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Biomedical subjects

M Hong

Publications and source records attributed to M Hong.

At least 37 records · Page 2Linked to original sources
PubMed2001

TAK1 is a ubiquitin-dependent kinase of MKK and IKK.

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Solid-state NMR investigation of the dynamics of the soluble and membrane-bound colicin Ia channel-forming domain.

Solid-state NMR spectroscopy was employed to study the molecular dynamics of the colicin Ia channel domain in the soluble and membrane-bound states. In the soluble state, the protein executes small-amplitude librations (with root-mean-square angular fluctuations of 0-10 degrees ) in the backbone and larger-amplitude motions (16-17 degrees ) in the side chains. Upon membrane binding, the motional amplitudes increase significantly for both the backbone (12-16 degrees ) and side chains (23-29 degrees ), as manifested by the reduction in the C-H and H-H dipolar couplings and (15)N chemical shift anisotropy. These motions occur not only on the pico- to nanosecond time scales, but also on the microsecond time scale, as revealed by the (1)H rotating-frame spin-lattice relaxation times. Average motional correlation times of 0.8 and 1.2 micros were extracted for the soluble and membrane-bound states, respectively. In comparison, both forms of the colicin Ia channel domain are completely immobile on the millisecond scale. These results indicate that the colicin Ia channel domain has enhanced conformational mobility in the lipid bilayer compared to the soluble state. This membrane-induced mobility increase is consistent with the loss of tertiary structure of the protein in the membrane, which was previously suggested by the extended helical array model [Zakharov et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4282-4287]. An extended structure would also facilitate protein interactions with the mobile lipids and thus increase the protein internal motions. We speculate that the large mobility of the membrane-bound colicin Ia channel domain is a prerequisite for channel opening in the presence of a voltage gradient.

Anisotropy↗
PubMed2001

Sugar-lectin interactions investigated through affinity capillary electrophoresis.

The affinity interactions of Concanavalin A (Con A) with various saccharide oligomers (dextrins, dextrans, and selected N-linked glycans from various glycoproteins) have been investigated through a capillary electrophoresis approach. Con A has shown a notable binding discrimination between the alpha-1,6-linked dextran and alpha-1,4-linked dextrin oligomers. Both the binding capacity and binding discrimination appear to decrease with an increase in sugar chainlength. While the core structure of N-linked glycans is deemed to be responsible for the overall binding of various glycans to Con A, the presence of mannose units at the non-reducing ends was found to be very beneficial to the affinity interaction with Con A. Finally, a connection between the glycan-lectin interaction and glycoprotein-lectin interaction has also been suggested.

Carbohydrates↗
PubMed2001

Short KIR haplotypes in pygmy chimpanzee (Bonobo) resemble the conserved framework of diverse human KIR haplotypes.

Some pygmy chimpanzees (also called Bonobos) give much simpler patterns of hybridization on Southern blotting with killer cell immunoglobulin-like receptor (KIR) cDNA probes than do either humans or common chimpanzees. Characterization of KIRs from pygmy chimpanzees having simple and complex banding patterns identified nine different KIRs, representing seven genes. Five of these genes have orthologs in the common chimpanzee, and three of them (KIRCI, KIR2DL4, and KIR2DL5) also have human orthologs. The remaining two genes are KIR3D paralogous to the human and common chimpanzee major histocompatibility complex A- and/or -B-specific KIRs. Within a pygmy chimpanzee family, KIR haplotypes were defined. Simple patterns on Southern blot were due to inheritance of "short" KIR haplotypes containing only three KIR genes, KIRCI, KIR2DL4, and KIR3D, each of which represents one of the three major KIR lineages. These three genes in pygmy chimpanzees or their corresponding genes in humans and common chimpanzees form the conserved "framework" common to all KIR haplotypes in these species and upon which haplotypic diversity is built. The fecundity and health of individual pygmy chimpanzees who are homozygotes for short KIR haplotypes attest to the viability of short KIR haplotypes, indicating that they can provide minimal, essential KIRs for the natural killer and T cells of the hominoid immune system.

Amino Acid Sequence↗
PubMed2001

Sensitivity-enhanced static 15N NMR of solids by 1h indirect detection.

A method for enhancing the sensitivity of 15N spectra of nonspinning solids through 1H indirect detection is introduced. By sampling the 1H signals in the windows of a pulsed spin-lock sequence, high-sensitivity 1H spectra can be obtained in two-dimensional (2D) spectra whose indirect dimension yields the 15N chemical shift pattern. By sacrificing the 1H chemical shift information, sensitivity gains of 1.8 to 2.5 for the 15N spectra were achieved experimentally. A similar sensitivity enhancement was also obtained for 2D (15)N-(1)H dipolar and 15N chemical shift correlation spectroscopy, by means of a 3D 1H/15N-1H/15N correlation experiment. We demonstrate this technique, termed PRINS for proton indirectly detected nitrogen static NMR, on a crystalline model compound with long 1H T(1rho) and on a 25-kDa protein with short 1H T(1rho). This 1H indirect detection approach should be useful for enhancing the sensitivity of 15N NMR of oriented membrane peptides. It can also be used to facilitate the empirical optimization of 15N-detected experiments where the inherent sensitivity of the sample is low.

Colicins↗
PubMed2001

Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state NMR spectroscopy.

The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.

Antimicrobial Cationic Peptides↗
PubMed2001

Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins.

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving 13C and 15N spins have been developed for this purpose, H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular 1H-13C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved 1H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR. simplifies the 1H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced 1H chemical shift resolution allows the assignment of overlapping H alpha and H beta resonances in Ser, identifies the 1H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.

Carbon Isotopes↗
PubMed2001

Bovine follicular fluid and serum share a unique isoform of matrix metalloproteinase-2 that is degraded by the oviductal fluid.

Whereas the mammalian fertilization environment consists of possible products of the mutual interaction between oviductal and follicular fluids in addition to both fluid components, little is known regarding the interaction. In the present study, we have demonstrated that a mutual interaction occurs, resulting in the biochemical changes of follicular fluid components. Gelatin zymographic analyses of bovine follicular fluid (bFF) showed consistently a distinct, gelatinolytic activity having a molecular weight of 110 kDa (GA110) in addition to other gelatinases, whereas bovine oviductal fluid (bOF) showed a lack of GA110. Surprisingly, when bFF was mixed with bOF before zymography, the GA110 of bFF mostly disappeared at a 1:1 (v/v) mixture, completely disappeared at a 1:10 mixture, as fast as within 30 min after mixing. Other bFF gelatinase activities were not affected by bOF at 1:1 or 10:1 mixtures. Addition of EDTA or phenanthroline, but not of phenylmethylsulfonyl fluoride or trypsin inhibitor, to the mixture greatly increased the gelatinolytic activity of bFF GA110. The increased activity of bFF GA110 by EDTA was again abolished by subsequent bOF treatment. Addition of aminophenylmercuric acetate to the EDTA-treated bFF also abolished GA110; however, this was accompanied by the disappearance of other gelatinases, except the 62-kDa gelatinase, the activity of which increased as the treatment continued up to 24 h. Addition of EDTA or phenanthroline to the gelatin gel incubation buffer after electrophoresis abolished almost all gelatinases of bFF, except those of 88-84 kDa, demonstrating that they were indeed gelatinases or isoforms. Bovine serum and fetal bovine serum also showed the presence of GA110, the activity of which was increased by EDTA. However, ovarian granulosa cell homogenate did not exhibit GA110. Immunoblot experiments using antibodies against matrix metalloproteinase (MMP)-2 and MMP-9 demonstrated that bFF GA110 was an isoform of MMP-2, and that the 62-kDa form was an active form of MMP-2. Disappearance of immunoreactive GA110 of bFF and serum by bOF was also observed. Based on these observations, we conclude that bFF and bovine serum share a unique isoform of MMP-2, and that bOF can specifically degrade the isoform, suggesting that a mutual interaction between bFF and bOF could occur at the time of ovulation.

Animals↗
PubMed2001

dksA is required for intercellular spread of Shigella flexneri via an RpoS-independent mechanism.

Pathogenesis of Shigella flexneri is dependent on the ability of the bacterium to invade and spread within epithelial cells. In this study, we identified dksA as a gene necessary for intercellular spread in, but not invasion of, cultured cells. The S. flexneri dksA mutant exhibited sensitivity to acid and oxidative stress, in part due to an effect of DksA on production of RpoS. However, an S. flexneri rpoS mutant formed plaques on tissue culture monolayers, thus excluding DksA regulation of RpoS as the mechanism responsible for the inability of the dksA mutant to spread intercellularly. Intracellular analysis of the dksA mutant indicates that it survived and divided within the Henle cell cytoplasm, but the dksA mutant cells were elongated, and some exhibited filamentation in the intracellular environment. Some of the S. flexneri dksA mutant cells showed aberrant localization of virulence protein IcsA, which may inhibit spread between epithelial cells.

Bacterial Proteins↗
PubMed2001

Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.

Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been suggested to participate in chronic disorders, such as diabetes and its complications. In contrast to the short and transient activation of NF-kappaB in vitro, we observed a long-lasting sustained activation of NF-kappaB in the absence of decreased IkappaBalpha in mononuclear cells from patients with type 1 diabetes. This was associated with increased transcription of NF-kappaBp65. A comparable increase in NF-kappaBp65 antigen and mRNA was also observed in vascular endothelial cells of diabetic rats. As a mechanism, we propose that binding of ligands such as advanced glycosylation end products (AGEs), members of the S100 family, or amyloid-beta peptide (Abeta) to the transmembrane receptor for AGE (RAGE) results in protein synthesis-dependent sustained activation of NF-kappaB both in vitro and in vivo. Infusion of AGE-albumin into mice bearing a beta-globin reporter transgene under control of NF-kappaB also resulted in prolonged expression of the reporter transgene. In vitro studies showed that RAGE-expressing cells induced sustained translocation of NF-kappaB (p50/p65) from the cytoplasm into the nucleus for >1 week. Sustained NF-kappaB activation by ligands of RAGE was mediated by initial degradation of IkappaB proteins followed by new synthesis of NF-kappaBp65 mRNA and protein in the presence of newly synthesized IkappaBalpha and IkappaBbeta. These data demonstrate that ligands of RAGE can induce sustained activation of NF-kappaB as a result of increased levels of de novo synthesized NF-kappaBp65 overriding endogenous negative feedback mechanisms and thus might contribute to the persistent NF-kappaB activation observed in hyperglycemia and possibly other chronic diseases.

Adult↗
PubMed2001

A novel zidovudine uptake system in microglia.

In the central nervous system (CNS), brain macrophages and microglia are the primary targets of productive human immunodeficiency virus 1 (HIV-1) infection. Zidovudine (ZDV), a thymidine derivative, has been reported to reduce the progression of the disease and prolong survival in patients with acquired immunodeficiency syndrome (AIDS) and AIDS dementia complex. Although a restricted ZDV distribution has been observed in the CNS, its accumulation in brain parenchyma has not been examined. We have investigated the uptake properties of radiolabeled ZDV by a continuous rat microglia cell line (MLS-9) grown as a monolayer on an impermeable surface. Although the organic cations verapamil, mepiperphenidol, quinidine, cimetidine, and N(1)-methylnicotinamide moderately inhibited ZDV uptake, the organic cation probes tetraethylammonium and 1-methyl-4-phenylpyridinium were weak inhibitors. ZDV uptake was significantly increased when the proton gradient was outward (pH(i) 6.3 < pH(o) 7.4; pH(i) approximately 7.1 < pH 8.0), whereas uptake decreased with extracellular acidification (pH(i) approximately 7.1 > pH(o) 6.0) or in the presence of the Na(+)/H(+) ionophore monensin. ZDV uptake was increased under depolarized membrane conditions (i.e., 138 mM K(+) in external medium) and decreased under hyperpolarized conditions (i.e., 2 mM K(+) in external medium), implying a membrane potential dependence. These results suggest that although ZDV transport system in microglia has some specificity features of an organic cation transporter, it involves a carrier, distinct from other cloned organic cation transporters, that is novel in its sensitivity to pH and membrane potential. This system may play a significant role in the transport of other weak organic cation substrates and/or metabolites in brain parenchyma.

Algorithms↗
PubMed2001

Drug transporters in the central nervous system: brain barriers and brain parenchyma considerations.

Drug transport in the central nervous system is highly regulated not only by the blood-brain and the blood-cerebrospinal fluid barriers but also in brain parenchyma. The novel localization of drug transporters in brain parenchyma cells, such as microglia and astrocytes, suggest a reconsideration of the present conceptualization of brain barriers as it relates to drug transport. That is, the cellular membranes of parenchyma cells act as a second "barrier" to drug permeability and express transporters whose properties appear similar to those localized at the conventional brain barriers. This review will focus on the molecular characteristics, localization, and substrate specificities of several classes of well known membrane drug transporters (i.e., the organic cation, organic anion, nucleoside, P-glycoprotein, and multidrug resistance proteins) in the brain. Comparisons to similar transporters localized within the peripheral system and clinical implications of the functional expression of specific drug transport families will be discussed when appropriate. Nutrient and neurotransmitter transporters, whose characteristics have been reviewed extensively elsewhere, will not be considered in this review.

Animals↗
PubMed2001

[Clinicopathologic analysis of rhinoscleroma].

OBJECTIVE: To investigate the clinicopathologic characteristics and reason of tissue injury caused by klebsiella rhinoscleromatis(KR) infection. METHODS: Twenty-four cases of rhinoscleromas in the nasal and pharyngeal region in this hospital from March 1983 to March 1998 were studied retrospectively. Warthin-Starry (W-S) stain was used in all cases and CD43, CD20, CD68, Lysozyme mark were observed in eleven specimens by ABC immunochemical method. Two cases were studied further with transmission electron microscopy. RESULTS: A great number of KR were found in Mikulicz cells and were confirmed further in the phagosomes in cytoplasm of Mikulicz cells, where a small number of endoplasmic reticulums and lysozymes was squeezed to the side of cells. A few of T-lymphocytes was found within the granuloma. CONCLUSION: Cellular immunity function of the patient with rhinoscleroma was depressed and the response to immunization of host which regards to KR was the major cause of tissue injury.

Adolescent↗
PubMed2000

Simultaneous intrastriatal and intranigral dopaminergic grafts in the parkinsonian rat model: role of the intranigral graft.

The current transplantation strategy in experimental and clinical Parkinson's disease (PD) has been to place nigral dopaminergic grafts not in their ontogenic site (substantia nigra) but in their target area (striatum). Although intrastriatal dopaminergic grafts are capable of reinnervating the striatum, they fail to reinnervate the nigra, which may be an important factor limiting the efficacy of fetal tissue transplantation in parkinsonian patients. We have previously shown that simultaneous intrastriatal and intranigral dopaminergic grafts (double grafts) may provide a more complete restoration of the nigrostriatal circuitry (Mendez et al. [1996] J Neurosci 16:7216-7227; Mendez and Hong [1997] Brain Res 778:194-205). In the present study, we investigated the contribution of the intranigral graft to functional recovery in double-grafted hemiparkinsonian rats. Twenty Wistar rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway were divided into two groups and received either double grafts (n = 10) or intrastriatal grafts alone (n = 10). Following transplantation, both intrastriatally and double-grafted animals had a significant decrease in rotational behavior. However, only animals with double grafts exhibited a significant increase in contralateral adjusting step performance. The intranigral graft was subsequently lesioned by a second 6-OHDA injection. Following the second lesion, animals with double grafts exhibited a significant reversal of rotational behavior and a 51% reduction in contralateral adjusting step performance. The reversal in functional recovery correlated with a significant loss of intranigral grafted neurons. These results suggest that the intranigral graft has an important role in the functional recovery of double-grafted animals. Restoration of dopaminergic innervation to both the nigra and the striatum may be crucial for optimizing graft efficacy and may be a superior strategy in neural transplantation for PD.

Amphetamine↗