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PubMed2000

Glycine receptors containing the alpha4 subunit in the embryonic sympathetic nervous system, spinal cord and male genital ridge.

Inhibitory glycine receptors (GlyRs) are known to mediate postsynaptic inhibition in spinal cord, brain stem and some higher brain regions. Several developmentally and regionally regulated GlyR isoforms exist, which result from a differential expression of the GlyR alpha (alpha1-alpha4) and beta subunit genes. Currently, very little is known about GlyRs containing the alpha4 subunit, whose existence was predicted from a partial genomic sequence. Here, we describe the isolation of complementary DNA (cDNA) sequences for the mouse and chick GlyR alpha4 subunits. We show that a mouse GlyR alpha4 subunit full-length cDNA directs the formation of functional homo-oligomeric strychnine-sensitive GlyRs in Xenopus laevis oocytes and mammalian cells, and that these resemble GlyRs composed of the alpha1 subunit in pharmacological profile and single-channel properties. In situ hybridization reveals high levels of GlyR alpha4 subunit transcripts in the embryonic (E13) chick spinal cord, lumbosacral sympathetic ganglia and dorsal root ganglia. The avian GlyR alpha4 subunit gene also shows male-specific expression in the developing genital ridge. The pharmacological profile of alpha4 subunit-containing receptors and deduced location of the avian GlyR alpha4 subunit are consistent with it being a component of the embryonic excitatory GlyRs previously identified in sympathetic neurons. Our data also suggest a novel role for GlyRs in the maturation of reproductive organs.

Amino Acid Sequence↗

PubMed2003

Autoantibodies in vitiligo patients recognize multiple domains of the melanin-concentrating hormone receptor.

Previously, we reported the identification of autoantibodies against the melanin-concentrating hormone receptor 1 in patients with vitiligo. In this study, the B cell epitopes on melanin-concentrating hormone receptor 1 that are recognized by these autoantibodies have been identified. Deletion derivatives of melanin-concentrating hormone receptor 1 complementary DNA were constructed and then translated in vitro with the concomitant incorporation of [35S]-methionine into the protein products. The [35S]-labeled melanin-concentrating hormone receptor 1 derivatives were subsequently used in radio-binding assays to investigate the reactivity of sera from nine vitiligo patients that were known to contain antibodies to the receptor. Analysis of the results obtained in the radio-binding assays suggested the existence of multiple antibody binding sites on melanin-concentrating hormone receptor 1, including regions between amino acids 1 to 138 and 139 to 298. Several patients exhibited autoantibodies to more than one melanin-concentrating hormone receptor 1 epitope indicating a heterogeneous humoral response to the receptor. Computer prediction of the potential B cell epitopes on melanin-concentrating hormone receptor 1 revealed that the epitope domains identified overlapped, at least in part, with regions predicted to be highly antigenic.

Adult↗

PubMed2003

Molecular cloning of p53 cDNA of Mongolian gerbil and establishment of yeast p53 functional assay system.

BACKGROUND: Epidemiological studies have shown a correlation between Helicobacter pylori infection and human gastric carcinogenesis. A Mongolian gerbil model has demonstrated that H. pylori infection induced gastric carcinoma. However, the disadvantage of this animal model is a lack of information regarding the cellular genes involved in oncogenesis. Mutation of the p53 gene is one of the most common steps in gastric carcinogenesis. In this study, we aimed to clone the p53 gene of the Mongolian gerbil and detect the functional mutations in H. pylori-infected animals. MATERIALS AND METHODS: The p53 complementary DNA (cDNA) of Mongolian gerbil was cloned by the methods of reverse-transcribed polymerase chain reaction and rapid amplification of cDNA ends. RESULTS: The p53 cDNA of Mongolian gerbil has a 78.8% homology to that of humans. A novel yeast p53 assay system was established and enabled to detect the functional mutations of the p53 gene in the stomach of the Mongolian gerbil. CONCLUSIONS: This is the first report of the complete sequence of wild-type p53 cDNA of the Mongolian gerbil. This genetic information and an assay system designed to detect the functional mutations of the p53 gene are useful for further investigations of gastric oncogenesis in this animal model.

Animals↗

PubMed2002

Efficient induction of minor histocompatibility antigen HA-1-specific cytotoxic T-cells using dendritic cells retrovirally transduced with HA-1-coding cDNA.

Cytotoxic T-cells (CTLs) specific for the hematopoietic system-restricted minor histocompatibility antigen (mHag) HA-1 efficiently lyse HA-1-positive leukemic cells without affecting nonhematopoietic cells. HA-1-specific CTLs are thus potential tools for adoptive immunotherapy of relapsed leukemia after HLA-matched-HA-1-mismatched stem cell transplantation (SCT). In vitro generation of HA-1-specific CTLs from SC donors is possible using dendritic cells (DCs) pulsed with synthetic HA-1 peptide as stimulator cells. However, this approach requires at least 6 weeks of in vitro culturing under GMP (good manufacturing practice) conditions. Our data show that in vitro induction of HA-1-specific CTLs is more rapid with the use of DCs that are retrovirally transduced with the HA-1 complementary DNA. Retrovirally transduced DCs showed functional and long-term stable expression of the HA-1 CTL epitope in primary CTL cultures. In 4 SC donors, HA-1-transduced DCs induced HA-1-specific CTLs in 14 to 21 days. The in vitro-generated CTL lines contained 6% to 9% T-cells that stained brightly with tetrameric HLA-A2/HA-1 peptide complexes (HA-1(A2) tetramer) and showed significant lysis of HA-1+ leukemic cells. The CTL induction procedure using peptide-pulsed DCs was less effective and required 28 to 35 days of T-cell culture. Thus, sustained presentation of mHag HA-1 by retrovirally transduced DCs facilitates the in vitro induction of HA-1-specific CTLs.

Antigen Presentation↗

PubMed1997

Functional impairment of rat enterochromaffin-like cells by interleukin 1 beta.

BACKGROUND & AIMS: Histamine-producing enterochromaffin-like (ECL) cells play an integrative role in the regulation of acid secretion. Decreased mucosal histamine concentrations and increased levels of interleukin (IL) 1 beta, IL-6, and IL-8 have been detected in the gastric mucosa inflamed with Helicobacter pylori. The aim of this study was to investigate the response of isolated ECL cells to these cytokines. METHODS: Enriched rat gastric ECL cells (85%-95%) were cultured for 2-4 days. RESULTS: Polymerase chain reaction showed IL-1 and IL-6, but not IL-8 receptors, in ECL cell complementary DNA. Positive receptor staining with biotinylated IL-1 beta corresponded to ECL cell enrichment (92%). IL-6 and IL-8 had no effect on histamine secretion. IL-1 beta (2 U/mL) stimulated basal histamine secretion and nitric oxide production within 60 minutes and cyclic guanosine monophosphate production within 20 minutes. Pretreatment for 20 minutes with IL-1 beta (2 U/mL) attenuated gastrin-stimulated histamine secretion by 40%-50%, reversed by the IL-1 receptor antagonist (10 U/ mL). Pretreatment for 20 minutes with IL-1 beta (2 U/mL) completely inhibited gastrin-stimulated (1 nmol/L) histidine decarboxylase activity. IL-1 beta (2 U/mL, 60 minutes) increased lactate dehydrogenase release to 25% of cell content. Cells pretreated with IL-1 beta did not respond to gastrin after a further 48-hour culture and showed decreased histamine content. CONCLUSIONS: ECL cells appear to express IL-1 receptors. IL-1 beta causes sustained functional impairment of ECL cells in vitro.

Animals↗

PubMed2001

Epidermal growth factor receptor mediates stress-induced expression of its ligands in rat gastric epithelial cells.

BACKGROUND & AIMS: Epidermal growth factor (EGF)-like growth factors are induced after acute gastric injury and may play an important role in mucosal repair. However, the mechanisms that trigger these growth factors are poorly understood. We determined the role of EGF receptor (EGFR) in stress-induced expression of heparin-binding EGF-like growth factor (HB-EGF) in a rat gastric epithelial cell line (RGM1 cells). METHODS: RGM1 cells were transfected with a plasmid containing complementary DNA encoding a dominant-negative human EGFR (HERCD533). Cells were treated with hydrogen peroxide (0-400 micromol/L) or sorbitol (600 mmol/L). Tyrosine phosphorylation of EGFR was determined by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. HB-EGF messenger RNA and protein were determined with Northern and Western blotting, respectively. Cell growth was evaluated by cell number and [(3)H]thymidine incorporation. RESULTS: Oxidative stress and osmotic stress induced tyrosine phosphorylation of EGFR within 2 minutes, followed by a marked increase in HB-EGF and amphiregulin transcripts in RGM1 cells. Introduction of HERCD533 into the cells inhibited not only tyrosine phosphorylation of EGFR but also growth response to EGF. Furthermore, oxidative stress-induced HB-EGF messenger RNA expression was impaired in HERCD533-expressing cells. CONCLUSIONS: EGFR plays a crucial role in the stress-induced expression of EGF-like growth factors in gastrointestinal epithelial cells.

Amphiregulin↗

PubMed1996

kan-1 (bile acid CoA:amino acid N-acyltransferase) messenger RNA as a novel predictive indicator for prognosis of hepatocellular carcinoma patients after partial hepatectomy.

We identified kan-1 complementary DNA (cDNA), the sequence of which is identical to bile acid CoA:amino acid N-acyltransferase (BAT), a liver enzyme that catalyzes the conjugation of bile acids with glycine or taurine. Kan-1(BAT) messenger RNA (mRNA) levels of the resected specimens obtained from 37 hepatocellular carcinoma (HCC) patients were studied in an attempt to evaluate prognostic significance in HCC patients after partial hepatectomy. Using Northern blot hybridization, kan-1(BAT) mRNA levels were quantified in tumorous and nontumorous tissues, and the ratio of the former to the latter was defined as the kan-1(BAT) ratio. Twelve patients had a kan-1(BAT) ratio < 0.5 (low kan-1[BAT] ratio), and 25 patients had a ratio >0.5 (high kan-1[BAT] ratio). The patients with a low kan-1(BAT) ratio demonstrated poorer survival than the patients with a high kan-1(BAT) ratio (P = .0013). The overall estimated hazard ratio for death in patients with a low kan-1(BAT) ratio was 68.05 according to a multivariate model (P = .0005). Thus, the kan-1(BAT) ratio may serve as a new molecular prognostic marker in HCC patients, following hepatic resection.

Acyltransferases↗

PubMed1997

Induction of hepatocyte growth factor activator messenger RNA in the liver following tissue injury and acute inflammation.

Hepatocyte growth factor activator (HGFA) is a serine protease that is responsible for localized activation of hepatocyte growth factor (HGF) in injured tissue. The activated HGF may be involved in regeneration of the injured tissue. HGFA is produced and secreted by the liver and circulates in the plasma as an inactive zymogen. In response to tissue injury, the HGFA zymogen is converted to the active form by limited proteolysis. In this study, we isolated a rat HGFA complementary DNA (cDNA) clone and analyzed the production of HGFA messenger RNA (mRNA) in response to tissue injury using this cDNA clone as a probe. The nucleotide sequence of the cDNA revealed that the amino acid sequences of rat and human HGFA showed a high degree of conservation in the regions of the characteristic domain structures, suggesting that rat and human HGFA are activated by a similar mechanism and have similar enzymatic activities in vivo. Tissue distribution analysis showed that the liver was the major site of rat HGFA mRNA synthesis. Moreover, the cells producing HGFA mRNA were identified as parenchymal liver cells. The level of HGFA mRNA increased in the liver after hepatotoxin or nephrotoxin treatment. This increase was also observed during acute inflammation induced by turpentine. These results suggest that the increase in production of HGFA mRNA in response to tissue injury is the result of an inflammatory response, and that HGFA is an acute phase protein.

Acute Disease↗

PubMed2001

A cellular gene up-regulated by hepatitis B virus-encoded X antigen promotes hepatocellular growth and survival.

Polymerase chain reaction (PCR) select complementary DNA (cDNA) subtraction of hepatitis B x antigen (HBxAg)-positive compared with -negative HepG2 cells resulted in the up-regulated expression of a cellular gene that encodes a transcript of 745 bases and a polypeptide 99 amino acids long. GenBank analysis revealed extensive homology with the amino terminal domain of cellular multidrug resistant proteins (MRP), although overexpression of this gene did not confer an MRP phenotype. In situ hybridization and immunostaining showed colocalized expression with HBxAg in the liver of hepatitis B carriers. Overexpression of this protein stimulated the growth of HepG2 cells in serum-free medium, and partially protected cells from anti-Fas-mediated killing, but did not promote growth in soft agar or tumor formation in nude mice. Introduction of the dominant negative inhibitor of nuclear factor kappaB (IkappaBalpha) into HBxAg-positive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, suggesting that its up-regulation is nuclear factor kappaB (NF-kappaB) dependent. Hence, HBxAg activation of NF-kappaB may result in the up-regulation of a cellular protein that promotes growth factor-independent survival and protects against Fas-mediated killing. This factor may contribute to the persistence of infected hepatocytes during chronic infection, which is important for the later development of hepatocellular carcinoma (HCC).

ATP-Binding Cassette Transporters↗

PubMed2003

A novel liver-specific zona pellucida domain containing protein that is expressed rarely in hepatocellular carcinoma.

We currently identified a liver-specific gene that encodes a novel zona pellucida (ZP) domain-containing protein named liver-specific ZP domain-containing protein (LZP). The full-length complementary DNA (cDNA) of human LZP has 2,255 bp with a complete open reading frame (ORF) of 1,635 bp. The gene is localized on chromosome 10q21.3 and spans 40 kb with 9 encoding exons and 8 introns. The deduced protein sequence has 545 amino acid residues, with an N-terminal signal peptide followed by 3 epidermal growth factor (EGF)-like domains and a ZP domain in C-terminal section. Interestingly, human LZP is expressed specifically in liver out of 23 tissues examined, and its mouse counterpart was detected at very early stage during embryo development. Moreover, LZP can be secreted into blood, albeit the protein was localized mainly on the nuclear envelop of hepatocytes. Most importantly, LZP is down-regulated in hepatocellular carcinoma (HCC) and HCC cell lines; meanwhile, the decreased level of hLZP messenger RNA (mRNA) could, at least in some HCC samples, be related to the methylation status of the putative LZP promoter. However, overexpression of hLZP in HCC cell line SMMC-7721 and human liver cell line L02 by stable cell transfection did not inhibit cell growth, implying that the down-regulation of hLZP in HCC might be a consequence of the dedifferentiation involved in hepatocarcinogenesis. In conclusion, these data suggest that LZP is a liver-specific protein involved possibly in hepatocellular function and development, and the protein could be used as potential negative biomarker for HCC pathologic diagnosis.

Amino Acid Sequence↗

PubMed2002

Vascular endothelial growth factor gene therapy in ischaemic rat skin flaps.

Gene therapy with the complementary DNA (cDNA) of the angiogenic cytokine vascular endothelial growth factor (VEGF) has emerged as a promising strategy in the treatment of myocardial and lower-limb ischaemia. The objective of this study was to determine whether these principles could be applied to a recognised model of skin-flap ischaemia. Plasmid vectors including the cDNA of green fluorescent protein (GFP) and one of three VEGF isoforms (A165, B167 or B186) were constructed, and their base sequences confirmed. GFP expression was used as a marker of successful in vitro transfection of human endothelial cells with each plasmid. The plasmids were then administered subcutaneously to rat abdominal skin flaps surgically rendered ischaemic, and the percentage of viable tissue was assessed at 1 week. Angiograms of the flaps and histological preparations of flap tissue were assessed for evidence of angiogenesis. The survival of flaps treated with VEGF A165 or B167 cDNA was significantly greater than that of controls (P < 0.05). The survival of flaps treated with VEGF B186 cDNA was greater than that of controls, but statistical significance was not reached. Angiograms and microvessel density counts failed to produce evidence of angiogenesis. With improved delivery strategies, VEGF may have a role in the management of surgical ischaemia.

Analysis of Variance↗

PubMed1994

Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder in which there is a deficiency in the synthesis by hematopoietic cells of the glycosyl-phosphatidylinositol molecules that anchor proteins to the cell membrane. Recently, we demonstrated that a gene termed PIG-A (for phosphatidylinositol glycan class A), a component of glycosyl-phosphatidylinositol biosynthesis, was responsible for PNH in two patients. The present study was undertaken to elucidate whether PIG-A is the gene responsible for all cases of PNH and to characterize further the somatically acquired abnormalities of this gene. METHODS: We studied granulocytes from 15 patients with PNH. The cell content of CD55 and CD59 was assessed by fluorescence-activated flow cytometry. PIG-A transcripts were reverse-transcribed, amplified by the polymerase chain reaction, and cloned into plasmids. The structure of the cloned complementary DNA was analyzed by nucleotide sequencing, and its function was assessed on the basis of its ability to restore to normal the abnormal phenotype of a PIG-A-deficient cell line after transfection. RESULTS: Three patients had size abnormalities of PIG-A transcripts with different patterns, and in one patient a very low level of the PIG-A transcript was found. Eleven patients had transcripts of normal size, but the transfection assay revealed that in each patient some of them were nonfunctional. The percentage of nonfunctional PIG-A transcripts was correlated with the percentage of affected granulocytes (P < 0.001). Sequence analysis demonstrated somatic mutations in two of the patients. CONCLUSIONS: PIG-A is the gene responsible for PNH in all patients studied to date.

Base Sequence↗

PubMed1996

Polymorphism of adhesion molecule CD31 and its role in acute graft-versus-host disease.

BACKGROUND: Graft-versus-host disease (GVHD) caused by poorly defined minor (i.e., other than HLA) histocompatibility antigens remains a serious problem in recipients of bone marrow transplants. We sought to determine whether the CD31 adhesion molecule is a minor alloantigen. METHODS: We directly sequenced samples of complementary DNA (cDNA) encoding CD31 molecules from 21 unrelated normal subjects. Sequence-specific primers were then designed to amplify alleles by the polymerase chain reaction, thereby permitting CD31 typing of genomic DNA from additional normal subjects. To assess the relevance of CD31 matching to bone marrow transplantation, we performed CD31 typing of 46 recipients of bone marrow (32 without GVHD and 14 with severe [grade III or IV] acute GVHD) and their HLA-identical sibling donors. The immunoreactivity of CD31 phenotypes with anti-CD31 monoclonal antibodies was compared by flow cytometry. RESULTS: Direct sequencing of cDNA for CD31 from the 21 normal subjects identified a single polymorphism, CTG-->GTG (Leu-->Val), at codon 125; we designated the resulting alleles CD31.L and CD31.V, respectively. The CD31 genotypes of these and 142 other unrelated subjects were of the expected frequencies. Among the transplant recipients, 71 percent of those with acute GVHD had CD31 genotypes that were not identical to the donor's genotype, as compared with 22 percent of the recipients without GVHD (P = 0.004). The binding of anti-CD31 monoclonal antibodies as measured by fluorescence-activated cell sorting correlated with the CD31 types of homozygous cell lines. CONCLUSIONS: The adhesion molecule CD31 is polymorphic. When donor and recipient genotypes are not identical, the risk of GVHD increases. Prospective CD31 typing may reduce the risk of acute GVHD.

Adolescent↗

PubMed1999

A serologic marker of paraneoplastic limbic and brain-stem encephalitis in patients with testicular cancer.

BACKGROUND: In patients with cancer, symptoms of limbic and brain-stem dysfunction may result from a paraneoplastic disorder. Paraneoplastic limbic or brain-stem encephalitis occurs more frequently with testicular cancer than with most other cancers. We sought antineuronal antibodies that might be used in a diagnostic test for this syndrome. METHODS: Immunohistochemical and immunoblotting techniques were used to detect serum and cerebrospinal fluid antibodies. Serologic screening of a complementary DNA library and Northern blotting were used to clone the target antigen and determine which tissues expressed it. RESULTS: Of 13 patients with testicular cancer and paraneoplastic limbic or brain-stem encephalitis (or both), 10 had antibodies in serum and cerebrospinal fluid against a 40-kd neuronal protein. These antibodies were used to clone a gene that we call Ma2, which codes for a protein (Ma2) that was recognized by serum from the 10 patients, but not by serum from 344 control subjects. Ma2 was selectively expressed by normal brain tissue and by the testicular tumors of the patients. Ma2 shares homology with Ma1, a "brain-testis-cancer" gene related to other paraneoplastic syndromes and tumors. CONCLUSIONS: The serum of patients with subacute limbic and brain-stem dysfunction and testicular cancer contains antibodies against a protein found in normal brain and in testicular tumors. Detection of these antibodies supports the paraneoplastic origin of the neurologic disorder and could be of diagnostic importance.

Animals↗

PubMed2000

Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene.

BACKGROUND: Myofibrillar myopathies, often referred to as desmin-related myopathies, are a heterogeneous group of inherited or sporadic distal-onset skeletal myopathies associated with cardiomyopathy. Among the myofibrillar proteins that characteristically accumulate within the muscle fibers of affected patients, the one found most consistently is desmin, a muscle-specific intermediate-filament protein responsible for the structural integrity of the myofibrils. Skeletal and cardiac myopathy develops in mice that lack desmin, suggesting that mutations in the desmin gene may be pathogenic. METHODS: We examined 22 patients from 8 families with dominantly inherited myofibrillar or desmin-related myopathy and 2 patients with sporadic disease and analyzed the desmin gene for mutations, using complementary DNA (cDNA) amplified from muscle-biopsy specimens and genomic DNA extracted from blood lymphocytes. Restriction-enzyme analysis was used to confirm the mutations. Expression vectors containing normal or mutant desmin cDNA were introduced into cultured cells to determine whether the mutant desmin formed intermediate filaments. RESULTS: Six missense mutations in the coding region of the desmin gene that cause the substitution of an amino acid were identified in 11 patients (10 members of 4 families and 1 patient with sporadic disease); a splicing defect that resulted in the deletion of exon 3 was identified in the other patient with sporadic disease. Mutations were clustered in the carboxy-terminal part of the rod domain, which is critical for filament assembly. In transfected cells, the mutant desmin was unable to form a filamentous network. Seven of the 12 patients with mutations in the desmin gene had cardiomyopathy. CONCLUSIONS: Mutations in the desmin gene affecting intermediate filaments cause a distinct myopathy that is often associated with cardiomyopathy and is termed "desmin myopathy." The mutant desmin interferes with the normal assembly of intermediate filaments, resulting in fragility of the myofibrils and severe dysfunction of skeletal and cardiac muscles.

Adult↗

PubMed2002

Adenovirus vector-mediated transfer of the vascular endothelial growth factor cDNA to healing abdominal fascia enhances vascularity and bursting strength in mice with normal and impaired wound healing.

BACKGROUND: We hypothesized that adenovirus-mediated transfer of the vascular endothelial growth factor (VEGF121) complementary DNA (cDNA) to murine laparotomy fascial wounds would enhance vascularity and bursting strength. METHODS: Microfibrillar collagen sponges saturated with adenovirus (Ad) vectors encoding for the human VEGF121 cDNA (Ad(CU)VEGF121.1), a control marker gene (Ad beta gal, AdLuc) or no transgene (AdNull) were sutured to fascial edges during laparotomy closure in normal mice and mice treated with dexamethasone. Endpoints addressed included transgene expression in the fascia and surrounding tissue, the number of blood vessels in the healing wound determined using immunostaining, and wound bursting strength using a calibrated tensinometer. RESULTS: Transgene expression was detected readily in the fascial edges, but only marginally detectable in neighboring tissues. In normal mice and mice treated with dexamethasone, no differences were observed at 7 days. Strikingly, however, 21 days after wound closure/therapy, significantly more blood vessels were present in the wounds that received the VEGF121 vector compared with controls (normal: AdNull: 4.2 +/- 1.8; Ad(CU)VEGF121.1: 11.2 +/- 1.2; P <.05; dexamethasone: AdNull: 1.4 +/- 0.8; Ad(CU)VEGF121.1: 5.4 +/- 1.2; P <.05), and bursting strength was significantly higher in VEGF121-treated wounds (normal: AdNull: 665 +/- 68 mN/mm; Ad(CU)VEGF121.1: 956 +/- 82 mN/mm; P <.0005; dexamethasone: AdNull: 234 +/- 111 mN/mm; Ad(CU)VEGF121.1: 592 +/- 121 mN/mm; P <.03). CONCLUSIONS: Adenovirus-mediated gene transfer to healing fascial wounds is achieved readily using a microfibrillar collagen sponge, with transfer of the human VEGF121 cDNA significantly enhancing wound vascularity and bursting strength in normal mice, as well as in mice treated with dexamethasone.

Adenoviridae↗

PubMed2003

Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 +/- 0.4 kcal.mol-1 and -52.3 +/- 1.4 cal.mol-1.K-1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 +/- 0.2 kcal.mol-1 and -23.0 +/- 0.8 cal.mol-1.K-1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 kBT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.

Base Composition↗

PubMed1995

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Alternative Splicing↗