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At least 271 records · Page 15Linked to original sources
PubMed2001

Selective gene expression and activation-dependent regulation of vasoactive intestinal peptide receptor type 1 and type 2 in human T cells.

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4(+) T cells and CD8(+) T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4(+) T cells than in CD8(+) T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4(+) T cells and CD8(+) T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4(+) T cell subpopulations is highly correlated with T cell activation.

Adult↗
PubMed2005

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA.

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1998

A single amino acid difference within the folate transporter encoded by the murine RFC-1 gene selectively alters its interaction with folate analogues. Implications for intrinsic antifolate resistance and directional orientation of the transporter within the plasma membrane of tumor cells.

The apparent Km, but not Vmax, for influx of methotrexate (MTX) mediated through the plasma membrane of S180 cells by the one-carbon, reduced folate transporter as well as the KD for binding to the transporter were 4-fold higher than in L1210 cells correlating with the greater intrinsic resistance of the former to this folate analogue. In contrast, no difference was observed between each cell type with regard to efflux of [3H]MTX mediated by this same transporter in ATP-depleted cells. The difference in influx Km in the case of this 10-methyl substituted N1O analogue of folic acid was not seen with more effective permeants, such as the unsubstituted N1O aminopterin or C1O analogues. Thus, values for influx Km for aminopterin, which were 1-1.2 microM in each cell type, increased as a result of substitution at N1O (MTX) 3-fold in L1210 cells but 12-fold in S180 cells. Nucleotide sequencing of reverse transcriptase-polymerase chain reaction-generated cDNA and of polymerase chain reaction-generated genomic DNA identified a single nucleotide difference between each cell type at +890 within exon 3 of the RFC-1 gene. This was in the form of a G (L1210 cells) to A (S180 cells) transition. Codon 297, the site of this transition, encodes either Ser or Asn in L1210 or S180 cells, respectively, which is located between the seventh and eight membrane-spanning helices. This amino acid difference had no effect on the electrophoretic mobility or amount of the transporter in each cell type that was shown by Western blotting with anti-RFC-1 peptide antibodies to migrate as 46 kDa in each case. Proof that this nucleotide difference alone accounted for the alteration in influx between each cell type was obtained by S180 RFC-1 cDNA versus L1210 RFC-1 cDNA transfection of an L1210 cell variant with undetectable MTX influx and RFC-1 gene expression. In this case, the higher Km for MTX influx associated with S180 cells was duplicated only in the S180 RFC-1 transfectants. These results appear to document the first example of a nucleotide alteration within the RFC-1 gene, which influences the interaction of MTX with the encoded plasma membrane transporter. An analysis of topology, in addition to other considerations, suggests that the site of the amino acid difference found in the transporter from L1210 and S180 cells occurs within or near the binding site on the external plasma membrane surface.

Amino Acid Sequence↗
PubMed1992

Genes selectively expressed in the infectious (metacyclic) stage of Leishmania major promastigotes encode a potential basic-zipper structural motif.

Complementary DNA clones representing transcripts selectively expressed in the non-dividing, infective (metacyclic) stage of Leishmania major promastigotes (MP) were identified by differential and subtractive screening. The majority of the selected clones hybridized on Northern blots to a set of transcripts highly expressed by MP, but to a much lower extent in proliferating and stationary-phase attenuated promastigotes. Stationary, but not log-phase cultures, of each of 5 L. major strains showing a potential for differentiation to metacyclics, expressed these transcripts (MAT-1; MP-associated transcripts). From sequence analysis of full-length cDNA clones corresponding to the predominating MAT-1 species, an open reading frame encoding a 139 aa polypeptide (15.4 kDa) was predicted and supported by immunoprecipitation by kala-azar sera of reticulocyte extract translation products using in vitro transcribed RNA. Although no significant primary sequence homology to database nucleic acid and protein sequences was found, the sequence displays similarities to the basic-zipper families of transcription regulatory proteins.

Amino Acid Sequence↗
PubMed2006

Selected gene polymorphisms and their interaction with maternal smoking, as risk factors for gastroschisis.

BACKGROUND: Gastroschisis is a severe birth defect in which the infant is born with a portion of the intestines extruding through a small tear in the abdominal wall, usually to the right of the umbilical cord. Its etiology is unknown, but the prevailing hypothesis is that it results from a vascular accident at the time of involution of the right umbilical vein or of the development of the superior mesenteric artery. METHODS: In a case-control study of 57 cases of gastroschisis and 506 controls, we tested DNA for polymorphisms of 32 genes representing enzymes involved in angiogenesis, blood vessel integrity, inflammation, wound repair, and dermal or epidermal strength. RESULTS: In logistic regression, controlling for maternal ethnicity, and using the homozygote wild-type as referent, the following gene polymorphisms were associated with an increased risk for a gastroschisis for heterozygotes: ICAM1 gly241arg (odds ratio [OR], 1.9; 95% confidence interval [CI], 1.1 -3.4); NOS3 glu298asp (OR, 1.9; 95% CI, 1.1-3.4); NPPA 2238T > C (OR, 1.9; 95% CI, 1.0-3.4); and ADD1 gly460trp (OR, 1.5; 95% CI, 0.8-2.8). Additionally, for the NPPA and ADD1 single-nucleotide polymorphisms (SNPs), the homozygote variants had a significantly higher risk than the heterozygotes (OR, 7.5; 95% CI, 1.7-33.5 and OR, 4.9; 95% CI, 1.9-12.9, respectively). Three SNPs showed a strong interaction with maternal smoking. The risk for smokers with 1 or 2 variant alleles compared to nonsmokers with the wild-type allele were: NOS3 (OR, 5.2; 95% CI, 2.4-11.4); ICAM1 (OR, 5.2; 95% CI, 2.1-12.7); and NPPA (OR, 6.4; 95% CI, 2.8-14.6). CONCLUSIONS: These results support the hypothesis of a vascular compromise as part of a multifactorial etiology of gastroschisis involving both genes and environmental factors.

Female↗
PubMed2005

Differential expression of 37 selected genes in hormone-refractory prostate cancer using quantitative taqman real-time RT-PCR.

Progression of prostate cancer to androgen independence remains the primary obstacle to improved survival. The development of more effective treatments depends on our understanding of the molecular events associated with the hormone-refractory stage. We quantified, among 90 screened genes, the expression of 37 target genes, using real-time quantitative RT-PCR. Gene expression was studied in 13 samples of HPRC compared to 33 clinically localised cancers and normal prostate tissue. We identify 19 genes with significant differential expression in HRPC compared to localised prostate cancer. Genes with decreased expression included receptors for growth factors, MMR genes and the serine protease hepsin. Analysis of increased gene expression confirmed the importance of AR upregulation and highlighted genes not previously linked to HRPC, including enzymes involved in steroid synthesis and the antiapoptotic factor survivin. Progression of prostate cancer to the hormone-refractory state is associated with differential gene expression, which may prove useful for both understanding disease progression and the development of new therapeutic approaches.

Androgens↗
PubMed1998

Analysis of selective gene activation in yeast by differential display.

TATA box binding protein-associated factors (TAF(II)s) are dispensable for transcription of most genes in yeast. To further investigate the in vivo functions of TAF(II)s, differential display was used to identify a small subset of yeast genes whose transcription is dependent on yeast TAF(II)145, the core TAF(II) component that contacts TATA box-binding protein (TBP). Messenger RNA profiles derived from a wild-type TAF(II)145 strain and a temperature-sensitive taf(II)1145 strain were analyzed. Those genes whose messenger RNA level was greatly reduced in the temperature-sensitive taf(II)145 strain were cloned and further characterized. The procedure for performing differential display described here is modified from protocols provided by the manufacturer (Display Systems) and optimized for the yeast system.

Binding Sites↗
PubMed2000

A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo.

Adenoviral (Ad) vectors are promising gene therapy vehicles due to their in vivo stability and efficiency, but their potential utility is compromised by their restricted tropism. Targeting strategies have been devised to improve the efficacy of these agents, but specific targeting following in vivo systemic administration of vector has not previously been demonstrated. The distinct aim of the current study was to determine whether an Ad-targeting strategy could maintain fidelity upon systemic vascular administration. We used a bispecific antibody to target Ad infection specifically to angiotensin-converting enzyme (ACE), which is preferentially expressed on pulmonary capillary endothelium and which may thus enable gene therapy for pulmonary vascular disease. Cell-specific gene delivery to ACE-expressing cells was first confirmed in vitro. Administration of retargeted vector complex via tail vein injection into rats resulted in at least a 20-fold increase in both Ad DNA localization and luciferase transgene expression in the lungs, compared to the untargeted vector. Furthermore, targeting led to reduced transgene expression in nontarget organs, especially the liver, where the reduction was over 80%. Immunohistochemical and immunoelectron microscopy analysis confirmed that the pulmonary transgene expression was specifically localized to endothelial cells. Enhancement of transgene expression in the lungs as a result of the ACE-targeting strategy was also confirmed using a new noninvasive imaging technique. This study shows that a retargeting approach can indeed specifically modify the gene delivery properties of an Ad vector given systemically and thus has encouraging implications for the further development of targetable, injectable Ad vectors.

3T3 Cells↗
PubMed1998

A point mutation in the human cytomegalovirus DNA polymerase gene selected in vitro by cidofovir confers a slow replication phenotype in cell culture.

In cell culture, cidofovir (CDV) was used to select a human cytomegalovirus (HCMV) strain with decreased drug susceptibility. The genotypic characterization of this virus revealed a single base substitution resulting in a K513N amino acid alteration in the viral DNA polymerase (UL54). Performed in parallel, the selection of HCMV for replication in the presence of ganciclovir (GCV) selected an M460V substitution in the phosphotransferase (UL97), as well as a K513N/V812L double substitution in DNA polymerase. Neither of the two DNA polymerase mutations has been previously identified in HCMV drug-resistant strains. To precisely elucidate their role in drug resistance, corresponding recombinant mutant viruses were generated by recombination of nine overlapping viral DNA fragments. The K513N recombinant virus showed 13- and 6.5-fold decreased susceptibility to CDV and GCV in vitro, respectively, compared with the wild-type recombinant virus. Mutation V812L was associated with a moderate (2-3-fold) decrease in susceptibility to CDV, GCV, foscarnet, and adefovir. A multiplicative interaction of the K513N and V812L mutations with regard to the profile and level of drug resistance was demonstrated in recombinant virus expressing both mutations. In vitro replication kinetic experiments revealed that the K513N mutation significantly decreased HCMV replication capacity. Consistent with this finding, the K513N mutant DNA polymerase exhibited reduced specific activity in comparison with the wild-type enzyme and was severely impaired in its 3'-5' exonuclease function. Unexpectedly, the K513N mutant enzyme showed no decrease in susceptibility to CDV-diphosphate or GCV-triphosphate. However, the K513N mutation decreased the susceptibility to CDV and GCV of the oriLyt plasmid replication in the transient transfection/infection assay, suggesting that the DNA replication of the K513N mutant virus is less sensitive to the corresponding inhibitors.

Amino Acid Sequence↗
PubMed2005

Sex differential in methylation patterns of selected genes in Singapore Chinese.

To date there have been few reports of a gender difference in methylation levels of genes. When examining the methylation levels of four autosomal genes (ESR1, MTHFR, CALCA and MGMT) in the white blood cells of a random sample of Singapore Chinese Health Study cohort participants (n = 291), we encountered an unexpected gender differential. Using MethyLight technology, we calculated a gene-specific percentage of methylated reference (PMR) value, which quantified the relative level of gene methylation for each study subject (134 males and 157 females). Two summary methylation indices were constructed by assigning gene-specific rank scores. We then used ANCOVA to compare logarithmically transformed individual PMR values and summary methylation indices by age and gender simultaneously. Adjustment was made for plasma homocysteine. For ESR1, for which a large proportion of subjects were negative for methylation, we also used polytomous regression to compare methylation across age and gender. Increasing age and the male gender independently predicted increasing PMR values for CALCA and MGMT. For the MTHFR gene, male gender was associated with higher PMR values (P = 0.002), while age was not (P = 0.75). Neither age nor gender had any statistically significant influence on the PMR values for ESR1 (P = 0.13 and 0.96, respectively). Our data suggest that gender is at least as strong a predictor of methylation level in the four genes under study as age, with males showing higher PMRs.

Age Factors↗
PubMed1992

Selective gene mutation in MEL cells.

MEL cells, undergoing erythroid differentiation and parasynchronized by dimethyl sulfoxide (DMSO) induction, were irradiated with a 3-s pulse of UV light at sublethal dose. A large number of clones deficient in different gene functions are found in the progeny of the treated cells, if the pulse irradiation is performed 18-24 h from the start of DMSO induction. Kinetics of thymidine incorporation into DNA show that the period of sensitivity corresponds to the S phase. The results show that the activities of the tested genes are differently affected depending on the exact time of cell irradiation. Maximum percent inhibition of cells not expressing glucose-6-phosphate dehydrogenase (G-6-PD) (70%) is produced by irradiating at 20 h from the start of DMSO induction; 6-phosphogluconate dehydrogenase (6-PGD) (55%), and hypoxanthine (guanine) phosphoribosyltransferase (HPRT) (33%), at 21 h; hemoglobin (50%), at 22 h. The time difference in the sensitivity to UV light is highly reproducible and has been exploited to isolate, with high efficiency, cellular clones deficient in any one of the tested functions. Determinations of enzymatic activities on cell lysates show that the expression of tested genes is actually altered in cells that, on the basis of cytochemical tests, appear unaffected by UV irradiation. While the production of mutant clones is observed only during the S phase of the cell cycle, immediate statistical damage of the cellular DNA is produced at all times of irradiation. This finding excludes that the two types of phenotypic alterations, blocked or altered gene expression, both propagated in the progeny of the cells as clonal properties, may derive from a preferential alteration of those functions during the S phase.

Cell Differentiation↗
PubMed1993

Odorant-selective genes and neurons mediate olfaction in C. elegans.

Olfaction is a versatile and sensitive mechanism for detecting volatile odorants. We show that the nematode C. elegans detects many volatile chemicals, which can be attractants, repellents, or attractants at low concentrations and repellents at high concentrations. Through laser ablation, we have identified chemosensory neurons that detect volatile odorants. Chemotaxis to volatile odorants requires different sensory neurons from chemotaxis to water-soluble attractants, indicating that C. elegans might have senses that correspond to smell and taste, respectively. Single neurons have complex sensory properties, since six distinguishable volatile odorants are sensed by only two types of sensory neurons. Chemotaxis to subsets of volatile odorants is disrupted by mutations in the odr genes, which might be involved in odorant sensation or signal transduction.

Animals↗