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Biomedical subjects

J Liao

Publications and source records attributed to J Liao.

At least 19 recordsLinked to original sources
PubMed2005

Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center.

INTRODUCTION: Canine malignant melanoma (CMM) is an aggressive neoplasm treated with surgery and/or fractionated RT; however, metastatic disease is common and chemoresistant. Preclinical and clinical studies by our laboratory and others have shown that xenogeneic DNA vaccination with tyrosinase family members can produce immune responses resulting in tumor rejection or protection and prolongation of survival. These studies provided the impetus for development of a xenogeneic DNA vaccine program in CMM. MATERIALS AND METHODS: Cohorts of three dogs each received increasing doses of xenogeneic plasmid DNA encoding either human tyrosinase (huTyr; 100/500/1500 mcg), murine GP75 (muGP75; 100/500/1500 mcg), murine tyrosinase (muTyr; 5 dogs each at 100/500 mcg), muTyr+/-HuGM-CSF (9 dogs at 50 mcg muTyr, 3 dogs each at 100/400/800 mcg HuGM-CSF, or 3 dogs each at 50 mcg muTyr with 100/400/800 mcg HuGM-CSF), or 50 mcg MuTyr intramuscularly biweekly for a total of four vaccinations. RESULTS: The Kaplan-Meier median survival time (KM MST) for all stage II-IV dogs treated with huTyr, muGP75 and muTyr are 389, 153 and 224 days, respectively. Preliminarily, the KM MST for stage II-IV dogs treated with 50 mcg MuTyr, 100/400/800 mcg HuGM-CSF or combination MuTyr/HuGM-CSF are 242, 148 and >402 (median not reached) days, respectively. Thirty-three stage II-III dogs with loco-regionally controlled CMM across the xenogeneic vaccine studies have a KM MST of 569 days. Minimal to mild pain was noted on vaccination and one dog experienced vitiligo. We have recently investigated antibody responses in dogs vaccinated with HuTyr and found 2- to 5-fold increases in circulating antibodies to human tyrosinase. CONCLUSIONS: The results of these trials demonstrate that xenogeneic DNA vaccination in CMM: (1) is safe, (2) leads to the development of anti-tyrosinase antibodies, (3) is potentially therapeutic, and (4) is an attractive candidate for further evaluation in an adjuvant, minimal residual disease Phase II setting for CMM.

Animals↗
PubMed2004

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.

Alcohol Dehydrogenase↗
PubMed2003

Acupuncture versus placebo versus sumatriptan for early treatment of migraine attacks: a randomized controlled trial.

OBJECTIVES: To investigate whether acupuncture is superior to placebo and equivalent to sumatriptan for the early treatment of an acute migraine attack. DESIGN: Randomized, partly double-blind (sumatriptan versus placebo) trial. SETTING: Two hospitals in Germany (one specialized in traditional Chinese medicine and one in the treatment of headache). SUBJECTS: A total of 179 migraineurs experiencing the first symptoms of a developing migraine attack. INTERVENTIONS: Traditional Chinese acupuncture, sumatriptan (6 mg subcutaneously) or placebo injection. MAIN OUTCOME MEASURE: Number of patients in whom a full migraine attack (defined as severe migraine headache) within 48 h was prevented. In patients who developed a migraine attack in spite of early treatment, acupuncture and sumatriptan were applied a second time, whilst patients initially randomized to placebo received sumatriptan. RESULTS: A full migraine attack was prevented in 21 of 60 (35%) patients receiving acupuncture, 21 of 58 (36%) patients receiving sumatriptan and 11 of 61 (18%) patients receiving placebo (relative risk of having a full attack 0.79 (95% CI, 0.64-0.99) for acupuncture versus placebo, and 0.78 (95% CI, 0.62-0.98) for sumatriptan versus placebo). Response to the second intervention in patients who developed a full attack was better with sumatriptan (17/31 patients who received sumatriptan twice and 37/46 patients who had had placebo first) than with acupuncture (4/31). The number of patients reporting side-effects was 14 in the acupuncture group, 23 in the sumatriptan group and 10 in the placebo group. CONCLUSIONS: In this trial acupuncture and sumatriptan were more effective than a placebo injection in the early treatment of an acute migraine attack. When an attack could not be prevented, sumatriptan was more effective than acupuncture at relieving headache.

Acupuncture Therapy↗
PubMed2001

Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects.

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is a congenital anomaly disorder associated with hemizygous 22q11 deletions. We previously showed that bacterial artificial chromosome (BAC) transgenic mice overexpressing four transgenes, PNUTL1, (CDCrel-1), GP1B beta, TBX1 and WDR14, had reduced viability, cardiovascular malformations and thymus gland hypoplasia. Since these are hallmark features of VCFS/DGS, we analyzed the mice for additional anomalies. We found that the mice have important defects in the middle and inner ear that are directly relevant to the disorder. The most striking defect was the presence of chronic otitis media, a common finding in VCFS/DGS patients. In addition, the mice had a hyperactive circling behavior and sensorineural hearing loss. This was associated with middle and inner ear malformations, analogous to Mondini dysplasia in humans reported to occur in VCFS/DGS patients. We propose that overexpression of one or more of the transgenes is responsible for the etiology of the ear defects in the mice. Based upon its pattern of expression in the ear and functional studies of the gene, TbX1 likely plays a central role. Haploinsufficiency of TBX1 may be responsible for ear disorders in VCFS/DGS patients.

Abnormalities, Multiple↗
PubMed2001

Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E.

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.

Base Sequence↗
PubMed2001

Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules.

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.

Animals↗
PubMed2001

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control.

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse-chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

3T3 Cells↗
PubMed2001

Heterologous expression of the single-mutation glucose isomerase (GIG138P) gene in Streptomyces lividans and its genetic instability.

A 1.3-kb PstI-BamHI fragment containing the single-mutation glucose isomerase (GIG138P, GI1) gene and its natural promoter was inserted into PstI-BglII linearized Streptomyces vector pIJ702. The ligation mixture was then introduced into Streptomyces lividans TK54 protoplasts; transformants were identified based on their thiostrepton resistance (ThR) and insertional inactivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme assay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the intracellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of the soluble proteins in S. lividans. Restriction analysis and Southern blot of pYH703 showed the existence of plasmid deletion, presumably owing to the interaction between the mel and GI1 sequences. Continuous liquid cultures of the recombinant strain demonstrated that the GI1 specific activity and GI1 expression in S. lividans decreased, and more obviously under non-selective conditions.

Aldose-Ketose Isomerases↗
PubMed2001

DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas.

The three human leukocyte antigen (HLA) class I antigens, HLA-A, HLA-B and HLA-C, play important roles in the elimination of transformed cells by cytotoxic T cells. Frequent loss of expression of these antigens at the cell surface has been observed in many human cancers. Various mechanisms for post-transcriptional regulation have been proposed and tested but the molecular mechanisms for transcriptional regulation are not clear. We show by immunohistochemistry that the HLA class I antigens are absent in 26 of 29 (89%) samples of human esophageal squamous cell carcinomas (ESCC). Eleven of the 26 ESCC samples lost mRNA expression for at least one of the HLA genes, as shown by RT-PCR. DNA from the 29 pairs of ESCC and neighboring normal epithelium were examined for CpG island hypermethylation, homozygous deletion, microsatellite instability (MSI) and loss of heterozygosity (LOH). DNA from normal epithelial tissues had no detectable methylation of the CpG islands of any of these gene loci. Thirteen of 29 ESCC samples (45%) exhibited methylation of one or more of the three HLA loci and six samples (21%) exhibited methylation of all three loci. The HLA-B gene locus was most frequently methylated (38%). HLA-B mRNA expression in an ESCC cell line, where HLA-B was hypermethylated and did not express mRNA, was activated after treatment with 5-aza-2'-deoxycytidine. Homozygous deletion of these three gene loci was not observed. Relatively low rates of LOH and MSI were observed for the microsatellite markers D6S306, D6S258, D6S273 and D6S1666, close to the HLA-A, -B and -C loci, although a high ratio of LOH was observed at a nearby locus (represented by the markers D6S1051 and D6S1560), where the tumor suppressor gene p21(Waf1) resides. A strong correlation between genetic alterations and mRNA inactivation was observed in the ESCC samples. Our results indicate that HLA class I gene expression was frequently down-regulated in ESCC at both the protein and mRNA levels and that hypermethylation of the promoter regions of the HLA-A, -B and -C genes is a major mechanism of transcriptional inactivation.

Azacitidine↗
PubMed2001

Effect of steroid-liposome on immunohistopathology of IgA nephropathy in ddY mice.

Immunopathological studies were performed to determine whether glomerular injuries in ddY mice, a spontaneous animal model for IgA nephropathy, are influenced by treatment with a newly developed liposome loaded with prednisolone phosphate (PSL-liposome). The newly synthesized novel cationic lipid 3,6-dipentadeciroxy-1-amizino-benzene (TRX-20) was employed to obtain selective affinity to the anionic cell surface and extracellular matrices in glomerular mesangial lesions. ddY mice were treated intravenously with 1.0 mg/kg of PSL-liposome once a week for 16 weeks (from 45 weeks to 61 weeks of age). ddY mice were also intravenously treated with 1.0 mg/kg of ordinary PSL once a week for the same duration. On immunofluorescence, depositions of IgA and C3 in the glomerular mesangial areas and capillary walls of PSL-liposome-treated ddY mice were markedly decreased as compared with those of ordinary PSL-treated and untreated control ddY mice. The mean intensity of IgA and C3 in glomeruli of PSL-liposome-treated ddY mice was decreased as compared with that in ordinary PSL-treated and untreated control ddY mice. Glomerular mesangial expansion in PSL-liposome-treated ddY mice was milder than that found in ordinary PSL-treated ddY mice or untreated control ddY mice. It appears that treatment with PSL-liposome is effective in improving glomerular IgA and C3 depositions and glomerular expansion in IgA nephropathy of ddY mice.

Animals↗
PubMed2001

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Adenoviridae↗
PubMed2001

[Antisense RNA for urokinase receptor inhibits the invasiveness and metastasis of lung carcinoma].

OBJECTIVE: To inhibit invasion and metastasis of human lung carcinoma cell line 95D by antisense RNA for urokinase receptor (uPAR). METHODS: Antisense RNA expression plasmid for uPAR was introduced into the highly metastatic human lung carcinoma cells 95D. Modified Boyden's chamber was used to detect the invasion ability, and nude mice were used to determine metastasis. RESULTS: Two transfected clones were found to integrate the antisense expression plasmid into genomic DNA and to express antisense RNA for uPAR. Antisense RNA blocked the uPAR expression in the transfected clones. The evident reductions of the invasiveness of antisense clones were observed by comparison with control cells, 95D cells and cells transfected with pcDNA3 vector. The metastatic potential of these two antisense clones decreased significantly in comparison with the controls (P < 0.05). CONCLUSIONS: The results suggest that antisense RNA for uPAR inhibits uPAR expression and leads to a decrease of metastatic potential of 95D cells. Antisense RNA technique may have a valuable application in anti-metastatic therapy of human lung carcinoma.

Animals↗
PubMed2001

[A clinical evaluation on reconstruction in extended partial laryngectomy for transglottic cancer].

OBJECTIVE: Transglottic cancer has been a definite indication for total laryngectomy in decades, compromising function of the larynx. The purpose of this study is to present a new radical surgery of extended partial laryngectomy and reconstruction to restore the essential function of larynx. METHODS: Forty-one cases of transglottic cancer were treated by extended vertical laryngectomy (26/41), extended frontolateral laryngectomy (5/41) and subtotal laryngectomy (10/41); the defects of larynx were reconstructed by rotary door myocutaneous flap in twenty-seven cases, by sternohyoideus flap in seven cases, by osteomuscular flap in five cases and by sternocleidomastoid flap in two cases. RESULTS: The 3 and 5 year survival rates were 85.7% (30/35) and 74.1% (20/27) respectively, those for stage III were 84.6% (22/26) and 76.2% (16/21), for stage IV were 3/4 and 1/2 respectively. All cases resumed acceptable voice, 92.7% (38/41) and enjoyed satisfactory phonation. The over-all decannulation rate was 87.8% (36/41), those with rotary door myocutaneous flap was 96.3% (26/27), with sternohyoideus flap was 5/7, with osteomuscular flap was 4/5 and with sternocleidomastoid flap was 1/2. All patients had normal swallow function. CONCLUSION: Extended partial laryngectomy is a kind of radical operation in selected cases of transglottic cancer. The method of reconstruction with rotary door myocutaneous flap can improve curative effect and the essential functions of the larynx.

Adult↗
PubMed2000

Interferon-alpha induces nmi-IFP35 heterodimeric complex formation that is affected by the phosphorylation of IFP35.

Nmi and IFP35 are interferon (IFN)-induced proteins. In cells treated with IFN-gamma, Nmi enhances the association of transcription co-activator CBP/p300 with signal transducer and activator of transcription proteins, and IFP35 forms a high molecular weight cytosolic complex of unknown constituents. Here we show that Nmi and IFP35 co-immunoprecipitate with an anti-keratin 19 antibody, which is due to cross-reaction of the antibody with Nmi, and suggests an Nmi-IFP35 physical association. In support of this, Nmi and IFP35 co-immunoprecipitate using anti-Nmi and anti-IFP35 antibodies, manifest enhanced colocalization as determined by immunofluorescence staining of IFN-treated cells, and form heterodimers as determined by chemical cross-linking. Nmi and IFP35 are primarily cytosolic proteins, and their interaction is increased after IFN-alpha treatment of cells as early as 1 h after exposure. Sucrose gradient sedimentation and size fractionation showed a shift of Nmi-IFP35 heterodimers toward a heavier fraction (100-200 kDa) in IFN-alpha-treated cells. This dynamic complex formation is reversed by pretreatment with okadaic acid. Two-dimensional gel analysis indicates that the IFN-induced complex formation correlates with IFP35 dephosphorylation. Our data demonstrate Nmi-IFP35 cytosolic localization and heterodimerization, and an IFN-alpha-regulated molecular event in which Nmi and IFP35 participate, reversibly and by a dephosphorylation dependent fashion, in a 100-200-kDa molecular complex formation.

Antibodies↗
PubMed2000

Unique carboxyl-terminal sequences of wild type and alternatively spliced variant forms of transforming growth factor-alpha precursors mediate specific interactions with ErbB4 and ErbB2.

We have previously reported that the human transforming growth factor-alpha (TGF-alpha) gene encodes three forms of TGF-alpha precursors, designated wild type (WT), variant I (VaI), and variant II (VaII), derived from alternative splicing. The two carboxyl-terminal valine residues of WT are replaced by 5 (GCRLY) or 4 (ATLG) amino acids in VaI or VaII, respectively. When overexpressed in Chinese hamster ovary (CHO) cells, VaI and ValI, but not WT, support autonomous growth. We detected tyrosine phosphorylation of ErbB2 in the absence of serum, in CHO cells expressing WT, VaI, or VaII, but not in mock transfectants. These observations prompted us to investigate possible interactions between the ErbBs and the TGF-alpha precursors in CHO cells. All TGF-alpha precursors were found to co-immunoprecipitate with the ErbBs, but with different specificity. WT co-immunoprecipitated with ErbB4, but not with ErbB1, ErbB2, or ErbB3. VaI and VaII co-immunoprecipitated with ErbB2, but not with ErbB1, ErbB3, or ErbB4. Confocal fluorescent microscopy analysis demonstrated that WT, VaI, and VaII all distribute equally to the cell surface while, as expected, a WT mutant lacking the two C-terminal valine residues does not. Point and deletion mutants involving the unique carboxyl-terminal residues of WT, VaI and VaII, indicated that the interactions between the three TGF-alpha precursors and the ErbBs were mediated by their carboxyl-terminal regions, which constitute distinct protein-binding motifs. A chimera of the intracellular domain of WT TGF-alpha linked to exogenous transmembrane and extracellular domains retained both the cell surface distribution and the specific interaction with ErbB4 of full-length WT, confirming that this interaction is mediated by the C-terminus of the TGF-alpha precursor. While interactions of WT and variant TGF-alpha with the ErbBs all result in ErbB2 activation, they produce different biological consequences, suggesting that the various TGF-alpha precursors differentially modulate ErbB signaling.

Alternative Splicing↗
PubMed2000

Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1-Stat1 interaction.

STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)-phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1-Stat1 interaction has not been understood. We report here that a region near the COOH terminus of PIAS1 (amino acids 392-541) directly interacts with the NH(2)-terminal domain of Stat1 (amino acids 1-191). A mutant PIAS1 lacking the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, we demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH(2)-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1-Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1.

Binding Sites↗