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H Li

Publications and source records attributed to H Li.

At least 1,225 records · Page 68Linked to original sources
PubMed1997

RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2.

Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.

Amino Acid Sequence↗
PubMed1997

Colocalization of GABA(A) and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat.

Changes in gastric motor activity are observed in response to glutamate and GABA in the DMV. We investigated the expression of GABA(A) and NMDA receptors within DMV neurons projecting to the stomach using pseudorabies virus (PRV). PRV immunoreactive (PRV-IR) cells expressing GABA(A) alpha1-IR, also expressed NMDAR1 suggesting that NMDA and GABA(A) receptors are colocalized. These results provide a neuroanatomical basis for these receptors jointly playing a role in gastric motor functions.

Animals↗
PubMed1997

Transfection of rat brain cells by electroporation.

Genetic intervention is a powerful means for answering basic biological questions about molecular events taking place in brain cells underlying complex behaviors. Traditional transfection method has proved difficult to apply to neuronal cells. We therefore described an alternative procedure for introducing DNA into cultured rat brain cells by electroporation. The various parameters involving the voltage, capacitance and electroporation buffer are investigated. We found that this transfection procedure is simple, reproducible and applicable to rat brain cells in vitro.

Animals↗
PubMed1997

Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have cancer chemopreventive properties by a mechanism that has been suggested to involve cyclooxygenase inhibition and reduction of prostaglandin (PGE2) levels in the target tissue. To test this hypothesis, we studied the effect of dietary sulindac sulfone (500-2000 ppm), a metabolite of sulindac reported to lack cyclooxygenase inhibitory activity, on tumor formation and PGE2 levels in the azoxymethane model of colon carcinogenesis. Rats treated with sulindac at 400 ppm and piroxicam at 150 ppm were used as positive controls. Rats received two s.c. injections of azoxymethane (15 mg/kg) for 2 weeks and were fed either experimental or control diets until necropsy. After 31 weeks of sulfone treatment, a dose-related increase in sulfone levels in both serum and cecal contents was measured; there was no evidence of metabolic conversion to sulindac or other metabolites. Rats treated with sulfone at 1000 and 2000 ppm, sulindac, and piroxicam had significantly fewer colonic adenomas and carcinomas compared with rats fed control diet as measured by tumor incidence, multiplicity, and tumor burden. Sulfone-treated rats also showed a dose-response relationship for inhibiting all tumor parameters. Colons from rats treated with sulindac or piroxicam contained PGE2 levels that ranged from approximately 16-49% of control levels. PGE2 levels in rats treated with sulfone up to 2000 ppm ranged from 78-118% of control levels. Moreover, the effects of sulindac sulfone on various enzymes responsible for regulating prostaglandin levels were evaluated. No significant inhibitory effects were observed for cyclooxygenase, lipoxygenase, or phospholipase A2. These results suggest that reduction of prostaglandin levels in the target tissue may not be necessary for the chemopreventive properties of sulindac.

Animals↗
PubMed1997

Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells.

Most cancer cells have increased levels of telomerase activity implicated in cell immortalization. Activation of telomerase, a ribonucleoprotein complex, catalyzes the elongation of the ends of mammalian chromosomal DNA (telomeres), the length of which regulates cell proliferation. Currently, how telomerase is regulated in cancer is not yet established. The present study shows that telomerase activity is regulated by protein phosphorylation in human breast cancer cells. Incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolished the telomerase activity; in contrast cytoplasmic telomerase activity was unaffected, and protein phosphatases 1 and 2B were ineffective. Inhibition of telomerase activity by PP2A was both concentration- and time-dependent and was prevented by the protein phosphatase inhibitor okadaic acid. In addition, nuclear telomerase inhibited by PP2A was reactivated by endogenous protein kinase(s) in the presence of ATP, but not in the presence of ATPgammaS. Furthermore, telomerase activity in cultured human breast cancer PMC42 cells was stimulated by okadaic acid, consistent with a role for PP2A in the regulation of telomerase activity in intact cells. These findings suggest that protein phosphorylation reversibly regulates the function of telomerase and that PP2A is a telomerase inhibitory factor in the nucleus of human breast cancer cells.

Breast Neoplasms↗
PubMed1997

Apoptosis primarily accounts for the growth-inhibitory properties of sulindac metabolites and involves a mechanism that is independent of cyclooxygenase inhibition, cell cycle arrest, and p53 induction.

Sulindac causes regression of and prevents recurrence of colonic adenomas in patients with familial adenomatous polyposis. Although cell cycle arrest and apoptosis have been proposed, the mechanism of action is poorly understood. In this study, we characterized the growth-inhibitory effects of active metabolites of sulindac in cultured colon adenocarcinoma cells by determining the contribution of apoptosis and cell cycle arrest and the requirement for cyclooxygenase (COX) inhibition and p53 involvement and compared the effects of sulindac metabolites with the chemotherapeutic drug, 5-fluorouracil (5-FU). Time course and dose-response experiments demonstrated that increased apoptosis paralleled the growth-inhibitory effects of the sulfide and sulfone. A relationship among a series of nonsteroidal anti-inflammatory drugs was observed between potency for growth inhibition and ability to induce apoptosis but not potency to inhibit COX. For example, the sulfone was at least 5000-fold less potent than the sulfide for inhibiting COX but only 6.5-fold less potent for inducing apoptosis. Moreover, the prostaglandin analogue, dimethyl-prostaglandin E2, failed to reverse the apoptosis-inducing effects of the sulfide. Sulindac metabolites caused G1 cell cycle arrest in proliferating cells but were comparably effective in nonproliferating cells. In contrast, 5-FU treatment was less effective in nonproliferating cells. Combined treatment with sulindac metabolites and 5-FU did not result in an additive apoptotic response. Treatment of cells with 5-FU increased p53 protein levels, whereas sulindac metabolites did not induce expression. Saos-2 cells, which lack p53, responded to sulindac metabolites but not 5-FU. These results show that apoptosis primarily contributes to growth inhibition by sulindac metabolites. The biochemical pathway does not require COX inhibition or p53 induction and appears to be fundamentally different from the apoptotic response to 5-FU.

16,16-Dimethylprostaglandin E2↗
PubMed1997

Dual conformations of a T cell receptor V alpha homodimer: implications for variability in V alpha V beta domain association.

The crystal structure of a mutant T cell receptor (TCR) V alpha domain containing a grafted third complementarity-determining region (CDR3) from a different V alpha was determined at 2.3 A resolution by molecular replacement using the wild-type V alpha structure as a search model. Like the wild-type V alpha domain, the mutant crystallized as a homodimer very similar to TCR V alpha V beta and antibody V(L)V(H) heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant V alpha homodimer differs from that in the wild-type by a rotation of 14 degrees such that the buried surface area in the dimer interface of the mutant is 140 A2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the V alpha and V beta domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.

Amino Acid Sequence↗
PubMed1997

Chemoenzymatic synthesis of a trimeric ganglioside GM3 analogue.

A trimeric beta-lactosyl cluster based on 2-nitro-2-(hydroxymethyl)propane-1,3-diol was prepared using the trichloroacetimidate method. Kinetic studies showed that this cluster was an effective acceptor for rat-liver alpha-(2-->3)-sialyltransferase. Its KM was comparable to those for monomeric lactose and N-acetyllactosamine acceptors, and its Vmax was 1% of that measured for the LacNAc acceptor. Preparative-scale sialylation using this enzyme afforded a trimeric cluster of the ganglioside GM3 oligosaccharide in good yield. The NMR spectra of the trimeric GM3 analogue suggest that the oligosaccharide conformation is not significantly perturbed by this level of clustering.

Amino Sugars↗
PubMed1997

The effect of M-stage on patterns of failure in posterior fossa primitive neuroectodermal tumors treated on CCG-921: a phase III study in a high-risk patient population.

PURPOSE: To analyze patterns of failure in patients (pts) with high-risk posterior fossa primitive neuroectodermal tumors (PF-PNETs) treated with combined modality therapy on a large, randomized multiinstitutional study. METHODS AND MATERIALS: One hundred eighty-eight prospectively staged pts with PF-PNET confirmed by central pathology review, with high-risk features, were treated on Children's Cancer Group Study 921 (CCG-921), comparing two chemoradiotherapy regimens. Patterns of initial sites of failure were analyzed, specifically evaluating the impact of Chang M-stage. RESULTS: Progression-free survival (PFS) correlated with the presence or absence of metastatic disease (p < 0.001), with 5-year PFS of 68 +/- 5.8% for M0 vs. 43 +/- 6.8% for M+ pts. The cumulative incidence functions (CIF) of recurrence were different (p = 0.005) and at 5 years were 29 +/- 4.7% for M0 pts and 48 +/- 5.5% for M+ pts. Involvement of the PF at time of initial failure as measured by CIF correlated with M-stage (p = 0.047) and occurred in 18 +/- 3.9% of M0 pts and 8 +/- 2.9% of M+ pts overall; PF as the only site of relapse also correlated with M-stage (p = 0.019) and was seen in 6 +/- 2.5 and 0% of M0 and M+ pts, respectively, at 5 years. Relapse in the spine and/or cerebrospinal fluid (CSF) at initial recurrence was correlated with M-stage (p < 0.002), with 5-year cumulative incidences of 14 +/- 3.7%, 26 +/- 8.2%, 40 +/- 15%, and 40 +/- 7.7% for M0, M1, M2, and M3 pts, respectively. Isolated spine/CSF recurrence correlated with M-stage (p = 0.034) and occurred in 2 +/- 1.5% of M0 and 9 +/- 3.2% of M+ pts by 5 years. The median time to relapse for pts who failed was 1.2 years (range 0.2-5.3). Ninety percent of all relapses occurred by 3 years. CONCLUSIONS: Original sites of disease are at the highest risk for relapse, but the entire neuraxis remains at significant risk, despite combined-modality treatment. M-Stage was prognostic for spine/CSF relapse as well as PFS and may be an important tool in guiding therapy. A more aggressive approach to local control in the neuraxis is warranted, especially in M+ patients.

Adolescent↗
PubMed1997

Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes.

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 --> Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the kcat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the kcat and increased specificity. The increases observed in kcat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

ABO Blood-Group System↗
PubMed1997

Identification of chemical synapses in the pharynx of Caenorhabditis elegans.

The rhythmic contraction of the Caenorhabditis elegans pharynx is unique in that the network of 12 neurons, including two M3 neurons, that regulate the contraction is known. The neurotransmitters secreted by these cells, and the target cells responding to these chemical signals, are not known. Here, we describe an approach to obtain this missing information and use the M3 cells as an example. Electrical recordings (electropharyngeograms) were used in conjunction with temporally and spatially defined application of neurotransmitters via photolysis of inactive, photolabile precursors. To illustrate the technique we used pharyngeal preparations in which the two M3 neurons are intact and preparations in which they were removed by laser irradiation. Removal of M3 neurons results in the loss of the small negative peaks in the electropharyngeograms and an increase in time during which the pharynx remains contracted. We demonstrate that the application of glutamate by photolysis of caged glutamate to a pharynx from which the two M3 neurons were removed produces effects similar to those observed before removal of the M3 neurons. In control experiments, photolytic release from photolabile precursors of carbamoylcholine, a stable and well characterized analog of acetylcholine, or of gamma-aminobutyric acid, from photolabile precursors did not have this effect. The response depended on the amount of glutamate released. By reducing the size of the photolytic beam, glutamate was released at several different locations of the pharynx. Two areas of the pharynx mainly respond to the application of glutamate; one corresponds to the pm4 muscle cells in the metacorpus, and the other to the junction between muscle cells pm5 in the isthmus and pm6 in the terminal bulb.

Animals↗
PubMed1997

Evidence for a dystrophin missense mutation as a cause of X-linked dilated cardiomyopathy.

BACKGROUND: X-linked dilated cardiomyopathy (XLCM) has previously been shown to be due to mutations in the dystrophin gene, which is located at Xp21. Mutations in the 5' portion of the gene, including the muscle promoter, exon 1, and the exon 1-intron 1 splice site, have been reported previously. The purpose of this study was to analyze the originally described family with XLCM (and other) for dystrophin mutations. METHODS AND RESULTS: Polymerase chain reaction (PCR) was used to amplify genomic DNA, and reverse-transcriptase PCR amplified cDNA from RNA obtained from heart and lymphoblastoid cell lines. Primers to the muscle promoter, brain promoter, and Purkinje cell promoter were designed, in addition to the exon 1 to exon 14 regions of dystrophin. Single-strand conformation polymorphism analysis was used for mutation detection, and DNA sequencing defined the mutation. Protein modeling was used for amino acid and secondary structure analysis. A missense mutation in exon 9 at nucleotide 1043 was identified that causes an alanine to be substituted for threonine, a highly conserved amino acid, at position 279 (T279A). This mutation results in a change in polarity in the evolutionarily conserved first hinge region (H1) of the protein and substitution of a beta-sheet for alpha-helix in this portion of the protein, destabilizing the protein. CONCLUSIONS: A novel missense mutation in exon 9 of dystrophin causing an abnormality at H1 leads to the cardiospecific phenotype of XLCM.

Amino Acid Sequence↗
PubMed1997

Chronic and recovered cases of sheep-associated malignant catarrhal fever in cattle.

Malignant catarrhal fever (MCF) is traditionally regarded as a disease with a short clinical course, low morbidity and high case fatality rate. Owing to the limitations of the assays used for laboratory diagnosis. It was difficult in characterise the clinical spectrum of sheep-associated MCF, particularly when the cattle recovered from an MCF-like clinical syndrome. Over a period of three years, 11 cattle that survived MCF for up to two-and-a-half years were identified on four premises. A clinical diagnosis of MCF was confirmed by the detection of ovine herpesvirus-2 DNA in peripheral blood leucocytes using a polymerase chain reaction (PCR) assay that detects a specific 238 base-pair fragment of viral genomic DNA. Of the 11 cattle examined, six recovered clinically with the exception of bilateral corneal oedema with stromal keratitis (four animals) and unilateral perforating keratitis (one animal). The 10 animals available for postmortem examination had disseminated subacute to chronic arteriopathy. Recovery was associated with the resolution of the acute lymphoid panarteritis that characterises the acute phase of MCF, and with the development of generalised chronic obliterative arteriosclerosis. Bilateral leucomata were due in part to the focal destruction of corneal endothelium secondary to acute endothelialitis. Formalin-fixed tissues and/or unfixed lymphoid cells from all 11 cattle were positive for sheep-associated MCF by PCR. These observations indicate that recovery and chronic disease are a significant part of the clinical spectrum of MCF and that such cases occur with some frequency in the area studied. The affected cattle remain persistently infected by the putative sheep-associated MCF gammaherpesvirus.

Animals↗
PubMed1997

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody.

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC), a CC chemokine, is chemotactic for cells that typically infiltrate the late-phase allergic reaction. We developed a mouse model of airway inflammation to study the role of MCP-3/FIC. The immunization of mice with OVA resulted in Ag-specific IgE Ab production and the expression of mRNA for IL-4 in the lung tissue. Two weeks after immunization mice were challenged with the allergen by inhalation. Lungs were lavaged, and the tissue was examined at 2 or 24 h. Allergen challenge resulted in the increased recovery of leukocytes in the lavage fluid, but saline challenge did not. There was a significant increase in eosinophils (29 +/- 8% vs 1.2 +/- 0.2%) and lymphocytes (25 +/- 4% vs 5 +/- 2%) in the bronchoaveolar lavage fluid. Histologic examination of the lung demonstrated intense airway inflammation following OVA challenge. The expression of MCP-3/FIC and other CC chemokines (MCP-1, macrophage inflammatory protein-1alpha, and RANTES) was investigated by reverse transcription-PCR followed by densitometric analyses. The allergen challenge up-regulated the expression of mRNA for MCP-1, MCP-3/FIC, and macrophage inflammatory protein-1alpha at 2 and/or 24 h. Immunocytochemical staining for MCP-3/FIC showed that the allergen challenge induced the expression of MCP-3/FIC predominantly in the airway epithelium. Pretreatment of mice with an anti-MCP-3/FIC Ab significantly inhibited the OVA-induced airway inflammation and the bronchoalveolar lavage eosinophilia (8 +/- 2% vs 46 +/- 11% after control Ab, p < 0.03). We conclude that MCP-3/FIC plays a significant role in the allergen-induced eosinophilic inflammation of the airways.

Animals↗
PubMed1997

Structural and functional analysis of the chick chondroitin sulfate proteoglycan (aggrecan) promoter and enhancer region.

Aggrecan is a large chondroitin sulfate proteoglycan, the expression of which is both tissue-specific and developmentally regulated. Here we report the cloning and sequencing of the 1.8-kilobase genomic 5' flanking sequence of the chick aggrecan gene and provide a functional and structural characterization of its promoter and enhancer region. Sequence analysis reveals potential Sp1, AP2, and NF-I related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CIIS1 and CIIS2. A number of these transcription factor binding motifs are embedded in a sequence flanked by prominent inverted repeats. Although lacking a classic TATA box, there are two instances in the 1.8-kb genomic fragment of TATA-like TCTAA sequences, as have been defined previously in other promoter regions. Primer extension and S1 protection analyses reveal three major transcription start sites, also located between the inverted repeats. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively reduced portions of the aggrecan promoter region allowed mapping of chondrocyte-specific transcription enhancer and silencer elements that are consistent with the sequence analysis. These findings suggest the importance of this regulatory region in the tissue-specific expression of the chick aggrecan gene.

Aggrecans↗
PubMed1997

Elimination of cyclin D1 in vertebrate cells leads to an altered cell cycle phenotype, which is rescued by overexpression of murine cyclins D1, D2, or D3 but not by a mutant cyclin D1.

DT40 lymphoma B-cells normally express cyclins D1 and D2 but not D3. When cyclin D1 expression was extinguished in these cells by gene knockout, specific alterations in their ability to transit the cell cycle were observed. These changes are exemplified by a delay of approximately 2 h in their progression through a normal 14-h cell cycle. This delay results in an increase in the number of cells in the G2/M phase population, most likely due to triggering of checkpoints in G2/M, inability to enter G1 normally, and/or alterations of crucial event(s) in early G1. The defect(s) in the cell cycle of these D1 "knockout" cells can be rescued by overexpression of any normal mouse D-type cyclin but not by a mutant mouse cyclin D1 protein that lacks the LXCXE motif at its amino terminus. These data suggest that the cell cycle alterations observed in the D1-/- cells are a direct effect of the absence of the cyclin D1 protein and support the hypothesis that the D-type cyclins have separate, but overlapping, functions. Elimination of cyclin D1 also resulted in enhanced sensitivity to radiation, resulting in a significant increase in apoptotic cells. Expression of any normal murine D-type cyclin in the D1-/- cells reversed this phenotype. Intriguingly, expression of the mutant cyclin D1 in the D1 -/- cells partially restored resistance to radiation-induced apoptosis. Thus, there may be distinct differences in cyclin D1 complexes and/or its target(s) in proliferating and apoptotic DT40 lymphoma B-cells.

Amino Acid Sequence↗
PubMed1997

Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab.

OspA (outer surface protein A) is an abundant immunogenic lipoprotein of the Lyme disease spirochete Borrelia burgdorferi. The crystal structure of a soluble recombinant form of OspA was solved in a complex with the Fab fragment of mouse monoclonal antibody 184.1 and refined to a resolution of 1.9 A. OspA has a repetitive antiparallel beta topology with an unusual nonglobular region of "freestanding" sheet connecting globular N- and C-terminal domains. Arrays of residues with alternating charges are a predominant feature of the folding pattern in the nonglobular region. The 184.1 epitope overlaps with a well conserved surface in the N-terminal domain, and a hydrophobic cavity buried in a positively charged cleft in the C-terminal domain is a potential binding site for an unknown ligand. An exposed variable region on the C-terminal domain of OspA is predicted to be an important factor in the worldwide effectiveness of OspA-based vaccines.

Amino Acid Sequence↗