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H Niu

Publications and source records attributed to H Niu.

At least 19 recordsLinked to original sources
PubMed1998

Antigen receptor signaling induces MAP kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor.

The bcl-6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B and T cells and required for GC formation and antibody affinity maturation. Deregulation of bcl-6 expression by chromosomal rearrangements and point mutations of the bcl-6 promoter region are implicated in the pathogenesis of B-cell lymphoma. The signals regulating bcl-6 expression are not known. Here we show that antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase (MAPK). Phosphorylation, in turn, targets BCL-6 for rapid degradation by the ubiquitin/proteasome pathway. These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation. This signaling pathway may be crucial for the control of B-cell differentiation and antibody response and has implications for the regulation of other POZ/zinc finger transcription factors in other tissues.

B-Lymphocytes↗
PubMed1998

Nonviral vector-mediated thymidine kinase gene transfer and ganciclovir treatment in leiomyoma cells.

OBJECTIVE: To test the hypotheses that ganciclovir is cytotoxic to leiomyoma cells transfected with herpes simplex virus thymidine kinase and that estrogen modulates the responsiveness of tumor cells to this gene therapy approach. METHODS: Human and rat cultured uterine leiomyoma cells were transfected with plasmids encoding the beta-galactosidase gene, thymidine kinase gene, or a control plasmid. Transfection efficiency was monitored by measuring beta-galactosidase enzyme activity. Ganciclovir cytotoxicity in thymidine kinase-transfected cells was assessed by monitoring cell viability using trypan blue exclusion. The "bystander effect," a phenomenon in which thymidine kinase-expressing cells exposed to ganciclovir are toxic to adjacent thymidine kinase-nonexpressing cells, was assessed when thymidine kinase vector-transfected cells were cocultured with control plasmid-transfected cells at various percentages before exposure to ganciclovir. The effect of estradiol on ganciclovir-thymidine kinase-mediated cytotoxicity was assessed in estrogen-responsive rat leiomyoma cells. RESULTS: A thymidine kinase-ganciclovir-mediated "bystander effect" was demonstrated, with 48.6% (human) and 65.6% (rat) cell death when 5% of the leiomyoma cells were transfected with the pNGVL1-tk vector, with 0.84% and 1.9% of the cells expected to express thymidine kinase as based on the 16.7% and 39.8% transfection efficiency determined by the reporter gene assay in human and rat leiomyoma cells, respectively. Estradiol promoted cell growth and enhanced the "bystander effect" in rat leiomyoma cells. CONCLUSION: These findings demonstrate the feasibility of using thymidine kinase gene therapy as a novel treatment for uterine leiomyomas. The effect of estrogen may provide a mechanism to enhance the tumor-suppressive effect of this approach.

Animals↗
PubMed1997

Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro.

Human single-stranded DNA-binding protein (HSSB, also called RPA), is a heterotrimeric complex that consists of three subunits, p70, p34, and p11. HSSB is essential for the in vitro replication of SV40 DNA and nucleotide excision repair. It also has important functions in other DNA transactions, including DNA recombination, transcription, and double-stranded DNA break repair. The p34 subunit of HSSB is phosphorylated in a cell cycle-dependent manner. Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro. In this study, we show that efficient phosphorylation of HSSB-p34 by DNA-PK requires Ku as well as DNA. The DNA-PK phosphorylation sites in HSSB-p34 have been mapped at Thr-21 and Ser-33. Kinetic studies demonstrated that a phosphate residue is first incorporated at Thr-21 followed by the incorporation of a second phosphate residue at Ser-33. We also identified Ser-29 as the major Cdc2 kinase phosphorylation site in the p34 subunit.

Amino Acid Sequence↗
PubMed1997

Dexamethasone regulates basic fibroblast growth factor, nerve growth factor and S100beta expression in cultured hippocampal astrocytes.

Glucocorticoids regulate hippocampal neuron survival during fetal development, in the adult, and during aging; however, the mechanisms underlying the effects are unclear. Since astrocytes contain adrenocortical receptors and synthesize and release a wide variety of growth factors, we hypothesized that glucocorticoids may alter neuron-astrocyte interactions by regulating the expression of growth factors in hippocampal astrocytes. In this study, three growth factors, which are important for hippocampal neuron development and survival, were investigated: basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and S100beta. Enriched type I astrocyte cultures were treated with 1 microM dexamethasone (DEX), a synthetic glucocorticoid, for up to 120 h. Cells and culture medium were collected and total RNA and protein were measured at 6, 12, 24, 48, 72, 96 and 120 h after the initiation of hormone treatment. Growth factor mRNA levels were measured and quantified using solution hybridization-RNase protection assays and protein levels were quantified using ELISA methods. We report that DEX stimulates the bFGF mRNA levels over the 120-h treatment. In contrast, DEX suppresses NGF mRNA continuously over the same period of treatment. DEX induces a biphasic response in S100beta mRNA levels. In addition, some of the changes in gene expression are translated into parallel changes in protein levels of these growth factors. Our results demonstrate that dexamethasone can differentially regulate the expression of growth factors in hippocampal astrocytes in vitro. This suggests that one of the mechanisms through which glucocorticoids affect hippocampal functions may be by regulating the expression of astrocyte-derived growth factors.

Animals↗
PubMed1996

Studies on the in vitro phosphorylation of HSSB-p34 and -p107 by cyclin-dependent kinases. Cyclin-substrate interactions dictate the efficiency of phosphorylation.

Cyclin-dependent kinases (Cdks) are required for cell cycle progression. Two potentially significant Cdk substrates in human cells are the human single-stranded binding protein (HSSB or RPA), which plays an essential role in DNA replication, repair, and recombination, and the tumor suppressor p107 which acts to negatively regulate cell growth. In this report we describe the in vitro phosphorylation of these two proteins by Cdks in an attempt to understand how cyclin-substrate interactions direct phosphorylation efficiencies. We show that cyclin A-Cdk2 efficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or as a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 that is active in phosphorylating histone H1, does not support the phosphorylation of the p34 subunit of HSSB. We provide evidence that this differential phosphorylation results from a specific interaction between HSSB-p34 and cyclin A, but not cyclin E. Thus the observed cell cycle-dependent phosphorylation of HSSB-p34 at the G1 to S transition is most likely catalyzed by cyclin A-Cdk2 initiated by the direct interaction between cyclin A and the HSSB-p34 subunit. These studies are consistent with our previous observation that p107, which directly binds cyclin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B-associated kinases. Here we further demonstrate that cyclin A only complexes with p107 in its unphosphorylated form. These data suggest a catalytic mechanism by which Cdk acts: substrate targeting by a cyclin-substrate interaction followed by dissociation of the Cdk upon phosphate incorporation allowing the Cdk to become available for the next cycle of phosphorylation.

Base Sequence↗
PubMed1996

Rat ribosomal RNA gene can utilize primate RNA polymerase I transcription machinery: lack of absolute species specificity in rDNA transcription.

The transcriptional activity of rodent ribosomal RNA gene (rDNA) in the primate cell was examined in the light of reported species specificity of eukaryotic ribosomal RNA synthesis. The present study showed that rat rDNA can be transcribed in HeLa nuclear extract whereas mouse rDNA was not transcribed in the heterologous extract. Rat and mouse rDNA transcription factors were interchangeable with respect to efficiency and accuracy of transcription. The initiation of rat ribosomal gene transcription by RNA polymerase I occurred at the +1 site in the heterologous extract. Initiation of transcription at the correct site also occurred in vivo following transfection of cloned rat rDNA into the primate (COS-7) cells. These data indicate that rat ribosomal RNA gene can be expressed in the primate system in vitro and in vivo. The absolute lack of species specificity in rDNA transcription has been discussed based on the present data and other reports.

Animals↗
PubMed1995

Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma.

The BCL6 gene codes for a zinc-finger transcription factor and is involved in chromosomal rearrangements in 30-40% of diffuse large-cell lymphoma (DLCL). These rearrangements cluster within the 5' regulatory region of BCL6 spanning its first non-coding exon. To determine the functional consequences of these alterations, we have analyzed the structure of the rearranged BCL6 alleles and their corresponding RNA and protein species in two DLCL biopsies and one tumor cell line which carried the t(3;14)(q27;q32) translocation involving the BCL6 and immunoglobulin heavy-chain (IgH) loci. In all three cases, the breakpoints were mapped within the IgH switch region and the BCL6 first intron, leading to the juxtaposition of part of the IgH locus upstream and in the same transcriptional orientation to the BCL6 coding exons. An analysis of cDNA clones showed that these recombinations generate chimeric IgH-BCL6 transcripts which initiated from IgH germline transcript promoters (I mu or I gamma 3), but retain a normal BCL6 coding domain. In the tumor cell line, the chimeric I gamma 3-BCL6 allele, but not the germline BCL6 gene, was transcriptionally active and produced a normal BCL6 protein. These findings indicate that t(3;14) translocations alter BCL6 expression by promoter substitution and imply that the consequence of these alterations is the deregulated expression of a normal BCL6 protein.

Alleles↗
PubMed1995

E1BF/Ku interacts physically and functionally with the core promoter binding factor CPBF and promotes the basal transcription of rat and human ribosomal RNA genes.

We have previously characterized an RNA polymerase (pol) I transcription factor, E1BF, from rat cells. This protein is immunologically related to Ku autoantigen and is required in pol-I directed transcription of rodent ribosomal RNA gene (rDNA). Glycerol density gradient fractionation and in situ UV cross-linking analysis of the purified factor showed directly that it consists of a heterodimer of 85 and 72 kDa polypeptides. E1BF also interacted with the human core promoter and augmented transcription of human rDNA as much as fivefold in HeLa nuclear extract, whereas transcription from adenovirus major late promoter, CMV or SV40 early promoters by pol II and of U6 and 5S RNA genes by pol III were either unaffected or minimally inhibited by the antibodies. Purified rat E1BF partially restored the suppression of human rDNA transcription by anti-Ku antibodies. Immunoprecipitation of rat cell extract with the anti-Ku antibodies followed by SDS-PAGE of the precipitated proteins and Southwestern analysis showed that E1BF interacts with CPBF, a core promoter binding factor. When the majority of CPBF and E1BF was removed from the reaction mixture by preincubation with a core promoter oligo nucleotide fragment, rDNA transcription was severely impaired. Addition of exogenous CPBF or E1BF to such a reaction resulted in significant restoration of the transcription, whereas inclusion of both factors caused further enhancement of rDNA transcription. These data demonstrate that E1BF is a basal pol I transcription factor that interacts with a core promoter binding factor both physically and functionally, and that is not a general pol II or pol III transcription factor.

Adenoviridae↗
PubMed1994

Enhancer 1 binding factor (E1BF), a Ku-related protein, is a growth-regulated RNA polymerase I transcription factor: association of a repressor activity with purified E1BF from serum-deprived cells.

Previous studies from this laboratory have demonstrated that the enhancer 1 binding factor (E1BF), a Ku-related protein, purified from the serum-enriched cells functions as a positive factor in an RNA polymerase (pol I) transcription system. We have now shown that E1BF purified from the serum-deprived cells (E1BFs) can inhibit rDNA transcription completely in a fractionated extract from the cells grown in serum-enriched medium. The suppression of transcription was overcome by the addition of control E1BF (E1BFc). Immunoprecipitation of purified E1BFs by the anti-Ku monoclonal antibody and addition of the supernatant to the transcription reaction mixture prevented the inhibition significantly, whereas immunoprecipitation with the control mouse IgG did not restore the transcription. The transcriptional repressor activity associated with the final DNA affinity column fractions copurified with E1BF. Neither the amount of E1BF nor its promoter binding activity was altered following serum depletion. E1BFs selectively inhibited the initiation of rDNA transcription. The inhibitory activity of E1BFs was not due to a nonspecific RNase activity. These data suggest that E1BF is post-translationally modified following serum starvation of cells, and that the repressor activity of E1BFs is largely responsible for the down-regulation of pol I transcription in serum-deprived cells.

Animals↗
PubMed1994

Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.

CDC2-CDC28 Kinases↗
PubMed1994

BCL-6 and the molecular pathogenesis of B-cell lymphoma.

The results presented identify the first genetic lesion associated with DLCL, the most clinically relevant form of NHL. Although no proof yet exists of a role for these lesions in DLCL pathogenesis, the feature of the BCL-6 gene product, its specific pattern of expression in B cells, and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of BCL-6 expression may contribute to DLCL development. A more precise definition of the role of BCL-6 in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express BCL-6 under heterologous promoters or lacking BCL-6 function due to targeted deletions. In addition to contributing to the understanding of DLCL pathogenesis, the identification of BCL-6 lesions may have relevant clinical implications. DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50% of patients experience long-term disease-free survival (Schneider et al. 1990). The fact that BCL-6 rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups. Furthermore, the BCL-6 rearrangements can be used to identify and monitor the malignant clone with sensitive PCR-based techniques. Since clinical remission has been observed in a significant fraction of DLCL cases, these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse (Gribben et al. 1993).

Animals↗
PubMed1994

Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells.

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant insertional mutants of C. jejuni 81-176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2-32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2-37 and K2-55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2-37 and K2-55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90,977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants. The differences in adherence between the two classes of flagellar mutant suggest that flagellin can serve as a secondary adhesion, although other adhesins mediate a motility-dependent internalization process. Characterization of the mutants at the molecular level and in animal models should further contribute to our understanding of the pathogenicity of these organisms.

Amino Acid Sequence↗
PubMed1972

Biophysical effects of adrenaline on the smooth muscle of the rabbit common carotid artery.

Effects of adrenaline on the smooth muscle of the rabbit common carotid artery were studied by the partitional chamber method. The experiments on excitation-contraction coupling were carried out in isotonic Krebs solution; the other experiments were carried out in hypertonic Krebs solution. Adrenaline (10(-7) g/ml) caused rhythmical electrical and mechanical activity of arterial strips in isotonic Krebs solution. By addition of adrenaline (10(-5) g/ml), the membrane was depolarized by about 10 mv and the amplitude of the electrotonic potential was decreased by 40-50% of the control in hypertonic Krebs solution. Present experimental results suggest that the depolarization of the membrane and the decrease of the amplitude of the electrotonic potential in the artery are due to the increase of Na and Cl conductance. Contraction appeared in all preparations exposed to 10(-8) g/ml adrenaline; at that concentration membrane potential and membrane resistance showed little or no change.

Animals↗