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PubMed1996

Heterologous expression of the cloned guinea pig alpha 2A, alpha 2B, and alpha 2C adrenoceptor subtypes. Radioligand binding and functional coupling to a CAMP-responsive reporter gene.

Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.

Adrenergic alpha-Antagonists↗
PubMed2006

Molecular characterization of a bHLH transcription factor involved in Arabidopsis abscisic acid-mediated response.

The suppression subtractive hybridization (SSH) method was used to isolate abscisic acid (ABA)-regulated genes in Arabidopsis. We report that the AtAIG1 gene is up-regulated by ABA and the AtAIG1 encodes a basic/helix-loop-helix (bHLH)-type transcription factor in Arabidopsis. The AtAIG1 was targeted into nucleus and bound to the E-box-DNA sequence, which is found in the promoter regions of many ABA-responsive genes. Functional analyses with the ataig1 knockout mutants revealed that the mutant plants show enhanced sensitivity to ABA. In addition, the ataig1 plants showed reduced expression of ABA-responsive genes, such as RD29A and RD22. Thus, these functional analyses suggest that the AtAIG1 protein plays an important role in ABA-mediated signal transduction pathway.

Abscisic Acid↗
PubMed1986

Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H (D. N. Nunn and M. E. Lidstrom, J. Bacteriol. 166:582-591, 1986). In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: phenazine methosulfate-dichlorophenolindophenol dye-linked methanol dehydrogenase activity; methanol-dependent whole-cell oxygen consumption; the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the absorption spectra of purified mutant methanol dehydrogenase proteins; and the presence or absence of the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome cL, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome cL, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol.

Alcohol Oxidoreductases↗
PubMed1994

Gene therapy for inflammatory diseases.

The concept of gene therapy may be broadened to include transient gene therapy (gene therapeutics) as a potential intervention in prevention and treatment of diseases which are a consequence of triggering the inflammatory response. Functioning genes can be delivered in vivo by a variety of technologies. Liposome technology is particularly attractive for gene therapeutics because plasmid DNA constructs can be delivered using liposomes which express transiently and do not readily incorporate into the host genome. In the lungs, DNA may be targeted by either intravenous or airway delivery. Airway delivery may be achieved either by direct injection into the airways or by aerosolizing liposome-DNA constructs. Expression of transgenes might also be targeted to areas of inflammation by choosing DNA constructs which contain the appropriate regulatory regions. Several genes have been cloned which are directly relevant to manipulating the inflammatory response and this technology could, in theory, by using either sense or antisense constructs, provide an opportunity to increase or decrease proteins relevant to the inflammatory response. Because of rapid progress in the technology of molecular biological techniques, and rapid progression of human trials using gene transfer methodologies, it is likely that extension of gene therapy to acute diseases such as those which are characterized by inflammation will open a new vista for pharmacological approaches to these complex diseases.

Animals↗
PubMed1992

Transcriptional control of vitamin D-regulated proteins.

Vitamin D is a physiological regulator of gene transcription associated with control of a broad spectrum of biological processes that include but is not restricted to growth, differentiation and calcium-mediated homeostatic control. Transcriptional regulation is mediated by sequence-specific interactions of a 1,25(OH)2D3-vitamin D receptor-accessory factor complex with vitamin D responsive elements (VDRE) residing in the promoters of hormone responsive genes. Functioning primarily as a transcription enhancer, activity at the VDRE is controlled by diverse and integrated cellular signalling pathways acting synergistically and/or antagonistically with a series of basal regulatory elements and other hormone regulated sequences that are components of modularly organized vitamin D-responsive gene promoters. Molecular mechanisms that integrate the activities at promoter elements contributing to vitamin D-related transcriptional control include overlapping transcription factor binding domains within regulatory elements and cooperative activities at independent regulatory sequences that determine the level of vitamin D responsiveness.

Animals↗
PubMed2005

Transcriptional regulation of neuronal genes and its effect on neural functions: gene expression in response to static magnetism in cultured rat hippocampal neurons.

We have previously shown a marked but transient increase in DNA binding of the nuclear transcription factor activator protein-1 after brief exposure to static magnetic fields in cultured rat hippocampal neurons, suggesting that exposure to static magnetism would lead to long-term consolidation as well as amplification of different functional alterations through modulation of de novo protein synthesis at the level of gene transcription in the hippocampus. Hippocampal neurons were cultured under sustained exposure to static magnetic fields at 100 mT, followed by extraction of total RNA for differential display (DD) analysis using random primers. The first and the second DD polymerase chain reaction similarly showed the downregulation of particular genes in response to sustained magnetism. Nucleotide sequence analysis followed by BLASTN homology searching revealed high homology of these 2 DD-PCR products to the 3' non-coding regions of the mouse basic helix-loop-helix transcription factor ALF1 and that of histone H3.3A, respectively. On Northern blot analysis using the 2 cloned differentially expressed fragments labeled with [alpha-(32)P]dCTP by the random primer method, a marked decrease was seen in expression of mRNA for ALF1 and histone H3.3A in hippocampal neurons cultured under sustained exposure to static magnetic fields at 100 mT. It thus appears that static magnetism may modulate cellular integrity and functionality through expression of a variety of responsive genes required for gene transcription and translation, proliferation, differentiation, maturation, survival, and so on in cultured rat hippocampal neurons.

Animals↗
PubMed2005

Single nucleotide polymorphisms in four functionally related immune response genes in the horse: CD14,TLR4, Cepsilon, andFcepsilon R1 alpha.

The objective of this study was to identify single nucleotide polymorphisms (SNPs) within four functionally related immune response genes in the horse, and to develop genotyping techniques that could be useful for future genomic studies of horse infectious and allergic diseases. The genes analysed were: the lipopolysaccharide (LPS) receptor gene CD14, the toll-like receptor 4 gene TLR4, the gene Cepsilon encoding the IgE heavy chain molecule and the gene FcepsilonR1 alpha coding for the alpha subunit of the IgE receptor molecule. Horse-specific primers amplifying selected gene regions were designed and SNPs were searched by selective resequencing and/or by PCR-SSCP (polymerase chain reaction-sequence specific conformational polymorphism) or PCR-RFLP (PCR-restriction fragment length polymorphism). Gene expression was analysed by RT-PCR (reverse transcriptase-PCR) of all four genes examined. For CD14, the cDNA sequence was determined and a novel sequence of the 5'UTR region was identified. The protein-coding sequence was identical to that previously deposited in GenBank. 5'UTR, intronic and both synonymous and non-synonymous exonic SNPs were identified. Three SNPs were found in the CD14 gene, four in the TLR4 gene; two SNPs were identified in the Cepsilon gene, and one SNP was found in the FcepsilonR1 alpha gene. PCR-RFLP was developed for genotyping eight of the SNPs identified. The RT-PCR assay showed that all the SNPs reported here are parts of expressed genes. The results showed that important immunity-related genes in horses are polymorphic and that even non-synonymous SNPs with potential functional impact may occur. The methods developed for genotyping and haplotyping the SNPs identified represent, along with markers described previously, a potentially useful tool for genomic analysis of the function and role of these genes in immunity and in mechanisms of disease.

Animals↗
PubMed1984

Enrichment of early HSV-induced proteins in phosphonoformate-treated cells.

The early HSV-specified proteins were selectively enriched in phosphonoformate (PFA)-treated Vero cells, i.e. in the absence of virus DNA synthesis. By pulse labelling, immunoprecipitation and enzyme immunoassay studies, the replication cascade of HSV-1 was shown to be blocked to a given stage allowing the production of early viral polypeptides only. With a prolonged treatment some of the early functions of the HSV genome seemed to cease as shown by the depletion of the respective polypeptides. Two suggestions rise from the presented experimental data: (i) the action of PFA on the production of early viral proteins modifies the recovery of HSV replication after the drug removal, (ii) the analysis of HSV polypeptide synthesis during a prolonged PFA treatment may be used for the evaluation of important viral gene functions responsible for the development of virus latency.

Antigens, Viral↗
PubMed1991

Multiple domains of the glucocorticoid receptor involved in synergism with the CACCC box factor(s).

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor-binding sequences with adjacent transcription factor-binding sites. To analyze the mechanism of synergy we tested different human glucocorticoid receptor mutants for synergistic function with another transcription factor in comparison with intrinsic trans-activation obtained with a single receptor binding site (glucocorticoid response element). Multiple domains were found to be involved in synergistic activity of the glucocorticoid receptor with the CACCC box factor. Deletions within the N-terminal receptor half affected simultaneously intrinsic trans-activation and synergism. However, deletion of the hormone-binding domain mainly impaired synergism rather than intrinsic trans-activation, clearly showing that this domain synergizes by a mechanism independent of intrinsic activation. A chimeric protein where the DNA-binding domain of the glucocorticoid receptor was replaced by that of the yeast GAL4 protein also showed functional synergism. These data suggest that some of the receptor domains outside the DNA-binding domain synergize by their intrinsic trans-activating property, but the hormone-binding domain contributes to synergism by a different mechanism.

Animals↗
PubMed2005

The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors.

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2'-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2'-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Antigens, Differentiation↗
PubMed1985

Transduction of virulence in herpes simplex virus type 1 from a pathogenic to an apathogenic strain by a cloned viral DNA fragment.

Herpes simplex virus (HSV) type 1 strain HFEM, the genome of which contains a deletion of about 4 kilobasepairs (kbp) between 0.7 and 0.8 viral map units, is apathogenic in the tree shrew when the animals are inoculated intravenously, intraperitoneally and/or subcutaneously. Similar results were obtained using Balb/c mice. Studies of the state of viral latency in animals infected with HSV-1 strain HFEM revealed that this strain was unable to colonize the ganglia of tree shrews. Infectious virus could be recovered only from the spleens of latently infected tree shrews. Thus, this system offers new opportunities for investigating the gene functions responsible for the virulence of HSV-1. Marker rescue experiments were performed by the contransfection technique using native DNA of HSV-1 strain HFEM and Bam H I DNA fragment B derived from the pathogenic HSV-1 strain F and cloned in a bacterial vector. A number of different intratypic recombinants were established in which the deleted region of HSV-1 strain HFEM had been repaired. The pathogenicity of these recombinants was examined in vivo. One of the recombinants (HSV-R-HFehx-C19) caused generalized and lethal herpes virus infection in juvenile and adult tupaias, indicating that the virulence of the pathogenic HSV-1 strain F can be transduced by the cloned Bam H I DNA fragment B to the apathogenic HSV-1 strain HFEM.

Animals↗
PubMed1991

Co-operative binding of the glucocorticoid receptor DNA binding domain is one of at least two mechanisms for synergism.

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor binding sequences with adjacent binding sites either for other transcription factors or for further steroid receptors. Analysis of the human glucocorticoid receptor revealed that the DNA-binding domain of the receptor is sufficient to mediate co-operative binding to adjacent receptor binding sites. This is a novel feature of the domain in addition to its DNA-binding, trans-activating and trans-repressing properties. Chimaeric proteins containing the N- or C-terminal receptor halves fused to the GAL4 DNA-binding domain do not co-operate in DNA-binding, however they do functionally synergize. Thus, at least two mechanisms contribute to the synergism of the human glucocorticoid receptor bound to two adjacent receptor binding sites.

Base Sequence↗
PubMed1976

On the lack of host-cell reactivation of UV-irradiated phage T5 II. Further characterization of the repair inhibition exerted by T5 infection.

Experiments reported in the preceding paper [4] had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predicitions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5. The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 mug/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occurring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the "second-step-transfer" region of T5 DNA (involving the remainder of the genome). Superinfection with T4v1 phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.

Caffeine↗
PubMed1982

B lymphocyte immune response gene phenotype is genetically determined.

We examined the effects of the developmental milieu on the capacity of B cells to undergo immune response gene-controlled, T cell-dependent polyclonal proliferation. Although I-Aq poly(Glu60 Ala30 Tyr10)n (GAT)-nonresponder T cells developing in a responder environment become phenotypic GAT-responders, I-Aq B cells remain unresponsive to GAT, even after maturation in a GAT-responder animal. Conversely, (B10.A x B10.Q)F1 ([GAT responder x GAT nonresponder]F1) T cells developing in a B10.Q GAT nonresponder host fail to respond to GAT, but F1 B cells from the same F1 leads to parent chimeras make excellent proliferative responses in the presence of GAT and responder T cells. Thus, by this assay, B cell immune response gene function is genetically determined and is not affected by the developmental milieu.

Animals↗
PubMed2004

Genetic evidence for the coexistence of pheromone perception and full trichromatic vision in howler monkeys.

Vertebrate pheromones are water-soluble chemicals perceived mainly by the vomeronasal organ (VNO) for intraspecific communications. Humans, apes, and Old World (OW) monkeys lack functional genes responsible for the pheromone signal transduction and are generally insensitive to vomeronasal pheromones. It has been hypothesized that the evolutionary deterioration of pheromone sensitivity occurred because pheromone communication became redundant after the emergence of full trichromatic color vision via the duplication of the X-chromosome-linked red/green opsin gene in the common ancestor of hominoids and OW monkeys. Interestingly, full trichromacy also evolved in the New World (NW) howler monkeys via an independent duplication of the same gene. Here we sequenced from three species of howler monkeys an essential component of the VNO pheromone transduction pathway, the gene encoding the ion channel TRP2. In contrast to those of hominoids and OW monkeys, the howler TRP2 sequences have none of the characteristics of pseudogenes. This and other observations indicate that howler monkeys have maintained both their systems of pheromone communication and full trichromatic vision, suggesting that the presence of full trichromacy alone does not lead to the loss of pheromone communication. We suggest that the ecological differences between OW and NW primates, particularly in habitat selection, may have also affected the evolution of pheromone perception.

Alouatta↗
PubMed2001

Antisense display: a new method for functional gene screen and its application to angiogenesis-related gene isolation.

We have developed an antisense-oriented method for functional gene screening, which we termed "antisense display." After evaluation in a cell-free system, we applied this method to a cellular system for isolating angiogenesis-related genes and came across a gene previously not described in humans. A screen of 10 mer antisense repertoire led to the identification of a unique 'angiostatic' antisense, and the gene corresponding to it was found to be preferentially expressed in vascular endothelial cells, coding for a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand-like domains. We named it vascular Rab-GAP/TBC domain-containing protein (VRP). The results obtained thus indicate that the antisense display method is useful for the isolation of functional genes responsible for phenotypes concerned.

Genetic Testing↗
PubMed2003

Research on the genetic variations of a1-fucosytransferase (FUT1) gene in 26 pig breeds.

Enterotoxigenic Escherichia coli F18(ECF18) is a main pathogen that causes edema disease and post-weaning diarrhoea in piglets, and al-fucosytransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ECF18 bacteria. The genetic variations at position 307 nucleotide in open reading frame of FUT1 gene in 26 pig breeds (total 1458 individuals) from 5 western commercial pig breeds and 21 Chinese native pig breeds were investigated by PCR-RFLP. The results showed that the genetic polymorphisms of the FUT1 locus were only detected in 5 western pig breeds and the Chinese Lingao pig breed, 5 western pig breeds possessed 3 different genotypes, and Lingao pig breed had two susceptible genotypes GG and AG, while all the other 20 Chinese native pig breeds only presented the susceptible genotype GG. The results indicated that if M307G-A point mutation in the coding region of FUT1 gene was the key factor determining the expression of the ECF18 receptor, most of Chinese native pig breeds were absent of the genetic background on the resistance to ECF18 bacteria. In this case, it was inferred that the resistance gene to ECF18 might be originated from western pig breeds. In addition, it is of great importance for the conservation of Lingao pig breed as it is the only found Chinese native pig breed possessing resistance M307A allele in FUT1 gene. Generally, compared with exotic pig breeds, Chinese native pig breeds have stronger resistance to edema disease and post-weaning diarrhoea in piglets. The results suggested that further study should be done to identify and characterize putative QTL (quantitative trait locus) or/and the functional gene responsible for the resistance to ECF18 in Chinese native pig breeds.

Alleles↗
PubMed1983

Separation of the immune response genes for LDH-B and MOPC-173. I. Description of an immune response defect in B10.BASR1.

By using the intra-I-region recombinant mouse strain, B10.BASR1 (H-2as4), the immune response (Ir) genes for LDH-B and MOPC-173 were genetically and serologically separated, as assayed by T cell proliferation. Previous work demonstrated that the H-2s and H-2b strains respond to LDH-B and MOPC-173, whereas the H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak-activated T helper cells by I-Ek-activated T suppressor cells. In the experiments reported here, B10.BASR1 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to MOPC-173 but not to LDH-B. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A alpha sA beta sE beta sJk) macrophages with A.TL anti-B10.HTT (anti-A beta sE beta sJs) serum absorbed with B10.BASR1 spleen cells blocked the LDH-B response but not the MOPC-173 response. Unabsorbed serum blocked both antigen responses. The primary immunogenic determinant recognized by LDH-B or MOPC-173 immune T cells was not present on both antigens, as MOPC-173-primed T cells and LDH-B-primed T cells responded only to the priming antigen. Lastly, by using the A beta mutant strain, B6CH-2bm12, it was shown that the Ir gene and Ia determinants affected by this mutation had no effect on the LDH-B and MOPC-173 proliferative responses. These results suggest the possibility of an intragenic recombinatorial event in either the A alpha or A beta chain resulting in the separation of these two immune response gene functions.

Animals↗