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PubMed2001

Emx2 directs the development of diencephalon in cooperation with Otx2.

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.

Animals↗

PubMed1995

Increased number of cells and metabolic activity in male human preimplantation embryos following in vitro fertilization.

The number of cells and metabolic activity of male and female human preimplantation embryos were examined to determine whether male embryos are more advanced than female embryos following in vitro fertilization (IVF). The metabolic activity of embryos fertilized normally was assessed daily by non-invasive measurement of pyruvate and glucose uptake and lactate production between days 2 and 6 after insemination. On day 6, the numbers of nuclei from the trophectoderm and inner cell mass of blastocysts were counted by differential labelling and fluorescence microscopy. Nuclei were then recovered and the sex of the embryos identified using nested primers to amplify the amelogenin gene and pseudogene sequences on the X and Y chromosomes, respectively. Development of male and female embryos were then compared retrospectively. From 69 of 178 (39%) embryos that developed to the blastocyst stage, the sex of 57 was determined; 21 (37%) were male and 36 (63%) female. The number of cells in male embryos was significantly greater on day 2 (P < 0.005), and this difference was maintained up to the blastocyst stage (in both the trophectoderm and the inner cell mass), although differences were not always significant. Pyruvate uptake was significantly higher by male embryos between days 2 and 5 (P < 0.05). Glucose uptake and lactate production were significantly higher in male embryos on days 4-5 (P < 0.05); this difference was not significant on days 5-6. Extrapolation from differences in the number of cells indicates that female embryos are approximately 4.5 h delayed in their development from day 2 onwards compared with male embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Blastocyst↗

PubMed2001

[VP7 typing of rotavirus from children with diarrhea in Chongqing Region].

OBJECTIVE: To study VP7 type of rotavirus from children with diarrhea in Chongqing area from 1998 to 2000. METHODS: Double-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. Nucleotide sequence analysis was used for VP7 typing. RESULTS: Among the 130 fecal specimens from pediatric patients with acute gastroenteritis during the epidemic season of 1998-1999, fifty specimens (38.46%) were identified as rotavirus-positive by RT-PCR. Of these, serotype G1 was found to be largely predominant and accounted for 88% (44/50), while serotype G3 accounted for 8% (4/50), coinfection of G1 and G3 accounted for 4% (2/50) respectively. While during the next epidemic season from 1999 to 2000, thirty-eight (33.93%) rotavirus positive specimens were detected from 112 stool samples by RT-PCR. Serotype G3 increased significantly-up to 78.95% (30/38) and became the most prevalent serotype in that season. It was found that only 13.16% (5/38) were serotype G1, 7.89% (3/38) were G1 and G3 mixture. The nucleotide sequence analysis results agreed with the RT-PCR typing assay. CONCLUSIONS: Serotype G1 and G3 were the prevalent VP7 types in Chongqing, China from 1998 to 2000. A shift from G1 to G3 was observed in the last two successive epidemic seasons of rotavirus epidemics.

Amino Acid Sequence↗

PubMed2002

Sequence analysis and genotypes of glutamate rich protein of Plasmodium falciparum isolates from different malaria endemic areas in China.

OBJECTIVE: To sequence the gene encoding glutamate rich protein (GLURP) and identify the genotypes of geographically different Plasmodium falciparum (P. f) isolates from China. METHODS: The gene of R2 repeat region of GLURP was amplified by nested polymerase chain reaction and cloned into T-vector. The nucleotide sequence of GLURP gene was determined by automatic sequencer (Dideoxy termination method) and analyzed by DNA Star software. RESULTS: At least 7 different GLURP genotypes ranging from 600 bp to 1,500 bp were found in Yunnan and Hainan provinces. R2 region of GLURP gene consisted of several repeat units. Each repeat unit was composed of 19-20 residues which were shown to be highly conserved. GLURP gene was also size polymorphic due to differences in the number of repeat units, whereas the repeat sequence was conserved. Sequence analysis showed that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or various isolates from the same geographical region. No obvious differences were found in the GLURP gene sequences among geographically different isolates. CONCLUSION: GLURP gene is highly structure conserved and size polymorphic, and so is useful in searching for malaria vaccine candidate antigen and developing a genotyping method for malaria research.

Amino Acid Sequence↗

PubMedDate not supplied

Comparison between Toxoplasma gondii DNA and specific immunoglobulins during pregnancy.

Early diagnosis of toxoplasmosis in pregnant women can be of great help in early intervention and prevention of congenital disorders that usually lead to fetal death. The purpose of the present study was to evaluate nested PCR amplification of the B1 gene of Toxoplasma gondii before and after treatment and in comparison to serological follow-up during treatment. The efficiency of treatment on the bases of PCR detection of T. gondii DNA was statistically significant, while it was insignificant when anti-toxoplasma specific IgM and IgG antibodies were used. PCR detection of T. gondii DNA when performed on whole blood is a rapid, sensitive and specific diagnostic procedure and is a valuable tool for establishing the diagnosis of T. gondii infection in women before or during pregnancy.

Adult↗

PubMed2004

[Amplification and cloning of the N gene of SARS-associated coronavirus].

OBJECTIVE: To amplify and clone the N gene of severe acute respiratory syndrome-associated coronavirus. METHOD: Using primer Premier 5.0 software, two pairs of nested PCR primers were designed to amplify the N gene. After purification, the amplified products were cloned into pMD18-T vectors, and the positive clones with the inserted fragments were identified by sequence analysis. RESULTS: The amplified products was about 1 375 bp in length, and sequence analysis demonstrated that the N gene fragments had been successfully inserted into pMD18-T vectors. CONCLUSION: The successful amplification and cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.

Base Sequence↗

PubMed2004

Evaluation of a new plate hybridization assay for the laboratory diagnosis of imported malaria in Italy.

A new molecular diagnostic method "Malaria-IBRIDOGEN" (Amplimedical S.p.A.--Bioline Division, Turin, Italy) based on a plate-hybridization assay for the simultaneous detection and identification of human malaria parasites was evaluated in this study. A target DNA sequence of the plasmodial 18S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and hybridized in microtiter wells with five biotinylated probes each specific for Plasmodium falciparum, P. vivax, P. malariae, P. ovale and the beta-globine human gene, respectively. Compared to the nested-PCR actually used in our laboratory for the molecular diagnosis of malaria, "Malaria-IBRIDOGEN" revealed an overall sensitivity of 100% (51/51) for the four human Plasmodium species testing 100 whole blood samples from people with malaria-like symptoms and fever. Specificity was 92% (45/49) considering four discordant samples as "false positive" by "Malaria-IBRIDOGEN". The assay showed a threshold of parasite density (detection limit) of 0.07 P. falciparum parasites/microliter, 0.15-1.5 P. vivax parasites/microliter, 0.3 P. malariae parasites/microliter and 0.4 P. ovale parasites/microliter of whole blood, respectively. This assay could be successfully applied to the laboratory diagnosis of malaria as a useful aid to microscopy.

Animals↗

PubMed1991

Detection of human herpesvirus 6 DNA in T and B cell lymphomas.

DNA extracted from tissues of 109 patients with lymphoproliferative disorders, including 40 T-cell lymphomas, 40 B-cell lymphomas, 8 Hodgkin's disease, 13 hyperplasia, 5 infectious mononucleosis, 2 histiocytic medullary reticulosis and 1 Kikuchi's disease, and peripheral blood lymphocytes (PBL) from 17 healthy donors were investigated for the presence of human herpesvirus 6 (HHV-6) DNA sequence by polymerase chain reaction (PCR) and Southern hybridization. The procedures for DNA amplification were composed of the first round of PCR using a pair of primers LL and LR, which were contained in the HHV-6 major capsid gene, and the second round of nested PCR using an internal pair of primers LLN and LRN. Results showed that the prevalence of HHV-6 DNA sequence in human tissues was similar in both B and T cell lymphoma patients and normal adults, and viral heterogeneity was detected in the amplified DNA sequence from local strain of Taiwan as compared to that of U1102 strain from Uganda.

DNA, Viral↗

PubMed2006

[Molecular detection and subtyping of Treponema pallidum subsp. pallidum in clinical specimens].

An in-house two-step nested PCR amplification targeting the tmpC gene (TP0319, encoding putative membrane lipoprotein) was used for detection of chromosomal DNA of Treponema pallidum subsp. pallidum in clinical specimens. We tested 138 blood serum samples from 111 adult patients with suspected, primary, secondary, early or late latent syphilis. T. p. pallidum DNA was not detected in any of the analyzed specimens. Out of 11 mucocutaneous swabs (7 genital and 4 pharyngeal), 6 collected from 3 patients with primary or secondary syphilis tested positive. One skin swab from a patient with early congenital syphilis was also positive as were his serum and cerebrospinal fluid samples. DNA sequencing of the genes TP0136 and TP0548 from the positive samples revealed two strains with DNA sequences identical to that of T. p. pallidum strain SS14 and two unique previously undescribed T. p. pallidum strains. The advances in molecular typing of T. p. pallidum in clinical specimens will be of relevance to the epidemiology of syphilis and will allow for clinical discrimination between reinfection and syphilitic reactivation.

Adult↗

PubMed1994

Can tenascin be redundant in cancer development?

Histological and biochemical analyses of tenascin in various human tumors have indicated that tenascin is expressed in various cancer stroma and increased in the serum, getting strong with advancement of its malignancy. Of interest, the prognostic analysis of breast and colon cancers revealed favorable survival and no lymphogenous metastasis in patients whose cancer expressed tenascin strongly. Injection of tenascin nonproducing A431 human epidermoid cancer cells into nude mice resulted in tenascin production by these cells, suggesting cancer cells can make tenascin if necessary. Thus, both carcinoma and adjacent stroma cells may produce tenascin to coordinate the microenvironment surrounding the cancer tissues. Several tenascin variants have been clearly demonstrated to date. With these findings in mind, we would propose that epithelial tenascin supports the carcinoma cell outgrowth, whereas stromal tenascin may block cancer invasion by covering the cancer nest. No obvious phenotype in tenascin gene knockout mice would indicate that tenascin is functionally redundant in developmental processes, yet it may well be very important in progression of cancer.

Animals↗

PubMed1993

[Detection of Helicobacter pylori in human saliva by using nested polymerase chain reaction].

Epidemiologic studies suggest person to person spread of Helicobacter pylori (H. pylori), but the exact mechanism is unknown. Spread through oral secretions has been suggested, however, it has proved very difficult to grow the organism from areas outside the stomach. A nested polymerase chain reaction (N-PCR) for the specific detection of H. pylori was developed with two primer pairs (nested primers) derived from the urease gene A of H. pylori. The N-PCR could detect all 21 H. pylori strains, including 20 isolated strains and 1 reference strain NCTC 14126, but could not detect other bacterial species, showing the N-PCR assay to be 100% specific. Tenfold serial dilution experiments revealed the detection of as little as 0.1 fg DNA by N-PCR. To evaluate the PCR assay for clinical samples, gastric biopsy and aspirate specimens were tested by N-PCR, and the results were compared with those of culture, urease test and histologic examination (reference standard, RS). Among 57 biopsy specimens, H. pylori sequence was detected by PCR in 39 of 39 (100%) positive tissues and in none of 18 negative tissues. H-pylori was detected in saliva of 11 out of 19 cases in which H. pylori was positive in gastric mucosa by PCR. Whereas, PCR was positive in saliva of only one out of 8 cases in which H. pylori was negative in gastric mucosa. Six gastric aspirate specimens were positive by N-PCR. PCR is an accurate and sensitive method that can detect the presence of H. pylori without the need of culture.

Adolescent↗

PubMed1997

[Chlamydia trachomatis infection in asymptomatic Chilean men and with urethritis. Usefulness of first catch urine samples].

BACKGROUND: Chlamydia trachomatis is responsible for 30 to 50% of genital tract infections and is present, without symptoms, in 20% of men and 60% of women. We have little information in Chile about the prevalence of this infection. AIM: To assess the prevalence of Chlamydia trachomatis in men, using first catch urine samples. SUBJECTS AND METHODS: Three hundred and fifty one first catch urine samples of asymptomatic men and 50 samples coming from men with a primary urethritis, were analyzed. Urethral discharge samples from the latter were simultaneously studied. Analysis was performed using an enzyme immuno analysis (MicroTrak Chlamydia EIA, Syva Co.) and a nested polymerase chain reaction towards the gene that codifies MOMP (PCR/OMP). RESULTS: Among asymptomatic men, two of 154 teenagers aged 18 to 19 years old (1.3%), 10 of 100 university students (10%) and eight of 97 adults over 30 years old (8.2%), were infected. The global prevalence of infection in these men was 5.7%. The prevalence of infection in men with urethritis was 12%. Urine EIA had a higher detection frequency than PCR/OMP, but according to another PCR assay, these results were false positives. EIA in first catch urine, had a sensitivity, specificity, positive and negative predictive value of 83.3, 75, 31.3 and 97% respectively, for the detection of Chlamydia trachomatis. The figures for PCR/OMP were 100% for all these parameters. CONCLUSIONS: The prevalence of Chlamydia trachomatis infection in Chilean men is similar to that reported in developed countries. Enzyme immuno assay in first catch urine had a good diagnostic accuracy and could be used in epidemiological studies in asymptomatic men.

Adolescent↗

PubMed1995

Evaluation of nested polymerase chain reaction for detecting mycobacterial DNA in pleural fluid.

A protocol based on the polymerase chain reaction (PCR) is the most sensitive method for detecting mycobacteria in clinical samples. However, few studies have assessed the usefulness of this method in the diagnosis of tuberculous effusion. We developed a highly sensitive and specific nested PCR method, that amplifies the bovine tuberculous MPB70 gene and the mycobacterial 16S rRNA gene for use in detecting Mycobacterium tuberculosis (M. tuberculosis) and mycobacteria, respectively, in clinical samples. We determined the sensitivity of this method for detecting mycobacteria in samples containing known amounts of mycobacterial DNA and in DNA extracted from pleural effusions obtained from 10 patients with pulmonary tuberculosis in whom standard microbiological techniques had detected mycobacteria in sputum but not in pleural effusion. The nested PCR method for the bovine tuberculous MPB70 gene and the mycobacterial 16S RNA gene was able to detect M. tuberculosis and mycobacterial genomes only if there were at least 2 copies per sample. Positive results for M. tuberculosis and the mycobacterial genomes were obtained by nested PCR in 2 of 10 and in 3 of 10 samples of pleural fluid, respectively but no mycobacteria were detected in malignant effusions obtained from 9 patients with lung cancer. The nested PCR method represents a rapid means for detecting mycobacteria in some pleural effusions previously found to be negative by culture. We speculate that the reaction of the host against mycobacteria is more important than the mycobacteria themselves in the pathogenesis of pleural effusion in which mycobacteria are not detected.

Base Sequence↗

PubMed2000

Temporal and Spatial Distribution of the nifH Gene of N(2) Fixing Bacteria in Forests and Clearcuts in Western Oregon.

Decomposition of plant litter is a primary mechanism of nutrient recycling and redistribution in most terrestrial ecosystems. Previously we demonstrated by a nested PCR protocol that 20 distinctive nifH (the gene encoding nitrogenase reductase) HaeIII restriction fragment length polymorphism (RFLP) patterns were derived from bulk DNA associated with samples of plant litter and soil collected at one Douglas Fir (DF) forest [33]. Five of the nifH DNA patterns (II-VI) were dominant types in DF litter with characteristic fragments of 237-303 bp length, whereas samples from soil contained primarily seven other patterns 131-188 bp length (IX-XV). Here we report that the 237-303 bp fragments characteristic for forest litter could generally not be detected in plant litter or soil samples collected in clearcuts that adjoin the forest sites. The same fragments (237-303 bp) were also found in the litter at this DF forest site over 16 months and were consistently found in litter at 12 other DF forest or recent (<2 yrs) clearcut sites. However, trace to none of these fragments were detected in 6 clearcut (5-10 yrs) or different forest types (oak, alder) collected over a 200 km east-west direction in western Oregon, USA. Data suggest that the logging practice in DF forests that creates a clearcut removes a unique gene pool of nitrogen-fixing microorganisms. These organisms could potentially contribute more to nitrogen fixation in forest litter than litter from natural or invasive plants that grow in clearcuts [26]. </hea

Journal Article↗

PubMed2005

Determination of gag and env subtypes of HIV-1 detected among injecting drug users (IDUs) in Manipur, India: evidence for intersubtype recombination.

The majority of HIV-1 transmission in Manipur, one of the northeastern states of India, is through the sharing of needles and syringes among the injecting drug users (IDUs). A total of 28 HIV seropositive samples were used to determine the HIV-1 subtypes with respect to both gag and envelope genes. The specific regions within gag and envelope genes were amplified from PBMC DNA by nested PCR using appropriate primers. These amplicons were used in heteroduplex mobility assay followed by DNA sequencing. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 89% of samples (25/28) were infected with subtype C with respect to both gag and envelope genes; however, 11% of the samples (3/28) showed subtype discordance with respect to the envelope (C2-V3) and gag (p24-p7) genomic regions. Simplot analysis of the discordant samples showed the presence of intersubtype recombination between subtype C and Thai B; two samples were found to be subtype C in envelope but Thai B in gag, whereas, one sample was found to be subtype Thai B in envelope and 'C' in gag region.

Adult↗

PubMed2006

Prognostic significance of gene expression profiles of metastatic neuroblastomas lacking MYCN gene amplification.

BACKGROUND: The aggressiveness of metastatic neuroblastomas that lack MYCN gene amplification varies with age--they are least aggressive when diagnosed in patients younger than 12 months and most aggressive when diagnosed in patients older than 24 months. However, age at diagnosis is not always associated with patient survival. We examined whether molecular classification of metastatic neuroblastomas without MYCN gene amplification at diagnosis using gene expression profiling could improve the prediction of risk of disease progression. METHODS: We used Affymetrix microarrays to determine the gene expression profiles of 102 untreated primary neuroblastomas without MYCN gene amplification obtained from children whose ages at diagnosis ranged from 0.1 to 151 months. A supervised method using diagonal linear discriminant analysis was devised to build a multigene model for predicting risk of disease progression. The accuracy of the model was evaluated using nested cross-validations, permutation analyses, and gene expression data from 15 additional tumors obtained at disease progression. RESULTS: An expression profile model using 55 genes defined a tumor signature that distinguished two groups of patients from among those older than 12 months at diagnosis and clinically classified as having high-risk disease, those with a progression-free survival (PFS) rate of 16% (95% confidence interval [CI] = 8% to 28%), and those with a PFS rate of 79% (95% CI = 57% to 91%) (P<.01). These tumor signatures also identified two groups of patients with PFS of 15% (95% CI = 7% to 27%) and 69% (95% CI = 40% to 86%) (P<.01) from among patients who were older than 18 months at diagnosis. The gene expression signature of untreated molecular high-risk tumors was also present in progressively growing tumors. CONCLUSION: Gene expression signatures of tumors obtained at diagnosis from patients with clinically indistinguishable high-risk, metastatic neuroblastomas identify subgroups with different outcomes. Accurate identification of these subgroups with gene expression profiles may facilitate development, implementation, and analysis of clinical trials aimed at improving outcome.

Adolescent↗

PubMed1998

[Cloning and sequencing of E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus].

OBJECTIVE: To clone E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus into mammalian expression vector and analyze its primary nucleotide structure. METHODS: Reverse transcription nested-PCR methods were employed for amplification E2/NS1 gene which was cloned into mammalian expression vector pcDNA3 by directed clone. Sequence of inserted gene was analyzed by Sanger's method. RESULTS: E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus was cloned into eukaroytic expression vector effectively. Sequence of E2/NS1 showed 88.37% identity in nucleotide and 89.29% in putative amino acid to that of a Japanese genotype III isolate of hepatitis C virus. Nevertheless it showed 70.69% and 75.55% identity to that of a Chinese genotype II isolate of hepatitis C virus. CONCLUSION: E2/NS1 gene from a Chinese genotype III isolate of hepatitis C virus was successfully cloned into mammalian expression vector by technique of DNA recombination. Sequence analysis showed that E2/NS1 gene has good homology intra-genotype and poor homology intergenotype of hepatitis C virus. Research of primary nucleotide structure of different genotype hepatitis C virus might be helpful for the development of vaccine against the virus.

Amino Acid Sequence↗

PubMed2003

[The early diagnosis and treatment of gastric mucosa-associated lymphoma].

OBJECTIVE: To determine the general markers for the diagnosis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma and select optimal cases to be interfered in early stage. METHODS: To investigate the changes of histology, Ki-67 expression, monoclonality of IgH gene rearrangement in gastric lymphoid hyperplasia (GLH) cases before and after Helicobacter pylori (Hp) eradication treatment. Histological changes were classified according to Isaacson's criteria of GLH; L26, UCHL-1, anti-Kappa, anti-Lambda and Ki-67 were applied for immunohistochemical staining; semi-nest PCR was used to detect IgH gene rearrangement. RESULTS: Thirty-one cases of GLH with predominant chronic gastritis and gastric ulcer were studied. The ratio of male to female in the 31 cases was 1.8 and the mean course 6.8 years. Twenty-nine cases had Hp infection. Most of the cases belonged to histological grade II and III. Only 1 case had lambda light chain restriction and 10 cases had monoclonal IgH gene rearrangement. Expression of Ki-67 and monoclonal IgH gene rearrangement were significantly increased with the crescendo of lymphoid hyperplasia grading (P < 0.05). 28 cases were given antibiotics with eradication of Hp in 24 cases and 2 cases had to be given another course of treatment. These cases were followed-up on average for 4.6 months. 18 cases showed complete regression both histologically and endoscopically, 4 cases partial regression and 6 cases no change. Four cases with histological grade II, III had the decrease of expression of Ki-67 and reversed to polyclonal IgH gene rearrangement, while there was no change in 4 cases with histological grade IV. CONCLUSIONS: General considerations of histology, expression of Ki-67 and combined clonality of IgH gene rearrangement help to screen cases which need intervention early and make a diagnosis of gastric MALT lymphoma in early stage. Monoclonal IgH gene rearrangement in cases with histological grade III or IV appears to carry more clinical significance.

Adolescent↗