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Biomedical subjects

C Huang

Publications and source records attributed to C Huang.

At least 325 records · Page 18Linked to original sources
PubMed1997

Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation.

Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

Actins↗
PubMed1997

Signal transduction through atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in immortal murine cells.

The exposure of mammalian cells to ultraviolet (u.v.) irradiation leads to activation of transcription factors, such as AP-1 and NFkappaB. It is postulated that the EGF receptor but not protein kinase C (PKC) is the major membrane mediator in UVC-induced signal transduction. We demonstrate here that the antisense oligonucleotides of PKC zeta and the dominant negative mutant of PKC lambda/iota as well as dominant negative PKC zeta markedly blocked UVC-induced AP-1 activity. In contrast, UVC-induced AP-1 activity in cells devoid of the EGF receptor (B82), is not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721), or wild-type EGF receptor (B82L). This was found at all UVC irradiation doses and time courses studied, while high levels of EGF-induced AP-1 activity were observed in B82L cells but not in B82 cells. This evidence strongly suggests that atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in JB6 and B82 cells.

Animals↗
PubMed1997

Inhibition of activator protein 1 activity and neoplastic transformation by aspirin.

Aspirin, along with its analgesic-antipyretic uses, is now also being considered for prevention of cardiovascular disease, cancer, and treatment of human immunodeficiency virus infection. Although many of aspirin's pharmacological actions are related to its ability to inhibit prostaglandin biosynthesis, some of its beneficial therapeutic effects are not completely understood. Transcription factor activator protein 1 (AP-1) is critical for the induction of neoplastic transformation and induction of multiple genes involved in inflammation and infection. We have used the JB6 mouse epidermal cell lines, a system that has been used extensively as an in vitro model for the study of tumor promotion and anti-tumor promotion, to study the anti-carcinogenesis effect of aspirin at the molecular level. Aspirin and aspirin-like salicylates inhibited the activation of AP-1 in the same dose range as seen for the inhibition of tumor promoter-induced transformation. The inhibition of AP-1 and tumor promoter-induced transformation in JB6 cells occurs through a prostaglandin independent- and an Erk1- or Erk2-independent pathway. The mechanism of AP-1 and transformation inhibition in this cell culture model may involve the elevation of H+ concentration. The inhibition effects on the activation of AP-1 activity by aspirin and aspirin-like salicylates may further explain the anti-carcinogenesis mechanism of action of these drugs.

Animals↗
PubMed1997

Folding of the conserved domain but not of flanking regions in the integrin beta2 subunit requires association with the alpha subunit.

We have used immunoprecipitation with mAbs to probe folding during biosynthesis of the beta2 integrin subunit of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) before and after association with the alphaL subunit. An evolutionarily conserved region is present in the beta2 subunit between amino acid residues 102 and 344. mAbs to one subregion before the conserved region, and two subregions after the conserved domain, immunoprecipitated both the unassociated beta'2 precursor and mature alphaL/beta2 complex, suggesting portions of these subregions are folded before association with alphaL. An activating mAb to the C-terminal cysteine-rich region, KIM127, preferentially bound to the unassociated beta subunit, suggesting that it may bind to an epitope that is in an alphabeta interface in unactivated LFA-1. By contrast, mAbs to five different epitopes in the conserved region did not react with unassociated beta'2 precursor, suggesting that this region folds after alphaL association and is intimately associated with the alphaL subunit in the alphaL/beta2 complex. mAbs to two different epitopes that involve the border between the conserved region and the C-terminal segment, were fully or partially reactive with the beta'2 precursor, suggesting that this region is partially folded before association with alphaL. The findings suggest that the conserved region is a distinct folding and hence structural unit, and is intimately associated with the alpha subunit.

Amino Acid Sequence↗
PubMed1997

Folding of the beta-propeller domain of the integrin alphaL subunit is independent of the I domain and dependent on the beta2 subunit.

We have studied the folding during biosynthesis of the lymphocyte function-associated antigen 1 (LFA-1) alphaL subunit using mAb to epitopes that map to seven different regions within the amino acid sequence. The N-terminal portion of alphaL is predicted to contain a beta-propeller domain, consisting of seven beta-sheets, and an I domain that is predicted to be inserted between beta-sheet 2 and beta-sheet 3 of the beta-propeller. The I domain of alphaL folds before association with the beta2 subunit, as shown by immunoprecipitation of the unassociated alphaL subunit by mAbs specific for four different sequence elements within the I domain. By contrast, the beta-propeller domain is not folded in unassociated alphaL after a chase of as long as 12 h after synthesis, but does fold upon association with beta2. This is shown with mAbs to regions of alphaL, that precede and follow the I domain in the primary structure. A mAb that maps near the junction of the C terminus of the I domain with the beta-propeller domain suggests that this region is partially folded before subunit association. The results show that the I domain and beta-propeller domains fold independently of one another, and suggest that the beta-propeller domain bears an interface for association with the beta subunit.

Antibodies, Monoclonal↗
PubMed1997

Phosphatidylinositol-3 kinase is necessary for 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and activated protein 1 activation.

Phorbol esters, which activate isoforms of protein kinase C, are general activators of the transcription factor activated protein 1 (AP-1). The pathway involved in this signal transduction is not very clear. Currently, little is known about whether phosphatidylinositol-3 (PI-3) kinase plays any role in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced signal transduction. We demonstrate here that TPA not only has markedly synergistic effects on insulin-induced PI-3 kinase activity, but it also can induce PI-3 kinase activity and the PI-3 phosphates by itself. We also found that insulin, a PI-3 kinase activator, enhanced TPA-induced AP-1 trans-activation and transformation in JB6 promotion-sensitive cells. Furthermore, wortmannin and LY294002, two PI-3 kinase inhibitors, markedly decreased AP-1 activity induced by insulin, TPA, or TPA and insulin and inhibited JB6 promotion-sensitive cell transformation induced by TPA or TPA and insulin. Most importantly, constitutive overexpression of the dominant negative PI-3 kinase P85 mutants completely blocked insulin- or TPA-induced AP-1 trans-activation and TPA-induced cell transformation. All evidence from present studies suggests that PI-3 kinase acts as a mediator in TPA-induced AP-1 activation and transformation in JB6 cells.

Androstadienes↗
PubMed1997

Polymer Dose Effects on Filtration Followed by Expression of Clay Slurries

Filtration followed by expression characteristics of cationic polymer conditioned clay slurries are reported for the first time in this work. As the polymer dosage increases, the resistance to filtration greatly decreases until a specific dose has been reached, where the zeta potential is close to zero. And further increase in polymer dose conversely raises the resistance. The expression data are interpreted by the Terzaghi-Voigt combined model, from which the model parameters are evaluated. The polymer conditioning has only a mild effect on the primary consolidation process but has, however, a considerable influence on secondary consolidation characteristics. When zeta potential changes from negative to positive, both the easiness of creeping of constituent particles within the sludge cake and the fraction of moisture removal by the secondary consolidation attain a maximum. The optimal polymer dose criterion considering the filtration and expression stages separately, or in combination, is

Journal Article↗
PubMed1997

Upregulation of cortactin expression during the maturation of megakaryocytes.

Cortactin is a potent filamentous actin-binding protein acting as a prominent substrate of Src tyrosine kinases. We have evaluated cortactin expression in a series of murine tissues and shown an abundant expression of cortactin in megakaryocytes and platelets. Cortactin, but not its related protein HS1, is upregulated during the phorbol 12-myristate 13-acetate (PMA)-mediated maturation of a human megakaryoblastic cell line CMK. Although the expression of Src-related kinases is also upregulated more rapidly than cortactin in PMA-treated CMK cells, tyrosine phosphorylation of cortactin appears to be only transiently elevated 4 days after PMA stimulation. In addition, cortactin expression is induced by thrombopoietin and interleukin-3 in megakaryocytes derived from murine bone marrow cells. Thus, cortactin represents a megakaryocyte-specific gene in bone marrow and the interaction of Src kinases with cortactin may be involved in the maturation of megakaryocytes.

Animals↗
PubMed1997

Influence of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the gel-to-liquid crystalline phase transition.

We have semisynthesized 19 species of mixed-chain phosphatidylethanolamines (PEs) in which the sn-1 acyl chain is derived from saturated fatty acids with varying chain lengths and the sn-2 acyl chain has different chain lengths but contains 0, 1, and 2 cis double bond(s). The gel-to-liquid crystalline phase transition temperatures (Tm) of lipid bilayers prepared from these 19 mixed-chain PEs were determined calorimetrically. When the Tm values are compared with those of saturated and monounsaturated counterparts, a common Tm profile is observed in the plot of Tm versus the number of cis double bonds. Specifically, a marked stepwise decrease in Tm is detected as the number of cis double bonds in the sn-2 acyl chain of the mixed-chain PE is successively increased from 0 to 1 and then to 2. The large Tm-lowering effect of the acyl chain unsaturation can be attributed to the increase in Gibbs free energy of the gel-state bilayer as a result of weaker lateral chain-chain interactions. In addition, we have applied molecular mechanics calculations to simulate the molecular structure of dienoic mixed-chain C(X):C(Y:2 delta n,n+3)PE in the gel-state bilayer, thus enabling the three independent structural parameters (N, delta C, and LS) to be calculated in terms of X, Y, and n, which are intrinsic quantities of C(X):C(Y:2 delta n,n+3)PE. When the Tm values and the corresponding N and delta C values of all dienoic mixed-chain PEs under study are first codified and then analyzed statistically by multiple regressions, the dependence of Tm on the structural parameters can be described quantitatively by a simple and general equation. The physical meaning and the usefulness of this simple and general equation are explained.

Calorimetry, Differential Scanning↗
PubMed1997

Probing the ethanol-induced chain interdigitations in gel-state bilayers of mixed-chain phosphatidylcholines.

Using high-resolution differential scanning calorimetry (DSC), we have studied the effects of ethanol concentrations, [EtOH], on the main phase transition temperatures (T[m]) of the following mixed-chain phosphatidylcholines (PCs): C(15):C(17)PC, C(17):C(15)PC, and C(12):C(20)PC. These lipids have a common molecular weight; however, their apparent acyl chain-length differences between the sn-1 and sn-2 acyl chains, delta C, are distinctively different. The delta C values for these three mixed-chain PCs are, respectively, 0.5, 3.5, and 6.5 C-C bond lengths. DSC results show that the T(m) profiles for C(15):C(17)PC and C(17):C(15)PC bilayers in the plot of T(m) versus [EtOH] are V-shaped biphasic curves, with the minimum T(m) occurring at 50 and 73 mg/ml of ethanol, respectively. In contrast, the C(12):C(20)PC bilayer exhibits a nearly linear decrease in T(m) with increasing [EtOH]. In addition, x-ray diffraction experiments were also performed to assess the structural changes of these three mixed-chain PCs in the gel-state bilayers, at 20 degrees C, in response to high concentrations of ethanol. X-ray diffraction data indicate that, in the absence of ethanol, these three lamellar lipids are all packed in the normal (L beta') gel phase in aqueous media. In the presence of 120 mg/ml of ethanol, however, the C(15):C(17)PC and C(17):C(15)PC lamellae are packed in the fully interdigitated (L beta[I]) gel phase. The V-shaped T(m) curves detected calorimetrically for these two lipids in response to [EtOH] can thus be explained by the ethanol-induced L beta' --> L beta[I] isothermal phase transition. Interestingly, the results of x-ray diffraction study reveal, for the first time, that an ethanol-induced L beta' --> L(MI) (mixed interdigitated phase) isothermal phase transition occurs in the gel-state bilayer of highly asymmetrical C(12):C(20)PC. Therefore, the chain asymmetry is recognized to play an important role in the ethanol-induced chain interdigitation at T < T(m).

Biophysical Phenomena↗
PubMed1997

Characterization of cartilage metabolic response to static and dynamic stress using a mechanical explant test system.

A new mechanical explant test system was used to study the metabolic response (via proteoglycan biosynthesis) of mature, weight-bearing canine articular cartilage subjected to static and dynamic compressive stresses. Stresses ranging from 0.5 to 24 MPa were applied sinusoidally at 1 Hz for intervals of 2-24 h. The explants were loaded in unconfined compression and compared to age-matched unloaded explants. Both static and dynamic compressive stress significantly decreased proteoglycan biosynthesis (range 25-85%) for all loading time intervals. The inhibition was proportional to the applied stress but was independent of loading time. After rehydration upon load removal, the measured water content of the loaded explants was not different from the unloaded explants for all test variables. Autoradiographic and electron microscopic analysis of loaded explants showed viable chondrocytes throughout the matrix. Our results suggest that the decreased metabolic response of cyclically loaded explants may be dominated by the static component (RMS) of the dynamic load. Furthermore, the observed decreased metabolism may be more representative of the in situ tissue response than that of unloaded explants, in which we found an increasing rate of metabolism for up to 6 days after explant removal.

Animals↗
PubMed1997

Immunolocalization of the pseudorabies virus immediate-early protein IE180 by immunoperoxidase staining.

The immediate-early (1E) gene of pseudorabies virus (PRV) expresses immediately upon infection, a phosphorylated protein (immediate-early protein, IE180) that can transactivate viral other genes and plays an essential role in regulating viral gene expression. In order to detect and localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate specific antibodies against IE180 protein. Two recombinant expression plasmids pN and pNB were constructed by cloning the IE gene onto pET 30a(+) expression vector via NcoI and BamHI sites. Plasmid pN contains the 1.8-kb NcoI-NcoI fragment of IE gene coding for the N-terminus of 616 amino acid residues, while pNB contains the 2.8-kb NcoI-Bam HI fragment coding for the rest of the IE180 protein. Both pN and pNB were transformed, respectively, into E. coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 protein. The specificities of the mice immune sera were confirmed by their abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay. Furthermore, immunoperoxidase staining of PRV infected cells undertaken with these antisera revealed the subcellular distribution of the IE proteins in the infected cells and also demonstrated their transportation from the cytoplasm to the nucleus during infection.

Animals↗
PubMed1997

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes.

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Aedes↗
PubMed1997

Factors associated with joint pain among postmenopausal women.

OBJECTIVES: To investigate potential factors associated with joint pain among postmenopausal women. DESIGN: Population-based cross-sectional study. SUBJECTS: Six hundred and ninety postmenopausal Japanese-American women from the Hawaii Osteoporosis Study (age: 55-93 y). MEASUREMENTS: Data for this study were collected at the 1992-1994 examination of the Hawaii Osteoporosis Study, except that data on spinal osteoarthritis were obtained based on radiographs at examinations before 1987. Information on painful joints at a variety of skeletal sites, smoking, and physical activity was collected by questionnaire. Bone density was measured at the lumbar spine, distal and proximal radius, and heel. Quantitative bone ultrasound was also measured at the heel. Prevalent vertebral fractures were identified on lateral spine radiographs using 3 s.d. below the normal population mean as the cutoff. Non-spine fractures were identified based on self-report, and were verified using medical records. RESULTS AND CONCLUSIONS: In this cross-sectional analysis, bone density, quantitative bone ultrasound, prevalent vertebral fractures, and non-spine fracture were not significantly associated with joint pain. Greater body weight or body mass index were significantly and positively associated with joint pain at most weight-bearing joints. The results suggest that a substantial proportion of joint pain at these sites could be prevented by avoidance of excess weight, provided the association is causative. Longitudinal studies are needed to confirm the observed cross-sectional associations.

Aged↗
PubMed1997

Organization of G proteins and adenylyl cyclase at the plasma membrane.

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein alpha and beta subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein alpha subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Adenylyl Cyclases↗