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Biomedical subjects

Y Wu

Publications and source records attributed to Y Wu.

At least 595 records · Page 33Linked to original sources
PubMed1998

Phospholipase cbeta4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum.

Elimination of excess climbing fiber (CF)-Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, Galphaq, and the gamma isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLCbeta4, one of two major isoforms expressed in Purkinje cells. PLCbeta4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLCbeta4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLCbeta4 mRNA but reciprocally high levels of PLCbeta3 mRNA are found. These results indicate that PLCbeta4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.

Animals↗
PubMed1998

A novel immediate early response gene, IEX-1, is induced by ultraviolet radiation in human keratinocytes.

Skin cancer is the most common human malignancy and is strongly associated with exposure to ultraviolet radiation (UVR). Several mechanisms including an increase in immediate early gene activation have been postulated to be involved in UVR-mediated carcinogenesis. We show that in a dose-dependent manner, UVR induces the expression of messenger RNA of a novel immediate early response gene, IEX-1, in human keratinocytes. Human keratinocytes and mouse fibroblasts transfected with an expression plasmid for IEX-1 grow at a faster rate than keratinocytes transfected with a similar plasmid that does not contain the IEX-1 sequence. IEX-1 protein is localized predominantly in the nucleus of keratinocytes by fluorescent antibody methods and by examination of the location of a green fluorescence IEX-1 fusion protein. Epidermal growth factor (EGF), a major mitogen of keratinocytes, and a tumor-promoting phorbol ester increase IEX-1 mRNA expression. IEX-1 may play a role in keratinocyte proliferation especially following UVR.

3T3 Cells↗
PubMed1998

[Identification of adhalin gene mutation in limb-girdle muscular dystrophy in Chinese].

OBJECTIVE: Limb-girdle muscular dystrophy (LGMD) is a group of severe genetic heterogeneity muscular diseases characterized by proximal muscular weakness of the pelvis and shoulder, affecting both male and female. This group of diseases involves four gene loci (13q12, 17q21,4q12,5q33). Up till now there is no research report about LGMD in Chinese. This study was intended to identify the pathogenic genes of LGMD in Chinese by mutation detecting. METHODS: The exons 2 and 3 of adhalin gene were analyzed in 13 Chinese LGMD patients and 20 controls by using PCR-SSCP and DNA sequencing. RESULTS: The R77C (Arg77Cys) missense mutation was found at the two alleles of a 9-year-old LGMD girl, which had not been found in the 40 wild type chromosomes. This is the first report on adhalin mutation that exists in LGMD in Chinese. CONCLUSION: Our results suggest that adhalin gene is one of the predisposing genes in LGMD in Chinese.

Child↗
PubMed1998

Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest melting temperature. When more than one melting domain is present the fragment is generally divided into several smaller ones. This, however, is not always necessary. We found that simple modifications of PCR fragments and primer sequences may substantially reduce the number of amplicons required. Furthermore, by plotting the (natural) melting curves of fragments without a GC-clamp, we could explain why fragments theoretically perfect for DGGE in practice failed to reveal mutations. Alternative fragment selection and the use of modified primers (addition of T/A or G/C tails) result in the detection of mutations that originally remained undetected. Our studies extend the utility of DGGE by using a minimum of PCR fragments and achieving a maximum of mutation detection.

Base Composition↗
PubMed1998

[Effect of Ciwujia (Radix Acanthopanacis senticosus) preparation on exercise performance under constant endurance load for elderly].

The effect of Ciwujia (Radix Acanthopanacis senticosus) preparation on human exercise performance was investigated by using 13 healthy volunteers aged 50-57. Under constant endurance load with 450 kg.m/min (75 W), the respiratory quotient after taking Ciwujia preparation was reduced to 0.88 from 0.96, which implied that the utilization of lipid increased with 27.2% as energy fuel during exercise. Meanwhile, the heart rate was reduced with 8.7%, and O2 uptake per heart beat increased with 16.18%. All results indicated that the Ciwujia preparation could increase O2 uptake, spare the glycogen in muscle and improve the exercise endurance and work performance in human subjects.

Animals↗
PubMed1998

Probing cellular protein targets of H2O2 with fluorescein-conjugated iodoacetamide and antibodies to fluorescein.

Recent studies suggest that H2O2, at subtoxic concentrations generated in response to the activation of a variety of cell surface receptors, functions as an intracellular messenger. However, the intracellular targets of H2O2 action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H2O2 is now described. This approach is based on the labeling of proteinaceous cysteine with 5-iodoacetamidofluorescein at pH 5.5 and immunoblot analysis of the labeled proteins with antibodies specific to fluorescein. With this procedure, many proteins in human A431 cells were shown to contain reactive cysteines and to be readily oxidized by H2O2 generated in response to cellular stimulation with epidermal growth factor. One of these H2O2-sensitive proteins was identified as protein tyrosine phosphatase 1B.

Antibodies↗
PubMed1998

PSTPIP 2, a second tyrosine phosphorylated, cytoskeletal-associated protein that binds a PEST-type protein-tyrosine phosphatase.

Although cytoskeletal regulation is critical to cell function during interphase and mitosis, the components of the cytoskeleton involved with its control are only beginning to be elucidated. Recently, we reported the identification of a cytoskeletal-associated protein, proline-serine-threonine phosphatase-interacting protein (PSTPIP), whose level of tyrosine phosphorylation was controlled by PEST-type protein-tyrosine phosphatases (PTPs) bound to a novel protein interaction site in the PSTPIP predicted coiled-coil domain. We also showed that the PSTPIP SH3 domain interacts with the Wiskott-Aldrich syndrome protein (WASP), a cytoskeletal regulatory protein, in a manner modulated by tyrosine phosphorylation. Here we describe the identification of PSTPIP 2, a widely expressed protein that is related to PSTPIP. PSTPIP 2 lacks an SH3 domain but contains a region predicted to bind to PEST-type PTPs, and structure-function analyses demonstrate that PSTPIP 2 interacts with the proline-rich C terminus of the PEST-type PTP hematopoietic stem cell factor in a manner similar to that previously demonstrated for PSTPIP. Confocal microscopy revealed that PSTPIP 2 colocalizes with PSTPIP in F actin-rich regions. PSTPIP 2 was found to be efficiently phosphorylated in v-Src-transfected or pervanadate-treated cells at two tyrosines conserved in PSTPIP, but in contrast to PSTPIP, tyrosine phosphorylated PSTPIP 2 was only weakly dephosphorylated in the presence of PTP HSCF. Finally, analysis of oligomer formation demonstrated that PSTPIP and PSTPIP 2 formed homo- but not heterodimers. These data suggest that a family of tyrosine phosphorylated, PEST PTP binding proteins may be implicated in cytoskeletal regulation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Comparison of conformational changes and inactivation of soybean lipoxygenase-1 during urea denaturation.

The unfolding and inactivation of soybean lipoxygenase-1 during urea denaturation has been compared. Equilibrium study indicates that inactivation of the enzyme occurs at low urea concentrations before significant conformational change of the molecule as a whole. In the presence of 6.0 M urea, the unfolding of soybean lipoxygenase-1, as monitored by fluorescence intensity, is a triphasic process, while the inactivation of the enzyme shows single-phase kinetics. The rate constant of inactivation is consistent with that of the fast conformational change of the enzyme. The results suggest that active sites of lipoxygenase-1 containing iron cofactor are situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole. The kinetic theory of substrate reactions catalyzed by unstable enzymes (Duggleby (1986) J. Theor. Biol. 123, 67-80) has been applied to study the effect of substrate on enzyme inactivation. On the basis of the kinetic equation of substrate reaction in the presence of urea, inactivation rate constants for the free enzyme and enzyme-substrate complex have been determined. The substrate, linoleic acid, has no effect on inactivation of the ferric form of lipoxygenase-1.

Binding Sites↗
PubMed1998

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria.

Heat shock proteins (Hsp) of four Rickettsia species, three Bartonella species, two Ehrlichia species, Orientia tsutsugamushi and seventeen other eubacterial species were characterized by the enhanced chemiluminescence Western blotting (WB) technique with antibodies raised against recombinant Hsp from Escherichia coli and purified GroES from R. typhi. Although E. coli DnaK and GroEL have epitopes that are highly conserved among the homologous proteins found in Rickettsia, Ehrlichia, O. tsutsugamushi, Bartonella and other Proteobacteria, anti-E. coli DnaK and GroEL monoclonal antibodies (Dasch et al. (1990) Ann. N.Y. Acad. Sci. 590, 352-369) recognize less conserved epitopes. In contrast, epitopes on E. coli DnaJ, GrpE and GroES are much less conserved since anti-E. coli DnaJ, GrpE and GroES polyclonal antibodies did not recognize DnaJ, GrpE or GroES homologues in Rickettsia, Bartonella, Orientia, Ehrlichia and Legionella. Polyclonal antiserum prepared against GroES from R. typhi reacted strongly with purified 10 kDa GroES peptide from Rickettsia and Bartonella, and strongly bound to proteins of varying electrophoretic mobility from Wolbachia, Legionella, Proteus and Shigella flexneri and more weakly to other GroES homologues including that found in E. coli. Consequently, commercially available anti-DnaJ, anti-GrpE and anti-GroES polyclonal antibodies and anti-DnaK monoclonal antibody raised against their respective recombinant E. coli Hsp are not suitable for detection and identification of homologues of these proteins in a wide range of eubacteria.

Alphaproteobacteria↗
PubMed1998

[Studies on the Ras p21 levels in workers exposed to coal tar pitch and coke oven volatiles].

To study the effects of serum p21 levels on the health of coal tar pitch(CTP) and coke oven volatiles (COV) workers recognized as the high risk population of pulmonary carcinoma, serum p21 levels among 24 cases of CTP workers, 25 cases of COV workers and 12 cases of normal control were detected with the method of Western Blotting. The results showed that the mean serum p21 level in exposed group was significantly higher than that in the normal control (366.8 vs 1270.7, P < 0.001). In the COV workers, serum p21 level in coke furnace workers was remarkably higher than that in the workers with other types of work. There were no differences for p21 among workers with different work years and between workers with or without smoking history. It indicates that CTP and COV can cause the p21 protein level increase in the occupational population.

Adult↗
PubMed1998

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1.

BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.

Animals↗
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

Methylprednisolone inhibition of TNF-alpha expression and NF-kB activation after spinal cord injury in rats.

Post-traumatic inflammatory reaction has been implicated in the secondary injury after SCI. TNF-alpha is a key inflammatory mediator, which plays a pathogenetic role in cell death in inflammatory disorders and traumatic brain injury. TNF-alpha exerts its effector actions, at least partially, through the activation of a pro-inflammatory transcription factor, NF-kB, which in turn upregulates such genes as iNOS, cytokines, adhesive molecules, and others. Consistent with a post-traumatic inflammatory reaction after SCI, we noted an increase in TNF-alpha expression by Western blotting (4.5-fold increase at 1 day after SCI, P<0.01) and immunohistochemistry in a rat SCI model. Post-traumatic TNF-alpha expression was accompanied by an increase in NF-kB binding activity in nuclear proteins isolated from the injured cord (3.9-fold increase, P<0.01). MP is the only drug proven effective in improving neurological function in patients with acute SCI. The mechanism of action of MP is not fully understood, but is thought to be related to its antioxidant effects. MP is also a potent anti-inflammatory agent, which has been recently shown to inhibit NF-kB binding activity. MP (30 mg/kg, i.v.) given immediately after SCI reduced TNF-alpha expression by 55% (P<0.01) and NF-kB binding activity. These findings suggest that post-traumatic inflammatory activity that is mediated by the TNF-alpha-NF-kB cascade can be suppressed by MP.

Animals↗
PubMed1998

Peptide containing the BCR oligomerization domain (AA 1-160) reverses the transformed phenotype of p210bcr-abl positive 32D myeloid leukemia cells.

We first showed that the introduction of a bcr-abl transcription unit into the 32D murine myeloid cell line (P210bcrabl32D) converts this cell line from an IL3 dependent cell line to an IL3 growth independent cell line. We next cloned a fragment of the bcr-abl cDNA, which codes for the bcr oligomerization domain and neighboring regions. To test for a transformation inhibitory effect of this oligomerization inhibitory peptide transcription unit on the p210bcr-abl mediated IL3 independent growth of the P210bcrabl32D cell line, we transiently co-electroporated into the growth factor dependent 32D cells, mixtures of plasmids which contained varying ratios of the plasmid expression vectors for the bcr oligomerization inhibitory peptide along with a smaller amount of the plasmid expression vector for the full length p210bcr-abl. (The P210bcr-abl protein converts the 32D from a growth factor dependent into a growth factor independent cell line.) We then showed that the oligomerization domain containing fragment from the bcr and bcr-abl proteins, can be used to inhibit the IL3 independent growth of p210bcr-abl positive 32D cells. These studies may be of eventual interest for those investigators whose goal is to design molecular therapeutic approaches to CML based on the use of peptidomimetic chemical functionalities, which mimic the structure and the inhibitory binding properties of the oligomerization domain containing fragment so as to inhibit the transforming function of the P210bcr-abl oncoprotein.

Animals↗
PubMed1998

A practical synthesis of 3-substituted delta 3,5(6)-steroids as new potential 5 alpha-reductase inhibitor.

A new and practical synthetic approach to 3-substituted delta 3,5(6)-Steroids, as potential 5 alpha-reductase inhibitor, is described. The key step involves Pd-catalyzed coupling reaction of steroid 3-enol 5H-3-oxo-octafluoropentanosulfonates. 3-Phenylacetylenyl substituted delta 3,5(6)-steroid 3g and 3-phosphate substituted delta 3,5(6)-steroid 3f in our synthesized compounds exhibited high 5 alpha-reductase inhibitory activity in vitro assay.

5-alpha Reductase Inhibitors↗
PubMed1998

A baculovirus single-stranded DNA binding protein, LEF-3, mediates the nuclear localization of the putative helicase P143.

The intracellular localization of the baculovirus Autographa californica nuclear polyhedrosis virus p143 gene product (P143) was investigated by immunofluoresence staining of infected and transfected cells. As expected for a protein essential for viral DNA replication, under these conditions, P143 was localized to the nucleus. However, when a plasmid directing the synthesis of P143 from its own promoter was co-transfected with a plasmid expressing IE-1, P143 was found in the cytoplasm. Cotransfection of cells with a series of plasmids expressing viral genes sufficient for stimulation of plasmid replication resulted in nuclear localization of P143 suggesting that one of the gene products required for viral DNA replication may also assist nuclear localization of P143. Sequential deletion of various genes from this assay revealed that when the plasmid expressing LEF-3 was deleted from the mixture, P143 remained in the cytoplasm. Plasmids were constructed where the synthesis of LEF-3 and P143 were directed by the ie-1 promoter. When these plasmids were cotransfected, both LEF-3 and P143 colocalized to the nucleus suggesting that an important function of LEF-3 is intracellular trafficking of P143 and directing it to the nucleus.

Animals↗
PubMed1998

Structure of an alpha-2,6-sialylated lipooligosaccharide from Neisseria meningitidis immunotype L1.

The recent cloning of the lipooligosaccharide (LOS) a-2,3-sialyltransferase from Neisseria meningitidis immunotype L3 permitted us to examine other immunotypes for this structural gene. We identified the gene and measured the enzyme activity in the L1 immunotype strain which had previously been reported to lack sialic acid in its LOS because it contains a terminal alpha-linked galactose which was thought not to be an acceptor for the sialyltransferase. This finding prompted us to re-examine the structure of the LOS from the L1 immunotype, which revealed the presence of sialic acid on the terminal alpha-linked galactose. Oligosaccharides derived from the LOS were shown to be sialylated by composition and methylation analysis, mass spectrometry and nuclear magnetic resonance. The detailed structural analysis showed the sialic acid to occur only at 06 of the terminal a-D-galactopyranose residue of the alpha-D-Gal-1,4-beta-D-Gal-1,4-beta-D-glc trisaccharide (Pk epitope) chain of the LOS, in the alpha-D configuration. These data are the first report of a alpha-2,6-linked sialic acid in a bacterial LOS or lipopolysaccharide, and also the first report of a sialylated Pk epitope.

Carbohydrate Conformation↗