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Biomedical subjects

H Wang

Publications and source records attributed to H Wang.

At least 289 records · Page 16Linked to original sources

High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer.

We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA binding protein. Binding was evaluated using a variety of single and double-stranded DNA molecules. Mlh1-Pms1 bound short substrates with low affinity and showed a slight preference for single-stranded DNA. In contrast, Mlh1-Pms1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative. High affinity binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow. However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting that Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be relevant to its participation in DNA mismatch repair, recombination and cellular responses to DNA damage.

Adaptor Proteins, Signal Transducing↗

Growth inhibition by keratinocyte growth factor receptor of human salivary adenocarcinoma cells through induction of differentiation and apoptosis.

We have reported that normal human salivary gland-derived epithelial cells exclusively express keratinocyte growth factor receptor (KGFR). In the process of malignant transformation of human salivary gland tumors, KGFR gene expression disappeared concomitantly with the de novo expression of the fibroblast growth factor receptor 1 (FGFR1) and FGFR4 genes. In the present study, we introduced wild-type KGFR cDNA or chimeric KGFR/FGFR1 cDNA, which encoded the extracellular domain of KGFR and the intracellular domain of FGFR1, into the HSY human salivary adenocarcinoma cell line. The KGFR tyrosine kinase suppressed the activity of FGF receptor substrate 2 (FRS2) and inhibited the growth of HSY by inducing differentiation and apoptosis in vitro and in vivo. Our results provided significant insight into the mechanism of KGFR tumor suppression and suggest that KGFR gene therapy might be a viable method of inhibiting human salivary adenocarcinoma growth.

Adaptor Proteins, Signal Transducing↗

Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT(388)-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF.

DT(388)-GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT(388)-GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT(388)-GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT(388)-GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C(6)-ceramide inhibited protein synthesis (EC(50) approximately 5 microM) and decreased viability (EC(50) approximately 6 microM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT(388)-GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.

Acute Disease↗

Differential regulation of IL-1beta and TNF-alpha RNA expression by MEK1 inhibitor after focal cerebral ischemia in mice.

Activation of the extracellular-signal-responsive kinase (ERK 1/2) by MAP kinase/ERK kinase (MEK1/2) following ischemia/reperfusion in the brain has been associated with cell death since inhibition of MEK1/2 provides neuroprotection in cerebral ischemia injury. Since inflammation has been implicated in ischemic brain injury, the present study investigated whether MEK1/2 modifies expression of two key inflammatory cytokines, IL-1beta and TNFalpha, that have been shown to exacerbate ischemic brain injury. A mouse model of transient cerebral ischemia was deployed to test the effect of selective MEK1/2 inhibitor (SL327) on infarct size and cytokine expression. SL327 (100 mg/kg, i.p.) administered 15 min prior to ischemia resulted in 64% reduction in infarct size over controls (n = 8, P < 0.01). Under the same condition, SL327 significantly reduced peak expression of IL-1beta mRNA (59% reduction compared to vehicle, P < 0.01, n = 4) but not TNF-alpha mRNA. A parallel reduction in IL-1beta protein (67%, P < 0.05, n = 6) was also observed using ELISA analysis. These data suggest that the neuroprotective effect of MEK1/2 inhibition may be mediated by suppression of IL-1beta. The study also demonstrates for the first time that these two cytokines are differentially regulated by kinase mediated signaling pathways.

Aminoacetonitrile↗

Dynamical relativistic effects in photoionization: spin-orbit-resolved angular distributions of xenon 4d photoelectrons near the Cooper minimum.

Two decades ago, it was predicted [Y. S. Kim et al., Phys. Rev. Lett. 46, 1326 (1981)] that relativistic effects should alter the dynamics of the photoionization process in the vicinity of Cooper minima. The present experimental and theoretical study of the angular distributions of Xe 4d(3/2) and 4d(5/2) photoelectrons demonstrates this effect for the first time. The results clearly imply that relativistic effects are likely to be important for intermediate- Z atoms at most energies.

Journal Article↗

Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture.

BACKGROUND: Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. METHODS: Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. RESULTS: Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P < 0.01). However, with extended co-culture, contraction of the collagen gels was greatly augmented (P < 0.01). In addition, with extended co-culture, degradation of collagen in the gels occurred. The addition of neutrophil elastase to the medium augmented both contraction and degradation (P < 0.01). Prostaglandin E2 production was significantly increased by co-culture and its presence attenuated collagen degradation. CONCLUSION: The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture.

Cell Line↗

Collaborative interactions between neutrophil elastase and metalloproteinases in extracellular matrix degradation in three-dimensional collagen gels.

BACKGROUND: Extended culture of monocytes and fibroblasts in three-dimensional collagen gels leads to degradation of the gels (see linked study in this issue, "Fibroblasts and monocytes contract and degrade three-dimensional collagen gels in extended co-culture"). The current study, therefore, was designed to evaluate production of matrix-degrading metalloproteinases by these cells in co-culture and to determine if neutrophil elastase could collaborate in the activation of these enzymes. Since co-cultures produce prostaglandin E2 (PGE2), the role of PGE2 in this process was also evaluated. METHODS: Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Matrix metalloproteinases (MMPs) were assessed by gelatin zymography (MMPs 2 and 9) and immunoblotting (MMPs 1 and 3). The role of PGE2 was explored by direct quantification, and by the addition of exogenous indomethacin and/or PGE2. RESULTS: Gelatin zymography and immunoblots revealed that MMPs 1, 2, 3 and 9 were induced by co-cultures of fibroblasts and monocytes. Neutrophil elastase added to the medium resulted in marked conversion of latent MMPs to lower molecular weight forms consistent with active MMPs, and was associated with augmentation of both contraction and degradation (P < 0.01). PGE2 appeared to decrease both MMP production and activation. CONCLUSION: The current study demonstrates that interactions between monocytes and fibroblasts can mediate tissue remodeling.

Blotting, Western↗

Regulation of prostate branching morphogenesis by activin A and follistatin.

Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.

Activin Receptors↗

Increased association of brain protein kinase C with the receptor for activated C kinase-1 (RACK1) in bipolar affective disorder.

BACKGROUND: Membrane protein kinase C (PKC) activity is increased in frontal cortex of subjects with bipolar affective disorder, and lithium was demonstrated to inhibit PKC translocation to membranes. Protein kinase C is anchored to the membrane via the receptor for activated C kinase-1 (RACK1), suggesting that interactions between these proteins may be altered in bipolar disease. METHODS: The levels of RACK1 coimmunoprecipitating with PKC isozymes were compared in homogenates of frontal cortex slices from postmortem bipolar subjects and matched control subjects. RESULTS: Receptor for activated C kinase-1 was located exclusively in membranes and, in control brains, the levels of RACK1 that coimmunoprecipitated with most PKC isozymes were increased by stimulation with the PKC activator, phorbol 12-myristate, 13-acetate (PMA). The association of RACK1 with membrane gammaPKC and zetaPKC was increased under basal conditions in bipolar relative to control brains. Stimulation with PMA increased the amount of RACK1 that coimmunoprecipitated with the alpha, beta, gamma, delta, and varepsilonPKC isozymes, but not zetaPKC, in bipolar tissues over that elicited in control tissues. CONCLUSIONS: These data suggest that the increased association of RACK1 with PKC isozymes may be responsible for the increases in membrane PKC and in its activation that were previously observed in frontal cortex of bipolar affective disorder brains.

Aged↗

The effect of ionizing radiation on intraocular lenses.

BACKGROUND: The native crystalline lens is the principal shield against ultraviolet radiation (UV), damage to the human retina. Every year in the United States, more than one million patients undergo removal of the natural lens in the course of cataract surgery (phakectomy), at which time an intraocular lens (IOL) is placed in the lens capsule. The IOL thenceforth serves as the principal barrier to ultraviolet radiation over the life of the implant, potentially for decades. The synthetic organic molecules of which IOLs are composed offer little UV protection unless ultraviolet-absorbing chromophores are incorporated into the lens material during manufacture. However, chromophores are alkenes potentially subject to radiolytic degradation. It is unknown whether ionizing radiation at clinical doses (e.g., to the brain or in the head-and-neck region) affects the UV-absorbing capacity of chromophore-bearing IOLs and consequently exposes the retina to potentially chronic UV damage. In addition, the polymers of which IOLs are composed are themselves subject to radiation damage, which theoretically might result in optical distortion in the visible light range. OBJECTIVE: To determine whether megavoltage photon ionizing radiation alters the absorption spectra of ultraviolet-shielding polymethylmethacrylate (PMMA) and organopolysiloxane (silicone) intraocular lenses (IOLs) in the UV (280 nm < or = lambda < 400 nm), visible (400 nm < or = lambda < or = 700 nm), and low-end near-infrared (700 nm < lambda < or = 830 nm) ranges. DESIGN: Prospective, nonrandomized trial of dose-paired IOL cohorts. METHODS: Fourteen IOLs, seven of PMMA (Chiron 6842B) and seven of silicone (IOLAB L141U), were paired and examined for absorption spectra in 1-nm intervals over the range lambda = 280-830 nm on a Cary 400 deuterium and quartz halogen source-lamp UV/visible spectrophotometer before and after undergoing megavoltage ionizing irradiation to doses of 2, 5, 10, 20, 40, 60, and 100 Gray, respectively. Because of artifactual aberrations inherent in analyzing convex lenses on a conventional flat-plate spectrophotometer, post-irradiation absorption spectra were subsequently reanalyzed on a Cary 300 spectrophotometer outfitted with a Labsphere Diffused Reflectance Accessory (DRA-CA-30-I) incorporating a Spectralon-coated integrating sphere. MAIN OUTCOME MEASURES: Primary: Changes in UV absorbance after irradiation. Secondary: Changes in visible and low-end near-infrared absorbance after irradiation. RESULTS: Photon ionizing radiation in the 2-Gy to 100-Gy range produced no detectable alterations in the UV (280 nm < or = lambda < 400 nm), visible (400 nm < or = lambda < or = 700 nm), or low-end near-infrared (700 nm < lambda < or = 830 nm) absorption spectra of any of the lenses irradiated. However, silicone IOLs as a group revealed peak post-irradiation UV absorption at a shorter wavelength than did PMMA IOLs, with marginally greater UV transmission at the uppermost extreme of the UV spectrum (lambda = 384.5-400 nm). CONCLUSIONS: At clinically relevant doses used in radiation therapy, megavoltage photon ionizing radiation produces no significant alterations in the absorption spectra of PMMA and silicone IOLs over the range lambda = 280- 830 nm. These findings indicate that, even at supraclinical doses, the UV-absorbing capacity of chromophore-bearing PMMA and silicone IOLs remains unimpaired. It is not clear whether the lower UV peak of silicone lenses represents a radiation effect or a peculiarity of the chromophore used in the lenses tested.

Humans↗

Strain differences in freezing behavior of PVG hooded and Sprague-Dawley rats: differential cortical expression of cholecystokinin2 receptors.

PVG hooded, but not Sprague-Dawley (SD), rats showed remarkable freezing behavior on cat exposure. Experiments using cDNA microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that cholecystokinin (CCK)2 receptors were expressed to a greater extent in the PVG hooded rats. A CCK2 antagonist, LY225910, inhibited freezing behavior in the PVG hooded rats while a CCK2 agonist, CCK-4, increased freezing behavior in the SD rats. These results strongly suggest that CCK2 receptors mediate the freezing behavior and the differential expression of these receptors underlie the strain difference in such behavior.

Animals↗

Different subtypes of alpha1-adrenoceptor modulate different K+ currents via different signaling pathways in canine ventricular myocytes.

Multiple subtypes (alpha1A, alpha1B, and alpha1D) of alpha1-adrenoreceptors (alpha1ARs) co-exist in the heart and mediate a variety of cellular functions. We studied alphaAR modulation of inward rectifier (IK1) and transient outward (Ito) K(+) currents in canine ventricular myocytes. Phenylephrine at 10 microM depressed only Ito without affecting IK1 and at 100 microM inhibited both Ito and IK1. The effect of phenylephrine on Ito was abolished by (+)niguldipine (10 nm) to inhibit alpha1AARs but not by chloroethyclonidine (10 microM) to inactivate alpha1BARs nor by BMY-7378 to antagonize alpha1DARs. In contrast, phenylephrine inhibition of IK1 was reversed only by BMY-7378 (1 nm). PDD (100 nm, phorbol ester activator of protein kinase C (PKC)) simulates and bisindolylmaleimide (50 nm, PKC inhibitor) weakens phenylephrine modulation of Ito but not IK1. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 and inhibitor peptides abolished the effects of phenylephrine on IK1. Enhancement of PKC or CaMKII activities was seen in alpha1aAR- or alpha1dAR-transfected HEK293 cells and in myocytes pretreated with 10 or 100 microM phenylephrine, respectively. Our data suggest that different subtypes of alpha1ARs selectively modulate different cardiac K(+) currents via different signal transduction mechanisms; alpha1AARs mediate Ito regulation via PKC, and alpha1DARs mediate IK1 regulation via CaMKII.

Adrenergic alpha-Agonists↗

Multiple phosphorylation sites in RGS16 differentially modulate its GAP activity.

Regulators of G-protein signaling (RGS) are GTPase-activating proteins (GAP) for activated Galpha subunits. We found that mouse RGS16, when expressed in HEK293T cells, is phosphorylated constitutively at serine 194 based on in vivo orthophosphate labeling experiments, while serine 53 is phosphorylated in a ligand-dependent manner upon stimulation by epinephrine in cells expressing the alpha2A adrenergic receptor. Phosphorylation on both sites impairs its GAP activity and subsequent attenuation on heterotrimeric G-protein-stimulated extracellular signal-regulated protein kinase activity. This is the first report of RGS functional downregulation by phosphorylation via a G-protein-coupled receptor.

Cell Line↗

Direct interaction of Arabidopsis cryptochromes with COP1 in light control development.

Arabidopsis seedling photomorphogenesis involves two antagonistically acting components, COP1 and HY5. COP1 specifically targets HY5 for degradation via the 26S proteasome in the dark through their direct physical interaction. Little is known regarding how light signals perceived by photoreceptors are transduced to regulate COP1. Arabidopsis has two related cryptochromes (cry1 and cry2) mediating various blue/ultraviolet-A light responses. Here we show that both photoactivated cryptochromes repress COP1 activity through a direct protein-protein contact and that this direct regulation is primarily responsible for the cryptochrome-mediated blue light regulation of seedling photomorphogenic development and genome expression profile.

Arabidopsis↗

Humanization and characterization of the anti-HLA-DR antibody 1D10.

1D10 is a previously described antibody that binds to cells from a majority of B-cell malignancies. The current studies were designed to further evaluate the antigen specificity of 1D10 and its potential as an immunotherapeutic agent. Studies with transfectants and immunoprecipitation demonstrated that 1D10 recognizes some, but not all, of the human HLA-DR beta chains. Both normal and malignant B cells can express the 1D10 antigen. A humanized version of 1D10 was produced using CDR grafting. The resulting antibody has an affinity that is similar to that of the parental murine antibody. In addition, the humanized antibody is capable of inducing complement-mediated cytotoxicity, antibody-dependent cell cytotoxicity, and direct apoptosis of 1D10-expressing B cells. Based on these in vitro anti-tumor activities, we conclude humanized 1D10 deserves further evaluation as an immunotherapeutic agent.

Amino Acid Sequence↗

Host marrow stem cell potential and engraftability at varying times after low-dose whole-body irradiation.

High levels of chimerism in syngeneic BALB/c transplants were reported when hosts were exposed to 1 Gy (100 cGy) whole body irradiation (WBI) and infused with 40 x 10(6) marrow cells. The recovery of host stem cells and alterations of enhanced host engraftability at varying times after 1 Gy WBI have now been evaluated in this study. Male BALB/c marrow (40 x 10(6) cells) was infused into female BALB/c hosts immediately or at 6, 12, and 24 weeks after 1 Gy WBI of host female BALB/c mice; engraftment percentages 8 weeks after cell injection at week 0, 6, 12, or 24 were 68% +/- 12%, 45% +/- 15%, 51% +/- 12%, or 20% +/- 8%, respectively. Eight-week engraftment levels in nonirradiated hosts average 7.7%. Conversely, engraftable stem cells measured at 8 weeks postengraftment in 1 Gy--exposed hosts were reduced to 8.6% +/- 3% of nonirradiated mice at time 0, 35% +/- 12% 6 weeks later, 49% +/- 10% at 3 months, and 21% +/- 7% at 6 months. Engraftment was still increased and stem cell decreased 1 year after 1 Gy. Furthermore, the primary cells transplanted into 1 Gy hosts can be serially transplanted, and the predominant effect of 1 Gy is directly on engrafting stem cells and not through accessory cells. These data show that transplantation in 1 Gy mice may be delayed until recovery of hematopoiesis, suggesting strategies in allogeneic transplantation to avoid the adverse effects of cytokine storm. The incomplete recovery of engraftable stem cells out to 12 months indicates that stem cell expansion, especially in patients previously treated with radiomimetic drugs, may not be feasible. (Blood. 2001;98:1246-1251)

Animals↗

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice.

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN(+/-) mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.

Alleles↗

Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.

Carcinoma, Embryonal↗