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PubMed1995

[The application of molecular biology to the diagnosis of mycobacteriosis].

The application of molecular biology techniques to the diagnosis of mycobacteriosis was evaluated. The hybridization protection assay (HPA) was found to be accurate and rapid in the identification of mycobacteria. The nested polymerase chain reaction (PCR) targeting the Pab gene was specific and sensitive enough for the rapid detection of Mycobacterium tuberculosis in clinical specimens. The overall sensitivity and specificity of the nested PCR were excellent, 97% and 92%, respectively. In addition, a novel method combining the PCR and the HPA for the rapid detection of MAC and M. tuberculosis was developed. This method was as useful as the nested PCR for M. tuberculosis above mentioned. The clinical usefulness of two commercially available mycobacteria detection kits, the MTD and the Amplicor, was evaluated and compared with that of the conventional smear and culture methods. The MTD showed the highest sensitivity, while the Amplicor showed the highest specificity. The HPA was also applied to drug susceptibility tests, which require 3 to 4 weeks by conventional methods. By this method, the results of resistance to isoniazid or rifampicin could be obtained after three days incubation. Another method for determining the drug resistance of mycobacteria is the detection of gene alterations related to the drug resistance. The deletion of the catalase-peroxidase gene related to isoniazid resistance was observed in 15% of isoniazid-resistant strains. On the other hand, point mutations in the RNA polymerase beta subunit (rpoB) gene relating to rifampicin resistance were detected in 31% of rifampicin-resistant strains by the non-radioisotope PCR-SSCP analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitubercular Agents↗

PubMed2003

[A molecular epidemiological study on the human immunodeficiency virus infection in Jiangsu province].

OBJECTIVE: To identify subtypes of human immunodeficiency virus 1 (HIV-1) strains and their distribution, infection sources, and the trends of HIV infection in Jiangsu province. METHODS: Anticongulated bloods from 46 infected persons were collected to separate previrus DNA. HIV-1 env genes were then amplified by nested-PCR and sequenced for their C2-V3 region so as to identify subtypes. The analysis of consensus sequence, genetic distance and phylogenetic tree were conducted with GCG software. RESULTS: By the end of 2001, there had been six subtypes of HIV-1 strains identified in Jiangsu province: A, B, B', C, D and E. The predominant subtypes were C (accounting for 40.48%) and B' (accounting for 38.10%). Subtype C accounted for 86.67% among injecting DUs while subtype B' accounted for 91.67% among commercial blood donors and receivers. CONCLUSION: Subtype B'among commercial blood donors was brought to Jiangsu from neighboring provinces. The outbreak of HIV-1 infection among local DUs was caused by subtype C from Xinjiang province. Findings from HIV/AIDS molecular epidemiologic study suggest that it is challenging for Jiangsu to treat patients, apply vaccine, prevent and control AIDS in the future.

China↗

PubMed1996

Tracing the development of single memory-lineage B cells in a highly defined immune response.

To study the development of B lymphocyte memory, we identified and isolated splenic B cells expressing a highly defined antibody variable region that constitutes a reproducible and predominant component of the memory antibody response to p-azophenylarsonate (Ars). Isolation was achieved during the primary immune response by surface staining and flow cytometry using a specific anti-idiotypic antibody called E4, which recognizes this canonical V region, encoded by one set of V gene segments. The isolated E4+ cells displayed all of the phenotypic characteristics of germinal center centrocytes, including a low level of surface Ig, a lack of surface IgD, a high level of receptor for peanut agglutinin, and expression of mutated antibody V genes. E4+ B cells were first detected in the spleen 7-8 d after primary immunization, reached peak numbers from days 10-13, and waned by day 16. Surprisingly, at their peak, E4+ cells comprised only 40,000 of all splenocytes, and half of these failed to bind Ars. Using this number, we estimate the total number of Ars-specific memory-lineage cells in the spleen to be no more than 50,000 (0.1%) at any one time, and presumably far fewer that are committed to the memory pool. Chromosomal copies of rearranged V genes from single E4+ cells were amplified by nested PCR, and the amplified products were sequenced directly without cloning, using standardized conditions that disclose virtually no Taq polymerase errors. V gene sequence analyses of E4+ cells isolated from single mice confirmed their canonical nature and revealed that they were derived from few precursors. In the average mouse, the E4+ pool was derived from fewer than five canonical precursors. Somatic mutations were found within the V genes of almost all cell isolates. At day 13, a significant fraction of E4+ cells had mutations known to increase antibody affinity for Ars, suggesting they were products of at least one cycle of post-mutational antigen-driven selection. However, the lack of shared mutations by clonally related cells indicated that the selective expansion of mutant subclones typical of memory responses had not yet taken place. This was supported by the observation that half of the E4+ cells failed to bind Ars. Collectively, our results indicate that the memory compartment is a highly selected entity, even at relatively early stages of the primary immune response when somatic mutation and clonal selection are still in progress. If germinal centers are the source of memory B cells, our data suggest that B cell memory may be derived from only a small fraction of all germinal centers.

Amino Acid Sequence↗

PubMed2003

[Detection of minimal residual disease in Ph+/bcr-abl+ acute lymphoblast leukemia by cytogenetic analysis, nested-PCR and flow cytometry].

To explore a simple and sensitive method to detect minimal residual disease (MRD) in Ph(+)/bcr-abl(+) ALL patients, the bone marrow samples from 84 de novo ALL patients were detected by cytogenetic analysis, nested-PCR and flow cytometry (FCM). Cytogenetic analysis method is used to detect Ph chromosome, nested-PCR and FCM are used to detect bcr/abl mRNA and an abnormal B-cell differentiation pattern in de novo and complete remission (CR) patients, respectively. The results showed that Ph chromosome has not been found in 14 cases of CR; bcr/abl fusion gene was detected in 11 of 14 CR patients by nested-PCR (78.57%) and bcr/abl fusion gene was positive in 5 of 14 in CR patients (35.71%) by FCM. The sensitivity of nested-PCR was 10(-6)-10(-7), and that of FCM was 10(-4)- 10(-5). It is concluded that the cytogenetic analysis is not sensitive for MRD detection, and the sensitivity of nested-PCR is higher than that of FCM in detecting MRD.

Adolescent↗

PubMed2006

Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events.

BACKGROUND: Platelets participate in events that immediately precede acute myocardial infarction. Because platelets lack nuclear DNA but retain megakaryocyte-derived mRNAs, the platelet transcriptome provides a novel window on gene expression preceding acute coronary events. METHODS AND RESULTS: We profiled platelet mRNA from patients with acute ST-segment-elevation myocardial infarction (STEMI, n=16) or stable coronary artery disease (n=44). The platelet transcriptomes were analyzed and single-gene models constructed to identify candidate genes with differential expression. We validated 1 candidate gene product by performing a prospective, nested case-control study (n=255 case-control pairs) among apparently healthy women to assess the risk of future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) associated with baseline plasma levels of the candidate protein. Platelets isolated from STEMI and coronary artery disease patients contained 54 differentially expressed transcripts. The strongest discriminators of STEMI in the microarrays were CD69 (odds ratio 6.2, P<0.001) and myeloid-related protein-14 (MRP-14; odds ratio 3.3, P=0.002). Plasma levels of MRP-8/14 heterodimer were higher in STEMI patients (17.0 versus 8.0 microg/mL, P<0.001). In the validation study, the risk of a first cardiovascular event increased with each increasing quartile of MRP-8/14 (Ptrend<0.001) such that women with the highest levels had a 3.8-fold increase in risk of any vascular event (P<0.001). Risks were independent of standard risk factors and C-reactive protein. CONCLUSIONS: The platelet transcriptome reveals quantitative differences between acute and stable coronary artery disease. MRP-14 expression increases before STEMI, and increasing plasma concentrations of MRP-8/14 among healthy individuals predict the risk of future cardiovascular events.

Acute Disease↗

PubMed1999

Purification of nuclear DNA from single hair shafts for DNA analysis in forensic sciences.

The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.

Journal Article↗

PubMed2004

Detection of Brazilian bovine respiratory syncytial virus strain by a reverse transcriptase-nested-polymerase chain reaction in experimentally infected calves.

A reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) was standardised to detect bovine respiratory syncytial virus (BRSV), using a Brazilian isolate, in three experimentally infected calves. This followed initial tests in infected chicken embryo related (CER) cells. One animal had lesions, characterized by interstitial multifocal pneumonia, severe interstitial and subpleural emphysema, and lung consolidated areas. Lung and tracheal tissues collected 6 days after infection were analysed by RT-nested-PCR. Primers, specific for the BRSV G and F glycoproteins genes, yielded amplification fragments of 371 and 481 bp, respectively, from the RNA of the cell-propagated virus. Using RNA extracted from organs of infected calves, RT-nested-PCR amplified the fragment of the G gene in all tracheal samples, but in only two of three lung samples analysed. These results suggest that RT-nested-PCR could be a promising assay for diagnosis and epidemiological analysis of BRSV in Brazil.

Animals↗

PubMed2003

[Comparison of the Epstein-Barr virus infection and 30 bp-deleted LMP1 gene among 4 histologic types of nasopharyngeal carcinoma].

OBJECTIVE: To compare the Epstein-Barr virus (EBV) infection rates and the frequencies of wt-LMP1 and del-LMP1 EBV variants detected singly or dually among the four types of nasopharyngeal carcinoma (NPC) and to illustrate the possible role of del-LMP1 gene in nasopharyngeal carcinogenesis. METHODS: EBER in situ hybridization was performed in 117 NPCs, including 48 non-keratinizing carcinomas (NKCs), 25 keratinizing squamous cell carcinomas (KSCCs), 5 adenosquamous carcinomas (ASCs), 6 mucoepidermoid carcinomas (MECs) and 33 adenocarcinomas (ACs). Nested PCR for demonstration of EBV LMP1 gene was performed on the tissue samples collected from 99 EBER-positive carcinoma cases and the peripheral blood mononuclear cells (PBMCs) of 53 healthy adults (HAs). RESULTS: As indicated by EBER in-situ hybridization, the EBV infection rates in both of 48 NKCs and 25 KSCCs were 100%; and the infection rates of 11 ASCs/MECs and 33 ACs were 9/11 and 51.5% (17/33), respectively. Worthy to note was that most of the NKC cells were EBER-positive while only a small number of EBER-positive neoplastic cells could be found in 17 ACs. The percentage of del-LMP1 EBV variant detected singly in NKCs (85.4%, 41/48) was not only significantly higher than that in PBMCs of 46 HAs (8.7%, 4/46) but also significantly higher than those detected in KSCCs (16.0%, 4/25). The dual infection rate of wt-LMP1 and del-LMP1 variants detected in KSCCs (56.0%, 14/25) was significantly higher than that of NKCs (12.5%, 6/48). The majority of the EBV detected in AC tissues (12/17) and HAs' PBMCs (34/46, 73.7%) were of dual wt-LMP1 and del-LMP1 variants. CONCLUSIONS: The EBV infection rates are significantly different among 3 major histological categories, namely, NKC/KSCC, ASC/MEC and AC. Though NKCs and KSCCs are always consistently associated with EBV, the single del-LMP1 EBV variant detected in NKCs is predominant over that in KSCCs and most of the KSCCs contain dual wt-LMP1 and del-LMP1 EBV variants. The EBV of the del-LMP1 variant might play a crucial role in carcinogenesis of NKC.

Adult↗

PubMed2004

Lack of evidence for reovirus infection in tissues from patients with biliary atresia and congenital dilatation of the bile duct.

BACKGROUND/AIMS: To clarify the association between the reovirus infection of the hepatobiliary tree and the development of infantile obstructive cholangiopathy (IOC) including biliary atresia (BA) and congenital dilatation of the bile duct (CBD). METHODS: We designed reovirus common primers for nested RT-PCR based on the L3 gene segment. The spectrum and the sensitivity of common primers were evaluated with purified reoviral RNAs and reovirus mixed with stool samples. Then, nested RT-PCRs were performed with hepatobiliary and fecal samples obtained from patients with BA, CBD, and control diseases. Additionally, electron microscopy of stool samples was performed. RESULTS: The L3 common primers could amplify cDNAs synthesized from RNAs of three prototypes of reovirus, and detect as much as 5.0x10(3) plaque forming unit of serotype 3 Dearing strain in 100 mg of fecal samples. However, no amplification product was detected in 136 hepatobiliary tissues taken from 67 patients including 26 BAs and 28 CBDs, or in 65 fecal samples obtained from 15 patients including 10 BAs and 1 CBD. Additionally, viral particles were not found in any stool specimens by the electron microscope. CONCLUSIONS: These data do not suggest that reoviruses play a major role in the etiology of IOC or BA.

Bile Ducts↗

PubMed1999

Gene conversion can create new MHC alleles.

It is possible to measure gene conversion of MHC genes with the help of a semi-nested PCR assay. Several considerations are of utmost importance when such an assay is set up. Using this assay, we have found that gene conversion occurs in MHC class II genes in mouse sperm, but not in somatic cells tested. Although this gene conversion occurs in germline cells, it is already completed in spermatogonia, and consequently is mitotic event unlinked to meiosis. The frequency of gene conversion events in MHC class II genes varies strongly from one allele to another, with the highest detected frequencies as high as 1/40,000 for an individual heterozygous for both donor and acceptor sequences. Deletions or insertions in one gene relative to the other seem to lower the efficiency of gene conversion considerably. Stretches within MHC genes amenable to gene conversion are located in CpG clusters, whereas MHC genes not involved in gene conversion have background CpG levels. DNA damage, either chemical or radiation induced, increases the frequency of gene conversion of MHC class II genes in cultured cells of the fibroblastoid lineage. The effect of chemical DNA damage seems roughly dose dependent, whereas irradiation has a maximal effect at low doses.

Alleles↗

PubMed1982

Genetic population structure in polygynous formica ants.

Genetic population structures, both mating and interaction structures, were investigated in three polygynous Formica ants by examining how genotype frequencies are distributed among the nests in their populations. The study is based on electrophoretically analysed enzyme polymorphisms. The patterns of genotypic variation among single-nest workers suggest that polygyny is functional in all the three species. The observed genotype frequencies indicate outbreeding within the study areas, and no spatial microdifferentiation in gene frequencies is detected. The coexistent gynes in nests of both F. transkaucasica and F. polyctena are genetically related to each other, and the same holds for worker nest mates. These results support the hypothesis that polygyny is favoured by kin selection. The lack of genetic relatedness among gyne nest mates in the highly polygynous F. aquilonia suggests that additional factors, such as mutualism or parental parasitism, are probably involved in the evolution of polygyny.

Animals↗

PubMed2001

PCR-dot blot hybridization based on the neuraminidase-encoding gene is useful for detection of Bacteroides fragilis.

Bacteroides fragilis is a Gram-negative obligate anaerobe frequently isolated from clinical specimens and sometimes causes severe septicemia in compromised hosts. Increasing interest has been shown in the enterotoxigenicity and drug resistance of B. fragilis in the field of medical microbiology. We previously reported rapid detection of this anaerobe by nested PCR targeting a neuraminidase-encoding gene nanH. In the present study, we synthesized a digoxigenin-labeled oligonucleotide probe, NH1, which is specific for nanH of B. fragilis, and we combined the hybridization assay using NH1 with the nanH-PCR to detect this anaerobe in a bacteremia model mice. In the specificity test, the oligonucleotide probe, NH1, hybridized only to amplification products from B. fragilis. PCR-dot blot hybridization based on nanH enabled detection of cells of B. fragilis in blood samples even when the number was as low as 2 x 10(3) colony-forming units/ml. These findings suggest that PCR-dot blot hybridization targeting nanH is a useful procedure for diagnosis of septicemia caused by B. fragilis when viable cells in blood cannot be detected by the traditional culture techniques.

Animals↗

PubMed2004

Common ataxia telangiectasia mutated haplotypes and risk of breast cancer: a nested case-control study.

INTRODUCTION: The ataxia telangiectasia mutated (ATM) gene is a tumor suppressor gene with functions in cell cycle arrest, apoptosis, and repair of DNA double-strand breaks. Based on family studies, women heterozygous for mutations in the ATM gene are reported to have a fourfold to fivefold increased risk of breast cancer compared with noncarriers of the mutations, although not all studies have confirmed this association. Haplotype analysis has been suggested as an efficient method for investigating the role of common variation in the ATM gene and breast cancer. Five biallelic haplotype tagging single nucleotide polymorphisms are estimated to capture 99% of the haplotype diversity in Caucasian populations. METHODS: We conducted a nested case-control study of breast cancer within the Nurses' Health Study cohort to address the role of common ATM haplotypes and breast cancer. Cases and controls were genotyped for five haplotype tagging single nucleotide polymorphisms. Haplotypes were predicted for 1309 cases and 1761 controls for which genotype information was available. RESULTS: Six unique haplotypes were predicted in this study, five of which occur at a frequency of 5% or greater. The overall distribution of haplotypes was not significantly different between cases and controls (chi2 = 3.43, five degrees of freedom, P = 0.63). CONCLUSION: There was no evidence that common haplotypes of ATM are associated with breast cancer risk. Extensive single nucleotide polymorphism detection using the entire genomic sequence of ATM will be necessary to rule out less common variation in ATM and sporadic breast cancer risk.

Adult↗

PubMed2004

Estrogen plus progestin and risk of venous thrombosis.

CONTEXT: Postmenopausal hormone therapy increases the risk of venous thrombosis. It is not known whether other factors influencing thrombosis add to this risk. OBJECTIVE: To report final data on incidence of venous thrombosis in the Women's Health Initiative Estrogen Plus Progestin clinical trial and the association of hormone therapy with venous thrombosis in the setting of other thrombosis risk factors. DESIGN, SETTING, AND PARTICIPANTS: Double-blind randomized controlled trial of 16,608 postmenopausal women between the ages of 50 and 79 years, who were enrolled in 1993 through 1998 at 40 US clinical centers with 5.6 years of follow up; and a nested case-control study. Baseline gene variants related to thrombosis risk were measured in the first 147 women who developed thrombosis and in 513 controls. INTERVENTION: Random assignment to 0.625 mg/d of conjugated equine estrogen plus 2.5 mg/d of medroxyprogesterone acetate, or placebo. MAIN OUTCOME MEASURES: Centrally validated deep vein thrombosis and pulmonary embolus. RESULTS: Venous thrombosis occurred in 167 women taking estrogen plus progestin (3.5 per 1000 person-years) and in 76 taking placebo (1.7 per 1000 person-years); hazard ratio (HR), 2.06 (95% confidence interval [CI], 1.57-2.70). Compared with women between the ages of 50 and 59 years who were taking placebo, the risk associated with hormone therapy was higher with age: HR of 4.28 (95% CI, 2.38-7.72) for women aged 60 to 69 years and 7.46 (95% CI, 4.32-14.38) for women aged 70 to 79 years. Compared with women who were of normal weight and taking placebo, the risk associated with taking estrogen plus progestin was increased among overweight and obese women: HR of 3.80 (95% CI, 2.08-6.94) and 5.61 (95% CI, 3.12-10.11), respectively. Factor V Leiden enhanced the hormone-associated risk of thrombosis with a 6.69-fold increased risk compared with women in the placebo group without the mutation (95% CI, 3.09-14.49). Other genetic variants (prothrombin 20210A, methylenetetrahydrofolate reductase C677T, factor XIII Val34Leu, PAI-1 4G/5G, and factor V HR2) did not modify the association of hormone therapy with venous thrombosis. CONCLUSIONS: Estrogen plus progestin was associated with doubling the risk of venous thrombosis. Estrogen plus progestin therapy increased the risks associated with age, overweight or obesity, and factor V Leiden.

Age Factors↗

PubMed2004

DNA methylation profiling of cervical squamous intraepithelial lesions using liquid-based cytology specimens: an approach that utilizes receiver-operating characteristic analysis.

BACKGROUND: Cervical carcinoma is a common malignancy among women worldwide, and its pathogenesis is related causally to human papillomavirus infection. The progression from precursor squamous intraepithelial lesions to cervical carcinoma requires additional genetic and epigenetic alterations that have not been characterized fully. The authors examined aberrant promoter methylation of multiple tumor suppressor genes in precursor squamous intraepithelial lesions. METHODS: A multiplex, nested, methylation-specific polymerase chain reaction approach was used to examine promoter methylation of 15 tumor suppressor genes in high-grade squamous intraepithelial lesions (HSIL, n = 11), low-grade squamous intraepithelial lesions (LSIL, n = 17), and negative tissues (n = 11) from liquid-based cytology samples. The area under the receiver-operating characteristic (ROC) curve was determined for individual methylated tumor suppressor genes and for gene combinations to evaluate test performance for the ability of methylation profiles to distinguish HSIL cytology samples from combined LSIL/negative cytology samples. RESULTS: Aberrant promoter methylation of DAPK1 and IGSF4 occurred at a high frequency in HSIL samples and was absent in LSIL and negative samples. There was a significant trend toward increased methylation with the increased severity of lesions, and the mean number of methylated genes was significantly higher in HSIL samples compared with LSIL and negative samples. Using the area under the ROC curve as a measure of test performance, the methylation of IGSF4 and DAPK1 had areas that were significantly greater than 0.5; thus, each had the ability to distinguish HSIL samples from combined LSIL/negative samples. The areas under the curve for the best two-gene combination (IGSF4/DAPK1) and the best three-gene combination (IGSF4/DAPK1/HIC1) were not statistically different from the best individual tumor suppressor gene (IGSF4) in distinguishing HSIL samples from combined LSIL/negative samples. CONCLUSIONS: Aberrant promoter methylation of tumor suppressor genes is an epigenetic alteration that occurs during neoplastic progression to cervical carcinoma. The methylation status of multiple tumor suppressor genes can be evaluated using ROC analysis to determine methylation profiles that can distinguish HSIL samples from combined LSIL/negative samples.

Adolescent↗

PubMed2003

Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA.

The evaluation of disseminated epithelial tumor cells in breast cancer patients has generated considerable interest due to its potential association with disease recurrence. Our work was performed to analyze the usefulness of 5 mucin genes expression (MUC2, MUC3, MUC5B, MUC6 and MUC7), using RT-PCR assays, to detect disseminated cancer cells in patients with operable breast cancer. The highest frequencies of positive RT-PCR tests in breast tumor extracts were observed for MUC5B (7/15) and MUC7 (5/12). The best specificity, negative results on all peripheral blood mononuclear (PBMN) cell samples from healthy donors, were shown for MUC2, MUC5B and MUC6 RT-PCR assays. Thus, we selected MUC5B as a target gene for further evaluation. Using a nested RT-PCR, MUC5B mRNA transcripts were detected in 16/31 primary breast tumors (but not in 36 samples of normal PBMN cells) and in the human MCF-7 breast cancer cell line but not in BT20, MDA, T47D and ZR-75 breast cancer cell lines, indicating that MUC5B mRNA is expressed in a population of breast cancer cells. Using this method, 9/46 patients (19.5%) who underwent curative surgery showed positive MUC5B mRNA in bone marrow aspirates obtained prior to surgery, including 5/24 patients (20.8%) with stage I or II breast cancer, without histopathologic lymph node involvement. These results indicate that MUC5B mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cell dissemination. A comparative evaluation between MUC5B mRNA, cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA in all bone marrow aspirates suggests a putative complementation for molecular detection of disseminated carcinoma cells. Considering that breast cancer is characterized by a great phenotypic heterogeneity, the use of multimarker approach could contribute to tumor cell detection in bone marrow and blood.

Bone Marrow↗

PubMed2001

Identification and tissue distribution of the novel human cytochrome P450 2S1 (CYP2S1).

With the aid of the htgs and dbEST databases, a novel cytochrome P450 cDNA was found by homology searches, and the corresponding gene was identified on chromosome 19. Nested PCR was used to amplify a full-length sequence of 1515 bp. The predicted 504 amino acid sequence displays 38--49% identity with CYP2 family members and the protein was designated CYP2S1. mRNA dot blot analysis demonstrated high expression levels in trachea, lung, stomach, small intestine, and spleen. The expression pattern was confirmed by Northern blot, which also revealed a single transcript of approximately 2.4 kb. Western blot analysis, using an antiserum directed against the C-terminus of the enzyme, detected a protein in human lung with the same mobility as recombinant CYP2S1. Subcellular fractionation and immunostaining revealed that CYP2S1 was localized in the endoplasmic reticulum. We conclude that CYP2S1 represents a novel abundantly expressed human P450 with potential importance for extrahepatic xenobiotic metabolism.

Amino Acid Sequence↗

PubMed2000

The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae.

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called "S" and "L." The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as "S" or "L" based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the "S" sclerotium phenotype, whereas group I strains consisted of both "S" and "L" isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.

Aflatoxins↗