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PubMed1991

Molecular organization of the Drosophila melanogaster Pig-1 gene.

The Pre-intermoult gene-1 (Pig-1) of Drosophila melanogaster maps on the X chromosome, at polytene bands 3C11-12, and is nested within the 79 kb intron of the dunce gene. Pig-1 has so far been characterized only preliminarily and its function is still unknown. We analysed the molecular organization of the gene by cDNA clone isolation and sequencing as well as S1 mapping and primer extension analyses. The results obtained reveal that the gene is colinear with its genomic sequence and define the usage of both 5' and 3' alternative sites for Pig-1 transcription; two continuous open reading frames (ORFs) are fully contained within the Pig-1 transcribed region, although several lines of evidence suggest that only the longer ORF is likely to be translated. We also report that the level of Pig-1 transcript is nearly fourfold reduced in a variant strain carrying a deletion within the Pig-1 upstream sequence, thus identifying a regulatory element required for high level gene expression.

Amino Acid Sequence↗

PubMed2006

Verification, characterization and tissue-specific expression of UreG, a urease accessory protein gene, from the amphioxus Branchiostoma belcheri.

UreG genes have been found in bacteria, fungi and plants but have not yet identified in animals, although a putative UreG-like gene has been documented in sea urchin. In the course of a large-scale sequencing of amphioxus gut cDNA library, we have identified a cDNA with high similarity to UreG genes. Both reverse transcription-polymerase chain reaction and nested polymerase chain reaction, as well as in situ hybridization histochemistry, verified that the cDNA represented an amphioxus UreG gene (AmphiUreG) rather than a microbial contaminant of the cDNA library. This is further supported by the presence of urease activity in amphioxus gut, gill and ovary. AmphiUreG encodes a deduced protein of 200 amino acid residues including a highly conserved P-loop, bearing approximately 46%-49%, 44%-48%, and 29%-37% similarity to fungal, plant and bacterial UreG proteins, respectively. It shows a tissue-specific expression pattern in amphioxus, and is especially abundant in the digestive system. This is the first UreG gene identified in animal species.

Amino Acid Sequence↗

PubMed1997

Identification of a cis-acting element in the class I major histocompatibility complex gene promoter responsive to activation by retroviral sequences.

The infection of cells with Moloney murine leukemia virus (M-MuLV) causes an increase in specific cellular gene products, including the major histocompatibility complex (MHC) class I antigens. This upregulation occurs through a transactivation process mediated by the long terminal repeat (LTR) of M-MuLV, and we show here that the gene activation response to the LTR requires at least one specific cis element within the MHC proximal promoter region. Nested deletions of MHC class I H-2Kb gene promoter sequence were subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector and then transiently introduced into BALB/c-3T3 cells expressing M-MuLV or cotransfected into BALB/c-3T3 cells with a vector containing subgenomic portions of the virus, including the LTR. CAT activity assays demonstrated that a minimal H-2Kb gene promoter (-64 to +12) contained elements sufficient for this transactivation. DNase I footprinting assays located a protein-binding site in the region of -64 to -34 bp from the transcriptional start site, and point mutation analysis confirmed the location of this cis-acting element, designated the let response element (LRE), and defined a binding motif. This LRE is distinct from binding sites for currently known transcription factors in the class I MHC gene promoter and is conserved in the promoters of human and murine MHC class I genes. Mutation of the LRE resulted in dramatic reduction in both DNA-protein binding activity in electrophoretic mobility shift assay and in the ability of the mutated promoter to respond to retroviral transactivation. Addition of the LRE to a heterologous promoter conferred the ability to respond to retroviral transactivation.

3T3 Cells↗

PubMed2003

[FHIT gene is abnormal in tongue carcinoma cell line].

To study the alteration of FHIT gene in tongue carcinoma Tca8113 cell line, total RNA of Tca8113 cells was extracted. The transcript of the FHIT gene of the Tca8113 cell line was detected with nest RT-PCR, and DNA was sequenced. The result showed that abnormal transcript (about 247 bp) of FHIT gene was detected in the Tca8113 cell line. The sequence analysis of the aberrant cDNAs revealed deletions of exons 1-8. Therefore, the deletion of the FHIT gene in Tca8113 cell line might support the hypothesis that the FHIT gene alteration is involved in the development of tongue carcinoma.

Acid Anhydride Hydrolases↗

PubMed1993

Herpes simplex encephalitis. Early diagnosis and immune activation in the acute stage and during long-term follow-up.

From a series of in all 93 patients with herpes simplex encephalitis (HSE), verified by biopsy and/or the demonstration of intrathecal synthesis of antibodies to the virus, cerebrospinal fluid (CSF) and serum samples were analysed and compared with samples from 80 patients with non-HSE, i.e. acute encephalitis of non-HSV origin (approximately 50% with other known aetiology, 50% of unknown origin) treated on the suspicion of HSE but in whom no signs of intrathecal HSV antibody synthesis were found, and samples from an additional 42 patients with other verified or suspected diseases of the CNS. To improve the early non-invasive diagnosis of HSE, a HSV IgG capture enzyme linked immunosorbent assay (ELISA) was developed to demonstrate intrathecal synthesis of antibodies to the virus and the results were compared to those of the indirect ELISA. The capture ELISA was found to be advantageous in detecting the early antibody response and yielded more clear-cut results. No correction for damage to the blood-CSF barrier was needed and the method was therefore less labour-intensive than the indirect ELISA. Furthermore, a polymerase chain reaction (PCR) assay, with two "nested" primers pairs selected in the glycoprotein D gene of HSV-1, was developed for the amplification of HSV DNA in CSF. The method was found to be a rapid and non-invasive means of diagnosing HSE in a very early stage of the disease; it was highly sensitive and specific. With a combination of nested PCR assays for HSV-1 and HSV-2 (primers in the glycoprotein G gene) in 10 microliters of CSF, HSV DNA was detected in CSF from 88 out of 93 patients (95%) with HSE. Evidence of HSV-2 aetiology was found in 6 of 93 consecutive cases of HSE in immunocompetent patients by type-specific assays for the demonstration of HSV-2 DNA (primers in the gG gene) and HSV-2 antibodies (to gG2 antigen) in the CSF. Five of the 6 patients with HSV-2 encephalitis exhibited a clinical picture of severe HSE indistinguishable from that of "classical" HSV-1 encephalitis. The combined use of PCR for the detection of HSV DNA in the CSF and the demonstration of intrathecal synthesis of antibodies to the virus will yield a reliable diagnosis and is now the method of choice for the diagnosis of HSE.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1995

[Human papillomavirus, human cytomegalovirus and p53 gene in cervical carcinoma].

OBJECTIVE: To investigate the role of human papillomavirus (HPV) human cytomegalovirus (HCMV) infections and p53 gene mutations in the oncogenesis of cervical cancer and to clarify the association between p53 inactivation and the presence of HPV DNA. METHODS: We examined 38 primary cervical carcinomas and 21 normal cervical specimens for the presence of HPV and HCMV DNA sequences by multiple primers PCR and nest primers PCR. The structure of p53 gene (exons 6-8) was also analyzed by PCR-SSCP silver staining method. RESULTS: Mutations of p53 gene (exon 7) were detected in 2 of 38 tumors. One of the cases with p53 mutation was positive for HPV 16 and two positive for HCMV. HPV 16 and 18 infections were noted in 63.2% (24/38) of the tumors, the positive rate of HCMV was 84.2% (32/38). However, HPV 16,18 and HCMV infection occurred in 4.8% and 38.1% respectively in 21 normal cervical specimens. 21 of the 24 HPV 16,18 positive tumors were also HCMV positive, but none of the normal cervical tissues was infected with both HPV and HCMV. CONCLUSION: Detection of p53 mutations in cervical carcinoma is infrequent and apparently independent of HPV infection. Cervical carcinoma is strongly associated with HPV 16 and 18 infection. A synergistic interaction may occur between HPV and HCMV infections in the oncogenesis of cervical cancer.

Carcinoma, Squamous Cell↗

PubMed2002

Detection of the gp43 gene and (1-3)-beta-D-glucan of Paracoccidioides brasiliensis in the blood of experimentally infected mice.

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. Diagnosis of PCM is sometimes difficult outside the endemic countries, thus a rapid and conclusive method for diagnosis of PCM has been anticipated. We compared the sensitivities of a nested PCR method for detecting the gp43 gene and a commercial kit for detecting (1-3)-beta-D-glucan in the blood of experimentally infected mice. Blood samples were collected from mice at 0 (soon after inoculation), 6, 12, 24, 48, and 72 hours and 5, 7, 10, 14, 17, 21, 24, 28 and 56 days after the intravenous inoculation of 10(6) yeast cells of P. brasiliensis, and were separated into clots and plasma. The (1-3)-beta-D-glucan detection kit in the plasma showed positive reactions in some samples within 7 days and 28 and 56 days after infections. In contrast, the PCR method was more sensitive than the (1-3)-beta-D-glucan detection kit throughout the observation period. The clot samples yielded more sensitive PCR-results than did the plasma samples. Although 24 hours is required for the PCR detection, it was confirmed to provide an accurate diagnosis of PCM.

Adult↗

PubMed2001

[Analysis of the non-deletion alpha-thalassemia mutations by PCR temperature gradient gel electrophoresis].

OBJECTIVE: To develop a PCR-temperature gradient gel electrophoresis(TGGE) method applied to screening for the point mutations causing the non-deletion alpha-thalassemia. METHODS: The entire alpha2-globin gene fragment (1085 bp) was selectively amplified from human genomic DNA with different genotypes of alpha-thalassemia and a 543 bp fragment spanning the exon 2 and exon 3 of the alpha2-globin gene was amplified with a pair of nested primers. The condition of perpendicular and parallel TGGE for the two different fragments in length was optimized and the candidate mutant was confirmed by DNA sequencing. In pilot study, 15 samples with suspected non-deletion alpha-thalassemia were screened for point mutations in alpha-globin gene. RESULTS: Hb Constant Spring (Hb CS) and Hb Quong Sze (Hb QS), two most commonly types of the non-deletion alpha-thalassemia distributed in Chinese, and a rare type of the Hb Westmead mutation, alpha2 CD122 CAC-->CAG(His-->Gln) could be detected by PCR-TGGE. Among those 15 samples screened for alpha-thalassemia mutation were 10 samples with Hb CS mutation, 2 samples with Hb QS mutation, 2 samples with Hb Westmead mutation, and a previously unreported one, alpha2 CD31 AGG-->AAG(Arg-->Lys), confirmed by DNA sequencing. CONCLUSION: The present PCR-TGGE method could be a useful tool for the molecular screening for the point mutations causing alpha-thalassemia and alpha-globin gene polymorphism.

Electrophoresis↗

PubMed1991

Detection of rubella virus gene sequences by enzymatic amplification and direct sequencing of amplified DNA.

We developed a rapid and sensitive polymerase chain reaction (PCR) assay for detecting and identifying rubella virus (RV). A segment of the RV gene which encodes the E1 membrane glycoprotein of RV was selected as a target for PCR amplification. Single-stranded viral RNA, extracted from infected cells or released from virions, was used as a template for reverse transcription followed by PCR amplification with two different sets of primer pairs, one nested within the other. The amplified E1 gene sequences were detected in ethidium bromide-stained agarose minigels, and their identities were verified by restriction enzyme digestion and hybridization to a probe directed at a site within the PCR target. Single-stranded DNA generated by asymmetric amplification of the target was directly sequenced by using fluorescent dideoxy-terminators and an automated procedure in order to confirm the target sequence. This PCR assay provides a rapid confirmatory test for the detection of RV by cell culture and appears to have considerable potential for the direct detection of RV in clinical specimens. The strategy used in the development of this PCR assay should be useful for developing other diagnostic PCR assays for viruses.

Animals↗

PubMed2002

Temporal arteritis and Chlamydia pneumoniae: failure to detect the organism by polymerase chain reaction in ninety cases and ninety controls.

OBJECTIVE: To examine the reported correlation between the presence of Chlamydia pneumoniae in temporal artery biopsy specimens and the diagnosis of temporal arteritis (TA). METHODS: Among 90 possible cases of TA identified at our institution between 1968 and 2000, 79 of the positive biopsy specimens (88%) demonstrated giant cells and the other 11 cases (12%) had other histopathologic features compatible with TA; by chart review, all 90 patients were confirmed to have met the American College of Rheumatology classification criteria for TA. Controls had negative temporal artery biopsy specimens during the same 32-year time period and their postbiopsy disease courses were not compatible with TA. Controls were matched with each case by sex, year of biopsy, and age within 10 years. The biopsy specimens from all cases and controls were reevaluated and readings were confirmed in a masked manner by an experienced eye pathologist. Polymerase chain reaction (PCR) analyses for C pneumoniae were performed on the 180 samples using 2 different sets of PCR primers (which target 2 different genes). A primer set targeting the ompA gene (CP1-CP2/CPC-CPD) was used to perform a nested PCR, followed by confirmation of the findings with primers targeting the 16S ribosomal RNA (rRNA) gene (Cpn90/Cpn91) in a touchdown-enzyme time-release PCR. We used positive and negative controls, as well as controls made from infected and noninfected HEp-2 cells, suspended in a formalin-fixed, paraffin-embedded matrix. RESULTS: Seventy-six percent of the 180 cases and controls were women. The mean age of the cases was 72.0 years (range 53-90), and that of the controls was 70.4 years (range 51-86). Eighty percent of the control samples were obtained by temporal artery biopsy performed within 1 year of the biopsies performed on the matched cases. Using the CP1-CP2/CPC-CPD primer set, only 1 TA case sample (1% of all case samples) was positive for the ompA gene. One control sample was also positive using these primers. With the Cpn90/Cpn91 primers, none of the cases and none of the controls were positive for the 16S rRNA gene. CONCLUSION: The results of this study using sensitive and specific PCR analyses do not support a role for C pneumoniae in the pathogenesis of TA.

Aged↗

PubMed1998

High frequency of fusion transcripts of exon 11 and exon 4/5 in AF-4 gene is observed in cord blood, as well as leukemic cells from infant leukemia patients with t(4;11)(q21;q23).

The MLL gene located on chromosome 11q23 and its translocation to the AF-4 gene located on chromosome 4q21 play a pivotal role in leukemogenesis in infancy. Studies of identical leukemic twins have provided evidence of the MLL rearrangement as a fetal event during pregnancy. We analyzed the presence and frequency of the MLL/AF-4 rearrangement in normal cord blood. Although no chimeric mRNA of MLL or AF-4 was detected in 65 cord blood samples, in-frame fusion transcripts of exon 11 and exon 4 or 5 of the AF-4 gene were detected in three of the samples by a nested polymerase chain reaction. When primers of exon 11 and exon 5 of the AF-4 gene were used, two forms of fusion transcripts (AF-4 exon 11/4 or exon 11/5) were detected in 20 of the 65 cord blood samples (31%) and also four of six leukemic cell samples with t(4;11) (67%), whereas such transcripts were not observed in any of 21 peripheral blood samples nor in fetal fibroblasts. These findings suggest that the in-frame fusion of exon 11 and exon 4 or 5 of the AF-4 gene frequently occurs in hematopoietic cells during the intrauterine period, even in a healthy fetus. Although it is unknown whether the proteins of the AF-4 fusion transcripts have some functions, the instability of the AF-4 gene may be associated with the leukemogenesis of infant leukemia.

Chromosomes, Human, Pair 11↗

PubMed2003

[SARS-associated coronavirus gene fragments were detected from a suspected pediatric SARS patient].

UNLABELLED: A Special "Fever and Cough" Clinic was set up at the Children's Hospital Affiliated to Capital Institute of Pediatrics for children with symptoms of fever and cough in late April when the severe acute respiratory syndrome (SARS) epidemic was at its peak in Beijing to separate the children with fever from others during their visit to the Outpatient Department. OBJECTIVE: For patients with fever, normal or low count of white blood cell and with suspected pneumonia suggested by X-ray, it was urgent to determine the etiological agents of the diseases before they were admitted to the hospital. METHODS: Throat swabs or nasopharyngeal aspirate specimens were collected from those patients and common respiratory virus antigens including influenza virus A and B, respiratory syncytial virus, adenovirus, parainfluenza virus types I, II, and III were tested by indirect immunofluorescent assay. The patients with atypical pneumonia diagnosed by X-ray and evidences of common respiratory virus infection were admitted to the regular ward for children with respiratory diseases. Children with pneumonia demonstrated by X-ray and negative for common respiratory viruses were admitted to the isolated ward for suspected SARS patients for the first step and further viral etiological studies were requested. RT-PCR was performed for those patients to detect gene fragments of human metapneumovirus (HMPV), rhinovirus (RhV) and enterovirus (EV) in their specimens. Nested RT-PCR was also developed to detect SARS coronavirus gene fragment from the specimens. Primer sequences for SARS virus detection with the PCR were selected according to the primer sequences published online by WHO on April 18, 2003. All the primers derived from the sequence at the 1b frame of coronavirus replicase gene and products with a size of 368 or 348 bp were expected with 2 different primer pairs. RESULTS: Amplicons with the sizes of 368 bp and 348 bp were obtained from a throat swab specimen collected from a 17 years old girl, who was admitted to the isolated ward because of high fever (39.5 degrees C) for 7 days, cough for 2 days, low WBC count, and pneumonia shown by X-ray when she visited the "Fever and Cough" Clinic, and without known history of contact with probable SARS patient. Antigens for the common respiratory viruses were all negative, RT-PCR for HMPV, RhV and EV were also negative while RT-PCR with different primer pairs for SARS virus were all positive which indicated that SARS coronavirus gene fragments were amplified from the specimen from this girl. The amplified fragment with a size of 368 bp was sequenced and the sequence was compared with those in the GenBank. The sequence shared 100% homology with the sequences from 1b frame of replicase genes from all 17 of SARS coronaviruses published in the GenBank so far, and shared very low homology with 2 reference strains of human coronavirus as well as other animal coronaviruses. The serum collected before her discharge from the hospital (19 days after the onset of the disease) showed SARS specific IgM and IgG antibodies. CONCLUSION: These data indicate that the patient was a confirmed case of SARS. It is of great importance to differentiate SARS patients from those infected with common respiratory viruses during SARS epidemic, especially for pediatric patients, because most of the patients visiting the outpatient department present with the symptoms of fever, cough and normal WBC count. The data mentioned above indicate that antigen and gene detections for those common respiratory viruses are useful methods for the differentiation to avoid the spread of SARS.

Adolescent↗

PubMed2004

Detection of free and plankton-associated Helicobacter pylori in seawater.

AIMS: To detect both free and plankton-associated Helicobacter pylori in seawater samples collected on the Italian coast of the Adriatic Sea using a nested-PCR. METHODS AND RESULTS: Dissolved oxygen, pH, salinity and chlorophyll 'a' were the parameters recorded together with the characterization of zooplanktonic organisms. Plankton-associated H. pylori DNA was searched for in water samples filtered through 200 and 64 microm nylon nets whereas free bacteria were retained with the subsequent filtration through 0.22 microm pore-size membranes. Nested-PCR using primers for the glmM (ureC) gene was performed to reveal the presence of H. pylori. The DNA sequencing of amplified products confirmed the specificity of the assay. The sensitivity of the nested-PCR assay for H. pylori detection was 62 CFU per 100 ml in spiked water samples. Helicobacter pylori either free or bound to planktonic organisms was found in seven of 12 monthly samples. In particular, free bacteria were detected during the summer sampling and in November, December and March associated to planktonic cells. CONCLUSIONS: The presence of free and plankton-associated H. pylori in seawater suggests that it can be a significant reservoir and a potential route of transmission for the microorganism. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study seems to provide a promising background to define new and effective strategies for surveillance of this human pathogen.

Animals↗

PubMed2006

Multi-factor dimensionality reduction applied to a large prospective investigation on gene-gene and gene-environment interactions.

It is becoming increasingly evident that single-locus effects cannot explain complex multifactorial human diseases like cancer. We applied the multi-factor dimensionality reduction (MDR) method to a large cohort study on gene-environment and gene-gene interactions. The study (case-control nested in the EPIC cohort) was established to investigate molecular changes and genetic susceptibility in relation to air pollution and environmental tobacco smoke (ETS) in non-smokers. We have analyzed 757 controls and 409 cases with bladder cancer (n=124), lung cancer (n=116) and myeloid leukemia (n=169). Thirty-six gene variants (DNA repair and metabolic genes) and three environmental exposure variables (measures of air pollution and ETS at home and at work) were analyzed. Interactions were assessed by prediction error percentage and cross-validation consistency (CVC) frequency. For lung cancer, the best model was given by a significant gene-environment association between the base excision repair (BER) XRCC1-Arg399Gln polymorphism, the double-strand break repair (DSBR) BRCA2-Asn372His polymorphism and the exposure variable 'distance from heavy traffic road', an indirect and robust indicator of air pollution (mean prediction error of 26%, P<0.001, mean CVC of 6.60, P=0.02). For bladder cancer, we found a significant 4-loci association between the BER APE1-Asp148Glu polymorphism, the DSBR RAD52-3'-untranslated region (3'-UTR) polymorphism and the metabolic gene polymorphisms COMT-Val158Met and MTHFR-677C>T (mean prediction error of 22%, P<0.001, mean CVC consistency of 7.40, P<0.037). For leukemia, a 3-loci model including RAD52-2259C>T, MnSOD-Ala9Val and CYP1A1-Ile462Val had a minimum prediction error of 31% (P<0.001) and a maximum CVC of 4.40 (P=0.086). The MDR method seems promising, because it provides a limited number of statistically stable interactions; however, the biological interpretation remains to be understood.

Drug Resistance, Multiple↗

PubMedDate not supplied

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: a preliminary study.

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR=?.58, 95% CI=1.32-23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.

Adult↗

PubMed2001

Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

Insulin and insulin-like growth factor I (IGF-I) can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein, and P450 cholesterol side-chain cleavage enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal interactions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act via concerted transcriptional control of promoter expression. To this end, we transiently transfected primary monolayer cultures of porcine granulosa-luteal cells with a reporter vector containing the putative 5'-upstream full-length (pLDLR1076/luc) regulatory region (-1076 to +11 bp) of the homologous LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH and insulin (or IGF-I) stimulated LDL receptor transcriptional activity maximally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luciferase. Further analysis of multiple 5'-nested deletional constructs of the LDL receptor gene promoter showed that deletion of -139 bp upstream of the transcriptional start site virtually abolished basal expression and promoter responsiveness to LH and insulin/IGF-I. In contrast, full basal activity and 60-80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other tissues is negatively regulated by the abundance of intracellular free cholesterol, we assessed the impact of concomitant pretreatment of granulosa-luteal cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 microM). Excess sterol markedly (50-70%) attenuated bihormonally and, in lesser measure, LH-stimulated and basal LDL receptor promoter expression, thus affirming a feedback-sensitive sterol-repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

Animals↗

PubMed2001

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes.

OBJECTIVE: To detect malaria DNA in mosquitoes. METHODS: A nested polymerase chain reaction (nested PCR) procedure which amplifies a 121 bp DNA of a SSUrRNA gene specific to Plasmodium vivax was used. RESULTS: In laboratory-infected mosquitoes, nested PCR could detect as few as 3 sporozoites or 1 infected mosquito mixed in a group of 99 normal ones. Furthermore, no specific 121 bp band was seen with DNA templates from other malaria parasites or negative mosquitoes. CONCLUSION: Sensitivity and specificity obtained indicated an advantage of the nested PCR over DNA probes or direct PCR for the detection of Plasmodium vivax sporozoites in mosquitoes with low-grade parasitic infections.

Animals↗

PubMed2002

[Single cell PCR for sex determination in preimplantation genetic diagnosis].

OBJECTIVE: To develop an efficient, rapid and reliable method for sex determination in preimplantation genetic diagnosis. METHODS: Nested PCR and fluorescent PCR were used to amplify amelogenin and IL-3 genes from human single lymphocytes and blastomeres. RESULTS: The amplification efficiencies of nested PCR and fluorescent PCR of man single lymphocyte/woman single lymphocyte/blastomere were 64.7%/65%/85.7%, and 72%/76%/86.9% respectively. The PCR products of the lymphocyte were exactly the same as those of the donor gDNA. The PCR products of blastomeres coming from the same embryo were all identical. CONCLUSION: The amplification efficiencies of nested PCR and fluorescent PCR are similar while fluorescent PCR is more conventional, time sparing and sensitive.

Adult↗