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PubMed1997

Polymorphisms within the human tumor necrosis factor-alpha promoter region in human immunodeficiency virus type 1-seropositive persons.

The question is addressed whether particular tumor necrosis factor-alpha (TNF-alpha) polymorphisms are associated with clinical course and outcome of human immunodeficiency virus type 1 (HIV-1) infection. The distribution of four TNF-alpha guanine (G) to adenosine (A) transition polymorphisms at positions -376, -308, -238, and -163 of the 5' promoter region of the TNF-alpha gene was studied in a nested case-control study among HIV-1-seropositive participants of the Amsterdam Cohort. None of the polymorphisms was significantly associated with long-term asymptomatic survival after HIV-1 infection compared with progression to clinical AIDS. Moreover, specific AIDS-defining illnesses or biologic phenotype of the HIV-1 virus were not associated with TNF-alpha alleles. The results of this study do not point toward a role for known TNF-alpha G to A transition polymorphisms in the clinical course of HIV-1 infection.

Acquired Immunodeficiency Syndrome↗

PubMed2001

A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome.

A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica. The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids. Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.

Alkaloids↗

PubMed2006

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56,419 completely sequenced and manually annotated full-length cDNAs.

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56,419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37,670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants.

Alternative Splicing↗

PubMed2006

H-DBAS: alternative splicing database of completely sequenced and manually annotated full-length cDNAs based on H-Invitational.

The Human-transcriptome DataBase for Alternative Splicing (H-DBAS) is a specialized database of alternatively spliced human transcripts. In this database, each of the alternative splicing (AS) variants corresponds to a completely sequenced and carefully annotated human full-length cDNA, one of those collected for the H-Invitational human-transcriptome annotation meeting. H-DBAS contains 38,664 representative alternative splicing variants (RASVs) in 11,744 loci, in total. The data is retrievable by various features of AS, which were annotated according to manual annotations, such as by patterns of ASs, consequently invoked alternations in the encoded amino acids and affected protein motifs, GO terms, predicted subcellular localization signals and transmembrane domains. The database also records recently identified very complex patterns of AS, in which two distinct genes seemed to be bridged, nested or degenerated (multiple CDS): in all three cases, completely unrelated proteins are encoded by a single locus. By using AS Viewer, each AS event can be analyzed in the context of full-length cDNAs, enabling the user's empirical understanding of the relation between AS event and the consequent alternations in the encoded amino acid sequences together with various kinds of affected protein motifs. H-DBAS is accessible at http://jbirc.jbic.or.jp/h-dbas/.

Alternative Splicing↗

PubMed1994

Protozyme: emerging evidence in nature.

The VMA1 locus in Saccharomyces cerevisiae contains a nested genetic element, the VDE gene, and expresses two functional proteins. A single VMA1 translational product seems to catalyze a self protein splicing in which an internal domain is excised out to produce a site-specific DNA endonuclease and the N- and C-domains are ligated by a transpeptidation reaction to yield a catalytic subunit of the vacuolar ATPase. Accumulating evidence in the past few years suggests that the VMA1 locus encodes a protozyme (for protos en zyme) which has dual roles in life as a protean catalyst for self protein splicing and for self gene homing. Four protozymes that share a common mechanism in protein splicing have been found in four organisms covering three major phylogenic trees.

Amino Acid Sequence↗

PubMed2001

Detection of SYT-SSX fusion gene in peripheral blood from a patient with synovial sarcoma.

This report describes a case involving a 22-year-old pregnant woman with synovial sarcoma in the thigh. The patient recognized an elastic hard mass accompanied by a dull pain in the anteromedial portion of the right thigh. Magnetic resonance imaging delineated a deep soft-tissue mass measuring 9 x 7 x 6 cm. Histologic diagnosis of poorly differentiated synovial sarcoma was made based the results of an open biopsy. In this patient, the SYT-SSX fusion gene transcript was detected by nested polymerase chain reaction (PCR) in the peripheral blood collected before biopsy. Two months after wide local resection of the tumor, multiple lung metastases developed. This is the first reported case in which tumor cells were detected by nested PCR in the peripheral blood of a patient with synovial sarcoma. These findings suggest that circulating tumor cells should be monitored because they may serve as a prognostic indicator for synovial sarcoma.

Adult↗

PubMed2003

A possible role of Helicobacter pylori in chronic rhinosinusitis: a preliminary report.

OBJECTIVE: The objective was to investigate the presence of Helicobacter pylori with polymerase chain reaction in the sinonasal mucosa of patients with or without chronic rhinosinusitis (CRS). STUDY DESIGN: A prospective clinical trial. METHODS: Mucosal tissue samples were collected from ethmoid cells of 12 patients with CRS and the removed mucosal part of the middle concha of 13 patients with concha bullosa who were treated surgically in our institution. DNA extracted from these samples was used for the amplification of 16S ribosomal RNA gene of H pylori by nested polymerase chain reaction. RESULTS: Helicobacter pylori DNA was detected in 4 of 12 patients with CRS, but it was not detected in patients with concha bullosa. Three of four patients with positive results for H pylori had gastroesophageal reflux-related complaints. CONCLUSIONS: It is possible to detect H pylori in the sinus mucosa of some patients with CRS. However, whether H pylori is a causative agent for CRS or a result of CRS is not known.

Adult↗

PubMed2000

Greater diversity of HIV-1 quasispecies in HIV-infected individuals with active tuberculosis.

OBJECTIVE: A continual increase in intrapatient HIV-1 heterogeneity is thought to contribute to evasion of host immune response and eventual progression to AIDS. Tuberculosis (TB) is diagnosed both early and late during the course of HIV-1 disease and may increase diversity of HIV-1 quasispecies by activating the HIV-1 immune response and increasing HIV-1 replication. We examined whether HIV-1 heterogeneity is altered in HIV-1-infected individuals with TB. METHODS: Blood samples were obtained from 7 HIV-1-infected patients with active TB (HIV/TB patients) and 9 HIV-1-infected patients (HIV patients) in Kampala, Uganda (CD4 counts of 0-650 cells/microl and HIV loads of 700-750,000 RNA copies/ml). The C2-C3 region of the HIV-1 envelope gene (env) was amplified by nested polymerase chain reaction (PCR) from lysed peripheral blood mononuclear cells (PBMCs) of each patient, and then subject to sequencing, clonal-quasispecies analysis and heteroduplex tracking analysis (HTA). RESULTS: HTA of env DNA fragments showed increased heterogeneity in the HIV/TB individuals compared with the HIV group. Further sequence and HTA analysis on ten individual env clones for each patient showed significantly greater HIV mutation frequencies in HIV/TB patients than in HIV patients. CONCLUSION: An increase in HIV-1 heterogeneity may be associated with a TB-mediated increase in HIV-1 replication. However, a diverse HIV-1 quasispecies population in HIV/TB patients as opposed to tight quasispecies clusters in HIV patients suggests a possible dissemination of lung-derived HIV-1 isolates from the TB-affected organ.

Adult↗

PubMed1996

PCR detection of Toxoplasma gondii DNA in CSF for the differential diagnosis of AIDS-related focal brain lesions.

To evaluate whether the detection of Toxoplasma gondii DNA in CSF could contribute to the differential diagnosis of AIDS-related focal brain lesions, CSF samples from 88 HIV-infected patients (56 with focal brain lesions and 32 without) were tested prospectively by a nested PCR for the B1 gene of T. gondii. The assay had a detection limit of 10 trophozoite equivalents. Six of 18 patients with toxoplasmic encephalitis, but none of the 70 patients with other disorders, were PCR-positive (33.3% sensitivity and 100% specificity). Considering only those patients with cerebral toxoplasmosis from whom CSF was collected before or during the first week of antitoxoplasmic therapy, sensitivity rose to 50%. This was higher than the sensitivity in patients whose CSF was collected after the first week of treatment (odds ratio (OR) of 7.0; 95% CI: 0.46-218.2). The administration of antitoxoplasmic prophylaxis did not affect the PCR results. Patients with a poor response to therapy had a higher probability of detectable T. gondii DNA in their CSF (OR of 5.0; 95% CI: 0.37-86.6). All patients with other central nervous system disorders were PCR-negative. Despite the moderate sensitivity, the high specificity and positive predictive value (100%) make this assay a useful tool in the differential diagnosis of AIDS-related focal brain lesions as part of a series of CSF and neuroradiological examinations.

AIDS-Related Opportunistic Infections↗

PubMed1995

Human cytomegalovirus DNA is not detectable with nested double polymerase chain reaction in healthy blood donors.

The PCR method was introduced to detect cytomegalovirus (CMV) DNA from 189 peripheral blood samples of volunteer donors. We adopted the nested double PCR method with primers specific for immediate early gene 1 followed by electrophoresis and ethidium bromide staining. This nested double PCR method was sensitive enough to detect approximately a single copy of CMV DNA. However, we failed to obtain positive amplification of CMV DNA from any of these donor samples. In contrast, CMV DNA could be detected in all 3 tested immunocompromised patients who had undergone bone marrow transplantation. These results support our previous report that the frequency of CMV DNA is of an order lower than 1 copy/10(5) leucocytes in the peripheral blood of healthy seropositive individuals.

Adolescent↗

PubMed2003

Influence of climatic factors on Pneumocystis carriage within a socially organized group of immunocompetent macaques (Macaca fascicularis).

As monkeys-derived Pneumocystis is closely related to P. jirovecii, simian populations should be considered as valuable models for the understanding of the epidemiology of human pneumocystosis. In the present study, the impact of environmental factors on the carriage of Pneumocystis was evaluated in socially organized group of immunocompetent macaques (Macaca fascicularis). The tribe, maintained in partial release at the Primatology Center of Strasbourg in France, comprised 29 animals at the end of the study. From December 2000 to November 2002, deep nasal swab samples were collected monthly from each animal under general anaesthesia. The presence of Pneumocystis DNA was assessed by nested PCR of mtLSU rRNA gene. No case of pneumocystosis was reported during the study. Pneumocystis DNA was detected in 166 out of 481 swab samples examined (34.5%). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. However, Pnemocystis carriage was clearly correlated to the mean precipitation rates.

Animals↗

PubMed2003

Evidence of Borrelia lonestari DNA in Amblyomma americanum (Acari: Ixodidae) removed from humans.

We used a nested PCR with Borrelia flagellin gene (flaB) primers and DNA sequencing to determine if Borrelia lonestari was present in Amblyomma americanum ticks removed from military personnel and sent to the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine. In our preliminary investigation, we detected Borrelia sequences in 19 of 510 A. americanum adults and nymphs from Ft. A. P. Hill, Va. During the 2001 tick season, the flaB primers were used to test all A. americanum samples as they were received, and 29 of 2,358 A. americanum samples tested individually or in small pools were positive. PCRs with 2,146 A. americanum samples in 2002 yielded 26 more Borrelia-positive samples. The positive ticks in 2001 and 2002 were from Arkansas, Delaware, Kansas, Kentucky, Maryland, New Jersey, North Carolina, Tennessee, and Virginia. The last positive sample of the 2001 season was a pool of larvae. To further investigate larval infection, we collected and tested questing A. americanum larvae from Aberdeen Proving Ground, Md.; 4 of 33 pools (40 larvae per pool) were positive. Infection of unfed larvae provides evidence of the maintenance of B. lonestari by means of transovarial transmission. Sequence analysis revealed that the amplicons were identical to sequences of the B. lonestari flaB gene in GenBank. Despite the low prevalence of infection, the risk of B. lonestari transmission may be magnified because A. americanum is often abundant and aggressive, and many tick bite victims receive multiple bites.

Animals↗

PubMed2003

Association of the presence of Helicobacter in gallbladder tissue with cholelithiasis and cholecystitis.

The presence of Helicobacter DNA species has been investigated in the biliary epithelium of patients with biliary diseases. However, conflicting results have been observed that may have been due to the small number of subjects studied, difficulty in obtaining a healthy control group, absence of controlling for confounding factors, or differences among populations. Therefore, we investigated the presence of Helicobacter species by culture and nested PCR of 16S rRNA genes in gallbladder tissue and bile from 46 Brazilian subjects with and 18 without cholelithiasis. The control group was mainly composed of liver donors and of patients who had submitted to cholecystectomy as part of the surgical treatment for morbid obesity. No Helicobacter species were grown from the bile or gallbladder tissues. Helicobacter DNA was detected in the gallbladder tissue and bile from 31.3 and 42.9% of the patients, respectively. In a logistic regression model, cholelithiasis was positively and independently associated with the female gender (P = 0.02), increasing age (P = 0.002), and the presence of Helicobacter DNA in the gallbladder tissue (P = 0.009). The presence of Helicobacter DNA in the bile was not associated with cholelithiasis (P = 0.8). A significant association between the presence of Helicobacter DNA in the gallbladder epithelium and histological cholecystitis, even after adjusting for gender and age (P = 0.002), was also observed. The sequences of the 16S rRNA genes were >99% similar to that of Helicobacter pylori. In conclusion, our results support the hypothesis that Helicobacter is associated with the pathogenesis of human cholelithiasis and cholecystitis.

Adult↗

PubMed2004

Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus.

Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 10(6) 50% egg infective doses (EID(50))/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID(50) was determined for all positive samples. The 50% tissue culture infective dose was determined using Madin-Darby canine kidney (MDCK) cells. Samples were also tested by an in vitro enzyme immunoassay test, Directigen Flu A, and by reverse transcription-PCR (RT-PCR) using nested primers from the nucleoprotein gene and a single set of primers from the matrix gene. RT-PCR using the matrix primers and virus isolation in embryonated eggs proved to be the most sensitive methods for the detection of virus. The Directigen Flu A test was the least sensitive method. The inclusion of 2% fetal calf serum in the viral transport medium inhibited the growth of virus from undiluted samples in MDCK cells but was essential for the maintenance of the virus titer in samples subjected to repeated freeze-thaw cycles.

Animals↗

PubMed2006

Investigation of pseudorabies virus latency in nervous tissues of seropositive pigs exposed to field strain.

The prevalence and quantity of latent pseudorabies virus (PrV) in nervous tissues of pigs exposed to field strain in Korea was investigated by nested and real-time PCR. Nervous tissues including trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS) were collected from 94 seropositive pigs. PrV latent infection in nervous tissues was initially investigated by nested PCR targeting three glycoprotein genes (gB, gE, and gG). Based on the obtained result, latent infection was detected in 95.7% of screened animals. Furthermore, it was revealed that the examined tissues harbored different copy numbers of latent PrV genome ranging from <10(2.0) to 10(7.1) copies per microgram of genomic DNA in real-time PCR analysis. These results show that under normal conditions, levels of latent PrV in the nervous tissues of pigs can vary across a wide range. Therefore, the data presented here provides information regarding control of the endemic state of PrV in Korea.

Animals↗

PubMed2004

Absence of hepatitis B virus DNA in patients with hepatitis C and non-A-E hepatitis in the State of São Paulo, Brazil.

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in Sao Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

Brazil↗

PubMed2005

Extraskeletal Ewing's sarcoma of the hard palate.

Extraskeletal Ewing's sarcoma (EES) is a rare soft tissue tumor morphologically indistinguishable from the more common Ewing's sarcoma of bone. We report a case of EES arising in the hard palate of 34-yr-old male patient. Microscopically, the monotonous small round cells without neuronal differentiation showed membranous positive immunoreactivity for MIC2/CD99 and vimentin. Ultrastructurally, the tumor cells showed a few intracytoplasmic organelles without evidence of neurosecretory granules or neurofilaments. The EWS-FLI1 chimeric gene was identified using the nested reverse transcriptase-polymerase chain reaction.

12E7 Antigen↗

PubMed2004

Establishment and assessment of two methods for quantitative detection of serum duck hepatitis B virus DNA.

AIM: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR). METHODS: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography, and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigenin-labeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigenin-labeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed. RESULTS: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay. After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis. Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle. The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P<0.01) between fluorescent qPCR and dot-blot hybridization. CONCLUSION: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.

Alkaline Phosphatase↗