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Biomedical subjects

Y Yang

Publications and source records attributed to Y Yang.

At least 721 records · Page 40Linked to original sources
PubMed1998

Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release.

Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.

Calcium↗
PubMed1998

Nopp140 functions as a molecular link between the nucleolus and the coiled bodies.

Coiled bodies are small nuclear organelles that are highly enriched in small nuclear RNAs, and that have long been thought to be associated with the nucleolus. Here we use mutational analysis, transient transfections, and the yeast two-hybrid system to show that the nucleolar phosphoprotein Nopp140 functions as a molecular link between the two prominent nuclear organelles. Exogenous Nopp140 accumulated in the nucleolus rapidly, but only after a lag phase in coiled bodies, suggesting a pathway between the two organelles. The expression of partial Nopp140 constructs exerted dominant negative effects on the endogenous Nopp140 by chasing it and other antigens that were common to both organelles out of the nucleolus. The alternating positively and negatively charged repeat domain of Nopp140 was required for targeting to both organelles. In addition, partial Nopp140 constructs caused formation of novel structures in the nucleoplasm and, in the case of the conserved carboxy terminus, led to the dispersal of coiled bodies. As a final link, we identified the coiled body-specific protein p80 coilin in a yeast two-hybrid screen with Nopp140. The interaction of the two proteins was confirmed by coimmunoprecipitation. Taken together, Nopp140 appeared to shuttle between the nucleolus and the coiled bodies, and to chaperone the transport of other molecules.

3T3 Cells↗
PubMed1998

T cell receptor (TCR) engagement leads to activation-induced splicing of tumor necrosis factor (TNF) nuclear pre-mRNA.

Inducible gene expression is primarily regulated at the level of transcription. Additional steps of "processing" pre-mRNA, involved in the regulation of induced gene expression, have not been previously reported. Here we report a novel mechanism of "activation-induced splicing" of preexisting tumor necrosis factor (TNF) message (pre-mRNA) in naive T lymphocytes after engagement of the T cell receptor (TCR), which still occurs after inhibition of transcription. Expression of TNF has been previously demonstrated to be regulated at both the transcriptional and translational levels. However, neither the large pool of TNF mRNA observed in activated T cells nor TNF protein production, which peaks very shortly after activation, can be solely attributed to increased transcription. Evidence is presented that activation-induced splicing of TNF pre-mRNA plays a significant role in the rapid production of TNF seen in activated T cells. Activation triggers processing of TNF pre-mRNA that has accumulated in naive T cells (before activation-induced transcription), and the mature TNF mRNA is translocated to the cytoplasm for rapid translation and protein production. This novel form of activation-induced splicing of TNF may allow T cells to mount an immediate response to activation stimuli under physiological conditions.

Animals↗
PubMed1998

Interleukin 1beta-converting enzyme (caspase-1) is overexpressed in adenocarcinoma of the pancreas.

We investigated the expression of interleukin 1beta-converting enzyme (ICE; caspase-1) in human adenocarcinomas of the pancreas. Immunohistochemistry and Western blot analyses revealed an overexpression of ICE in 71 and 80% of tumor cells, respectively. Also, on a mRNA level, ICE mRNA was overexpressed in 45% of the cases, as compared to normal pancreatic tissue. Interestingly, the overexpression of ICE in tumor cells correlated significantly with the overexpression of cyclin D1, epidermal growth factor, and epidermal growth factor receptor (P < 0.0005, P < 0.05, and P < 0.002, respectively), which are involved in cell cycle progression and proliferation in human pancreatic carcinoma. This is the first report concerning ICE expression in human carcinomas; however, the exact mechanism underlying these close correlations warrant further research.

Adenocarcinoma↗
PubMed1998

Targeted integration of a recombinant globin gene adeno-associated viral vector into human chromosome 19.

Transfer of a globin gene into stem cells along with the regulatory elements required to achieve high level expression in maturing erythroid cells would provide effective gene therapy for Cooley's Anemia. We have explored the use of recombinant adeno-associated viral (rAAV) vectors for this purpose. A vector designated rHS32A gamma*3'RE that contains regulatory elements from the locus control and flanking regions, integrates as a stable head-to-tail concatamer in erythroleukemia cells at a high multiplicity of infection and exhibits high level, regulated gamma globin gene expression. Inducible expression of the non-structural Rep proteins of wild-type AAV in HeLa cells transduced with rAAV vectors does not increase overall integration frequency, but targeted integration of rHS32A gamma*'3'RE into human chromosome 19 was documented.

Chromosome Mapping↗
PubMed1998

Evidence of a functional interaction between serine 3 and serine 25 Mos phosphorylation sites. A dominant inhibitory role of serine 25 phosphorylation on Mos protein kinase.

Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.

3T3 Cells↗
PubMed1998

The X11alpha protein slows cellular amyloid precursor protein processing and reduces Abeta40 and Abeta42 secretion.

Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Abeta peptides, including Abeta40 and Abeta42-the major component of amyloid plaques in Alzheimer's disease brain. The phosphotyrosine binding (PTB) domain of X11 binds to a peptide containing a YENPTY motif found in the carboxyl terminus of APP. We have cloned the full-length X11 gene now referred to as X11alpha. Coexpression of X11alpha with APP results in comparatively greater levels of cellular APP and less APPs, Abeta40, and Abeta42 recovered in conditioned medium of transiently transfected HEK 293 cells. These effects are impaired by a single missense mutation of either APP (Y682G within the YENPTY motif) or X11alpha (F608V within the PTB domain), which diminishes their interaction, thus demonstrating specificity. The inhibitory effect of X11alpha on Abeta40 and Abeta42 secretion is amplified by coexpression with the Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulse-chase analysis demonstrates that X11alpha prolongs the half-life of APP from approximately 2 h to approximately 4 h. The effects of X11alpha on cellular APP and APPs recovery were confirmed in a 293 cell line stably transfected with APP. The specific binding of the PTB domain of X11alpha to the YENPTY motif-containing peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Abeta40, and Abeta42 in conditioned medium of transfected HEK 293 cells. X11alpha may be involved in APP trafficking and metabolism in neurons and thus may be implicated in amyloidogenesis in normal aging and Alzheimer's disease brain.

Alzheimer Disease↗
PubMed1998

Study of relapsing remitting experimental allergic encephalomyelitis SJL mouse model using MION-46L enhanced in vivo MRI: early histopathological correlation.

MION-46L, a superparamagnetic iron oxide contrast agent, was investigated for its ability to increase the sensitivity of in vivo 3D MRI in the detection of brain lesions in a chronic experimental allergic encephalomyelitis (crEAE) mouse model. Lesion conspicuity on postcontrast 3D MRI was dramatically enhanced as compared to precontrast images corresponding to areas of inflammatory and demyelinating lesions. MION-46L could be detected on Prussian blue iron stain in the vascular endothelium, the perivascular space, and in macrophages within perivascular cuffs and areas of inflammation and demyelination. By taking advantage of the MION-46L induced macroscopic susceptibility effect, acute early lesions measuring only 100 microm in diameter could be detected. MION-46L enhanced MRI may be used to 1) provide a unique sensitivity in EAE lesion detection and correlate imaging to histopathology; 2) help to understand EAE lesion development and its underlying pathophysiology; and 3) eventually assist in preclinical screening of new experimental therapies directed at patients with multiple sclerosis (MS).

Animals↗
PubMed1998

Synaptic reorganization following kainic acid-induced seizures during development.

Prolonged seizures in the adult brain causes neuronal loss in the hippocampus and aberrant growth (sprouting) of granule cell axons (mossy fibers) in the supragranular zone of the fascia dentata and stratum infrapyramidale of CA3. There is considerable evidence that these changes in neuronal growth following seizures are age related, with younger animals having fewer reactive changes following prolonged seizures than older animals. However, there is little information available regarding the age at which seizures in the developing brain result in alterations in axonal growth and synapse formation. In this study, we evaluated the effects of kainic acid (KA)-induced seizures during development on synaptic reorganization using the expression of growth-associated protein-43 (GAP-43), a marker for synaptogenesis and Timm stain which detects the presence of zinc in granule cell axons. Age specific doses of KA were used to induce seizures of similar intensity at various ages (postnatal days (P) 12, 21, 25, 35, 45, 60) in Sprague-Dawley rats. Up to the age of P25, there were no differences in either Timm or GAP-43 staining between animals with KA seizures and controls. In P25 and older KA-treated rats, Timm staining was found in the supragranular layer of the dentate gyrus. This staining increased with age at the time of KA injection. Seizures in adult (P60), but not younger rats also resulted in increased staining in the suprapyramidal layer of the CA3 subfields. Changes in GAP-43 were delayed compared to the Timm staining with no differences between KA-treated animals and controls until P35 when a band of GAP-43 immunostaining appeared in the supragranular inner molecular layer, progressively increasing in intensity and thickness with time. This study demonstrates that seizure-induced reactive synaptogenesis is age-related. Since both Timm and GAP-43 reflect different aspects of reactive synaptogenesis, used in combination these methods provide useful information about the structural changes following seizures during development.

Aging↗
PubMed1998

The structural basis of ankyrin-like repeat function as revealed by the solution structure of myotrophin.

BACKGROUND: Myotrophin is a 12.5 kDa protein that appears to have a key role in the initiation of cardiac hypertrophy, a central process in many heart diseases. Myotrophin primarily comprises ankyrin-like (ANK) repeats, the 33 amino acid motifs involved in a wide range of protein-protein interactions. As a first step in the structure-based search for cardiac hypertrophy antagonists and in order to gain insight into the molecular basis of action of the ubiquitous and multifunctional ANK repeat motif, we have determined the solution structure of myotrophin using multidimensional heteronuclear NMR spectroscopy. RESULTS: The myotrophin structure determination was based on 2786 experimental NMR restraints, and the precision of the coordinates for the final 45 simulated-annealing structures is 0.43 A for the backbone atoms and 0.87 A for all atoms. The structure of myotrophin is well defined and is ellipsoidal: approximately 46 A long and 21 A wide. The ANK repeats, which constitute the main part of the myotrophin structure, are characteristic of a hairpin-like protruding tip followed by a helix-turn-helix motif. The V-shaped helix-turn-helix of the ANK repeats stack sequentially in bundles and are stabilized by compact hydrophobic cores, whereas the protruding tips are less ordered. This arrangement is quite different to the continuous beta-sheet topology observed in the corresponding regions of another ANK protein, 53BP2, the structure of which was determined in complex with p53. CONCLUSIONS: The solution structure of myotrophin provides important insights into the structural and dynamic features of the ANK motif, and suggests that the protruding tips with highly variable sequences may be critical to facilitate diverse protein-protein recognition. The present structure also provides a molecular basis for the further functional characterization of myotrophin and the development of therapeutics for hypertrophy-related heart diseases.

Amino Acid Sequence↗
PubMed1998

Immunocytochemical analysis of sex differences in calcitonin gene-related peptide in the rat dorsal root ganglion, with special reference to estrogen and its receptor.

Previous studies have shown that the calcitonin gene-related peptide (CGRP) immunoreactivity in the central nervous system (CNS) of adult rats is sexually dimorphic and regulated by sex steroid. In the present study, we used immunocytochemistry to investigate the sex difference in CGRP-immunoreactive (IR) neurons in rat dorsal root ganglia (DRG). The numbers of CGRP-IR neurons at the cervical, lumbar and sacral levels in the female rats were significantly lower than those of the male rats. We also found that the number of CGRP-IR neurons at the lumbar level was increased in ovariectomized (OVX) rats, but was decreased in estradiol (E2)-treated rats (OVX+E2). A large number of estrogen receptor (ER)-IR neurons at the lumbar level were found in the female rats, and its number was greater than that in the male rats. We also investigated the change in the number of ER-IR neurons of OVX rats after estrogen treatment. The number of ER-IR neurons in the OVX+E2 rats was consistent with that of the intact female rats, but was significantly increased in the OVX rats. As shown by a double-labeling immunocytochemical method, over 80% of the CGRP-IR neurons at the lumbar level showed ER immunoreactivity in the female, OVX and OVX+E2 rats, compared to only about 46% in the male rats. These results indicate that there is a gender difference in CGRP expression in the rat DRG, and that this CGRP expression might be downregulated by estrogen (at least in part) through its receptor.

Animals↗
PubMed1998

Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor.

The CC-chemokine receptor CCR5 has been shown to be the major coreceptor for HIV-1 entry into cells, and humans with homozygous mutation in the ccr5 gene are highly resistant to HIV-1 infection, despite the existence of many other HIV-1 coreceptors. To investigate the physiologic function of CCR5 and to understand the cellular mechanisms of these clinical observations, we generated a CCR5-deficient mouse model (ccr5[-/-]) by targeted deletion of the ccr5 gene. We found that although developed normally in a pathogen-free environment, CCR5-deficient mice showed reduced efficiency in clearance of Listeria infection and exert a protective effect against LPS-induced endotoxemia, reflecting a partial defect in macrophage function. In addition, CCR5-deficient mice had an enhanced delayed-type hypersensitivity reaction and increased humoral responses to T cell-dependent antigenic challenge, indicating a novel role of CCR5 in down-modulating T cell-dependent immune response.

Animals↗
PubMed1998

Solid-phase microextraction of polychlorinated biphenyls.

The extraction and analysis of 21 polychlorinated biphenyls (PCBs) ranging from di- to decachlorobiphenyls in ocean, wetland and leachate water samples were achieved using solid-phase microextraction (SPME) with a 100-micron poly(di-methylsiloxane) (PDMS) fiber and gas chromatography-electron-capture detection (GC-ECD). Severe carryover between samples (e.g., 20%) occurs on both stir bars and the SPME fibers demonstrating that it is important to use a new stir bar for each sample, as well as to perform SPME-GC blanks between samples to avoid quantitative errors. The equilibrium partitioning coefficients of individual PCB congeners between PDMS and water were found to be surprisingly different compared to their octanol-water partitioning coefficient (Kow), demonstrating that Kow cannot be used to estimate the partitioning behavior of PCBs in the SPME process. Using a 15-min SPME extraction, SPME analysis with GC-ECD was linear (r2 > or = 0.97) from approximately 5 pg/ml to the solubility limit of each congener. Concentrations in water samples obtained by 15-min SPME extractions compared favorably with those obtained by toluene extractions, demonstrating that SPME combined with GC is a useful technique for the rapid determination of PCBs in water samples.

Absorption↗
PubMed1998

A novel extracellular domain variant of the human integrin alpha 7 subunit generated by alternative intron splicing.

The integrin alpha 7 beta 1 laminin receptor, which is expressed on replicating myoblasts, and upregulated during myogenic differentiation, is involved in cell adhesion and communication between muscle cells and the extracellular matrix. It is a major cell-surface substrate in skeletal muscle cells for the cell-surface, argininespecific, ADP-ribosyltransferase. Both the extracellular and cytoplasmic domains of the mouse alpha 7 subunit undergo alternative splicing during development, generating differentially expressed variants with presumably unique ligand-binding and signalling properties. Here human cDNA clones isolated from a fetal heart lambda gt10 cDNA library encoded the complete sequence of the alpha 7 subunit and hybridised to a single major 4.4 kb alpha 7 subunit transcript abundantly expressed in human skeletal muscle, moderately expressed in heart, and weakly expressed in most other tissues. One clone out of four contained a novel 225-nucleotide in-frame deletion corresponding to 75 amino acids in the C-terminal region of the extracellular domain. The variant, whose expression appears to be tissue-specific, is created by alternative splicing at sites flanking an intron in the alpha 7 gene. A related mouse form was identified in P19 embryonal carcinoma cells. Deletion of the spliced region, which either contains or is in very close proximity to the major ADP-ribosylation site of the alpha 7 subunit, may serve to modulate the effects of ADP-ribosylation, or alternatively molecular associations, and receptor-ligand affinity.

Alternative Splicing↗
PubMed1998

Paradoxical effect of GM-CSF gene transfer on the tumorigenicity and immunogenicity of murine tumors.

The effect of granulocyte/macrophage colony-stimulating factor (GM-CSF) gene transfer on the tumorigenicity and immunogenicity of 2 different murine tumor lines was determined. Transduction of B16 melanoma cells with the GM-CSF gene rendered the cells more immunogenic. In contrast, transduction of NG4TL4 fibrosarcoma in FVB/N mice (NG) with the GM-CSF gene showed increased tumorigenicity in a high producer line (NG-MGh). The parent NG or NG-MG cells induced the same level of cytotoxic T-lymphocyte (CTL) response and the same magnitude of tumor transplantation immunity. However, the proliferation of the NG-MGh cells was increased 2- to 10-fold. There was no increase in apoptosis in the NG cells and there was no increase of NG-MGh cells in S-phase, hence the increase of the proliferative activity appeared to be indeed inherent to the cells. Mixing the splenocytes from the NG-MGh tumor bearers with the NG tumor cells did not increase tumorigenicity but totally inhibited the growth of the NG tumor, indicating that suppressor cells were not present. Mixing 10,000 rad X-irradiated NG-MGh cells with viable NG tumor cells resulted in 3- to 10-fold increased NG tumor growth rate. The in vitro proliferation of NG cells was increased by adding both GM-CSFs and macrophages and not by either one alone, suggesting that interaction between macrophages and GM-CSFs resulted in the production of tumor growth enhancing factor(s). Our findings suggest that transduction of NG tumor cells with the GM-CSF gene increases tumorigenicity, which is attributed both to an increased inherent proliferative ability of the tumor cells and to the in vivo production of a tumor growth enhancing factor(s) at the tumor site.

3T3 Cells↗
PubMed1998

Analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene found in rat urine by nano-electrospray mass spectrometry.

Nano-electrospray has been used in combination with high resolution and tandem mass spectrometry in the analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene (BP). Accurate mass measurements indicate that the [M-H]- ion of the major metabolite has a chemical formula C25H22NO6S, which corresponds to the deprotonated form of tetrahydro-trihydroxy-BP-S-N-acetylcysteine. Tandem mass spectrometry of this [M-H]- ion results in a collision induced dissociation spectrum identical to that of synthetic 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine.

Acetylcysteine↗
PubMed1998

Is canine hepatocerebellar degeneration syndrome an animal model for carbohydrate-deficient glycoprotein syndrome in humans? An example of sequencing glycoprotein glycans with mass spectrometry.

The clinical symptoms and morphologic features of canine hepatocerebellar degeneration syndrome (CHD) bear striking resemblance to those of human carbohydrate-deficient glycoprotein syndrome (CDGS). The characteristic biochemical and molecular features of human CDGS lie in the truncated carbohydrate side chains of serum transferrin and numerous other glycoproteins of affected persons. Therefore, to explore the biochemical similarities between CHD and CDGS, we compared the structures of the carbohydrate side chains of canine serum transferrin isolated from a normal and a CHD-affected dog. Because of the very small quantity of serum transferrin available from the CHD-affected dog, we used analytical procedures that minimize sample consumption. In this scheme, we used microbore liquid chromatography interfaced to electrospray tandem mass spectrometry to identify and purify the glycopeptides from the tryptic digest of canine serum transferrin. This was followed by a series of exoglycosidase digestions coupled with mass spectrometric detection to sequence the carbohydrate side chains of the glycopeptides. With these procedures we completely characterized the carbohydrate chains attached to serum transferrin isolated from both a normal and a CHD-affected dog. However, we found no discernible differences in glycosylation of the carbohydrate side chains of serum transferrin from these two animals, suggesting that the biochemical defect in puppies with CHD differs from that in children with CDGS.

Alkylation↗