Biomedical subjects

Shaohua Yang

Publications and source records attributed to Shaohua Yang.

12 recordsLinked to original sources

PubMed2006

Neuroprotective effects of 17beta-estradiol and nonfeminizing estrogens against H2O2 toxicity in human neuroblastoma SK-N-SH cells.

Neuroprotective effects of estrogens have been shown in various in vitro and in vivo models, but the mechanisms underlying protection by estrogen are not clear. Mounting evidence suggests antioxidant effects contribute to the neuroprotective effects of estrogens. In the present study, we assessed the protective effects of estrogens against H2O2-induced toxicity in human neuroblastoma cells and the potential mechanisms involved in this protection. We demonstrate that 17beta-estradiol (17beta-E2) increases cell survival against H2O2 toxicity in human neuroblastoma cells. 17beta-E2 effectively reduced lipid peroxidation induced by 5-min H2O2 exposure. Furthermore, 17beta-E2 exerts the protective effects by maintaining intracellular Ca2+ homeostasis, attenuating ATP depletion, ablating mitochondrial calcium overloading, and preserving mitochondrial membrane potential. Two nonfeminizing estrogens, 17alpha- and ent-estradiol, were as effective as 17beta-E2 in increasing cell survival, alleviating lipid peroxidation, preserving mitochondrial function, and maintaining intracellular glutathione levels and Ca2+ homeostasis against H2O2 insult. Moreover, the estrogen receptor antagonist fulvestrant (ICI 182,780) did not block effects of 17beta-E2, but increased cell survival and blunted intracellular Ca2+ increases. However, these estrogens failed to reduce cytosolic reactive oxygen species, even at concentrations as high as 10 microM. In conclusion, estrogens exert protective effects against oxidative stress by inhibiting lipid peroxidation and subsequently preserving Ca2+ homeostasis, mitochondrial membrane potential, and ATP levels.

Calcium↗

PubMed2005

Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats.

Recent evidence indicates that cell-cycle regulating proteins are involved in apoptotic process in post-mitotic neurons. In this study, we examined cell-cycle regulators for G1/S cell-cycle progression after a transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. In the cerebral frontoparietal cortex, we observed a marked induction of Cyclin D1 (a coactivator of Cdks), and proliferating cell nuclear antigen (PCNA), together with upregulated Cdk kinase activities. This process is accompanied with multiple phosphorylation of retinoblastoma (Rb) protein at Cdk phosphorylation sites in neurons from the ischemic cortex. We further examined DNA synthesis by the incorporation of BrdU, a nucleotide analog that incorporates into newly synthesized DNA. Within 24-h of reperfusion after 60-min occlusion, substantial BrdU-positive neurons were observed in the ischemic cortex. Inhibition of Cdk4 activity during this ischemia/reperfusion is highly neuroprotective. These results suggest that ischemia/reperfusion cerebral damage induces signalings at the G1/S cell-cycle transition, and may constitute a critical step in the neuronal apoptotic pathway in ischemia/reperfusion induced neuronal damage.

Animals↗

PubMed2005

Structure-activity study of the interaction of bioreductive benzoquinone alkylating agents with DNA topoisomerase II.

PURPOSE: Quantitative structure-activity studies were performed on a series of benzoquinone mustard (BM) bifunctional alkylating agents to determine whether DNA topoisomerase II (topo II) inhibition was responsible for cell growth inhibition. METHODS: Topo II inhibition was evaluated by decatenation and agarose gel electrophoresis assays. RESULTS: The BM compounds were shown to potently inhibit the decatenation activity of topo II. Though BM compounds promoted the formation of protein-DNA complexes in isolated nuclei and cells, this effect was undiminished when levels of topo II varied. The BM compounds had little activity in a topo II-mediated DNA cleavage assay, suggesting that they do not function as topo II poisons. Rather, BM-induced protein-DNA complex formation was likely due to the bifunctional alkylating reactivity of these compounds. Finally, the growth inhibitory properties of these compounds did not correlate with their ability to inhibit topo II, indicating that these compounds did not exert their cellular activity through inhibition of topo II. Some BM compounds reacted very quickly with glutathione and cysteine, likely initially through an electrophilic Michael addition. In the absence of cysteine, the growth inhibitory effects of BM were increased tenfold, indicating the modulatory effect of cysteine sulfhydryl adducts. EPR studies showed that a semiquinone-free radical was produced by some BM compounds. CONCLUSIONS: BM compounds likely exert their action through DNA cross-linking and/or by inducing oxidative stress. Although topo II is not a direct target of these agents, this enzyme may play a role in processing the consequences of direct DNA adduction and/or oxidative DNA damage.

Alkylating Agents↗

PubMed2005

Role of nonfeminizing estrogens in brain protection from cerebral ischemia: an animal model of Alzheimer's disease neuropathology.

Estrogens are potent and efficacious neuroprotectants both in vitro and in vivo in a variety of models of neurotoxicity. We determined the structural requirements for neuroprotection in in vitro assays using a library of more than 70 novel estratrienes, synthesized to reduce or eliminate estrogen receptor (ER) binding. We observed that neuroprotection could be enhanced by as much as 200-fold through modifications that positioned a large, bulky group at the C2 and/or C4 position of the phenolic A ring of the estratriene. Further, substitutions on the B, C, or D rings either reduced or did not markedly change neuroprotection. For this library of compounds, there was a negative correlation between ER binding and neuroprotection, as the more potent compounds showed weaker or no ER binding. In an in vivo model for neuroprotection, transient cerebral ischemia, efficacious compounds were active in protection of brain tissue from this pro-oxidant insult. Finally, estradiol protected brains from insult-induced Alzheimer's disease (AD) neuropathology, including activation of apoptosis, stimulation of Abeta production, hyperphosphorylation of tau, activation of cyclin-dependent kinases, and activation of catastrophic attempts at neuronal mitosis. Collectively, these results demonstrate that nonfeminizing estrogens are neuroprotective and protect the brain from the induction of AD-like neuropathology in an animal model. These features of nonfeminizing estrogens make them attractive compounds for assessment of efficacy in AD and stroke, because they are not expected to show the side effects of chronic estrogen therapy that are ER mediated in the liver, uterus, and breast.

Alzheimer Disease↗

PubMed2004

Biochemical and proteomics approaches to characterize topoisomerase IIalpha cysteines and DNA as targets responsible for cisplatin-induced inhibition of topoisomerase IIalpha.

Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase IIalpha. This inhibition was not accompanied by stabilization of a covalent topoisomerase IIalpha-DNA intermediate. Pretreatment of kinetoplast plasmid DNA (kDNA) or pBR322 DNA with submicromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase IIalpha catalytic assay. Cisplatin nearly equally inhibited growth of a parental K562 and an etoposide-resistant K/VP.5 cell line that contained decreased topoisomerase IIalpha levels, a result consistent with isolated enzyme experiments demonstrating that cisplatin was not a topoisomerase IIalpha poison. Because cisplatin is known to react with protein sulfhydryl groups, the 13 cysteine groups in the topoisomerase IIalpha monomer were evaluated by mass spectrometry to determine which cysteines were free and disulfide-bonded to identify possible sites of cisplatin adduction. High-pressure liquid chromatography-matrix-assisted laser desorption ionization mass spectrometry showed that topoisomerase IIalpha contained at least five free cysteines (170, 216, 300, 392, and 405) and two disulfide-bonded cysteine pairs (427-455 and 997-1008). Cysteine 733 was also disulfide-bonded, but its partner cysteine could not be identified. Cisplatin antagonized the formation of a fluorescence adduct between topoisomerase IIalpha and the sulfhydryl-reactive maleimide reagent 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic acid methyl ester (ThioGlo-1). Dithiothreitol, which was shown by spectrophotometry to react rapidly with cisplatin (6-min half-time), diminished the capacity of cisplatin to interfere with ThioGlo-1 binding to topoisomerase IIalpha. The results of this study suggest that cisplatin may exert some of its cell growth inhibitory and antitumor activity by inhibition of topoisomerase IIalpha through reaction with critical enzyme sulfhydryl groups and/or by forming DNA adducts that render the DNA substrate refractory to topoisomerase IIalpha.

Antigens, Neoplasm↗

PubMed2004

An improved palladium-based DMFCs cathode catalyst.

A novel carbon-supported palladium-rich Pd3Pt1/C catalyst prepared by a modified polyol process showed a better cell performance than Pt/C in direct methanol fuel cells, which may be attributed to palladium's inactivity to methanol electro-oxidation while exhibiting good performance to oxygen reduction reaction.

Journal Article↗

PubMed2004

Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein.

Neurofibrillary tangles (NFTs) are a pathological hallmark of many neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17). However, the cellular origin and the consequence of the NFT formation are poorly understood. Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the pathogenesis of sporadic AD, which accounts for the majority of AD occurrence. Here we show that after a transient cerebral ischemia, hyperphosphorylated tau accumulates in cortical neurons in a site-specific manner. The hyperphosphorylated tau presents a conformation similar to those present in human tauopathies, and colocalizes largely with signs of apoptosis. Our current study suggests that tau hyperphosphorylation may contribute to the brain damage induced by transient cerebral ischemia, and may be involved in the pathogenesis of neurodegenerative disorders in patients after stroke. Further, these results indicate that ischemic neuronal damage and apoptosis associates with tau hyperphosphorylation, and potentially NFTs formation. Finally, our results also suggest that neuronal apoptosis may be a therapeutic target in preventing tauopathy-related neurodegenerative diseases.

Analysis of Variance↗

PubMed2004

Increased beta-secretase activity and expression in rats following transient cerebral ischemia.

The aberrant proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases is key to amyloid plaque formation in Alzheimer's disease (AD). Identification of an aspartyl protease as the beta-secretase (beta-site APP cleaving enzyme, BACE) involved in APP processing provides a pharmaceutical target for potential AD treatment. In the present studies, we demonstrate that transient cerebral ischemia in female rats caused a 30% increase in beta-secretase activity. alpha-Secretase activity did not increase significantly. We examined protein levels of BACE1, and its analogue BACE2, in ischemic brain extracts. BACE1 protein levels increased 67%, while BACE2 protein level did not change after such a transient ischemia. Immunohistochemical studies demonstrated that BACE1 protein was increased in the ischemic neocortex, when compared with its contralateral cortex. Further, colocalization assessment indicated that BACE1 strongly associated with staining for the apoptotic marker, TUNEL. These results may partially explain epidemiological study, which demonstrate a higher incidence of dementia after stroke. Further, our results support the hypothesis that apoptosis and aberrant APP processing are correlated events in AD brain, and suggest that inhibition of BACE may have a therapeutic effect in the prevention of dementia after stroke recovery.

Amyloid Precursor Protein Secretases↗

PubMed2004

Estrogen attenuates nuclear factor-kappa B activation induced by transient cerebral ischemia.

The protective effects of estrogens have been widely reported in a number of animal and cell culture models, but the molecular mechanisms of this potent neuroprotective activity are not well understood. Both in vitro and in vivo studies indicate that in the central nervous system and peripheral tissues, estrogen treatment reduces cytokine production and inflammatory responses. Nuclear factor-kappa B (NFkappaB) plays an essential role in the regulation of post-ischemic inflammation, which is detrimental to recovery from an ischemic stroke. We investigated the role of NFkappaB in neuronal survival in rats that received transient middle cerebral artery (MCA) occlusion, and observed that this transient cerebral ischemia induced substantial apoptosis and inflammatory responses, including IkappaB phosphorylation, NF-kappaB activation and iNOS over-expression. 17 beta-estradiol (E2) treatment produced strong protective effects by reducing infarct volume, neuronal apoptosis, and inflammatory responses. These findings provide evidence for a novel molecular and cellular interaction between the sex hormone and the immunoresponsive system. These studies also provide evidence that suppression of post-ischemic inflammation may play a critical role in estrogen-mediated neuroprotection.

Actins↗

PubMed2004

Transient cerebral ischemia induces aberrant neuronal cell cycle re-entry and Alzheimer's disease-like tauopathy in female rats.

Aberrant mitosis occurs in many tauopathy-related neurodegenerative diseases and is believed to precede the formation of neurofibrillary tangles. In this study, we report for the first time that transient cerebral ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tangle-like conformational epitopes in adult female rat cortex. Following transient cerebral ischemia in rats, initiation of apoptosis precedes and is potentially integrated with subsequent aberrant mitosis and tau hyperphosphorylation. Furthermore, inhibition of mitosis-related cyclin-dependent kinases (Cdks) by roscovitine significantly reduced the hyperphosphorylation of tau. Administration of the female sex steroid and potent neuroprotective agent, 17beta-estradiol, reduced ischemia-reperfusion-induced cerebral damage and the subsequent aberrant mitosis and tauopathies. These results provide a neuropathological basis for the higher prevalence of dementia in stroke patients and support the hypothesis that apoptosis and aberrant mitosis are integrated pathological events in neurons that may play a critical role in the development of Alzheimer's disease and other tauopathy-related neuropathology.

Alzheimer Disease↗

PubMed2003

Cyclic mechanical stretching of human tendon fibroblasts increases the production of prostaglandin E2 and levels of cyclooxygenase expression: a novel in vitro model study.

Prostaglandin E(2) (PGE(2)) is a known inflammatory mediator of tendinitis, for which mechanical loading on tendons is believed to be one of the most prominent causation factors. Previous in vitro studies have shown that cyclic mechanical stretching of cells can cause changes in cell morphology and alteration of both DNA and protein syntheses. In our study, a novel system was used whereby tendon fibroblasts are cultured on microgrooved silicone surfaces and are subjected to cyclic uniaxial stretching along their long axes to mimic in vivo conditions. Using this unique model system, the cell shape and alignment can be controlled. Further, this study was designed to test the hypotheses that PGE(2) production increases in a stretching magnitude-dependent manner and that cyclooxygenase (COX) is responsible for the increased PGE(2) production in tendon fibroblasts. Human patellar tendon fibroblasts were cultured on the microgrooved silicone membranes and cyclically stretched at 4%, 8%, or 12% of nominal dish length for 24 hr. PGE(2) production was found to be increased 1.7-fold at 8% cyclic stretching and 2.2-fold at 12% cyclic stretching compared with nonstretched controls. In addition, human tendon fibroblasts had increased expression of both COX-1 and COX-2 for all three applied stretching magnitudes, with the exception of COX-1 at 4% cyclic stretching. Also, cellular PGE(2) production, after 8% cyclic stretching, was significantly decreased with the addition of indomethacin (25 microM), a COX competitive inhibitor, compared with stretched cells without indomethacin treatment. These findings suggest that the increase in PGE(2) production by the human tendon fibroblasts is stretching magnitude-dependent, and that the increase in COX expression contributes to the increased production of PGE(2) after cyclic stretching. As PGE(2) is a known inflammatory mediator of tendinitis, the contribution of COX-1 and COX-2 to PGE(2) production and their roles in tendon inflammation are clearly indicated.

Adult↗

PubMed2002

Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site.

Growth hormone-binding protein (GHBP) is complexed to a substantial fraction of circulating GH. In humans, rabbits, and other species, GHBP derives from proteolytic shedding of the GH receptor (GHR) extracellular domain. In cell culture studies, stimuli such as phorbol ester, platelet-derived growth factor, or serum induce GHR proteolysis, which concomitantly yields shed GHBP in cell supernatants and a cell-associated cytoplasmic domain-containing GHR remnant. This process is sensitive to metalloprotease inhibition, and genetic reconstitution studies identify tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), a transmembrane metalloprotease, as a GHR sheddase. Stimuli that induce GHR proteolysis render cells less responsive to GH, but the mechanism(s) of this desensitization is not yet understood. In this study, we mapped the rabbit (rb) GHR cleavage site. We adenovirally expressed a C-terminal epitope-tagged rbGHR lacking most of its cytoplasmic domain, purified the remnant protein induced by the phorbol ester, PMA, and derived the cleavage site by N-terminal sequencing of the purified remnant. The N-terminal sequence, (239)FTCEEDFR(246), matched perfectly the rbGHR and suggests that cleavage occurs eight residues from the membrane in the proximal extracellular domain stem region. Deletion and alanine substitution mutagenesis indicated that, similar to other TACE substrates, the spacing of residues in this region, more than their identity, influences GHR cleavage susceptibility. Further, we determined that PMA pretreatment desensitized a cleavage-sensitive GHR mutant, but not a cleavage-insensitive mutant, to GH-induced JAK2 activation. These results suggest that inducible GHR proteolysis can regulate GH signaling.

Amino Acid Sequence↗