Biomedical subjects

K Wu

Publications and source records attributed to K Wu.

At least 19 recordsLinked to original sources

PubMed2000

The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation.

ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Here we present evidence suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed.

Amino Acid Sequence↗

PubMed2000

Genetic and cellular defects contributing to benign tumor formation in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common inherited cancer predisposition syndrome. The NF1 gene product, neurofibromin, is hypothesized to function as a tumor suppressor and nearly all NF1 patients develop benign peripheral nerve tumors. These neurofibromas presumably arise from NF1 inactivation in S100(+)Schwann cells, but there is no formal proof for this mechanism. We demonstrate that fibro-blasts isolated from neurofibromas carried at least one normal NF1 allele and expressed both NF1 mRNA and protein, whereas the S100(+)cells typically lacked the NF1 transcript. Our findings further indicate that additional molecular events aside from NF1 inactivation in Schwann cells and/or other neural crest derivatives contribute to neurofibroma formation.

Adult↗

PubMed2000

Expression and subcellular localization of a membrane protein related to Hsp30p in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae YDR033w gene product is homologous to Hsp30p and Yro2p, both of which are induced during heat shock. To investigate the subcellular localization of the YDR033w gene product, hemagglutinin (HA) epitope-tagged protein was expressed, detected on immunoblots, and localized by immunofluorescence to cell membranes, primarily the plasma membrane. A punctuate immunofluorescence pattern was observed within cell buds. The nuclear envelope, but not the vacuole or mitochondrial membranes, were also immunostained. We refer to YDR033w as MRH1 to denote that it encodes a membrane protein related to Hsp30p.

Amino Acid Sequence↗

PubMed2000

Inverted urothelial papilloma: is ploidy, MIB-1 proliferative activity, or p53 protein accumulation predictive of urothelial carcinoma?

BACKGROUND: Inverted urothelial papilloma is an unusual neoplasm of the urinary tract. Although the association between inverted urothelial papilloma and urothelial carcinoma is not entirely clear, many studies indicate that patients with inverted papilloma are at increased risk for the development of urothelial carcinoma. In addition, aneuploid inverted papillomas have been associated with the subsequent development of urothelial carcinoma. The objective of this study was to determine whether ploidy, MIB-1 proliferative activity, or p53 protein staining in inverted papilloma were predictive of urothelial carcinoma. METHODS: Fifty-one cases of inverted papilloma were retrieved from the Tissue Registry of the Mayo Clinic. Clinical records were reviewed for patient age, length of follow-up, and history of urothelial carcinoma (defined as carcinoma prior to, concurrent with, or subsequent to the diagnosis of inverted papilloma). DNA ploidy analysis was determined using Feulgen stained sections from paraffin embedded tissues using an image analysis system. Quantitation of MIB-1 proliferative activity and p53 immunostaining was determined similarly using immunoperoxidase stained sections from paraffin embedded tissues. RESULTS: The mean age at diagnosis of inverted papilloma was 63.9 years (range, 37-87 years), and there were 39 men and 12 women. Patients were followed for a mean of 56.5 months (range, 1-252 months). Tumors ranged in size from 0.2 to 4.3 cm (mean, 0.9 cm). Eight patients (15.7%) had a prior, concurrent, or subsequent noninvasive World Health Organization and International Society of Urologic Pathology (WHO/ISUP) papillary neoplasm of low malignant potential or papillary carcinoma of low grade (formerly WHO Grade 1 or 2 papillary urothelial carcinoma). Inverted papillomas in patients with a history of urothelial carcinoma were all diploid and had a mean MIB-1 activity of 6.3% (range, 0.04-24.8%) and mean p53 protein staining of 12.6% (range, 0.5-24.9%). These inverted papillomas ranged in size from 0.3 to 1.0 cm (mean, 0.5 cm). Inverted papillomas in patients without a history of urothelial carcinoma were aneuploid in 6 cases (14.3%) and diploid in the remaining cases. These inverted papillomas had a mean MIB-1 activity of 1.6% (range, 0.06-9.0%) and mean p53 protein staining of 9.7% (range, 0.05-38.0%). Tumor size ranged from 0.2 to 4.3 cm (mean, 1.0 cm). There were no statistically significant differences in MIB-1 activity, p53 protein staining, ploidy, and morphologic features between inverted papillomas in patients with and without a history of urothelial carcinoma. CONCLUSIONS: Ploidy, MIB-1 proliferative activity, and p53 immunostaining in inverted urothelial papilloma were not useful in identifying patients who had a history of urothelial carcinoma.

Adult↗

PubMed2000

Localization of brain-derived neurotrophic factor and TrkB receptors to postsynaptic densities of adult rat cerebral cortex.

Although neurotrophins are critical for neuronal survival and differentiation, recent studies suggest that they also regulate synaptic plasticity. Brain-derived neurotrophic factor (BDNF) rapidly increases synaptic transmission in hippocampal neurons, and enhances long-term potentiation (LTP), a cellular and molecular model of learning and memory. Loci and precise mechanisms of BDNF action remain to be defined: evidence supports both pre- and postsynaptic sites of action. To help elucidate the synaptic mechanisms of BDNF action, we used antisera directed against the extracellular and intracellular domains of trkB receptors, anti-trkBout and anti-trkBin, respectively, to localize the receptors in relation to synapses. Synaptic localization of BDNF was examined in parallel using anti-BDNF antisera. By light microscopy, trkBin and trkBout immunoreactivities were localized to hippocampal neurons and all layers of the overlying visual cortex. Immunoelectron microscopic analysis of the cerebral cortex revealed that trkB and BDNF localize discretely to postsynaptic densities (PSD) of axo-spinous asymmetric synaptic junctions, that are the morphological correlates of excitatory, glutamatergic synapses. TrkB immunoreactivity was also detected in the nucleoplasm by light and electron microscopy. Western blot analysis indicated that both anti-trkBout and anti-trkBin antisera react with a protein band in the PSD corresponding to the molecular weight expected for trkB; however, molecular species distinct from that for trkB were recognized in the nuclear fraction by both anti-trkBin and anti-trkBout antisera, indicating that the nuclear immunoreactivity, seen by immunocytochemistry, reflects cross-reactivity with proteins closely related to, but distinct from, trkB. The PSD localization of both BDNF and trkB supports the contention that this receptor/ligand pair participates in postsynaptic plasticity.

Animals↗

PubMed2000

Obituary

Explore the source record for details and available documents.

Journal Article↗

PubMed2000

DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene.

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.

Animals↗

PubMed2000

Membrane-bound macrophage colony-stimulating factor and its receptor play adhesion molecule-like roles in leukemic cells.

Membrane-bound macrophage colony-stimulating factor (m-M-CSF) is the membrane form M-CSF by alternative splicing. J6-1 leukemic cell line spontaneously forms cell clusters, whose growth depends on the auto-juxtacrine mediated by m-M-CSF and its receptor (M-CSFR). In this study, M-CSFR isolated from J6-1 cells and recombinant human M-CSF soluble receptor (rh-M-CSFsR) were used to study their effects on J6-1 cells. Both receptors inhibited cell proliferation. Use of M-CSFR monoclonal antibodies, M-CSFR or rh-M-CSFsR to block either M-CSFR or m-M-CSF on cell surface inhibited the cluster forming process, while both receptors stimulated cells adhering to culture plate. Furthermore, M-CSFR and/or rh-M-CSFsR caused multiple cellular changes including cytoplasmic pH, multinuclear cell ratio, antigen expression and cell diameter. A [Ca(2+)] rise was induced within 90 s by both receptors. Western blot experiments showed that rh-M-CSFsR caused tyrosine phosphorylation on multiple cytoplasmic proteins of 45 kDa and 55-90 kDa, which could be blocked by H7. These observations suggested that m-M-CSF and M-CSFR mediate J6-1 cell intercellular adhesion with bi-directional signal transduction, and Ca(2+), protein tyrosine kinases, PKC and/or other H7 sensitive kinase(s) involve in the counter-directional signal transduction.

Cell Adhesion↗

PubMed2000

A prospective study of plasma ascorbic acid concentrations and breast cancer (United States).

OBJECTIVES: To investigate the association between prediagnostic plasma ascorbic acid concentrations and subsequent breast cancer risk in a nested case-control study. METHODS: Female volunteer residents of Washington County, MD, donated 14,625 non-fasting blood samples in 1989. Incident breast cancer cases (n = 115) and controls (n = 115) were matched by age, menopausal status at donation, and date and hour of blood donation. RESULTS: Median ascorbic acid concentrations were similar between cases and controls (1.44 mg/dl vs. 1.39 mg/dl. p = 0.78). There was no evidence for a dose-response relationship between higher plasma ascorbic acid concentrations and breast cancer risk [highest vs. lowest fifths: ORadjusted = 0.90, Ptrend = 0.98). CONCLUSIONS: Findings from this prospective study do not suggest a protective association between prediagnostic plasma ascorbic acid concentrations and breast cancer risk in the subsequent 5 years of follow-up.

Ascorbic Acid↗

PubMed2000

Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana.

Post-translational modification of histones, in particular acetylation, is an important mechanism in the regulation of eukaryotic gene expression. Histone deacetylases are enzymes that remove acetyl groups from the core histones and play a key role in the repression of transcription. HD2 is a maize histone deacetylase, which shows no sequence homology to the histone deacetylases identified from other eukaryotes. We have identified two putative HD2-like histone deacetylase cDNA clones, AtHD2A and AtHD2B, from Arabidopsis thaliana by screening the expressed sequence tag database. AtHD2A and AtHD2B encode putative proteins of 246 and 305 amino acids, and share 44% and 46% amino acid identity to the maize HD2, respectively. Northern blot analysis indicated that AtHD2A was highly expressed in flowers and young siliques of Arabidopsis plants, whereas AtHD2B was widely expressed in stems, leaves, flowers and young siliques. AtHD2A repressed transcription when directed to a promoter containing GAL4-binding sites as a GAL4 fusion protein. Deletion of the extended acidic domain or the domain containing predicted catalytic residues of AtHD2A resulted in the loss of gene repression activity, revealing the importance of both domains to AtHD2A function. Arabidopsis plants were transformed with a gene construct comprising an AtHD2A cDNA in the antisense orientation driven by a strong constitutive promoter, -394tCUP. Silencing of AtHD2A expression resulted in aborted seed development in transgenic Arabidopsis plants, suggesting that the AtHD2A gene product was important in the reproductive development of Arabidopsis thaliana.

Amino Acid Sequence↗

PubMed2000

The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation.

We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IkappaBalpha. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF(HOS/beta-TRCP)-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/beta-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IkappaB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2000

[The relationship of polymorphism of angiotensinogen and angiotensin converting enzyme with essential hypertension].

OBJECTIVE: In order to investigate the candidate genes for essential hypertension(EH), the authors determined the frequencies of M235T allele variation in exon 2 of angiotensinogen(AGT) gene and angiotensin converting enzyme (ACE) gene in 95 normotensives and 87 hypertensives in Chinese. METHODS: Polymerase chain reaction combined with restriction enzyme digestion was used to detect the variation AGT gene while PCR was used to determine ACE gene polymorphism. RESULTS: T235 allele frequency was significantly higher in hypertensive patients than in controls (0.45 vs 0.33, P<0. 05) and it was noticeably higher in male hypertensives. The frequency of ACE D allele in the EH patients with family history was higher than that in controls(0.59 vs 0.41,P<0.05). CONCLUSION: The variation of AGT gene was involved in the pathogenesis of hypertension, especially in male. The ACE D allele was associated with EH with family history.

Alleles↗

PubMed2000

Prospects for treating acquired pendular nystagmus with servo-controlled optics.

PURPOSE: To determine whether a device featuring electronically controlled motor-driven prisms can reduce oscillopsia and improve acuity in patients with acquired pendular nystagmus (APN). METHODS: A device was developed that senses eye movements and, by the use of motor-driven prisms, oscillates the image of the world in lockstep with the pathologic nystagmus, to negate its deleterious visual effects. Unlike existing optical and surgical treatments for nystagmus, the device negates only the pathologic movements. Voluntary and normal reflex eye movements required for normal vision are unaffected. The benefits of the device were assessed by its impact on acuity in five patients with medication-refractory APN. RESULTS: All patients reported decreases in oscillopsia when the device was in operation. Averaged across patients, the device increased the percentage of time in which retinal image velocity was within +/-4 degrees/sec from 12.8% to 33.3%. Acuities improved in four of five patients, by an average of 0.21 logMAR units. CONCLUSIONS: The symptoms of pendular nystagmus can be treated with a servomechanical device. Further refinements in the device should result in greater improvements in acuity, and a portable, wearable version is feasible using existing technologies.

Adult↗

PubMed1999

Ion penetration of the water-Oil interface

Ions typically pass with difficulty from water into organic phases because of water's superior solvation power. This inhibits such processes as ion transport in batteries or in lipid bilayers of cells. Ion penetration across such an interface was studied with unusual structural control. Hydronium ions were soft-landed at 1 electron volt on cold films of 3-methylpentane ("oil") on a metal substrate. The field produced by these ions drove them through the films when warmed. Coadsorption of water (0.14 to 35 bilayers) inhibited the ion penetration by creating a solvation energy trap. A Born solvation model successfully predicted the trapping energies (0 to 38 kilojoules per mole).

Journal Article↗

PubMed1999

A Novel pH-Sensitive MRI Contrast Agent.

A tetrasubstituted derivative of 1,4,7,10-tetraazacyclododecane with amide coordinating groups and extended noncoordinating phosphonate groups forms a complex with gadolinium(III) (shown in the picture) which contains one slowly exchanging inner-sphere water molecule (tau(M)=21 µs). The 20-MHz water proton relaxivity of the complex was found to be highly pH dependent. Protonation of the noncoordinating phosphonate groups appears to catalyze prototropic exchange of the bound water protons, thereby providing a mechanism for enhanced water contrast below pH 7.

Journal Article↗

PubMed1999

Telomerase activity and telomere length in lymphocytes from patients with cutaneous T-cell lymphoma.

BACKGROUND: Telomeres shorten with successive cell divisions in normal somatic cells. Telomerase is a ribonucleoprotein enzyme associated with cellular proliferation and plays an important role in maintaining the stability of chromosomes and the length of DNA telomeres. Telomerase activity has been detected in tissues from many human tumors, but is not present in the majority of normal tissues. Thus, measurement of telomerase activity and telomere length may contribute to understanding the mechanism of tumorigenesis and provide useful diagnostic or prognostic information. The aim of this study was to investigate the telomerase activity and telomere length from patients with cutaneous T-cell lymphoma (CTCL). METHODS: Eighteen skin-homing T-cell lines were established from skin biopsies and 10 peripheral blood mononuclear cells (PBMC) were isolated from patients with various stages of CTCL together with 22 PBMC from healthy donors. For each sample an identical amount of cellular protein was measured quantitatively for telomerase activity using the telomerase polymerase chain reaction-enzyme-linked immunosorbent assay based on the telomeric repeat amplification protocol method. Telomere length was assayed using a commercial kit. RESULTS: Eight of ten PBMC and 16 of 18 skin-homing T-cell lines from patients with CTCL showed moderate to strong telomerase activity. Freshly obtained PBMC from healthy donors showed weak levels of telomerase activity. A shorter telomere length was found in cell lines and PBMC from patients with CTCL compared with healthy controls. Four skin-homing T-cell lines going into growth crisis showed sharply reduced telomerase activity. CONCLUSIONS: The results of the current study indicate that both skin-homing T-cells and PBMC from CTCL have high telomerase activity and short telomere length. These changes are similar to the changes observed in the majority of malignant cells including other types of T-cell lymphoma. It is interesting to note that even in the very early stages of CTCL such as parapsoriasis (which is a clinically benign disease) the changes already are present, indicating that a significantly high level of telomerase activity frequently occurs in CTCL and may be an important event in tumorigenesis. Telomerase activity and telomere length are useful markers for CTCL risk assessment.

Aged↗

PubMed1999

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study.

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193-->Ala) and H193N (His193-->Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor d-threo-1-phenyl-2- decanoylamino-3-morpholinopropan-1-ol, at concentrations which inhibited the wild-type enzyme by >80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

Amino Acid Sequence↗

PubMed1999

Brain-derived neurotrophic factor enhances association of protein tyrosine phosphatase PTP1D with the NMDA receptor subunit NR2B in the cortical postsynaptic density.

Our recent studies revealed that brain-derived neurotrophic factor (BDNF) rapidly enhances tyrosine phosphorylation and dephosphorylation of the NMDA receptor subunit, NR2B, in the postsynaptic density (PSD), potentially regulating synaptic plasticity. To explore the molecular mechanisms underlying synaptic NR2B signaling, we examined the protein tyrosine phosphatase, PTP1D; BDNF reportedly increases association of PTP1D with tyrosine phosphorylated proteins in cortical neurons and PC 12 cells. We now report that PTP1D is an intrinsic component of the rat cerebrocortical PSD, based on Western blot analysis using specific anti-PTP1D antibodies. In addition, NR2B was co-immunoprecipitated with PTP1D using anti-NR2B antibodies or anti-PTP1D antibodies, indicating physical association of the subunit with PTP1D. Moreover, treatment of the purified PSD with BDNF for 5 min elicited a two-fold increase in the association of NR2B with PTP1D. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, did not alter the association. Finally, an overlay assay revealed that BDNF caused a two-fold increase in binding of blotted PSD NR2B proteins to PTP1D-SH2 domains, revealing molecular mechanisms mediating the PTP1D-NR2B binding. Taken together, our results raise the possibility that PTP1D participates in BDNF-mediated NR2B signaling cascades at the postsynaptic site, thereby regulating synaptic plasticity.

Animals↗