at an artificial insemination centre, had presented no clinical signs, indicating that bovine semen should be screened for BoHV-5 to prevent transmission of the virus.

Agglutination Tests↗