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Biomedical subjects

W Sun

Publications and source records attributed to W Sun.

At least 73 records · Page 4Linked to original sources
PubMed2000

MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway.

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2000

MEKK2 gene disruption causes loss of cytokine production in response to IgE and c-Kit ligand stimulation of ES cell-derived mast cells.

Ligation of the high-affinity IgE receptor (FcepsilonRI) or of c-Kit stimulates cytokine production in mast cells. We show that MEK kinase 2 (MEKK2), a MAPK kinase kinase (MAP3K) that regulates the JNK and ERK5 pathways, is required for cytokine production in embryonic stem (ES) cell-derived mast cells (ESMC). Targeted disruption of the MEKK2 or MEKK1 gene was used to abolish expression of the respective kinases in ESMC. Transcription of specific cytokines in response to IgE or c-Kit ligand was markedly reduced in MEKK2(-/-) ESMC relative to wild-type ESMC. Cytokine production in MEKK1(-/-) ESMC was similar to that of wild-type ESMC, demonstrating the specificity of MEKK2 in signaling cytokine gene regulation. MEKK2(-/-) ESMC also lost receptor-mediated stimulation of JNK. In contrast, JNK activation in response to UV irradiation was normal, showing that MEKK2 is required for receptor signaling but not for cellular stress responses. MEKK2 is the first MAP3K shown to be required for mast cell tyrosine kinase receptor signaling controlling cytokine gene expression.

Animals↗
PubMed2000

Restoration of positioning control following Disabled-2 expression in ovarian and breast tumor cells.

The physical interaction of epithelial cells with the basement membrane ensures correct positioning and acts as a survival factor for epithelial cells. Cells that detach from the basement membrane often undergo apoptosis; however, in carcinomas, this positional control is absent, permitting disorganized cell proliferation. In the majority of breast and ovarian carcinomas (85-90%), the expression of a candidate tumor suppressor, Disabled-2 (Dab2), is frequently lost. The Dab2-negative tumor cells are no longer in contact with an intact basement membrane, as indicated by the absence of collagen IV (in about 90% of cases). However, in the subset (10-15%) of ovarian tumors in which Dab2 expression is positive, the presence of a basement membrane-like structure around tumor cells was observed. Recombinant adenovirus-mediated expression of Dab2 was used in Dab2-negative ovarian and breast cancer cells, and re-expression of Dab2 was found to lead to cell death or growth arrest. Dab2 expression suppressed MAPK activation and c-fos expression. Plating the infected cells on a basement membrane matrigel rescued the cells from death and growth arrest. Thus, Dab2 exhibits a negative activity for cell growth and survival, which can be countered by attachment of the cells to basement membrane matrix. We conclude that Dab2 functions in cell positioning control and mediates the exigency for basement membrane attachment of epithelial cells. Loss of Dab2 may contribute to the basement membrane-independent, disorganized proliferation of tumor cells in ovarian and breast carcinomas.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Survival of bifidobacteria in yogurt and simulated gastric juice following immobilization in gellan-xanthan beads.

A novel acid-stable bead made of gellan gum and xanthan gum was used to immobilize Bifidobacteria. The beads (0.75% gellan and 1% xanthan gum) had an average diameter of 3 mm and did not shrink in 25% lactic acid solution, pH 1.5, or 20% acetic acid solution, pH 1.5, after storage at 4 degrees C for 4 weeks. Bifidobacterium infantis ATCC 15697, the most acid-tolerant strain tested, was immobilized in gellan-xanthan beads and its survival in peptone water, pH 4, pasteurized yogurt, and simulated gastric juice was monitored. In peptone water, pH 4, the reduction in cell count of immobilized cells of B. infantis ATCC 15697 was not significantly different from that obtained with free cells during 6 weeks of storage at 4 degrees C. However, counts of immobilized cells of B. infantis ATCC 15697 remained significantly higher than free cells (P < 0.0001) when both were exposed to simulated gastric juices at pH 2.5, 2.0 and 1.5. At pH 2.5, the viable count of free cells dropped from 1.23 x 10(9) CFU/ml to an undetectable level (< 10 CFU/ml) in 30 min, while the viable count of immobilized cells decreased by only 0.67 log cycle in the same time period. Immobilized cells also survived significantly better than free cells (P <0.05) in pasteurized yogurt after refrigerated storage for 5 weeks.

Bifidobacterium↗
PubMed2000

Trigemino-autonomic connections in the muskrat: the neural substrate for the diving response.

Stimulation of the anterior ethmoidal nerve of the muskrat produces a cardiorespiratory depression similar to the diving response. This includes an apnea, a parasympathetic bradycardia, and a selective increase in sympathetic vascular tone. However, the brainstem circuitry that links the afferent stimulus to the efferent autonomic responses is unknown. We used the anterograde transneuronal transport of the herpes simplex virus (HSV-1), strain 129, after its injection into the anterior ethmoidal nerve to determine the primary, secondary, and tertiary brainstem relays responsible for this cardiorespiratory response. In an effort to check the validity of this relatively untested tracer, we also injected the medullary dorsal horn with biotinylated dextran amine to determine the secondary trigemino-autonomic projections. Approximately 1 microl (6x10(6) PFU) of the HSV-1 virus was injected directly into the anterior ethmoidal nerve of muskrats. After 2-6 days, their trigeminal ganglions, spinal cords and brainstems were cut and immunohistologically processed for HSV-1. Initially (2 days), HSV-1 was observed only in the trigeminal ganglion. After approximately 3 days, HSV-1 was observed first in many brainstem areas optimally labeled between 4 and 4.5 days. In these cases, the ventrolateral superficial medullary dorsal horn, the ventral paratrigeminal nucleus and the interface between the interpolar and caudal subnuclei were labeled ipsilaterally. The nucleus tractus solitarius (NTS), especially its ventrolateral, dorsolateral, and commissural subnuclei were labeled as well as the caudal, intermediate and rostral ventrolateral medulla. Within the pons, the superior salivatory nucleus, the A5 area, the ventrolateral part of the parabrachial nucleus and the Kölliker-Fuse nucleus were labeled. Only after a survival of 4 days or more, the locus coeruleus, the nucleus raphe magnus, the nucleus paragigantocellularis, pars alpha, and the pontine raphe nucleus were labeled. Injections of biotinylated dextran amine were made into the medullary dorsal horn (MDH) in a location similar to that labeled after the viral injections. Fine fibers and terminals were labeled in the same brainstem areas labeled after injections of HSV-1 into the anterior ethmoidal nerve. This study outlines the potential brainstem circuit for the diving response, the most powerful autonomic reflex known. It also confirms the efficacy for using HSV-1, strain 129, as an anterograde transneuronal transport method.

Animals↗
PubMed2000

MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase.

MEK kinase 2 (MEKK2) is a 70-kDa protein serine/threonine kinase that has been shown to function as a mitogen-activated protein kinase (MAPK) kinase kinase. MEKK2 has its kinase domain in the COOH-terminal moiety of the protein. The NH(2)-terminal moiety of MEKK2 has no signature motif that would suggest a defined regulatory function. Yeast two-hybrid screening was performed to identify proteins that bind MEKK2. Protein kinase C-related kinase 2 (PRK2) was found to bind MEKK2; PRK2 has been previously shown to bind RhoA and the Src homology 3 domain of Nck. PRK2 did not bind MEKK3, which is closely related to MEKK2. The MEKK2 binding site maps to amino acids 637-660 in PRK2, which is distinct from the binding sites for RhoA and Nck. This sequence is divergent in the closely related kinase PRK1, which did not bind MEKK2. In cells, MEKK2 and PRK2 are co-immunoprecipitated and PRK2 is activated by MEKK2. Similarly, purified recombinant MEKK2 activated PRK2 in vitro. MEKK2 activation of PRK2 is independent of MEKK2 regulation of the c-Jun NH(2)-terminal kinase pathway. MEKK2 activation of PRK2 results in a bifurcation of signaling for the dual control of MAPK pathways and PRK2 regulated responses.

Amino Acid Sequence↗
PubMed2000

PAL31, a novel nuclear protein, expressed in the developing brain.

We cloned a cDNA encoding a novel protein (PAL31) predominantly expressed in the fetal rat brain by differential display. PAL31 contains leucine-rich repeat domains, a highly acidic region and a putative nuclear localization signal. PAL31 has 50-70% similarity to SSP29, APRIL, LANP, PHAP I, and PP32. Expression of PAL31 mRNA in the brain was high during the fetal period and decreased after birth. Immunohistochemical studies showed that PAL31 is expressed in the entire embryonic brain, whereas in the adult brain its expression is restricted to the subventricular zone where there are neural progenitor cells. It was also revealed that PAL31 is colocalized with PCNA in the nucleus, indicating that the PAL31 expression is developmentally regulated. Considering the primary structure of PAL31 and its spatiotemporal expression pattern, PAL31 is a novel nuclear protein related to the development of the brain through the proliferation of neuronal cells.

Amino Acid Motifs↗
PubMed2000

Protective immunity induced by the full-length cDNA encoding paramyosin of Chinese Schistosoma japonicum.

The full-length cDNA encoding paramyosin of Chinese S. japonicum (Sjc97) has been cloned and sequenced for the first time. The homology of the nucleotide sequence of paramyosin and the deduced amino acid sequence between Chinese, Philippine, Japanese strains and S. mansoni (Sm97) were 99.7, 99.8 and 96% at amino acid level, respectively; 99.4, 99.2 and 91% at nucleotide level, respectively. The immunogenicity and protective efficacy of a DNA vaccine encoding Sjc97 was evaluated in C57BL/6 mice. Mice immunized intramuscularly with pCMV-Sjc97 resulted in predominantly IgG2a and IgG2b immune responses, and immune sera were able to mediate antibody-dependent macrophage mediated cytotoxicity in vitro. Cytokine profiles in immunized C57BL/6 mice demonstrated Th1 bias, with IFN-gamma and IL-2 production and lack of IL-4 and IL-5. Immunization with pCMV-Sjc97 conferred a significant level of protection against cercariae in C57BL/6 mice. These results demonstrate that the nucleic acid encoding Sjc97 was able to induce a Th1 type immune response and confer protective efficacy in C57BL/6 mice when administrated via the intramuscular route.

Animals↗
PubMed2000

Vasoactive intestinal peptide (VIP) inhibits TGF-beta1 production in murine macrophages.

Vasoactive intestinal peptide (VIP) and the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), produced and/or released in the lymphoid microenvironment act primarily as macrophage- and T cell-deactivating agents. In the present study we investigate the effect of VIP and PACAP on the production of TGF-beta1 in the macrophage cell line Raw 264.7 and in peritoneal macrophages. The two neuropeptides do not affect the baseline TGF-beta1 production by unstimulated macrophages, but reduce dramatically TGF-beta1 production by LPS-stimulated macrophages. The effects are mediated through the specific receptors VPAC1, VPAC2, and PAC1. The effect of VIP is mediated primarily through the cAMP pathway, whereas PACAP activates both the cAMP and the protein kinase C pathway. VIP reduces the TGF-beta1 steady-state mRNA levels in both peritoneal macrophages and Raw 264.7 cells treated with LPS. A similar effect is observed upon the in vivo administration of VIP. This report adds VIP and PACAP to the only other neuropeptide, substance P, known to regulate TGF-beta1 production in immune cells.

Animals↗
PubMed2000

Chronic effects of therapeutic irradiation for localized prostatic carcinoma on anorectal function.

PURPOSE: To evaluate prospectively the prevalence and pathophysiology of anorectal dysfunction following radiation therapy (RTH) for localized carcinoma of the prostate. METHODS AND MATERIALS: The following parameters of anorectal function were evaluated in each of 35 patients (aged 55-82 years) with localized prostatic carcinoma treated with RTH either to a dose of 55 Gy/20 fractions/4 weeks (18 patients) or 64 Gy/32 fractions/6.5 weeks (17 patients), before RTH and 4-6 weeks and at a mean (+/- SD) of 1.4 (+/- 0.2) years after its completion: (1) anorectal symptoms (questionnaire), (2) anorectal pressures at rest and in response to voluntary squeeze and increases in intra-abdominal pressure (multiport anorectal manometry), (3) rectal sensation (balloon distension) and (4) anal sphincteric morphology (endoanal ultrasound). RESULTS: All but 1 patient completed three series of measurements. RTH had no effect on anal sphincteric morphology. The increase in frequency of defecation and fecal urgency and incontinence scores previously reported in the patients 4-6 weeks after RTH were sustained 1 year later (p < 0.001, p < 0.001, and p < 0.05, cf. baseline, respectively). At this time, 56% (19 of 34), 50% (17 of 34) and 26% (9 of 34) of the patients had increased frequency of defecation, fecal urgency, and incontinence, respectively. Decreases in anal sphincteric pressures at rest and in response to voluntary squeeze recorded in the patients 4-6 weeks after RTH were not sustained 1 year later but the volumes of rectal distension associated with perception of the stimulus and desire to defecate were lower compared with baseline volumes (p < 0.01 and p < 0.05, respectively), reflecting heightened rectal sensitivity in the patients. There was no difference in measurements between the two radiation dose regimens. Univariate logistical regression analysis was performed on patients who had experienced increased symptom scores or decreases in recorded motor and sensory manometric parameters at 1 year, cf. baseline. The predictor variables used included individual patient tumor and treatment characteristics as well as individual patient symptom scores and parameters of anorectal motor and sensory function at baseline and 4-6 weeks after RTH. The results of the univariate logistical regression analysis showed that (1) frequency of defecation at 4-6 weeks and (2) rectal volumes at baseline both for (a) perception (p < 0.001) and (b) desire to defecate (p < 0.001), predicted significantly for the patients who had symptoms and signs of anorectal dysfunction at 1 year. Individual patient tumor and treatment-related variables tested, in contrast, had no predictive significance. CONCLUSIONS: Anorectal symptoms following RTH for prostatic carcinoma are common and persist at least until 1 year after its completion and are associated with objective evidence of heightened rectal sensitivity.

Aged↗
PubMed2000

Regulation of biodegradability and drug release behavior of aliphatic polyesters by blending.

Polyester blending of poly(epsilon-caprolactone) (PCL) with poly(D, L-lactide) (PLA) and their random copolymers (R(CL/LA)) was found to be a convenient approach to regulate the degradation and drug release behaviors of the polyesters. The blend composition and compatibility both affected its degradation and drug release behavior. A DSC study showed that PCL was compatible with 50:50 poly(CL-CO-D,L-LA) (R(50/50)) but incompatible with 25:75 poly(CL-CO-LA) (R(25/75)) and PLA homopolymer. The hydrolysis experiments indicated that with the same CL/LA segment proportion, compatible blends (PCL/R(50/50)) had higher water content and faster weight loss than incompatible blends (PCL/PLA, PCL/R(25/75)). In the compatible blends the PCL degradation rate was increased while that of R(50/50) was decreased. The controlled release kinetics, diffusion constants, and permeation coefficients of the polymer blends were measured by using northindrone (NTD) as a model. The NTD release rates from the polyester blends increased as the CL unit fraction increased but decreased with increasing the LA unit fraction in the blends. With the same CL/LA unit ratios, the NTD release rate from the compatible blend was slower than that from the incompatible blend. The NTD release from the polyester blend was controlled by the diffusion process in the early stage, but the degradation-caused NTD release was later involved. By tailoring the blend composition to such an extent that the degradation-caused release compensated the decline of the diffusion-caused release, a zero-order NTD release was achieved.

Biocompatible Materials↗
PubMed2000

Interaction of neuronal Cdc2-like protein kinase with microtubule-associated protein tau.

Neuronal Cdc2-like protein kinase (NCLK), a approximately 58-kDa heterodimer, was isolated from neuronal microtubules (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901). The biochemical nature of NCLK-microtubule association is not known. In this study we found that NCLK is released from microtubules upon microtubule disassembly as a 450-kDa species. The 450-kDa species is an NCLK.tau complex, and NCLK-bound tau is in a nonphosphorylated state. Tau phosphorylation causes NCLK.tau complex dissociation, and phosphorylated tau does not bind to NCLK. In vitro, the Cdk5 subunit of NCLK binds to the microtubule-binding region of tau and NCLK associates with microtubules only in the presence of tau. Our data indicate that in brain extract NCLK is complexed with tau in a tau phosphorylation-dependent manner and that tau anchors NCLK to microtubules. Recently NCLK has been suggested to be aberrantly activated and to hyperphosphorylate tau in Alzheimer's disease brain (Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P, and Tsai, L.-H. (1999) Nature 402, 615-622). Our findings may explain why in Alzheimer's disease NCLK specifically hyperphosphorylates tau, although this kinase has a number of protein substrates in the brain.

Animals↗
PubMed2000

2-amino-2-oxazolines as subtype selective alpha(2) adrenoceptor agonists.

Cyclohexylamino oxazoline 1 (AGN 190837), an analogue of 2 (Bay a6781), is a potent alpha(2) adrenoceptor agonist. On the basis of a design generated by receptor-ligand modeling, a number of cyclohexyl and norbornyl analogues were synthesized wherein the propyl group of 1 was replaced by phenylalkyl subsituents. This resulted in compound 6 being an alpha(2c) selective agonist, as well as 7 and 9 being alpha(2a)/alpha(2c) selective.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2000

Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage.

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.

Amino Acid Sequence↗
PubMed2000

Potential Energy Curves of Some Electronic Excited States of Metal Diatomic Molecules Using the Energy Consistent Method.

An analytical five-term expansion potential is suggested to improve the recently proposed energy consistent method (ECM) potential based on a three-term expansion [Weiguo Sun, Mol. Phys. 92, 105 (1997) and Weiguo Sun and Hao Feng, J. Phys. B: At., Mol. Opt. Phys. 32, 5109 (1999)]. Applications of the new ECM to the electronic excited state 3(1)Pi of NaK, the excited state 2(3)Sigma(+)(g) of Na(2), and the excited states 3(1)Sigma(+)(u) and 3(1)Pi(u) of K(2) molecules show that the new ECM potential not only agrees excellently with the known Rydberg-Klein-Rees (RKR) data or accurate configuration interaction (CI) studies but also gives the potential data at the molecular asymptote and the dissociation region where experimental and quantum mechanical studies may have difficulties. The new ECM can correct the artifact which appeared in the three-term expansion potentials of both the previous ECM and the Huxley-Murrell-Sorbie (HMS) potential and can describe some states to which both the previous ECM and the HMS fail. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Sensitive detection of a plant virus by electrochemical enzyme-linked immunoassay.

The electrochemical enzyme-linked immunoassay increases the sensitivity of the detection of cucumber mosaic virus (CMV) by 5-fold compared with the spectrophotometric o-phenylenediamine (OPD) enzyme-linked immunosorbent assay (ELISA). The detection limit for the purified CMV is 1.0 ng/mL and the highest dilution ratio of the infected leaf sap is 1:5.0 x 10(4). The method is based on coupling the oxidation reaction of o-aminophenol (OAP)-H2O2 catalyzed by HRP-IgG conjugate with the electro-reduction of the enzymatic product. The enzymatic product 2-aminophenoxazine-3-one exhibits a sensitive second order derivative linear-sweep voltammetric response at the potential of -0.65 V (vs. Ag/AgCl) in pH 8.0 Britton-Robinson (B-R) buffer solution. So it can be applied to the detection of the plant virus with highly improved sensitivity.

Cucumovirus↗
PubMed2000

Effects of AC and DC stimulation on chinchilla SOAE amplitude and frequency.

The effects of AC and DC current on spontaneous otoacoustic emissions (SOAEs) were studied in normal chinchillas and chinchillas with selective inner hair cell (IHC) loss. Electrical stimulation was delivered through an electrode on the round window or through an electrode in scala media. SOAE frequencies ranged from 4 to 11 kHz and amplitudes ranged from 13 to 51 dB SPL. AC simulation suppressed SOAE amplitude. The suppression contours had a narrowly tuned, low-threshold tip located above the frequency of the SOAE. AC suppression contours were similar to acoustic suppression contours except that the AC suppression contours lacked a high-threshold, low frequency tail. The lowest threshold of the AC suppression contour was 3.9 microA rms whereas the lowest acoustic suppression threshold was 19 dB SPL. AC stimulation, which induced an electrically evoked otoacoustic emission, interacted with the SOAE to generate distortion product otoacoustic emissions (DPOAEs) of up to 26 dB SPL at 2f(S)-f(AC) (f(S)=SOAE). DPOAE amplitude increased with AC current, but saturated at high levels. DC current steps affected both SOAE frequency and amplitude. Positive current at the round window decreased SOAE amplitude and frequency whereas negative current increased SOAE frequency, but had little effect on amplitude. The effects of AC and DC current on SOAEs in animals with IHC loss were similar to those in normal chinchillas.

Animals↗