Biomedical subjects

K Hong

Publications and source records attributed to K Hong.

At least 37 records · Page 2Linked to original sources

PubMed2001

Intracellular signaling molecules activated by Epstein-Barr virus for induction of interferon regulatory factor 7.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is the principal oncogenic protein in the EBV transformation process. LMP-1 induces the expression of interferon regulatory factor 7 (IRF-7) and activates IRF-7 protein by phosphorylation and nuclear translocation. LMP-1 is an integral membrane protein with two regions in its C terminus that initiate signaling processes, the C-terminal activator regions 1 (CTAR-1) and CTAR-2. Here, genetic analysis of LMP-1 has determined that the PXQXT motif that governs the interaction between LMP-1 CTAR-1 and tumor necrosis factor receptor-associated factors (TRAFs) is needed to induce the expression of IRF-7. Mutations in the PXQXT motif in CTAR-1 that disrupt the interaction between LMP-1 and TRAFs abolished the induction of IRF-7. Also, dominant-negative mutants of TRAFs inhibited the induction of IRF-7 by CTAR-1. The last three amino acids (YYD) of CTAR-2 are also important for the induction of IRF-7. When both PXQXT and YYD were mutated (LMP-DM), the LMP-1 mutant failed to induce IRF-7. Also, LMP-DM blocked the induction of IRF-7 by wild-type LMP-1. These data strongly suggest that both CTAR-1 and CTAR-2 of LMP-1 independently induce the expression of IRF-7. In addition, NF-kappaB is involved in the induction of IRF-7. A superrepressor of IkappaB (sr-IkappaB) could block the induction of IRF-7 by LMP-1, and overexpression of NF-kappaB (p65 plus p50) could induce the expression of IRF-7. In addition, we have found that human IRF-7 is a stable protein, and sodium butyrate, a modifier of chromatin structure, induces IRF-7.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Solid-state fermentation of phytase from cassava dregs.

Phytases produced by numerous microorganisms and plants degrade phytic acid that has chelated with metal ions in food and feed. It is important to study phytase for the role of metal ions in nutrition of animals and humans as well as in the reduction of organic phosphate content of aqueous environment. This article reports on solid-state fermentation of phytase from a new substrate of cassava dregs. Large quantities of cassava dregs are produced in tropical areas as a byproduct of cassava starch processing. Protein and inorganic salts were found to be low in cassava dregs. Cassava dregs could be employed for phytase synthesis after the addition of a nitrogen source and mineral salts. Ammonium nitrate was the best nitrogen source among the nitrogen sources investigated, including beef extract, yeast extract, urea, ammonium nitrate, sodium nitrate, and ammonium sulfate. Sodium dodecyl sulfate promoted phytase production from cassava dregs. A maximum phytase yield of 6.73 U/g of dry mass was obtained. The obtained phytase was stable at feed-processing temperature, since 70% of initial enzyme activity was maintained after 30 min of treatment at 75 degrees C.

6-Phytase↗

PubMed2000

Calcium stores regulate the polarity and input specificity of synaptic modification.

Activity-induced synaptic modification is essential for the development and plasticity of the nervous system. Repetitive correlated activation of pre- and postsynaptic neurons can induce persistent enhancement or decrement of synaptic efficacy, commonly referred to as long-term potentiation or depression (LTP or LTD). An important unresolved issue is whether and to what extent LTP and LTD are restricted to the activated synapses. Here we show that, in the CA1 region of the hippocampus, reduction of postsynaptic calcium influx by partial blockade of NMDA (N-methyl-D-aspartate) receptors results in a conversion of LTP to LTD and a loss of input specificity normally associated with LTP, with LTD appearing at heterosynaptic inputs. The induction of LTD at homo- and heterosynaptic sites requires functional ryanodine receptors and inositol triphosphate (InsP3) receptors, respectively. Functional blockade or genetic deletion of type 1 InsP3 receptors led to a conversion of LTD to LTP and elimination of heterosynaptic LTD, whereas blocking ryanodine receptors eliminated only homosynaptic LTD. Thus, postsynaptic Ca2+, deriving from Ca2+ influx and differential release of Ca2+ from internal stores through ryanodine and InsP3 receptors, regulates both the polarity and input specificity of activity-induced synaptic modification.

Animals↗

PubMed2000

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Antibody-Dependent Cell Cytotoxicity↗

PubMed2000

Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo.

Liposomes have been proposed as a vehicle to deliver proteins to antigen-presenting cells (APC), such as dendritic cells (DC), to stimulate strong T cell-mediated immune responses. Unfortunately, because of their instability in vivo and their rapid uptake by cells of the mononuclear phagocyte system on intravenous administration, most types of conventional liposomes lack clinical applicability. In contrast, sterically stabilized liposomes (SL) have increased in vivo stability. It is shown that both immature and mature DC take up SL into neutral or mildly acidic compartments distinct from endocytic vacuoles. These DC presented SL-encapsulated protein to both CD4(+) and CD8(+) T cells in vitro. Although CD4(+) T-cell responses were comparable to those induced by soluble protein, CD8(+) T-cell proliferation was up to 300-fold stronger when DC had been pulsed with SL-encapsulated ovalbumin. DC processed SL-encapsulated antigen through a TAP-dependent mechanism. Immunization of mice with SL-encapsulated ovalbumin led to antigen presentation by DC in vivo and stimulated greater CD8(+) T-cell responses than immunization with soluble protein or with conventional or positively charged liposomes carrying ovalbumin. Therefore, the application of SL-encapsulated antigens offers a novel effective, safe vaccine approach if a combination of CD8(+) and CD4(+) T-cell responses is desired (ie, in anti-viral or anti-tumor immunity).

Adoptive Transfer↗

PubMed2000

In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit.

Mechanosensory signaling mediated by mechanically gated ion channels constitutes the basis for the senses of touch and hearing and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. We report a genetically based structure-function analysis of the candidate mechanotransducing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. We identify molecular lesions in 40 EMS-induced mec-4 alleles and further probe residue and domain function using site-directed approaches. Our analysis highlights residues and subdomains critical for MEC-4 activity and suggests possible roles of these in channel assembly and/or function. We describe a class of substitutions that disrupt normal channel activity in touch transduction but remain permissive for neurotoxic channel hyperactivation, and we show that expression of an N-terminal MEC-4 fragment interferes with in vivo channel function. These data advance working models for the MEC-4 mechanotransducing channel and identify residues, unique to MEC-4 or the MEC-4 degenerin subfamily, that might be specifically required for mechanotransducing function. Because many other substitutions identified by our study affect residues conserved within the DEG/ENaC channel superfamily, this work also provides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large number of eukaryotic channel classes, we suggest that similar genetic-based structure-activity studies might be generally applied to generate insight into the in vivo function of diverse channel types.

Amino Acid Sequence↗

PubMed2000

The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits.

LPG2 (a gene involved in lipophosphoglycan assembly) encodes the Golgi GDP-Man transporter of the protozoan parasite Leishmania and is a defining member of a new family of eukaryotic nucleotide-sugar transporters (NSTs). Although NST activities are widespread, mammalian cells lack a GDP-Man NST, thereby providing an ideal heterologous system for probing the LPG2 structure and activity. LPG2 expression constructs introduced into either mammalian cells or a Leishmania lpg2(-) mutant conferred GDP-Man, GDP-Ara, and GDP-Fuc (in Leishmania only) uptake in isolated microsomes. LPG2 is the first NST to be associated with multiple substrate specificities. Uptake activity showed latency, exhibited an antiport mechanism of transport with GMP, and was susceptible to the anion transport inhibitor DIDS. The apparent K(m) for GDP-Man uptake was similar in transfected mammalian cells (12.2 microM) or Leishmania (6.9 microM). Given the evolutionary distance between protozoans and vertebrates, these data suggest that LPG2 functions autonomously to provide transporter activity. Using epitope-tagged LPG2 proteins, we showed the existence of hexameric LPG2 complexes by immunoprecipitation experiments, glycerol gradient centrifugation, pore-limited native gel electrophoresis, and cross-linking experiments. This provides strong biochemical evidence for a multimeric complex of NSTs, a finding with important implications to the structure and specificity of NSTs in both Leishmania and other organisms. Inhibition of essential GDP-Man uptake in fungal and protozoan systems offers an attractive target for potential chemotherapy.

Animals↗

PubMed2000

In vitro characterization of a novel polymeric-based pH-sensitive liposome system.

This study demonstrates rapid and pH-sensitive release of a highly water-soluble fluorescent aqueous content marker, pyranine, from egg phosphatidylcholine liposomes following incorporation of N-isopropylacrylamide (NIPA) copolymers in liposomal membranes. The pH-sensitivity of this system correlates with the precipitation of the copolymers at acidic pH. In vitro release can be significantly improved by increasing the percentage of anchor in the copolymer and thus favoring its binding to the liposomal bilayer. In the case of liposomes containing a poly(ethylene glycol)-phospholipid conjugate, the insertion of the pH-sensitive copolymer in the liposomal membrane appears to be sterically inhibited. Dye release from these formulations at acidic pH can still be achieved by varying the anchor molar ratio and/or molecular mass of the polymers or by including the latter during the liposome preparation procedure. Removal of unbound polymer results in decreased leakage only when the copolymer is inserted by incubation with preformed liposomes, but can be overcome by preparing liposomes in the presence of polymer. Aqueous content and lipid mixing assays suggest contents release can occur without membrane fusion. The results of this study indicate that the addition of pH-sensitive copolymers of NIPA represents promising strategy for improving liposomal drug delivery.

Acrylamides↗

PubMed2000

Calcium signalling in the guidance of nerve growth by netrin-1.

Pathfinding by growing axons in the developing nervous system is guided by diffusible or bound factors that attract or repel the axonal growth cone. The cytoplasmic signalling mechanisms that trigger the responses of the growth cone to guidance factors are mostly unknown. Previous studies have shown that the level and temporal patterns of cytoplasmic Ca2+ can regulate the rate of growth-cone extension in vitro and in vivo. Here we report that Ca2+ also mediates the turning behaviour of the growth cones of cultured Xenopus neurons that are induced by an extracellular gradient of netrin-1, an established diffusible guidance factor in vivo. The netrin-1-induced turning response depends on Ca2+ influx through plasma membrane Ca2+ channels, as well as Ca2+-induced Ca2+ release from cytoplasmic stores. Reduction of Ca2+ signals by blocking either of these two Ca2+ sources converted the netrin-1-induced response from attraction to repulsion. Activation of Ca2+-induced Ca2+ release from internal stores with a gradient of ryanodine in the absence of netrin-1 was sufficient to trigger either attractive or repulsive responses, depending on the ryanodine concentration used. These results support the model that cytoplasmic Ca2+ signals mediate growth-cone guidance by netrin-1, and different patterns of Ca2+ elevation trigger attractive and repulsive turning responses.

Animals↗

PubMed2000

Liposome targeting to tumors using vitamin and growth factor receptors.

Liposome-encapsulated anticancer drugs reveal their potential for increased therapeutic efficacy and decreased nonspecific toxicities due to their ability to enhance the delivery of chemotherapeutic agents to solid tumors. Advances in liposome technology have resulted in the development of ligand-targeted liposomes capable of selectively increasing the efficacy of carried agents against receptor-bearing tumor cells. Receptors for vitamins and growth factors have become attractive targets for ligand-directed liposomal therapies due to their high expression levels on various forms of cancer and their ability to internalize after binding to the liposomes conjugated to receptors' natural ligands (vitamins) or synthetic agonists (receptor-specific antibodies and synthetic peptides). This chapter summarizes various strategies and advances in targeting liposomes to vitamin and growth factor receptors in vitro and in vivo with special emphasis on two extensively studied liposome-targeting systems utilizing folate receptor and HER2/neu growth factor receptor.

Antineoplastic Agents↗

PubMed2000

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources.

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C6), 3-hydroxyoctanoate (HO or C8), and/or 3-hydroxydecanoate (HD or C10) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97-99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.

Glucose↗

PubMed2000

Nicorandil attenuates both temporal and spatial repolarization alternans.

T-wave alternans (TWA) on the electrocardiogram have been frequently associated with long QT syndrome (LQTS) and abrupt rate change. The present study investigated the effect of the potassium channel opener nicorandil on the repolarization alternans at the endocardium and the epicardium in the left ventricle. Electrocardiogram and transmural monophasic action potentials from the endocardium and the epicardium were simultaneously recorded in Langendorff-perfused guinea pig hearts. The hearts were paced at a basic cycle length (BCL) of 240 ms and the cycle length (CL) was abruptly shortened to 170 ms to induce repolarization alternans. Disopyramide and nicorandil were used to increase or attenuate repolarization alternans, respectively. Repolarization alternans were numerically expressed as the sum of the absolute difference between consecutive monophasic action potential durations at 90% repolarization (MAPD90) in the first 10 beats. In the control hearts, the MAPD90 alternans were 78.6 +/- 14.9 ms at the endocardium, and 49.8 +/- 58 ms at the epicardium (P = .03 endocardium vs epicardium). Disopyramide (2 microg/mL) increased the MAPD90 alternans to 186.6 +/- 30.6 ms at the endocardium and 116.4 +/- 16.5 ms at the epicardium, and enhanced the difference of repolarization alternans between the endocardium and the epicardium (transmural dispersion) from 28.8 +/- 11.3 ms to 70.2 +/- 18.7 ms (P = .02 vs controls). Nicorandil (400 ng/mL) suppressed the MAPD90 alternans to 79.6 +/- 16.3 ms at the endocardium and 56.0 +/- 11.8 ms at the epicardium, and attenuated the transmural dispersion to 23.6 +/- 6.0 ms (P = .02 vs disopyramide-administrated hearts). Our results suggest that nicorandil attenuates both temporal (beat-to-beat) and spatial (between the endocardium and the epicardium) repolarization alternans induced by the combination of cycle length changes and disopyramide administration.

Animals↗

PubMed2000

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

Animals↗

PubMed2000

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient.

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia eutropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal sigma 70 promotor. The pKS/PHB was the most efficient plasmid for phb operon expression among the three plasmids used: i.e., pKS-, pAED4, and pJM9131. It was observed that isopropyl-beta-D-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkanoate content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.

Cupriavidus necator↗

PubMed2000

Effect of C:N molar ratio on monomer composition of polyhydroxyalkanoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria. In this study, the effect of C:N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkanoate produced by Pseudomonas pseudoalkaligenus YS1. It was observed that there were some fixed ranges of C:N molar ratio that affect the monomer composition of PHA independently of the substrate. For strain 0806, the ranges were C:N < 20, 20 < C:N < 200, and C:N > 200. The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate. For strain YS1, the ranges were C:N < 20, 20 < C:N < 45, and C:N > 45. These results are useful for controlling monomer composition in PHA production.

3-Hydroxybutyric Acid↗

PubMed2000

Optimal production of polyhydroxyalkanoates in activated sludge biomass.

Polyhydroxyalkanoates (PHAs) have been recognized as good candidates for biodegradable plastics, but their high price compared with conventional plastics has limited their use. In this study, activated sludge microorganisms from a conventional wastewater treatment process were induced, by controlling the carbon:nitrogen (C:N) ratio in the reactor liquor, to accumulate PHAs. In addition, an intermittent nitrogen feeding program was established to optimize the volumetric PHA productivity in a wastewater treatment process. The optimal overall polymer production yield of 0.111 g of polymer/g of carbonaceous substrate consumed was achieved under a C:N ratio of 96:1 by feeding nitrogen in the reactor liquor once every four cycles. At the same time, the amount of excess sludge generated from the wastewater treatment process was reduced by 22.9%.

Bacteria↗

PubMed2000

Involvement of Ca(2+) in antiarrhythmic effect of ischemic preconditioning in isolated rat heart.

We investigated the relationship between the effects of ischemic preconditioning (IPC) and Ca(2+) preconditioning (CPC) on reperfusion-induced arrhythmias. In the control group (noPC), Langendorff-perfused rat hearts were subjected to 5-min zero-flow global ischemia (I) followed by 15-min reperfusion (I/R). In ischemic preconditioning groups (IPC), the hearts were subjected to three cycles of 3-min global ischemia and 5-min reperfusion. In the CPC group, the hearts were exposed to three cycles of 3-min perfusion of higher Ca(2+) (2.3 mmol/l Ca(2+)) followed by 5-min perfusion of normal 1.3 mmol/l Ca(2+), and the hearts were then subjected to I/R. Verapamil was administered in several hearts of the IPC group (VR+IPC). Ventricular arrhythmias upon reperfusion were less frequently seen in the IPC and CPC groups than in the noPC and VR+IPC groups. IPC and CPC could attenuate conduction delay and enhance shortening of the monophasic action potential duration during ischemia. The ventricular fibrillation threshold measured at 1-min reperfusion was significantly higher in the IPC and CPC groups than in the noPC and VR+IPC groups. Verapamil completely abolished the salutary effects of IPC. These results demonstrate that Ca(2+) plays an important role in the antiarrhythmic effect of IPC during reperfusion.

Animals↗

PubMed2000

Emerging antibody-based HER2 (ErbB-2/neu) therapeutics.

Targeting HER2(ErbB-2/neu) overexpressing tumor cells to selectively deliver anticancer agents and thereby reduce host toxicity represents a rational and emerging strategy for the treatment of breast and other epithelial cancers. The extracellular domain of the HER2 receptor tyrosine kinase is readily accessible to systemically administered antibody-based therapeutics, including growth-inhibiting monclonals such as rhuMAbHER2 (trastuzmab/Herceptin) as well as anti-HER2 immunotoxins, antibody-dependent enzyme prodrug therapy (ADEPT), and immune cell recruiting bispecific antibodies. In addition to summarizing recent advances in these antibody-based strategies, this review focuses on preclinical advances in the development of anti-HER2 immunoliposomes (ILs) as a platform technology for targeted drug delivery. Extensive in vitro and in vivo testing including efficacy and tumor uptake studies in multiple human tumor xenograft models now provide conclusive evidence for the superior therapeutic efficacy of anti-HER2 ILs-doxorubicin (dox) over free dox or liposomal (Ls)-dox, and even over combinations of dox and Ls-dox with rhuMAbHER2. As anti-HER2 ILs-dox approaches clinical testing in patients with advanced HER2 overexpressing breast cancer, future applications of this novel targeting strategy will also broaden to include intracellular delivery of other anticancer agents as well as therapeutic nucleic acids (oligonucleotides, genes).

Journal Article↗