Biomedical subjects

X Yang

Publications and source records attributed to X Yang.

At least 631 records · Page 35Linked to original sources

PubMed1998

Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7.

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.

Apoptosis↗

PubMed1998

[Molecular cloning and characterization of novel protein kinase gene DYRK3].

OBJECTIVE: To isolate full length cDNA of a novel protein kinase and to deduce the protein kinase's classification position and functions. METHODS: cDNA libraries gDNA library was screened with a partial cDNA clone which is homologous to human protein kinase DYRK2 as probe. FISH mapping was performed. RESULTS: Two full length cDNAs of a novel protein kinase from human muscle cDNA library and human testis cDNA library were isolated. The full length cDNA from muscle has an open reading frame which is predicted to encode a protein of 588 amino acid residues and the cDNA from testis to encode a protein of 568 amino acid residues. CONCLUSION: Because the sequence from the 27th codon to the 3' end of the cDNA from muscle is identical to that from the 7th codon to the 3' end of the cDNA from testis, they should be different transcripts of the same gene. As the gene is highly homologous to human protein kinase DYRK2, the present authors termed the gene DYRK3. DYRK3 is homologous to many serine/threonine protein kinases such as yeast Yak1, human Clk1, human Mnb, drosophila melanogaster Mnb and Cdk2. DYRK3 should belong to the Clk family in CMGC group of serine/threonine protein kinase. DYRK3 has been mapped to chromosome 1q32 by FISH.

Amino Acid Sequence↗

PubMed1998

The quinone-binding site in succinate-ubiquinone reductase from Escherichia coli. Quinone-binding domain and amino acid residues involved in quinone binding.

When purified ubiquinone (Q)-depleted succinate-ubiquinone reductase from Escherichia coli is photoaffinity-labeled with 3-azido-2-methyl-5-methoxy-[3H]6-geranyl-1,4-benzoquinone ([3H]azido-Q) followed by SDS-polyacrylamide gel electrophoresis, radioactivity is found in the SdhC subunit, indicating that this subunit is responsible for ubiquinone binding. An [3H]azido-Q-linked peptide, with a retention time of 61.7 min, is obtained by high performance liquid chromatography of the protease K digest of [3H]azido-Q-labeled SdhC obtained from preparative SDS-polyacrylamide gel electrophoresis on labeled reductase. The partial N-terminal amino acid sequence of this peptide is NH2-TIRFPITAIASILHRVS-, corresponding to residues 17-33. The ubiquinone-binding domain in the proposed structural model of SdhC, constructed based on the hydropathy plot of the deduced amino acid sequence of this protein, is located at the N-terminal end toward the transmembrane helix I. To identify amino acid residues responsible for ubiquinone binding, substitution mutations at the putative ubiquinone-binding region of SdhC were generated and characterized. E. coli NM256 lacking genomic succinate-Q reductase genes was constructed and used to harbor the mutated succinate-Q reductase genes in a low copy number pRKD418 plasmid. Substitution of serine 27 of SdhC with alanine, cysteine, or threonine or substitution of arginine 31 with alanine, lysine, or histidine yields cells unable to grow aerobically in minimum medium with succinate as carbon source. Furthermore, little succinate-ubiquinone reductase activity and [3H]azido-Q uptake are detected in succinate-ubiquinone reductases prepared from these mutant cells grown aerobically in LB medium. These results indicate that the hydroxyl group, the size of the amino acid side chain at position 27, and the guanidino group at position 31 of SdhC are critical for succinate-ubiquinone reductase activity, perhaps by formation of hydrogen bonds with carbonyl groups of the 1,4-benzoquinone ring of the quinone molecule. The hydroxyl group, but not the size of the amino acid side chain, at position 33 of SdhC is also important, because Ser-33 can be substituted with threonine but not with alanine.

Affinity Labels↗

PubMed1998

A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase.

Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.

Acid Anhydride Hydrolases↗

PubMed1998

Mitogen-activated protein kinase phosphorylates and regulates the HIV-1 Vif protein.

The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in virus replication and infectivity. Here we show that Vif is phosphorylated and regulated by p44/42 mitogen-activated protein kinase (MAPK). Vif phosphorylation by MAPK was demonstrated in vitro as well as in vivo and was shown to occur on serine and threonine residues. Two-dimensional tryptic phosphopeptide mapping indicated that Vif is phosphorylated by MAPK on the same sites in vitro and in vivo. Radioactive peptide sequencing identified two phosphorylation sites, Thr96 and Ser165. These phosphorylation sites do not correspond to the known optimum consensus sequences for phosphorylation by MAPK (PX(S/T)P) nor to the minimum consensus sequence ((S/T)P), indicating that MAPK can phosphorylate proteins at sites other than those containing the PX(S/T)P or (S/T)P motifs. Synthetic Vif peptides corresponding to the local sequences of the phosphorylation sites were not phosphorylated by MAPK, suggesting that recognition of these sites by MAPK is likely to require structural determinants outside the phosphorylation site. Mutations of the Thr96 site, which is conserved among Vif sequences from HIV-1, HIV-2, and SIV, resulted in significant loss of Vif activity and inhibition of HIV-1 replication. These results suggest that MAPK plays a direct role in regulating HIV-1 replication and infectivity by phosphorylating Vif and identify a novel mechanism for activation of HIV-1 replication by mitogens and other extracellular stimuli.

Amino Acid Sequence↗

PubMed1998

Molecular cloning of human er1 cDNA and its differential expression in breast tumours and tumour-derived cell lines.

We recently cloned and characterized a novel immediate-early gene, called er1, from Xenopus embryos whose expression levels were increased during mesoderm induction by fibroblast growth factor (FGF). We describe here the isolation and expression pattern of the human er1 sequence. Human ER1 and Xenopus ER1 proteins display 91% similarity; the amino acid sequence motifs, including the putative DNA-binding SANT domain, the predicted nuclear localization signals (NLS) and the putative SH3 binding domain share 100% identity. er1 mRNA expression was negligible in all 50 normal human tissues analyzed. Examination of nine breast carcinoma-derived cell lines and eight breast tumour tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) revealed that human er1 was consistently expressed in all tumour cell lines and tumour tissue while remaining undetectable in normal breast cell lines and breast tissue. These data suggest that er1 expression is associated with the neoplastic state in human breast carcinoma.

Amino Acid Sequence↗

PubMed1998

Role of EP2 receptors and cAMP in prostaglandin E2 regulated expression of type I collagen alpha1, lysyl oxidase, and cyclooxygenase-1 genes in human embryo lung fibroblasts.

In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin-1beta((IL-1beta) and transforming growth factor-beta (TGFbeta) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase-1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 19961. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11-deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11-deoxy PGE1 decreases basal and TGF-beta induced type I collagen alpha1 (COL) mRNA, basal and IL-1beta induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2 or 11-deoxy PGE1. It suppresses both basal and TGF-beta induced COL mRNA levels. Both PGE2 and 11-deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat-1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat-1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP.

Alprostadil↗

PubMed1998

Pro-caspase-3 is a major physiologic target of caspase-8.

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s-1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.

Apoptosis↗

PubMed1998

[Deiodinase].

The members of deiodinase family including type I, type II and type III deiodinase are seleno-proteins. They play an important role in the regulation of thyroid hormone metabolism. They are different in substrate specificity and sensitivity to inhibitor and kinetics mechanism. Their cDNAs exhibit limited regions of homology, but they have common conserved sequence, TGA codon of selenocysteine and some structures and sequences which are essential for selenocysteine insertion.

Animals↗

PubMed1998

Inactivation of S-adenosyl-L-homocysteine hydrolase and antiviral activity with 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosine analogues (4'-haloacetylene analogues derived from adenosine).

Treatment of a protected 9-(5, 6-dideoxy-beta-D-ribo-hex-5-ynofuranosyl)adenine derivative with silver nitrate and N-iodosuccinimide (NIS) and deprotection gave the 6'-iodo acetylenic nucleoside analogue 3c. Halogenation of 3-O-benzoyl-5,6-dideoxy-1, 2-O-isopropylidene-alpha-D-ribo-hex-5-enofuranose gave 6-halo acetylenic sugars that were converted to anomeric 1,2-di-O-acetyl derivatives and coupled with 6-N-benzoyladenine. These intermediates were deprotected to give the 6'-chloro 3a, 6'-bromo 3b, and 6'-iodo 3c acetylenic nucleoside analogues. Iodo compound 3c appears to inactivate S-adenosyl-L-homocysteine hydrolase by a type I ("cofactor depletion") mechanism since complete reduction of enzyme-bound NAD+ to NADH was observed and no release of adenine or iodide ion was detected. In contrast, incubation of the enzyme with the chloro 3a or bromo 3b analogues resulted in release of Cl- or Br- and Ade, as well as partial reduction of E-NAD+ to E-NADH. Compounds 3a, 3b, and 3c were inhibitory to replication of vaccinia virus, vesicular stomatitis virus, parainfluenza-3 virus, and reovirus-1 (3a < 3b < 3c, in order of increasing activity). The antiviral effects appear to correlate with type I mechanism-based inhibition of S-adenosyl-L-homocysteine hydrolase. Mechanistic considerations are discussed.

Adenosine↗

PubMed1998

Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx.

The Fas death receptor can activate the Jun NH2-terminal kinase (JNK) pathway through the receptor-associated protein Daxx. Daxx was found to activate the JNK kinase kinase ASK1, and overexpression of a kinase-deficient ASK1 mutant inhibited Fas- and Daxx-induced apoptosis and JNK activation. Fas activation induced Daxx to interact with ASK1, which consequently relieved an inhibitory intramolecular interaction between the amino- and carboxyl-termini of ASK1, activating its kinase activity. The Daxx-ASK1 connection completes a signaling pathway from a cell surface death receptor to kinase cascades that modulate nuclear transcription factors.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p.

The regulation of vesicular transport in eukaryotic cells involves Ras-like GTPases of the Ypt/Rab family. Studies in yeast and mammalian cells indicate that individual family members act in vesicle docking/fusion to specific target membranes. Using the two-hybrid system, we have now identified a 248 amino acid, integral membrane protein, termed Yip1, that specifically binds to the transport GTPases Ypt1p and Ypt31p. Evidence for physical interaction of these GTPases with Yip1p was also demonstrated by affinity chromatography and/or co-immunoprecipitation. Like the two GTPases, Yip1p is essential for yeast cell viability and, according to subcellular fractionation and indirect immunofluorescence, is located to Golgi membranes at steady state. Mutant cells depleted of Yip1p and conditionally lethal yip1 mutants at the non-permissive temperature massively accumulate endoplasmic reticulum membranes and display aberrations in protein secretion and glycosylation of secreted invertase. The results suggests for a role for Yip1p in recruiting the two GTPases to Golgi target membranes in preparation for fusion.

Amino Acid Sequence↗

PubMed1998

Biological and molecular basis of human breast cancer.

Human breast cancer remains the most common malignancy in the American women. The ultimate cure of this disease relies on a better understanding of the mechanisms underlying the initiation and progression of this disease. The neoplastic transformation of HBEC in vitro represents a successful model for obtaining knowledge on the molecular and biological alterations that may contribute to the tumorigenic mechanisms. We have presented here a current understanding of chemically transformed HBEC in the following aspects: 1. Factors affecting the transformation of HBEC such as genetic predisposition and differentiation status and prior immortalization; 2. New targets for studying the mechanism of cell immortalization such as alterations in telomerase activity and differential expression of cell cycle dependent genes as well as others recently isolated through differential cloning such as H-ferritin, and a calcium binding protein; 3. Epigenetic and genetic mechanisms underlying cell transformation; 4. The association of microsatellite instability in specific loci on chromosomes 11, 13, and 16 with the progression of cell transformation; and 5. The application of microcell mediated chromosome transfer technique as an approach to testing the functional role of specific genes whose dysregulation or loss of function may contribute to the ultimate cell transformation. Further efforts in this cell system will be directed to determine the roles of identified molecular changes as well as the mapping/cloning of tumor suppressor or senescence genes such as those that may reside on chromosomes 11 or 17.

Breast Neoplasms↗

PubMed1998

Essential role of CED-4 oligomerization in CED-3 activation and apoptosis.

Control of the activation of apoptosis is important both in development and in protection against cancer. In the classic genetic model Caenorhabditis elegans, the pro-apoptotic protein CED-4 activates the CED-3 caspase and is inhibited by the Bcl-2-like protein CED-9. Both processes are mediated by protein-protein interaction. Facilitating the proximity of CED-3 zymogen molecules was found to induce caspase activation and cell death. CED-4 protein oligomerized in cells and in vitro. This oligomerization induced CED-3 proximity and competed with CED-4:CED-9 interaction. Mutations that abolished CED-4 oligomerization inactivated its ability to activate CED-3. Thus, the mechanism of control is that CED-3 in CED-3:CED-4 complexes is activated by CED-4 oligomerization, which is inhibited by binding of CED-9 to CED-4.

Apoptosis↗

PubMed1998

Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells.

Previously, a Bcl-2-interacting protein, BAG-1, was cloned from mouse cells and was shown to interact with several other proteins and to be important for inhibition of apoptosis. Human BAG-1 (hBAG-1) cDNA, recently isolated by us and two other groups, has been shown to be identical to a hormone receptor-binding protein, RAP46. However, different molecular masses of hBAG-1 protein products were noted by these three groups. Here we demonstrated that hBAG-1 protein was expressed as four isoforms, designated p50, p46, p33 and p29, with apparent molecular masses of 50 kDa, 46 kDa, 33 kDa and 29 kDa, respectively. Deletion, site-directed mutagenesis and in vitro transcription/translation analysis showed that the four protein products of hBAG-1 were expressed by alternative initiation from four different start codons through a leaky scanning mechanism. Furthermore, we demonstrated that the distinct forms of hBAG-1 have different subcellular localizations, suggesting that they may have distinct functions in the cells. Characterization of hBAG-1 RNA and protein also showed that hBAG-1 was overexpressed in human cervical, breast and lung cancer cell lines. Taken together, these data clarify the conflicting observations reported in the literature and suggest that hBAG-1 is expressed as four forms of protein products, which may play a differential role in apoptosis and oncogenesis of human cells.

Alternative Splicing↗

PubMed1998

NMR identification of epitopes of Lyme disease antigen OspA to monoclonal antibodies.

Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi has been a focus of vaccine development. We have identified epitopes of OspA to two monoclonal antibodies (mAbs) by comparing NMR chemical shifts of free OspA and those in Fab complexes. Deuteration of non-labile protons in OspA extended the size limit of this technique so that it was applicable to the 78 kDa complexes of OspA and the Fab fragment. The epitope identified by NMR to an mAb, 184.1, agrees well with that previously defined by the crystal structure of the same complex, indicating the ability of the NMR method to accurately map an epitope in a large protein complex. The technique mapped the epitope to mAb 336, a mAb of clinical interest, to a region centered at the C-terminal alpha-helix. The results provides a basis for rational design of OspA-based Lyme disease vaccines.

Amino Acid Sequence↗

PubMed1998

Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2.

smad genes constitute a family of nine members whose products serve as intracellular mediators of transforming growth factor beta signals. SMAD2, which is a tumor suppressor involved in colorectal and lung cancer, has been shown to induce dorsal mesoderm in Xenopus laevis in response to transforming growth factor beta and activins. The smad2 gene is expressed ubiquitously during murine embryogenesis and in many adult mouse tissues. Animals that lacked smad2 died before 8.5 days of development (E8.5). E6.5 homozygous mutants were smaller than controls, lacked the extraembryonic portion of the egg cylinder, and appeared strikingly similar to E6.5 smad4 mutants. This similarity was no longer evident at E7.5, however, because the smad2 mutants contained embryonic ectoderm within their interiors. Molecular analysis showed that smad2 mutant embryos did not undergo gastrulation or make mesoderm. The results demonstrate that smad2 is required for egg cylinder elongation, gastrulation, and mesoderm induction.

Animals↗

PubMed1998

Gene knockout B cell-deficient mice demonstrate that B cells play an important role in the initiation of T cell responses to Chlamydia trachomatis (mouse pneumonitis) lung infection.

T cell-mediated immunity as measured by delayed-type hypersensitivity, and IFN-gamma production has been shown to be critical for host defense against Chlamydia trachomatis infection in both human and animal studies. Using gene-targeted B cell-deficient mice, we examined the role of B cells in protective immunity to C. trachomatis (mouse pneumonitis) (MoPn) lung infection. B cell-deficient mice were observed to have a significantly higher mortality rate and in vivo chlamydial growth than did wild-type mice following MoPn lung infection. Interestingly, B cell-deficient mice not only lacked Ab responses but also failed to mount an efficient delayed-type hypersensitivity response following chlamydial lung infection. In contrast to results obtained from MoPn-infected wild-type C57BL/6 mice, spleen cells from infected B cell-deficient mice failed to produce Th1-related (IFN-gamma) or Th2-related (IL-6 and IL-10) cytokines after Chlamydia-specific in vitro restimulation. Moreover, unlike wild-type mice, B cell-deficient mice were not immune to rechallenge infection following recovery from primary chlamydial infection. The data indicate that B cells play an important role in host defense to primary and secondary chlamydial infection and suggest that B cells are crucial for the initiation of early T cell responses to chlamydial infection. This study provides evidence for the role of B cells in the in vivo priming of T cells during infection with the intracellular bacterial pathogen, C. trachomatis.

Animals↗