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Biomedical subjects

L Hong

Publications and source records attributed to L Hong.

At least 19 recordsLinked to original sources
PubMed2001

The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site.

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c(552) with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c(552) from the apo state on the addition of heme has been demonstrated using NMR techniques. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.

Bacteria↗
PubMed2001

Structure-based design: potent inhibitors of human brain memapsin 2 (beta-secretase).

Memapsin 2 (beta-secretase) is one of two proteases that cleave the beta-amyloid precursor protein (APP) to produce the 40-42 residue amyloid-beta peptide (Abeta) in the human brain, a key event in the progression of Alzheimer's disease. On the basis of the X-ray crystal structure of our lead inhibitor (2, OM99-2 with eight residues) bound to memapsin, we have reduced the molecular weight and designed potent memapsin inhibitors. Structure-based design and preliminary structure-activity studies have been presented.

Amyloid Precursor Protein Secretases↗
PubMed2001

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design.

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].

Alzheimer Disease↗
PubMed2001

MUP-4 is a novel transmembrane protein with functions in epithelial cell adhesion in Caenorhabditis elegans.

Tissue functions and mechanical coupling of cells must be integrated throughout development. A striking example of this coupling is the interactions of body wall muscle and hypodermal cells in Caenorhabditis elegans. These tissues are intimately associated in development and their interactions generate structures that provide a continuous mechanical link to transmit muscle forces across the hypodermis to the cuticle. Previously, we established that mup-4 is essential in embryonic epithelial (hypodermal) morphogenesis and maintenance of muscle position. Here, we report that mup-4 encodes a novel transmembrane protein that is required for attachments between the apical epithelial surface and the cuticular matrix. Its extracellular domain includes epidermal growth factor-like repeats, a von Willebrand factor A domain, and two sea urchin enterokinase modules. Its intracellular domain is homologous to filaggrin, an intermediate filament (IF)-associated protein that regulates IF compaction and that has not previously been reported as part of a junctional complex. MUP-4 colocalizes with epithelial hemidesmosomes overlying body wall muscles, beginning at the time of embryonic cuticle maturation, as well as with other sites of mechanical coupling. These findings support that MUP-4 is a junctional protein that functions in IF tethering, cell-matrix adherence, and mechanical coupling of tissues.

Animals↗
PubMed2001

Inhibition of endothelial cell migration by cerivastatin, an HMG-CoA reductase inhibitor: contribution to its anti-angiogenic effect.

Recent studies have suggested that inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (statins) can play a role in protection against vascular risk, which is independent of cholesterol reduction. It could act by inhibiting the synthesis of isoprenoids (farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)), which are respectively essential for membrane attachment and biological activity of GTPases Ras and RhoA. This study demonstrates that a statin (cerivastatin) inhibits angiogenesis. This effect was due to a decrease in endothelial cell locomotion which was reversed by GGPP. It was mainly related to delocalization of RhoA from cell membrane to cytoplasm, responsible for the disorganization of actin stress fibers. Furthermore, a decrease in MMP-2 secretion, involved in cell invasion, was also observed. This effect is rather due to Ras inhibition as it was reversed by FPP. This anti-angiogenic activity could explain the beneficial effect of statins on atherosclerosis and on cancer prevention as shown by clinical studies.

Actins↗
PubMed2001

Determination of assay and impurities of gamma irradiated chloramphenicol in eye ointment.

A sample preparation method was developed to isolate chloramphenicol and its radiolytic products from an oily ointment base. The isolation method suspended the eye ointment in n-hexane at 45 degrees C, and isolated the target compounds as residue by centrifugation. It was found that the main element to ensure a satisfactory isolation was keeping the sample solution at 45 degrees C during sample preparation. Linearity, precision, accuracy and suitability of the method were confirmed valid for both assay and impurity tests. This isolation method was ideal for assay, unique for extraction of unexpected and complex radiolysis products, and had a number of advantages compared to the pretreatment methods described in The United Stares Pharmacopoeia and British Pharmacopoeia, in terms of accuracy, precision, and easy handling. The effect of gamma-irradiation on chloramphenicol eye ointment was studied by HPLC, after applying the developed sample preparation method. The present assay and impurity test methods with HPLC were confirmed to be suitable for irradiated chloramphenicol in eye ointment. Formation of radiolytic products induced by gamma-irradiation was evidenced by the impurity test. The assay test showed that active ingredient of chloramphenicol eye ointment decreased by 3.3% at an irradiation dose of 25 kGy and by 11.1% at 50 kGy.

Centrifugation↗
PubMed2001

Enhanced formation of fibrosis in a rabbit aneurysm by gelatin hydrogel incorporating basic fibroblast growth factor.

OBJECTIVE: This study was undertaken to analyze whether the controlled release of basic fibroblast growth factor (bFGF) can promote intrasaccular thrombosis in an experimental aneurysmal model. METHODS: Carotid aneurysms were constructed in 80 rabbits with venous pouches and treated by placing gelatin hydrogels into each aneurysm incorporating 0, 25, 50, or 100 microg of bFGF or incorporating 100 microg of bFGF with different water contents. In the controls, the venous pouches either were not treated or were treated with gauze alone. Gelatin hydrogel was used for the controlled release of bFGF into the aneurysms. The formation of fibrosis in the aneurysms was histologically viewed to assess the area occupied by the fibrous tissues at 3 and 6 weeks after the hydrogel application. The effect of the bFGF dose and water content on obliterating the aneurysm by the hydrogels incorporating bFGF was also investigated. RESULTS: Six weeks after the application of gelatin hydrogels with a water content of 95 wt% incorporating 100 microg of bFGF, the lateral pouch orifice was completely closed, obliterating the aneurysm at the level of tissue appearance, in contrast to hydrogels incorporating lower doses of bFGF and other control agents. The venous pouch aneurysm was histologically occupied with the newly formed fibrous tissue, and the fibrous tissue area and percentage of the aneurysmal lumen occupied by the fibrosis-gauze complex were significantly larger than those of other hydrogel applications (P < 0.05). The neointima tissue was homogeneously covered with a monolayer of Factor VIII-positive cells. The fact that there was no difference in the water content in the fibrosis formation induced by the bFGF-incorporated gelatin hydrogels indicated that the hydrogel biodegradability did not affect the obliteration of the aneurysm. CONCLUSION: Local controlled release of bFGF stimulated the formation of in vivo fibrosis, resulting in obliteration of the aneurysm. The long-term results of the fibrous organization remain speculative.

Animals↗
PubMed2001

A trial to prepare biodegradable collagen-hydroxyapatite composites for bone repair.

This paper is a trial to prepare collagen-hydroxyapatite composites in vitro by an alternate immersion method. Collagen sponges of different biodegradabilities were prepared through chemical cross-linking of Type I collagen with glutaraldehyde (GA) at concentrations of 0.2, 1.0, and 2.0 wt%. The sponges were immersed at 37 degrees C in Tris-HCl-buffered solution containing 200 mM CaCl2 (pH 7.4) for 2 h and then in an aqueous solution of 120 mM Na2HPO4 (pH 9.3) for a 2 h further (one immersion cycle). The alternate immersion cycle was repeated for different times to obtain collagen-hydroxyapatite composites. The characterization of the resulting composites was performed by Fourier transform infrared spectroscopy (FT-IR). X-ray diffraction (XRD), and scanning electron microscopy (SEM). The weight of composites increased with an increase in immersion cycles and the rate of increase became greater with higher GA cross-linking levels for collagen sponge preparation. The pH of the phosphate solution decreased with the immersion cycle, which suggests H+ generation accompanied hydroxyapatite formation. Irrespective of the GA concentration and immersion cycle, every composite showed IR absorption bands attributable to phosphate and hydroxyl groups at 950-1100 or 550-650 and 3000-3500 cm(-1) and broad peaks specific to hydroxyapatite on the XRD charts. SEM study revealed small white clusters of hydroxyapatite interspersed uniformly on/in the collagen framework without any preferential orientation. The composite prepared from 0.2 wt% GA cross-linked collagen sponge which showed favourable characteristics was applied to a rat skull defect to evaluate its osteoconductivity as well as biodegradability. The formation of new bone tissue was histologically observed at the defect 12 weeks after application in marked contrast to the collagen sponge alone. The composite degraded without any inflammation reaction. It is concluded that the collagen-hydroxyapatite composite prepared by the present method is a biodegradable biomaterial of osteoconductivity applicable to bone repair.

Animals↗
PubMed2001

Inhibitory effects of nitric oxide on estrogen production and cAMP levels in rat granulosa cell cultures.

Previous studies demonstrated inhibitory effects of nitric oxide (NO) and cGMP on ovarian steroidogenesis. This study examined the effects of NO on estrogen levels and cAMP accumulation from immature cultured rat granulosa cells. Granulosa cells were incubated with media alone (control), FSH or FSH plus increasing concentrations of the NO generator, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO). While FSH increased estrogen levels 15-fold compared with controls, DETA/NO inhibited FSH-stimulated aromatase activity in a dose-dependent manner. Time-course studies revealed that the inhibitory effects of DETA/NO on aromatase activity persisted throughout the 72 h culture period. Treatment with DETA/NO also inhibited the stimulatory effects of forskolin on estrogen production, indicating that NO can influence steroidogenesis by actions downstream of the FSH receptor. Incubation of cells with FSH plus DETA/NO increased cGMP accumulation over 100-fold, compared with cells treated with media or FSH alone. In this regard, a cGMP analog mimicked the inhibitory effects of NO on FSH- and forskolin-stimulated estrogen production, indicating a potential mechanism of NO action. NO also decreased FSH-stimulated (cAMP) accumulation from cultured cells, indicating an antagonistic effect of NO on the second messenger mediating FSH actions. These findings demonstrate that NO inhibits estrogen production from rat granulosa cells, potentially reflecting actions on the second messengers cGMP and cAMP.

Animals↗
PubMed2001

Regional difference of muscle oxygen saturation and blood volume during exercise determined by near infrared imaging device.

UNLABELLED: Using a near infrared (NIR) imaging device, we tested the hypothesis that regional differences in oxygen status could be detected in the gastrocnemius muscle during exercise and recovery. Six healthy subjects performed the standing plantar flexion exercises for 2 min; the frequency was one contraction per second. The NIR imaging device was placed over the medial head of the right gastrocnemius muscle and the signals from two optical sensors situated on the middle proximal and middle distal portions were used. The NIR-O(2) saturation (difference between deoxygenated and oxygenated Hb signals) and NIR-blood volume (sum of the oxygenated and deoxygenated Hb signals) were calculated in optical density units. Plantar flexion resulted in more deoxygenation during exercise and more reoxygenation during recovery in the distal portion compared with the proximal portion. The changes in NIR-O(2) between rest and a 2 min exercise, and between a 2 min exercise and a 3 min recovery were 0.11 and -0.23, respectively, in the distal portion, which were significantly larger than proximal values (0.05 and -0.10, p < 0.05). Plantar flexion resulted in lower NIR-blood volumes during exercise and greater recovery of blood after exercise in the distal portion compared with the proximal portion. The changes in NIR blood volume between rest and a 2 min exercise and between a 2 min exercise and a 3 min recovery were -0.19 and 0.31, respectively, in the distal portion, significantly larger than proximal values (-0.07 and 0.12, p < 0.05 for all comparisons). These findings indicate that the distal portion of the medial gastrocnemius had larger changes in NIR-O(2) saturation and NIR-blood volume than the proximal portion had. This is consistent with the distal portion having a greater impairment of blood flow possibly because of the higher intramuscular pressure during exercise. IN CONCLUSION: (1) regional differences in oxygen status in the gastrocnemius muscle were detected with exercise, with the distal portion having greater NIR-O(2) saturation and NIR-blood volume changes, and (2) the NIR imaging device might be a useful method to detect the regional differences of oxygen status in the muscle.

Adult↗
PubMed2000

Synthesis and biological activity of novel 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs.

A series of novel haloethyl and piperidyl phosphoramidate FdUMP prodrug analogues has been synthesized, and the growth inhibitory activity of these compounds has been evaluated against L1210 mouse leukemia cells. All compounds exhibited potent inhibition of L1210 cell proliferation with IC(50) values in the nanomolar range. Growth inhibition was reversed by the addition of 5 microM thymidine, suggesting a mechanism of action involving the intracellular release of FdUMP. (31)P NMR studies carried out on model haloethyl phosphoramidates confirm the release of nucleotide via cyclization of the phosphoramidate anion to the aziridinium ion intermediate followed by hydrolysis of the P-N bond. The data suggests that <50% of the prodrug is converted to FdUMP intracellularly by this pathway. Piperidyl phosphoramidate analogues are also converted to nucleotide intracellularly, presumably by the action of an endogenous phosphoramidase.

Amides↗
PubMed2000

High-throughput scanning of the rat genome using interspersed repetitive sequence-PCR markers.

We report the establishment of a hybridization-based marker system for the rat genome based on the PCR amplification of interspersed repetitive sequences (IRS). Overall, 351 IRS markers were mapped within the rat genome. The IRS marker panel consists of 210 nonpolymorphic and 141 polymorphic markers that were screened for presence/absence polymorphism patterns in 38 different rat strains and substrains that are commonly used in biomedical research. The IRS marker panel was demonstrated to be useful for rapid genome screening in experimental rat crosses and high-throughput characterization of large-insert genomic library clones. Information on corresponding YAC clones is made available for this IRS marker set distributed over the whole rat genome. The two existing rat radiation hybrid maps were integrated by placing the IRS markers in both maps. The genetic and physical mapping data presented provide substantial information for ongoing positional cloning projects in the rat.

Animals↗
PubMed2000

Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor.

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease involved in the production of beta-amyloid peptide in Alzheimer's disease and is a major target for drug design. We determined the crystal structure of the protease domain of human memapsin 2 complexed to an eight-residue inhibitor at 1.9 angstrom resolution. The active site of memapsin 2 is more open and less hydrophobic than that of other human aspartic proteases. The subsite locations from S4 to S2' are well defined. A kink of the inhibitor chain at P2' and the change of chain direction of P3' and P4' may be mimicked to provide inhibitor selectivity.

Amyloid Precursor Protein Secretases↗
PubMed2000

IL-11 selectively inhibits aeroallergen-induced pulmonary eosinophilia and Th2 cytokine production.

IL-11 is a pleiotropic cytokine that induces tissue remodeling with subepithelial fibrosis when expressed in the airway. Its effects on the Th2-dominated airway inflammation that is characteristic of asthma, however, are poorly understood. To characterize the effects of IL-11 on Th2 tissue inflammation, we compared the inflammatory responses elicited by OVA in sensitized mice in which IL-11 is overexpressed in a lung-specific fashion (CC10-IL-11) with that in transgene- wild-type littermate controls. Transgene- and CC10-IL-11 transgene+ mice had comparable levels of circulating Ag-specific IgE after sensitization. OVA challenge of sensitized transgene- mice caused airway and parenchymal eosinophilic inflammation, Th2 cell accumulation, and mucus hypersecretion with mucus metaplasia. Exaggerated levels of immunoreactive endothelial cell VCAM-1, mucin (Muc) 5ac gene expression and bronchoalveolar lavage and lung IL-4, IL-5, and IL-13 protein and mRNA were also noted. In contrast, OVA challenge in CC10-IL-11 animals elicited impressively lower levels of tissue and bronchoalveolar lavage inflammation, eosinophilia, and Th2 cell accumulation, and significantly lower levels of VCAM-1 and IL-4, IL-5, and IL-13 mRNA and protein. IL-11 did not cause a comparable decrease in mucus hypersecretion, Muc 5ac gene expression, or the level of expression of RANTES, monocyte chemoattractant protein-2, or monocyte chemoattractant protein-3. In addition, IL-11 did not augment IFN-gamma production demonstrating that the inhibitory effects of IL-11 were not due to a shift toward Th1 inflammation. These studies demonstrate that IL-11 selectively inhibits Ag-induced eosinophilia, Th2 inflammation, and VCAM-1 gene expression in pulmonary tissues.

Administration, Inhalation↗
PubMed2000

Global distribution of the CCR2-64I/CCR5-59653T HIV-1 disease-protective haplotype.

OBJECTIVES: Several natural polymorphisms in the genes for the human CC-chemokine receptors CCR5 and CCR2 are associated with HIV-1 disease. The CCR2-64I genetic variant [a G to A substitution resulting in a valine (V) to isoleucine (I) change at position 64] is in strong linkage disequilibrium with a mutation within the CCR5 regulatory region (CCR5-59653T). Individuals with two CCR2-64I alleles are not resistant to sexual transmission of HIV-1, but progress significantly more slowly to HIV-1 disease. It is therefore important to determine the global distributions of CCR2-64I and CCR5-59653T genetic variants and define the degree of linkage between them. DESIGN AND METHODS: We have developed molecular beacon-based, real-time PCR allele discrimination assays for all three chemokine receptor mutations, and used these spectral genotyping assays to genotype 3923 individuals from a globally distributed set of 53 populations. RESULTS: CCR2-64I and CCR5-59653T genetic variants are found in almost all populations studied: their allele frequencies are greatest (approximately 35%) in Africa and Asia but decrease in Northern Europe. We confirm that CCR2-64I is in strong linkage disequilibrium with CCR5-59653T (96.92% of individuals had the same genotype for both CCR2-64I and CCR5-59653T polymorphisms). CONCLUSIONS: The greater geographical distribution of the CCR2-64I/CCR5-59653T haplotype compared with that of CCR5-delta32 suggests that it is a much older mutation whose origin predates the dispersal of modern humans.

Alleles↗
PubMed2000

A Drosophila complementary DNA resource.

Collections of nonredundant, full-length complementary DNA (cDNA) clones for each of the model organisms and humans will be important resources for studies of gene structure and function. We describe a general strategy for producing such collections and its implementation, which so far has generated a set of cDNAs corresponding to over 40% of the genes in the fruit fly Drosophila melanogaster.

3' Untranslated Regions↗