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Biomedical subjects

S M Hwang

Publications and source records attributed to S M Hwang.

At least 19 recordsLinked to original sources
PubMed2002

Differential expression of a WD protein during squamous differentiation of tracheal epithelial cells.

The lining of the trachea consists of a pseudostratified, mucociliary epithelium that under a variety of conditions, such as vitamin A deficiency, toxic and mechanical injury, becomes a stratified squamous epithelium. Several in vitro cell culture models have been established to study the process of differentiation of airway epithelium. Such studies have indicated that mucosecretory differentiation of tracheal epithelial cells can be modulated by substratum. This study was undertaken to understand molecular mechanisms of squamous differentiation in tracheal epithelia. Primary cultured tracheal cells grown on uncoated filters were differentiated to single layer of squamous cells, whereas cells were grown as stratified columnar cells on collagen-I coated filters. The responses to secretagogues were altered according to culture conditions. DD-PCR revealed that FAK and a WD protein expression was increased in squamous tracheal epithelia. Expression of a WD protein was changed by the treatment of retinoic acid in various epithelial cells. These results indicated that squamous differentiation of tracheal cells changes the expression of a variety of genes, and that the experimental model for this study can be employed to study molecular mechanisms of squamous differentiation in airway epithelial cells.

Animals↗
PubMed2002

A potent small molecule, nonpeptide inhibitor of cathepsin K (SB 331750) prevents bone matrix resorption in the ovariectomized rat.

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.

Animals↗
PubMed2001

Azepanone-based inhibitors of human and rat cathepsin K.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

Administration, Oral↗
PubMed2001

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro.

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K(i) = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situ cytochemical assay (IC(50) > 1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50) > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K(i) = 0.052 nm) and K (K(i) = 1.57 nm) was also active in both assays (IC(50) = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.

Bone Resorption↗
PubMed2001

Functional and structural changes of the epidermal barrier induced by various types of insults in hairless mice.

Acute barrier disruption, regardless of the method of induction, depletes the stratum corneum intercellular lipids and this stimulates a series of lipid/ DNA synthesis activities which lead to barrier recovery. After barrier disruption by tape stripping, occlusion with a water vapor-impermeable membrane inhibits barrier repair. In this study, we investigated the changes in the murine epidermis after barrier perturbation by tape stripping and three different types of surfactants (Emalex NP-12, ENP-12; sodium dodecyl sulfate, SDS; benzalkonium chloride, BKC). To examine the effect of an artificial barrier, we covered the animals with a water vapor-impermeable membrane for 3 days following barrier disruption and then exposed them to the air for 2 days. The histological findings after occlusion or air exposure were similar. However, after air exposure for 2 days, the thickness of the epidermis including the stratum corneum and the stratum granulosum layers decreased to about half that of the epidermis after occlusion. Ultrastructural examination revealed obvious distortion of the lamellar bilayers within the stratum corneum interstices immediately after barrier disruption. After 3 days of occlusion, extensive disorganization was evident in the intercellular domain of the stratum corneum, whereas 2 days after removal of the occlusion, the normal basic unit structure of the lamellar bilayers had partially reappeared. Our findings provide evidence that the kinetic pattern of barrier repair and the morphological changes are similar after occlusion following barrier disruption regardless of the mechanism of disruption.

Animals↗
PubMed2001

Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae.

Androstadienes↗
PubMed2001

Basis of occlusive therapy in psoriasis: correcting defects in permeability barrier and calcium gradient.

BACKGROUND: Although occlusive dressings have great potential in the management of psoriasis vulgaris, the therapeutic mechanism is not completely understood. Occlusion artificially restores and corrects the defective barrier in psoriasis plaques. Additionally, occlusion is know to normalize the epidermal calcium gradients in hyperproliferative murine skin models. METHODS: To investigate the basis of the therapeutic effect of occlusion on psoriatic plaques, we investigated the ultrastructural morphology of intercorneocyte lipid layers, lamellar bodies, and calcium gradient in chronic plaque-type psoriasis after occlusion with a water vapor-impermeable membrane. The specimens were processed for electron microscopy using: (i) ruthenium tetroxide postfixation; and (ii) ion-capture cytochemistry for calcium localization. RESULTS: Occlusion for 7 days resulted in a nearly mature pattern of intercellular multilamellar structures, re-establishment of the near-normal epidermal calcium gradient, and disappearance of calcium precipitates from the stratum corneum interstices. CONCLUSIONS: The normalization of the permeability barrier and epidermal calcium gradient may play important roles in the therapeutic effects of occlusive dressings in chronic plaque-type psoriasis.

Calcium↗
PubMed2001

The effects of famciclovir and epidural block in the treatment of herpes zoster.

In our previous study, we concluded that an epidural blockade combined with intravenous acyclovir is very effective in treating the acute pain in herpes zoster and postherpetic neuralgia. We evaluated the efficacy of oral famciclovir and epidural blockade on the pain of herpes zoster, compared to acyclovir administered intravenously and epidural blockade. For this purpose, we examined a new group treated with famciclovir and epidural blockade to compare with the group treated with acyclovir and epidural blockade in our previously study. The changes in the intensity of pain, the number of days required for relief of pain, and the total duration of pain were checked. We compared the days required for relief of pain (DRP) and the total duration of pain (TDP) of this group with those of the previous studied group treated with acyclovir and epidural blockade. DRP was significantly less, but TDP was similar. DRP and TDP were significantly lower, if the patients were treated within 7 days of symptom onset. The patients had a shorter DRP regardless of pain type than the previously studied group treated with acycolvir and epidural blockade. For the severe and moderate pain grades, there was a shorter DRP from 100 to 10. TDP was not significantly different for the groups regardless of pain type or grade. We believe that famciclovir and epidural blockade are very effective in treating the pain of herpes zoster, with a view to shortening the period of acute pain, providing similar effects on the prevention of postherpetic neuralgia, and being convenient to administer, compared to intravenous acyclovir and epidural blockade in our previous study.

2-Aminopurine↗
PubMed2001

Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat.

An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic alpha(v)beta3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds alpha(v)beta3 and the closely related integrin alpha(v)beta5 with low nanomolar affinity but binds only weakly to the related integrins alpha(IIb)beta3, and alpha5beta1. Compound 1 inhibited alpha(v)beta3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 approximately 20 microM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (microCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the alpha(v)beta3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.

Animals↗
PubMed2001

Human osteoclast cathepsin K is processed intracellularly prior to attachment and bone resorption.

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.

Biochemistry↗
PubMed2000

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages.

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.

Animals↗
PubMed2000

Structural and functional effects of oleic acid and iontophoresis on hairless mouse stratum corneum.

The aim of this study was to assess the effects of chemical and electrical modes of percutaneous penetration enhancement on the intercellular lipid lamellae of the stratum corneum. Hairless mice were treated with either oleic acid/propylene glycol and iontophoresis separately or together. Permeability barrier function was evaluated by measuring transepidermal water loss and correlated with the structure of stratum corneum intercellular lamellae, as evaluated by electron microscopy, using ruthenium tetroxide postfixation. Transepidermal water loss levels did not change following 1 h iontophoresis alone. In contrast, topical applications of 0.3 M oleic acid in propylene glycol for 1 h increased transepidermal water loss significantly. Moreover, the combined use of iontophoresis plus 0.3 M oleic acid for 1 h further increased transepidermal water loss at equivalent time points. Ultrastructural observations demonstrated both marked disorganization of the intercellular lipid lamellae, as well as the presence of distended lacunae within the stratum corneum in oleic acid/propylene glycol plus or minus iontophoresis-treated stratum corneum. This study provides direct evidence that the oleic acid/propylene glycol system can disrupt the stratum corneum lipid lamellar structures, and that coapplications of oleic acid with iontophoresis further enhance the effects of oleic acid. The synergy between chemical and physical enhancement may afford a new approach to promote transdermal drug delivery.

Animals↗
PubMed1999

Characterization of the DNA binding and bending HMG domain of the yeast hypoxic repressor Rox1.

The yeast Rox1 hypoxic transcriptional repressor protein binds to and bends a specific DNA sequence through an HMG domain located at the N-terminus. To better understand the structure of Rox1 and how it interacts with DNA, 38 missense mutations in the HMG domain were isolated through a combination of random and site-directed mutageneses, the latter directed to two Ile residues that play an important role in DNA recognition and bending by HMG domains. The mutants were characterized in terms of their ability to repress the hypoxic gene ANB1 and the auto-repressed ROX1 gene in vivo. The mutant HMG domains were fused to maltose binding protein and expressed in and purified from Escherichia coli and their relative affinities for DNA and ability to bend DNA were determined. A model of the structure of the Rox1 HMG domain was derived using sequence similarities between Rox1 and the human protein SRY, the structure of which has been determined. The results of the mutational analysis are interpreted in terms of the model structure of Rox1.

Amino Acid Sequence↗
PubMed1999

Orally bioavailable nonpeptide vitronectin receptor antagonists containing 2-aminopyridine arginine mimetics.

A peptide RGD analog containing a novel 2-aminopyridine arginine mimetic was discovered to have good affinity and selectivity for the vitronectin receptor. Incorporation of the 2-aminopyridine arginine mimetic into the 3-oxo-1,4-benzodiazepine-2-acetic acid integrin antagonist series led to novel and potent nonpeptide vitronectin receptor antagonists with promising levels of oral bioavailability.

Administration, Oral↗
PubMed1999

Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions.

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

Amino Acid Sequence↗
PubMed1999

Conformational preferences in a benzodiazepine series of potent nonpeptide fibrinogen receptor antagonists.

Previously, we reported the direct design of highly potent nonpeptide 3-oxo-1,4-benzodiazepine fibrinogen receptor antagonists from a constrained, RGD-containing cyclic semipeptide. The critical features incorporated into the design of these nonpeptides were the exocyclic amide at the 8-position which overlaid the Arg carbonyl, the phenyl ring which maintained an extended Gly conformation, and the diazepine ring which mimicked the gamma-turn at Asp. In this paper, we investigate conformational preferences of the 8-substituted benzodiazepine analogues by examining structural modifications to both the exocyclic amide and the seven-membered diazepine ring and by studying the conformation of the benzodiazepine ring using molecular modeling, X-ray crystallography, and NMR. We found that the directionality of the amide at the 8-position had little effect on activity and the (E)-olefin analogue retained significant potency, indicating that the trans orientation of the amide, and not the carbonyl or NH groups, made the largest contribution to the observed activity. For the diazepine ring, with the exception of the closely analogous 3-oxo-2-benzazepine ring system described previously, all of the modifications led to a significant reduction in activity compared to the potent 3-oxo-1, 4-benzodiazepine parent ring system, implicating this particular type of ring system as a desirable structural feature for high potency. Energy minimizations of a number of the modified analogues revealed that none could adopt the same low-energy conformation as the one shared by the active (S)-isomer of the 3-oxo-1, 4-benzodiazepines and 3-oxo-2-benzazepines. The overall data suggest that the features contributing to the observed high potency in this series are the orientation of the 3-4 amide and the conformational constraint imposed by the seven-membered ring, both of which position the key acidic and basic groups in the proper spatial relationship.

Benzodiazepines↗