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Biomedical subjects

Daming Zhu

Publications and source records attributed to Daming Zhu.

11 recordsLinked to original sources
PubMed2005

A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli.

Residual host cell protein impurities in recombinant proteins intended for human use must be accurately quantified to help establish their safety. We describe a novel means of host cell protein quantitation, in which a slot blot system was employed together with scanning laser densitometry to allow picogram level sensitivity in detection of residual host cell proteins in unpurified fermentation products and final purified bulk samples. Two allelic forms of merozoite surface protein 1, a promising malaria vaccine candidate antigen currently undergoing evaluation in clinical trials, were expressed in E. coli as clinical grade proteins, refolded, and carried through several chromatographic purification steps. Several lots of these proteins were analyzed with this generic quantitative assay that uses rat polyclonal antibodies generated against soluble and insoluble E. coli proteins. The assay had a detection range of 6.1-1562 ng/mL, with a detection limit of 6.1 ng/mL, comparable to reported ELISA-based methods. This assay proved simple yet very sensitive and accurate, giving highly reproducible results. Thus it is suitable for evaluating host cell protein levels in clinical grade recombinant proteins expressed in E. coli.

Animals↗
PubMed2005

N-methyl-D-aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal-regulated kinase pathway.

N-Methyl-D-aspartate (NMDA) at a subtoxic concentration (100 microM) promotes neuronal survival against glutamate-mediated excitotoxicity via a brain-derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3-K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration- and time-dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK-801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 microM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3-K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl-2, bcl-X(L), and bax mRNA levels,. NMDA increases Bcl-2 and Bcl-X(L) protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate-mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl-2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.

Animals↗
PubMed2005

Montanide ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations.

Montanide ISA 720 is an experimental adjuvant, formulated as water-in-oil emulsions, that induces high antibody titers in several animal species. It has been used in human vaccine trials with malaria and HIV vaccines. The heightened response is likely due, in part, to the formation of a depot at the injection site. However, post-formulation modifications were seen with seven proteins tested during storage of ISA 720 formulations at 37 degrees C for 1 week and two proteins stored longer at 4 degrees C. Potency studies in mice, in which the stored vaccines were diluted into placebo emulsions for appropriate dosing, indicated that this instability could lead to loss of immunogenicity in the post-injection depot, limiting the allowable storage time of preformed vaccines. We describe point-of-injection formulation for ISA 720 vaccines that meets the requirement for in vitro stability. For preformed vaccines, addition of glycine or glycylglycine prevented antigen modification on storage at 37 degrees C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot.

Adjuvants, Immunologic↗
PubMed2004

Niacin noncompetitively inhibits DGAT2 but not DGAT1 activity in HepG2 cells.

Niacin is a widely used lipid-regulating agent in dyslipidemic patients. Previously, we have shown that niacin inhibits triacylglycerol synthesis. In this report, using HepG2 cells, we have examined the effect of niacin on the mRNA expression and microsomal activity of diacylglycerol acyltransferase 1 and 2 (DGAT1 and DGAT2), the last committed but distinctly different enzymes for triglyceride synthesis. Addition of niacin to the DGAT assay reaction mixture dose-dependently (0-3 mM) inhibited DGAT activity by 35-50%, and the IC(50) was found to be 0.1 mM. Enzyme kinetic studies showed apparent K(m) values of 8.3 microM and 100 microM using [(14)C]oleoyl-CoA and sn-1,2-dioleoylglycerol as substrates, respectively. A decrease in apparent V(max) was observed with niacin, whereas the apparent K(m) remained constant. A Lineweaver-Burk plot of DGAT inhibition by niacin showed a noncompetitive type of inhibition. Niacin selectively inhibited DGAT2 but not DGAT1 activity. Niacin inhibited overt DGAT activity. Niacin had no effect on the expression of DGAT1 and DGAT2 mRNA. These data suggest that niacin directly and noncompetitively inhibits DGAT2 but not DGAT1, resulting in decreased triglyceride synthesis and hepatic atherogenic lipoprotein secretion, thus indicating a major target site for its mechanism of action.

Acyltransferases↗
PubMed2004

CTRD: a fast applet for computing signed translocation distance between genomes.

CTRD is a software for computing translocation distance between genomes. It takes two genomes as its input and tests whether one genome can be transformed into the other. If possible, it computes the translocation distance between two genomes, and gives the translocation operation serial. We adopt the fastest known O(n(2)log n) algorithm. Our contributions include (1) give a necessary and sufficient condition to ensure that one genome can be transformed into the other for translocation operations, and (2) develop a software using the fastest known O(n(2)log n) algorithm.

Algorithms↗
PubMed2004

Inhibition of protein kinase C promotes neuronal survival in low potassium through an Akt-dependent pathway.

Cerebellar granule cell neurons undergo apoptotic cell death when subjected to serum-free conditions at physiological concentrations of potassium (5 mM). Protein kinase C (PKC) is known to play a role in preventing neuronal apoptosis under trophic factor deprivation, but its role in protecting cerebellar neurons from cell death under conditions of low potassium is unknown. This study sought to determine the involvement of PKC in neuronal survival and to determine if PKC regulated the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in low physiologic concentrations of potassium. Incubation with a pan-PKC inhibitor, Ro-31-8220 (2 microm), or a specific PKCAlpha inhibitor, Gö6976, protected cerebellar granule cell neurons from low potassium-mediated cell death. In contrast, phorbol ester (TPA, 100 nm), a PKC activator, increased cell death. Incubation with, Ro-31-8220 rescued neurons from cell death induced by the PI 3-K inhibitor, LY294002, suggesting that Ro-31-8220 may affect Akt phosphorylation. Western blot analysis showed that serum-free, low potassium conditions decreased Akt phosphorylation, which was exacerbated by treatment with LY294002. In contrast, PKC inhibitors, Gö6976 or Ro-31-8220, increased Akt phosphorylation approximately two and four-fold, respectively in low potassium conditions. Because Akt activation appears to be critical in promoting neuronal survival under these culture conditions, increased Akt phosphorylation brought about by inhibiting PKC promotes neuronal survival.

Animals↗
PubMed2004

AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression.

The signal transduction and molecular mechanisms underlying alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-mediated neuroprotection are unknown. In the present study, we determined a major AMPA receptor-mediated neuroprotective pathway. Exposure of cerebellar granule cells to AMPA (500 microM) + aniracetam (1 microM), a known blocker of AMPA receptor desensitization, evoked an accumulation of brain-derived neurotropic factor (BDNF) in the culture medium and enhanced TrkB-tyrosine phosphorylation following the release of BDNF. AMPA also activated the src-family tyrosine kinase, Lyn, and the downstream target of the phosphatidylinositol 3-kinase (PI3-K) pathway, Akt. Extracellular signal regulated kinase (ERK), a component of the mitogen-activated protein kinase (MAPK) pathway, was also activated. K252a, a selective inhibitor of neurotrophin signaling, blocked the AMPA-mediated neuroprotection. The involvement of BDNF release in protecting neurons by AMPA was confirmed using a BDNF-blocking antibody. AMPA-mediated neuroprotection is blocked by PP1, an inhibitor of src family kinases, LY294002, a PI3-K inhibitor, or U0126, a MAPK kinase (MEK) inhibitor. Neuroprotective concentrations of AMPA increased BDNF mRNA levels that was blocked by the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX). The increase in BDNF gene expression appeared to be the downstream target of the PI3-K-dependent activation of the MAPK cascade since MEK or the PI3-K inhibitor blocked the AMPA receptor-mediated increase in BDNF mRNA. Thus, AMPA receptors protect neurons through a mechanism involving BDNF release, TrkB receptor activation, and a signaling pathway involving a PI3-K dependent activation of MAPK that increases BDNF expression.

Animals↗
PubMed2004

Role of brain-derived neurotrophic factor and NF-kappaB in neuronal plasticity and survival: From genes to phenotype.

PURPOSE: Brain-derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and promotes diverse effects in neurons including development, maintenance of function, synaptic plasticity, and survival in different animal models. We present advances in our understanding of the genomics of the BDNF gene (bdnf) and its regulation by calcium-activated transcription factors, including cAMP response element binding protein (CREB) and more recently, nuclear factor kappaB (NF-kappaB) and discuss these findings in the context of neuronal plasticity and survival. METHODS: We used amplified bdnf complementary DNAs (cDNAs) and genomic DNA templates for direct sequencing and sequence variant discovery, information mining of public databases, and conventional molecular and cellular biology approaches to screen bdnf for novel regulatory elements, alternatively spliced exons, and functional sequence variants. RESULTS: We discovered a candidate NF-kappaB site in promoter 3 of bdnf and showned that activation of N-methyl-D-aspartate (NMDA) inotropic glutamate receptors increased bdnf expression through an NF-kappaB-dependent pathway and extended the finding to show that NF-kappaB was required for NMDA neuroprotection in vitro. In addition, sequence analysis of bdnf cDNAs from different brain regions predicted at least three pre-pro-BDNF protein isoforms, two of which were previously unknown. Each isoform differs at the amino terminus and may have functional importance. CONCLUSIONS: Given the central role that BDNF plays in the developing and adult nervous system, understanding how BDNF is regulated and how it functions will enhance our knowledge of its diverse effects, which may lead to more effective treatments for neurodegenerative disorders and reveal the role of BDNF in complex phenotypes related to behavior.

Animals↗
PubMed2003

N-methyl-D-aspartate and TrkB receptor activation in cerebellar granule cells: an in vitro model of preconditioning to stimulate intrinsic survival pathways in neurons.

Delineating the mechanisms of survival pathways that exist in neurons will provide important insight into how neurons utilize intracellular proteins as neuroprotectants against the causes of acute neurodegeneration. We have employed cultured rat cerebellar granule cells as a model for determining the mechanisms of these intraneuronal survival pathways. Glutamate has long been known to kill neurons by an N-methyl-d-aspartate (NMDA) receptor-mediated mechanism. Paradoxically, subtoxic concentrations of NMDA protect neurons against glutamate-mediated excitotoxicity. Because NMDA protects neurons in physiologic concentrations of glucose and oxygen, we refer to this phenomenon as physiologic preconditioning. One of the major mechanisms of NMDA neuroprotection involves the activation of NMDA receptors leading to the rapid release of brain-derived neurotrophic factor (BDNF). BDNF then binds to and activates its cognate receptor, receptor tyrosine kinase B (TrkB). The efficient utilization of these two receptors confers remarkable resistance against millimolar concentrations of glutamate that kill more than eighty percent of the neurons in the absence of preconditioning the neurons with a subtoxic concentration of NMDA. Exactly how the neurons mediate neuroprotection by activation of both receptors is just beginning to be understood. Both NMDA and TrkB receptors activate nuclear factor kappaB (NF-kappaB), a transcription factor known to be involved in protecting neurons against many different kinds of toxic insults. By converging on survival transcription factors, such as NF-kappaB, NMDA and TrkB receptors protect neurons. Thus, crosstalk between these very different receptors provides a rapid means of neuronal communication to upregulate survival proteins through release and transcriptional activation of messenger RNA.

Animals↗
PubMed2002

Effect of gemfibrozil on apolipoprotein B secretion and diacylglycerol acyltransferase activity in human hepatoblastoma (HepG2) cells.

The mechanism of action of a widely used drug gemfibrozil to reduce triglycerides (TG) and apolipoprotein B (apo B) is incompletely understood. Using human hepatoblastoma (HepG2) cells, we examined the effect of gemfibrozil on apo B secretion and TG synthesis catalyzed by diacylglycerol acyltransferase (DGAT), primary processes associated with the secretion of LDL. Gemfibrozil significantly decreased apo B secretion by HepG2 cells. It decreased oleate-induced stimulation of apo B secretion, suggesting that gemfibrozil-mediated inhibition of apo B secretion may be dependent on the synthesis of TG catalyzed by DGAT. Pre-incubation of HepG2 cells with gemfibrozil (200-400 micromol/l for 48 h) significantly inhibited microsomal DGAT activity. When added directly to the DGAT assay system containing control microsomes, gemfibrozil significantly inhibited the activity of DGAT by 14-25%. Gemfibrozil (200-400 micromol/l) inhibited TG synthesis by 47-50% as measured by the incorporation of 3H-oleic acid into TG. The data indicate that gemfibrozil inhibits DGAT activity resulting in decreased synthesis of TG and its availability for apo B lipidation rendering it susceptible to intracellular apo B degradation leading to the decreased secretion. These in-vitro data suggest a novel additional mechanism by which gemfibrozil lowers plasma TG and atherogenic apo B lipoproteins in dyslipidemic patients.

Acyltransferases↗