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Biomedical subjects

S H Chiou

Publications and source records attributed to S H Chiou.

At least 19 recordsLinked to original sources
PubMed2005

Using human plasma supplemented medium to cultivate human bone marrow-derived mesenchymal stem cell and evaluation of its multiple-lineage potential.

The objective of this study was to evaluate the proliferation and the multiple-lineage differentiation capacity when bone marrow mesenchymal stem cells (BMSCs) were cultured short-term in autologous serum/plasma instead of fetal calf serum (FCS). The BMSCs from 12 donors were cultivated individually in 10% autogenic plasma or serum, with or without bFGF and EGF growth factors. Cell proliferation was examined by a Tetrazolium assay (MTT) after passages 1, 3, and 5. A medium supplemented with 10% human plasma or serum was sufficient to propagate BMSCs. However, no significant proliferation was shown when bFGF and EGF (20 ng/mL each) were added into the medium with autologous serum/plasma. We examined, inductions of adipogenesis, osteogenesis, and chondrocytogenesis, as capacities of multiple-lineage differentiation of cultivated BMSCs (passages 8). Differentiation was investigated by both RT-PCR and immunohistochemistry staining (IHC). Qualitative evidence demonstrated the differentiation capacity was preserved in cultivated BMSCs with autologous serum/plasma.

Bone Marrow Cells↗
PubMed2001

Up-regulation of Fas ligand expression by human cytomegalovirus immediate-early gene product 2: a novel mechanism in cytomegalovirus-induced apoptosis in human retina.

Human CMV (HCMV) is an important pathogen that causes widespread diseases in immunocompromised individuals. Among the opportunistic HCMV infections, HCMV retinitis is most common in transplant recipients and AIDS patients. It often leads to blindness if left untreated. The question as to how HCMV infection causes retinal pathogenesis remains unresolved. Here, we report that viral immediate-early gene product 2 (IE2), but not IE1, up-regulates the Fas ligand (FasL) expression in HCMV-infected human retinal pigment epithelium cells. Increased secretion of FasL from virally infected cells into cultured medium was observed upon HCMV infection. The capability of such cell-free medium to induce apoptosis of Fas (CD95)-expressing Jurkat cells further implies that Fas-FasL interaction might mediate cell death in the lesion of HCMV retinitis. To support this idea, we observed augmented soluble FasL levels in vitreous from AIDS patients with HCMV retinitis as compared with that from AIDS patients without HCMV infection. In addition, by in situ hybridization and immunohistochemistry, we detected enhanced signals of FasL, the existence of viral IE Ags and apoptotic cells at the same sites in the lesion of HCMV-infected retina. These results strongly suggest that IE2 induction of FasL expression in human retina might be an important event that takes place in the early stage of infection and finally leads to visual loss in individuals affiliated with HCMV retinitis.

AIDS-Related Opportunistic Infections↗
PubMed2001

Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus.

Bungarus fasciatus fraction IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of B. fasciatus by ion-exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10(-8) m with alpha-chymotrypsin as measured by a BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The three-dimensional NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculations. The final set of 10 NMR structures was well defined, with average root mean square deviations of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A for residues The side chains of Phe(23), Tyr(24), Tyr(25), Phe(35), and Phe(47) exhibited many long-range nuclear Overhauser effects and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among proteins in the BPTI-like superfamily, we compared the three-dimensional structure of BF9 with three BPTI-like proteins that possess distinct biological functions. These proteins possessed similar secondary structure elements, but the loop regions and beta-turn were different from one another. Based on residues at the functional site of each protein, we suggest that the flexibility, rigidity, and variations of the amino acid residues in both the loop and beta-turn regions are related to their biological functions.

Amino Acid Sequence↗
PubMed2001

Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities.

Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.

Amino Acid Sequence↗
PubMed2001

Growth of endothelial cells on different concentrations of Gly-Arg-Gly-Asp photochemically grafted in polyethylene glycol modified polyurethane.

To improve endothelial cell adhesion and growth on the surface of polyethylene glycol modified polyurethane (PU-PEG), cell adhesive peptide Gly-Arg-Gly-Asp (GRGD) was photochemically grafted to the surface. The surface grafted GRGD-N-Succinimidyl-6-[4'-azido-2'-nitrophenylamino]hexanoate (SANPAH) on a PU-PEG surface was performed by adsorption and subsequent ultraviolet irradiation. Fourier transform infrared spectra (FTIR) and electron spectroscopy for chemical analysis (ESCA) confirmed the GRGD grafted to form a PU-PEG-GRGD surface. The composition fraction of nitrogen calculated from ESCA analysis for the PU-PEG-GRGD surface was well correlated with the concentration of GRGD to be immobilized. Human umbilical vein endothelial cells (ECs) were well adhered and growing on the PU-PEG-GRGD surface. Moreover, the viability of ECs growing on PU-PEG-GRGD surfaces, analyzed by MTT test, was also well correlated with the GRGD concentrations immobilized on the surface. With photochemical techniques, we could manipulate different contents of GRGD to form multiple regions of PU-PEG-GRGD surface that could enhance the growth of ECs on the surface, and the enhancement efficiency was well correlated with GRGD contents.

Cell Adhesion↗
PubMed2001

Effect of platelet activating factor and butyrate on the expression of interleukin-2 receptor alpha in nasopharyngeal carcinoma cells.

Serum level of soluble interleukin-2 receptor alpha (sIL-2Ralpha) has been shown to correlate with disease progression and prognosis of cancer patients. However, the available information about the source and the pathophysiological regulation of IL-2Ralpha in cancer cells is limited. This study addressed the questions of prognostic value and the source of sIL-2Ralpha in patients with nasopharyngeal carcinoma (NPC). Biological regulation of IL-2Ralpha was characterized in NPC cell lines. Serum sIL-2Ralpha levels of 113 NPC patients were measured by enzyme-linked immunosorbent assay (ELISA). Levels of sIL-2Ralpha in NPC patients were significantly higher than that in the healthy controls, and sIL-2Ralpha levels were correlated with disease progression and patient survival. IL-2Ralpha was identified in cancer cells by immunocytochemistry. In vitro, IL-2Ralpha expression was markedly increased following treatment with platelet activating factor and/or n-sodium butyrate. Increased secretion of IL-2Ralpha was also detected in the culture media. The secreted IL-2Ralpha could functionally bind IL-2. These results indicate that elevated sIL-2Ralpha was often detected in patients with advanced NPC. The elevated sIL-2Ralpha could be shed from NPC cells by a yet to be determined mechanism and IL-2Ralpha expression in NPC cells could be upregulated by platelet activating factor and butyrate.

Adult↗
PubMed2001

Non-penetrating trabeculectomy for open angle glaucoma.

BACKGROUND: Non-penetrating trabeculectomy is used as the surgical intervention for open angle glaucoma. It has advantage of creating gradual filtration through the thin trabeculo-Descemet membrane to reduce markedly the postoperative complications typical for penetrating operation. We described our experience of the non-penetrating trabeculectomy for open angle glaucoma. METHODS: This is a retrospective study of 28 eyes of 28 patients with primary open glaucoma (POAG). Non-penetrating trabeculectomy was performed by one surgeon from July 1998 to March 2000. Examinations were performed preoperatively and postoperatively in 1 and 7 days and 1, 3, 6, 12 and 21 months. RESULTS: The mean follow-up was 12.5 +/- 6.3 months (S.D.). The mean preoperative intraocular pressure (IOP) was 27.8 +/- 6.7 mmHg. The mean postoperative IOP was 11.5 +/- 6.2 mmHg in 1 day and 15.7 +/- 5.8 mmHg in 1 week, and it remained stable in the following 12 months. IOP of 71.3% (20/28) of the eyes could be controlled below or equal to 21 mmHg with no or only topical antiglaucoma medication. There were only few postoperative complications. CONCLUSIONS: Non-penetrating trabeculectomy is efficient in controlling intraocular pressure of open angle glaucoma. No entering the anterior chamber diminishes the postoperative complications typical of the penetrating trabeculectomy.

Aged↗
PubMed2000

Expression of a kallikrein-like protease from the snake venom: engineering of autocatalytic site in the fusion protein to facilitate protein refolding.

In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E. coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression. The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved. In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E. coli system. Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly. The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease. Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease. Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.

Amidohydrolases↗
PubMed2000

Characterization of Pen n 13, a major allergen from the mold Penicillium notatum.

Penicillium notatum is a well-known indoor aeroallergen and is frequently included in skin test panels for allergic diagnosis. On two-dimensional immunoblotting using patients' sera containing IgE and monoclonal antibody D7B8 specific for Pen c 1 of P. citrinum, two allergens with a molecular mass of 33 kDa but different isoelectric points were identified. A novel cDNA coding for Pen n 13 was cloned and sequenced. The nucleotide sequence codes for a protein 397 amino acids including a putative signal peptide of 25 amino acids and a propeptide of 90 amino acids. The allergen is an alkaline serine protease that shares more than 39% identical residues with other kinds of mold allergens. The coding cDNA of Pen n 13 was cloned into vector pQE-30 and expressed in E. coli M15 as a His-tag fusion protein and purified to homogeneity. The fusion protein reacted with monoclonal antibodies of Pen c 1 and with IgE from Penicillium-allergic patients. Furthermore, it also cross-reacted strongly with IgE specific for the natural Pen c 1, indicating that similar IgE binding epitopes may exist in the allergens of P. notatum and P. citrinum. Antigenicity index plots indicated that there are several similar epitope regions of high antigenic indices in Pen c 1 and Pen n 13, corroborating that mold allergens belonging to the alkaline serine protease family possess similar protein structure and strong antigenic cross-reactivity.

Allergens↗
PubMed2000

Using conjunctival swab with polymerase chain reaction to aid diagnosis of cytomegalovirus retinitis in AIDS patients.

Conjunctival swabs were used in combination with polymerase chain reactions (PCR) or virus culture to aid to confirm the diagnosis of cytomegalovirus (CMV) retinitis in AIDS patients. Based on ophthalmoscopic findings, 13 AIDS patients were diagnosed as having CMV retinitis and treated with systemic ganciclovir from January 1997 to December 1998. Nine of 13 AIDS patients with CMV retinitis were found to be positive for CMV DNA by using a conjunctival swab with PCR (CS-PCR). CMV was also isolated from the conjunctival swab culture in 2 AIDS patients with CMV retinitis. Following 1 month of systemic ganciclovir treatment, except for 1 patient who had disease progression and remained positive for CMV DNA by using CS-PCR, 12 patients were negative for CMV with a concordant lesion regression. Results of this study also indicate that this CS-PCR method is valuable for confirming the diagnosis of CMV retinitis and monitoring the ganciclovir effect on the treatment of CMV retinitis in AIDS patients.

AIDS-Related Opportunistic Infections↗
PubMed2000

Detection of human cytomegalovirus retinitis and monitoring of ganciclovir treatment using conjunctival swab with polymerase chain reaction in AIDS patients.

This report studies the accuracy of conjunctival swab polymerase chain reaction (CS-PCR) for the diagnosis of human cytomegalovirus retinitis (HCMV) in AIDS patients. PCR and virus culture were used for the detection of HCMV in conjunctival swab, serum, and urine specimens from 38 AIDS patients between April 1996 and April 1998. The clinical utility of the identification of HCMV retinitis by these 6 different methods was demonstrated by their prediction power to estimate AIDS patients at risk of contracting HCMV retinitis. The sensitivity, specificity, positive predictive value, and negative predictive value of CS-PCR for the detection of HCMV retinitis were 91.5%, 80.9%, 60.8%, and 92.7%, respectively; for serum PCR were 74.3%, 81.7%, 57.2%, and 90.3%; for urine PCR were 100%, 17.3%, 20.4%, and 100%; for conjunctival swab culture were 22.7%, 100%, 100%, and 86%; for serum culture were 27.3%, 98.1%, 75%, and 86.4%; and for urine culture were 90.9%, 44.2%, 25.6%, and 95.8%.

AIDS-Related Opportunistic Infections↗
PubMed2000

Ophthalmic findings in patients with acquired immunodeficiency syndrome.

Ocular manifestations have been reported in up to 60% of individuals infected with human immunodeficiency virus (HIV) in the United States, and it is becoming increasing apparent that these ocular manifestations almost invariably reflect extent of progression of the disease. The prevalence of ocular abnormalities among acquired immunodeficiency syndrome (AIDS) patients in Taiwan has not been reported. In the present study, we examined and followed up the ophthalmic conditions of a total of 274 HIV-infected patients during the period from March 1993 to May 1999. The results show that cotton-wool spots was the most common ocular finding in this series of patients with AIDS, occurring in 22 (32.8%) of 67 AIDS patients. Cytomegalovirus (CMV) retinitis was the most commonly seen opportunistic ocular infection, occurring in 14 (20.8%) of 67 AIDS patients. These findings suggest that AIDS patients should be closely followed for signs of opportunistic ocular disease which may initially be asymptomatic. Close co-operation between the ophthalmologist and the internist is essential to ensure timely therapeutic intervention, which can decrease the risk of further complications including visual impairment and blindness.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Comparative study of timolol gel versus timolol solution for patients with glaucoma.

BACKGROUND: The timolol gel was developed for the simplification of multi-dose medications for glaucoma patients. This gel can be used once per day instead of twice per day of the aqueous form. It is necessary to understand the clinical effects of intraocular pressure maintained in glaucoma by there two forms of timolol. METHODS: Fifty-two eligible patients were included and completed the 12-week study. In this randomized, open-label, two-period crossover study design, patients received either the gel form of 0.5% timolol once daily or the aqueous form of 0.5% timolol twice daily for the first 6 weeks, then crossed over to the other treatment form for the remaining 6 weeks. Intraocular pressure was measured at baseline, week 6 and week 12 at the end of the treatment period. The incidences of all the adverse events were recorded and summarized in each follow-up visit. RESULTS: No statistically significant difference in intraocular pressure was observed between the two different control treatment groups (p>0.05). However, stickiness and transient blurred vision was reported more often by patients using the gel form than the aqueous form of timolol. CONCLUSIONS: The gel form of 0.5% timolol used once daily offers a new alternative that is probably more convenient than the aqueous form of 0.5% timolol given twice daily in maintaining the intraocular pressure of patients with glaucoma.

Adrenergic beta-Antagonists↗
PubMed1999

Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus).

A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus.

Amino Acid Sequence↗
PubMed1999

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.

Allergens↗
PubMed1999

Comparison of three classes of snake neurotoxins by homology modeling and computer simulation graphics.

We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.

Amino Acid Sequence↗
PubMed1999

Homology modeling of cephalopod lens S-crystallin: a natural mutant of sigma-class glutathione transferase with diminished endogenous activity.

The soluble S-crystallin constitutes the major lens protein in cephalopods. The primary amino acid sequence of S-crystallin shows an overall 41% identity with the digestive gland sigma-class glutathione transferase (GST) of cephalopod. However, the lens S-crystallin fails to bind to the S-hexylglutathione affinity column and shows very little GST activity in the nucleophilic aromatic substitution reaction between GSH and 1-chloro-2,4-dinitrobenzene. When compared with other classes of GST, the S-crystallin has an 11-amino acid residues insertion between the conserved alpha4 and alpha5 helices. Based on the crystal structure of squid sigma-class GST, a tertiary structure model for the octopus lens S-crystallin is constructed. The modeled S-crystallin structure has an overall topology similar to the squid sigma-class GST, albeit with longer alpha4 and alpha5 helical chains, corresponding to the long insertion. This insertion, however, makes the active center region of S-crystallin to be in a more closed conformation than the sigma-class GST. The active center region of S-crystallin is even more shielded and buried after dimerization, which may explain for the failure of S-crystallin to bind to the immobilized-glutathione in affinity chromatography. In the active site region, the electrostatic potential surface calculated from the modeled structure is quite different from that of squid GST. The positively charged environment, which contributes to stabilize the negatively charged Meisenheimer complex, is altered in S-crystallin probably because of mutation of Asn99 in GST to Asp101 in S-crystallin. Furthermore, the important Phe106 in authentic GST is changed to His108 in S-crystallin. Combining the topological differences as revealed by computer graphics and sequence variation at these structurally relevant residues provide strong structural evidences to account for the much decreased GST activity of S-crystallin as compared with the authentic GST of the digestive gland.

Algorithms↗