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Biomedical subjects

R C Chang

Publications and source records attributed to R C Chang.

At least 19 recordsLinked to original sources
PubMed2001

Effects of tumor necrosis factor alpha on taurine uptake in cultured rat astrocytes.

Taurine is known to play a major role in volume regulation in astrocytic swelling associated with stroke and brain trauma. Apart from brain edema, the severity of brain injury is related to the levels of inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha). TNFalpha had been shown to be closely associated with brain edema formation since the neutralization of TNFalpha reduced brain edema. Considering taurine has osmoregulatory functions in astrocytes, experiments were performed to study the effects of TNFalpha on taurine uptake in cultured astrocytes. Astrocytes exposed to 20 ng/ml of TNFalpha for 48 h showed a 91% increase in taurine uptake and significant increase was observed after 24 h exposure. This cytokine caused neither significant changes in cell volume nor taurine release. The increased in taurine uptake induced by TNFalpha was unlikely resulted from the modification of Na(+) movement because TNFalpha decreased tyrosine uptake, Na(+)-dependent transport system. In contrast to TNFalpha, interferon-gamma (IFNgamma) did not significantly affect taurine uptake. Taken together, our results did not support a suggestion that TNFalpha affects cell volume regulation via modulating taurine uptake in astrocytes. Increasing lines of evidence have demonstrated that taurine has anti-inflammatory and anti-oxidative effects, these findings therefore suggested that the increase in taurine uptake might be an adaptive response or a tool for astrocytes against oxidative stress.

Animals↗
PubMed2001

Synthesis of fatty acid esters by recombinant Staphylococcus epidermidis lipases in aqueous environment.

Various flavor esters were obtained by using recombinant lipases from Staphylococcus epidermidis as a catalyst in an aqueous environment. These esters were enzymatically synthesized to overcome the problems associated with chemical processes. This study showed that the S. epidermidis lipases could catalyze ester synthesis from decyl alcohol and fatty acids of different chain length. The wild-type and mutant lipases (M419A and V649I) could efficiently catalyze the synthesis of decyl alcohol esters of unsaturated fatty acids. In contrast, the yield of decyl laurate was better by wild-type and mutant enzyme V6491, but mutant enzyme M419A only favored the synthesis of decyl myristate. The esterification of oleic acid and various carbon-chain-length alcohols from ethanol to hexadecanol increased up to decanol by wild-type and M419A mutant enzymes and reached an optimum for dodecanol by V6491 mutant enzyme. The enzyme is potentially useful in food industries such as dairy product flavoring.

Esterification↗
PubMed2001

Neurons reduce glial responses to lipopolysaccharide (LPS) and prevent injury of microglial cells from over-activation by LPS.

The microenvironment of the CNS has been considered to tonically inhibit glial activities. It has been shown that glia become activated where neuronal death occurs in the aging brain. We have previously demonstrated that neurons tonically inhibit glial activities including their responses to the bacterial endotoxin lipopolysaccharide (LPS). It is not clear whether activation of glia, especially microglia in the aging brain, is the consequence of disinhibition due to neuronal death. This study was designed to determine if glia regain their responsiveness to LPS once the neurons have died in aged cultures. When cultured alone, glia from postnatal day one rat mesencephalons stimulated with LPS (0.1-1000 ng/mL) produced both nitric oxide (NO) and tumor necrosis factor alpha (TNFalpha), yielding a sigmoid and a bell-shaped curve, respectively. When neuron-containing cultures were prepared from embryonic day 14/15 mesencephalons, the shape of the dose-response curve for NO was monotonic and the bell-shaped curve for TNFalpha production was shifted to the right. After 1 month of culture under conditions where neurons die, the production curves for NO and TNFalpha in LPS-stimulated glia shifted back to the left compared to mixed neuron-glia cultures. Immunostaining of rat microglia for the marker CR3 (the receptor for complement component C3) demonstrated that high concentrations of LPS (1 microg/mL) reduced the number of microglia in mixed-glial cultures. In contrast, reduction of CR3 immunostaining was not observed in LPS-stimulated mixed neuron-glia cultures. Taken together, the results demonstrate that disinhibition of the glial response to LPS occurs after neurons die in aged cultures. Once neurons have died, the responsiveness of glia to LPS is restored. Neurons prevented injury to microglia by reducing their responsiveness to LPS. This study broadens our understanding of the ways in which the CNS microenvironment affects cerebral inflammation.

Animals↗
PubMed2001

In vitro cellular response of retinoic acid treated human oral cancer cell lines.

BACKGROUND: The purpose of this study is to identify the cellular response ofretinoic acid-treated human oral cancer cell lines. METHODS: Seven human oral cancer cell lines KB, SCC4, SCC9, SCC15, SCC25, OEC-M1, OC1 and OC2 were used for cell culture experiments. Direct cell number counting method was utilized to evaluate cellular response of these human oral cancer cells at the presence or absence of all-trans RA at 1 mM. RESULTS: Through 7-day observation, the cell population of SCC9, SCC15 and SCC25 of RA-treated groups decreased when compared with the non RA-treated groups. These three cell lines were further verified using [3H] thymidine incorporation DNA synthesis assay. KB, SCC4, OC1, OC2 and OEC-M1 cell lines did not show growth inhibition at the presence of RA at 1 mM. CONCLUSIONS: The molecular event of how SCC9, SCC15 and SCC25 are inhibited by RA and how KB, OC1, OC2 and OECM1 are resistant to RA can be further explored on the basis of this study.

Carcinoma, Squamous Cell↗
PubMed2001

p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease.

p16 regulates the G(1)-S cell cycle transition by inhibiting the cyclin D-cyclin-dependent kinase (CDK)4/CDK6-mediated phosphorylation of retinoblastoma protein (pRb). We examined the possible derangement of the p16-CDK/cyclin D-pRb pathway in 40 primary neuroblastomas including 18 samples in the unfavorable stages (C and D) and 22 in the favorable stages (A, B, and Ds) by PCR, reverse transcription-PCR, Western blot, and immunohistochemistry and correlated the results with clinical outcome. No samples harbored alterations of the p16 gene. Interestingly, the samples in the unfavorable stages exhibited expression of p16 mRNA and protein more frequently than those in the favorable stages [mRNA, 9 of 18 (50%) versus 2 of 22 (9%), P = 0.006; protein, 5 of 16 (31%) versus 0 of 18 (0%), P = 0.013]. Alterations of the downstream components of the pathway were infrequent. pRb was deregulated in the majority of samples investigated [27 of 33 (82%), 24 with hyperphosphorylated pRb and 3 with no pRb protein]. The phosphorylation status of pRb did not correlate with p16 protein expression, suggesting that the elevated p16 protein may not be functioning properly to regulate the pathway. Among patients of all stages, p16 expression was significantly associated with a lower overall survival. There was no overexpression of MDM2, and loss of p14(ARF) expression and p53 mutation were infrequent events. Taken together, these findings suggest that up-regulated p16 expression may represent a unique feature of aggressive neuroblastoma.

Cell Cycle↗
PubMed2000

In vitro sensitivity of T-cell lymphoblastic leukemia to UCN-01 (7-hydroxystaurosporine) is dependent on p16 protein status: a Pediatric Oncology Group study.

p16 regulates the cell cycle pathway by inhibiting the cyclin Ds-cyclin-dependent kinase (CDK) 4/6-mediated phosphorylation of retinoblastoma protein (pRb). Previously, we reported that most primary T-cell acute lymphoblastic leukemia (T-ALL) harbored p16 inactivation and hyperphosphorylated pRb without cyclin Ds or CDK4/6 alterations. Therefore, inhibiting CDK4/6 may be an ideal therapeutic approach for p16 (-) T-ALL. UCN-01 (7-hydroxystaurosporine) is a potent antitumor agent that exerts its effects through the inhibition of CDKs. We now report that p16 protein expression status of T-ALL cells influences their sensitivity to UCN-01. In 36 primary T-ALL cells, the IC50s of UCN-01 in the 27 p16 (-) cells (43+/-52 nM) was significantly lower than that in the 9 p16 (+) cells (258+/-260 nM). Our results suggest that agents like UCN-01 may be useful as a p16-selective therapy for T-ALL.

Alkaloids↗
PubMed2000

Measured and calculated effects of mutations in bacteriophage T4 lysozyme on interactions in solution.

Understanding the molecular determinants of protein interactions in solution has fundamental implications for understanding protein solution thermodynamics and, hence, processes as diverse as separations performance and cellular self-organization. Our earlier theoretical calculations indicate that the protein-protein interactions are dominated by a small number of configurations in which highly complementary surface regions are apposed, rather than by the overall colloidal interactions. To examine this paradigm more explicitly, we investigated the effects of protein structural modifications on protein-protein interactions. Experimental measurements are presented of B(22)(') values of a set of mutants of Ser44 in bacteriophage T4 lysozyme. Effects are seen with both charged and uncharged substitutions. The results with the charged substitutions follow the expected trends, whereas those with the uncharged substitutions may be explained by the impact of the mutations on the local protein geometry, which directly affects the complementarity of protein interactions. These effects are also captured well by molecular calculations that account for the mutations. The interaction energetics between protein pairs could provide information on the propensity for adventitious interactions, which can have important implications for separations and for normal and pathological self-assembly. Thus, protein structural data implicit in genomic information, coupled with appropriate calculational and experimental tools, can ultimately provide insights into protein interactions in vivo and in bioprocessing.

Bacteriophage T4↗
PubMed2000

Immune modulatory effects of neural cell adhesion molecules on lipopolysaccharide-induced nitric oxide production by cultured glia.

Activation of glial cells often occurs at sites of neuronal injury or death and where there is disruption of communication between glia and neurons. We have previously reported that neurons exert an inhibitory influence on LPS-stimulated nitric oxide (NO) production in glial cells. We hypothesized that neural cell adhesion molecules (NCAM) might mediate this inhibitory effect, and this study was designed to elucidate the role of NCAM on lipopolysaccharide (LPS)-induced NO production. We found that soluble NCAMs reduced LPS-stimulated NO production by cultured glia. A monoclonal antibody that recognizes the third immunoglobulin (Ig) domain and can mimic the functions of NCAMs reduced LPS-stimulated NO production, whereas another antibody that binds to other regions of the NCAM did not modulate NO production. Using a 10-amino acid peptide from the third Ig domain of the NCAM, a peptide fragment within the region recognized by the NCAM antibody, mimics the effect of the molecule in reducing NO production. This study demonstrated that NCAMs could modulate LPS-stimulated NO production, most likely via interaction between NCAMs. These results suggest that neuron-glia interactions via NCAMs play an important role in regulating the activities of glial cells in the brain.

Adjuvants, Immunologic↗
PubMed2000

Post-transcriptional inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by Gö6976 in murine microglia.

Glia in the brain respond to various toxins with an increased expression of inducible nitric oxide synthase (iNOS) and an increased production of nitric oxide (NO). Here, we report that lipopolysaccharide (LPS)-induced expression of iNOS was down-regulated post-transcriptionally through the destabilization of iNOS mRNA by the indolocarbazole compound, Gö6976, in murine microglia. This Gö6976 effect is specific for iNOS since tumor necrosis factor alpha was unaffected by the compound. Interestingly, the post-transcriptional effects ascribed to Gö6976 were not observed with other inhibitors of protein kinase A, C (PKC), G, or protein tyrosine kinases. Instead, these kinases appear to affect the iNOS/NO system at the transcriptional level. In the past, Gö6976 has been reported to be a rather specific inhibitor of PKC in vitro. Results from our experiments, through prolonged treatment with phorbol esters and with the various PKC inhibitors including phorbol ester-insensitive PKC isotype inhibitor, suggest that the Gö6976-mediated post-transcriptional regulation of iNOS gene expression and NO production in microglia is not mediated through its reputed effects on PKC activity. Since the effects of various neurotoxins and certain neurodegenerative diseases may be manifested through alterations in the iNOS/NO system, post-transcriptional control of this system may represent a novel strategy for therapeutic intervention.

Animals↗
PubMed2000

A novel effect of an opioid receptor antagonist, naloxone, on the production of reactive oxygen species by microglia: a study by electron paramagnetic resonance spectroscopy.

Microglia as the first line of defensive cells in the brain produce free radicals including superoxide and nitric oxide (NO), contributing to neurodegeneration. An opioid receptor antagonist, naloxone, has been considered pharmacologically beneficial to endotoxin shock, experimental cerebral ischemia, and spinal cord injury. However, the mechanisms underlying these beneficial effects of naloxone are still not clear. This study explores the effects of naloxone on the production of superoxide and NO by the murine microglial cell line, BV2, stimulated with lipopolysaccharide (LPS) as measured by electron paramagnetic resonance (EPR). The production of superoxide triggered by phobol-12-myristate-13-acetate (PMA) resulted in superoxide dismutase (SOD)-inhibitable, catalase-uninhibitable 5,5-dimethyl-1-pyrroline N-oxide (DMPO) hydroxyl radical adduct formation. LPS enhanced the production of superoxide and triggered the formation of non-heme iron-nitrosyl complex. Cells pre-treated with naloxone showed significant reduction of superoxide production by 35%. However, it could not significantly reduce the formation of non-heme iron-nitrosyl complex and nitrite. Taken together, the results expand our understanding of the neuroprotective effects of naloxone as it decreases superoxide production by microglia.

Animals↗
PubMed2000

Influence of neurons on lipopolysaccharide-stimulated production of nitric oxide and tumor necrosis factor-alpha by cultured glia.

Cerebral inflammation often originates in a region where neuronal death occurs and thereafter slowly spreads outward. This study aimed to elucidate the roles of neurons in modulating the production of inflammatory factors stimulated by the bacterial endotoxin lipopolysaccharide (LPS). Culturing neurons with mixed glia reduced nitrite and tumor necrosis factor-alpha (TNF-alpha) production compared to cultures with only mixed glia, and shifted the dose-response curve to the right. The decreased nitrite and TNF-alpha production were not due to the cytotoxicity of LPS. Immunocytochemical analysis of glia-neuron co-cultures revealed the morphological changes in the activated microglia. Culturing PC12 cells with rat mixed-glia also reduced nitrite production. The influence of neurons on glial inflammation was partly due to the cell-cell contacts between neurons and glia via neural cell adhesion molecules (NCAM) because NCAM significantly reduced LPS-stimulated nitrite production. These results demonstrate that neurons reduce the production of inflammatory factors by glia. Since cerebral inflammation is important in many neurological disorders, this study might provide insight about the role of glia-neuron interactions in inflammatory responses in the brain.

Animals↗
PubMed2000

Reduction by naloxone of lipopolysaccharide-induced neurotoxicity in mouse cortical neuron-glia co-cultures.

An inflammatory response in the CNS mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Using mouse cortical mixed glia cultures, we have previously demonstrated that the bacterial endotoxin lipopolysaccharide induces the activation of microglia and the production of proinflammatory factors. Naloxone, an opioid receptor antagonist, inhibits the lipopolysaccharide-induced activation of microglia and the production of proinflammatory factors. Using neuron-glia co-cultures, we extended our study to determine if naloxone has a neuroprotective effect against lipopolysaccharide-induced neuronal damage and analysed the underlying mechanism of action for its potential neuroprotective effect. Pretreatment of cultures with naloxone (1 microM) followed by treatment with lipopolysaccharide significantly inhibited the lipopolysaccharide-induced production of nitric oxide and the release of tumor necrosis factor-alpha, and significantly reduced the lipopolysaccharide-induced damage to neurons. More importantly, both naloxone and its opioid-receptor ineffective enantiomer (+)-naloxone were equally effective in inhibiting the lipopolysaccharide-induced generation of proinflammatory factors and the activation of microglia, as well as in the protection of neurons. These results indicate that the neuroprotective effect of naloxone is mediated by its inhibition of microglial activity and may be unrelated to its binding to the classical opioid receptors.

Animals↗
PubMed2000

High concentrations of extracellular potassium enhance bacterial endotoxin lipopolysaccharide-induced neurotoxicity in glia-neuron mixed cultures.

A sudden increase in extracellular potassium ions (K(+)) often occurs in cerebral ischemia and after brain trauma. This increase of extracellular K(+) constitutes the basis for spreading depression across the cerebral cortex, resulting in the expansion of neuronal death after ischemic and traumatic brain injuries. Besides spreading depression, it has become clear that cerebral inflammation also is a key factor contributing to secondary brain injury in acute neurological disorders. Experiments to validate the relationship between elevated levels of extracellular K(+) and inflammation have not been studied. This study aims to elucidate the roles of high concentrations of extracellular K(+) in bacterial endotoxin lipopolysaccharide-induced production of inflammatory factors. Increased concentration of KCl in the medium (20mM) significantly enhanced neurotoxicity by lipopolysaccharide in glia-neuron mixed cultures. To delineate the underlying mechanisms of increased neurotoxicity, the effects of high extracellular K(+) were examined by using mixed glial cultures. KCl at 20mM significantly enhanced nitrite, an index for nitric oxide, production by about twofold, and was pronounced from 24 to 48h, depending on the concentration of KCl. Besides nitric oxide production of tumor necrosis factor-alpha was also enhanced. The augmentative effects of high KCl on the production of inflammatory factors were probably due to the further activation of microglia, since high KCl also enhanced the production of tumor necrosis factor-alpha in microglia-enriched cultures. The increased production of nitrite by high K(+) was eliminated through use of a K(+)-blocker. Taken together, the results show that increases of extracellular K(+) concentrations in spreading depression augment lipopolysaccharide-elicited neurotoxicity, because production of inflammatory factors such as nitric oxide and tumor necrosis factor-alpha are potentiated. Since spreading depression and cerebral inflammation are important in acute neurological disorders, the present results suggest a biochemical mechanism: elevated extracellular K(+) concentrations augment glial inflammatory responses, and thus the neurotoxicity.

Animals↗
PubMed2000

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells.

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Biomechanical and histological studies of particulate hydroxylapatite implanted in femur bone defects of adult dogs.

The purpose of this study was to investigate the biomechanical behavior and the histology of particulate hydroxylapatite (HA) implanted in bone defects created in femurs of 48 adult mongrel dogs. The bone defects, measuring 10 x 10 x 25 mm with or without HA implantation, were allowed to heal until the end of the 1st, 2nd, 3rd, 6th, 9th and 12th months after surgery. The harvested tissue specimens from the implant sites were processed into long cubic or rectangular prismatic forms. Their mechanical strengths were assessed using compression and shearing tests to measure the maximal compressive and shear stress by force loading on the middle portions of the specimens. Histological sections of each stage were processed with both decalcified and nondecalcified methods. The results showed that the mechanical strength of the tested specimens was closely related to the tissue regeneration within the bone defect. Progressive regeneration of new bone was observed at each stage, with complete bone formation at the 9-month period. A consistent increase in both maximal compressive stress and maximal shear stress was noted at each stage, responsive to the ossification and maturation of the regenerating tissue within the bone defect. This animal model provides a novel approach to directly estimate the tissue strength of the HA-implanted bone defect.

Animals↗
PubMed2000

Universal inactivation of both p16 and p15 but not downstream components is an essential event in the pathogenesis of T-cell acute lymphoblastic leukemia.

p16/p15 regulate the cell cycle pathway by inhibiting the cyclin Ds-CDK4/6 mediated phosphorylation of pRb. We reported previously that in T-cell acute lymphoblastic leukemia (T-ALL), p16 and p15 were frequently (approximately 70%) inactivated at the DNA level by deletion, mutation, or hypermethylation. Therefore, we hypothesize that inactivation of the cell cycle regulatory pathway may be essential in the pathogenesis of T-ALL, and that the remaining T-ALL with a wild-type p16/p15 gene likely harbor inactivation of these genes at RNA or protein levels. Alternatively, the downstream components of the pathway including CDK4/6, cyclin Ds, and pRb may be deregulated. In 124 primary T-ALLs, we found inactivation of the p16 and p15 genes at the DNA level in 79 (64%) and 64 (52%) samples, respectively. Only 9 of the 45 samples with wild-type p16 expressed p16 protein, whereas the remaining 36 lacked p16 expression at the RNA or protein level. In the 60 samples with an intact p15 gene, only 2 expressed p15 mRNA, and the only one analyzed lacked p15 protein. Overall, the abrogation rates for p16 and p15 at DNA/RNA/protein levels were 93% (115 of 124) and 99% (123 of 124), respectively. Although no alterations were evident in cyclin Ds or CDK4/6, pRb was hyperphosphorylated in the majority of samples investigated. These findings strongly support that both p16 and p15 are specific targets in the deregulation of the cell cycle pathway in T-ALL and that the inactivation of these genes is most likely essential in the pathogenesis of this disease.

Blotting, Western↗
PubMed1999

Long-term increase of Sp-1 transcription factors in the hippocampus after kainic acid treatment.

Systemic administration of kainic acid (KA), a glutamate receptor agonist, causes robust seizures and has been used as an excellent rodent model for human temporal lobe epilepsy. Recently, we have demonstrated that a single injection of KA increases the steady-state levels of proenkephalin (PENK) mRNA in the rat hippocampus for at least one year. However, the molecular mechanisms underlying this long-term increase in PENK mRNA levels have not been clearly defined. To determine the possible involvement of the Sp-1 transcription factors in this regulation, electrophoresis mobility-shift assays were used to study the expression of Sp-1 factors in the hippocampus after KA treatment. The results showed that there are long-lasting increases in Sp-1 DNA-binding activity. The Sp-1 DNA-binding complexes were only competed by the non-radioactive Sp-1 element and not by ENKCRE2, AP-1 or CRE elements, indicating the specificity of Sp-1 DNA-binding activity. Since the expression of Sp-1 parallels the time course of long-lasting increase in the expression of PENK mRNA and mossy fiber sprouting after KA treatment, we hypothesize that the increase in Sp-1 activity may be associated with the long-term changes in the plasticity of hippocampal function after KA-induced seizures.

Animals↗