Biomedical subjects

H Chao

Publications and source records attributed to H Chao.

At least 19 recordsLinked to original sources

PubMed2002

Parvovirus-mediated gene transfer for the haemophilias.

Gene therapy may revolutionize the treatment of haemophilia. Effective gene therapy requires sustained therapeutic levels of factors IX (FIX) and VIII. Adeno-associated virus (AAV) is a member of the parvovirus family, is a nonpathogenic virus with a broad host cell range, and does not provoke a significant immune response upon infection. These favourable characteristics make AAV a suitable gene transfer vector for factor deficient patients. A new understanding of AAV biology coupled with novel AAV vector designs suggest that the goal of effective gene transfer is within reach. We review here recent advances in AAV vectors used for gene transfer of the haemophilias.

Blotting, Western↗

PubMed2001

Induction of tolerance to human factor VIII in mice.

This paper reports loss of human factor VIII (hFVIII) inhibitory antibody in immunocompetent C57BL/6 mice. High-titer anti-hFVIII antibody developed in the mice within 7 to 14 days of intraportal administration of adeno-associated virus (AAV) carrying FVIII that coincided with a reduction in plasma hFVIII antigen. Bethesda titers (> 100 units) persisted relatively unchanged for 9 to 10 months. Unexpectedly, at 10 months after injection of the virus, hFVIII protein (up to 59 ng/mL) was detected in 3 mice at the same time as disappearance of hFVIII inhibitor. The level of hFVIII was similar to that found in immunodeficient mice receiving the same dose of recombinant AAV carrying hFVIII without hFVIII inhibitor. These results suggest that tolerance to hFVIII can be induced by sustained expression of hFVIII in a mouse model. Further elucidation of this observation may affect use of FVIII gene transfer in the treatment of inhibitor-positive patients with hemophilia A.

Animals↗

PubMed2001

Determination of sialic acids by liquid chromatography-mass spectrometry.

Sialic acids are widely found in nature as components of oligosaccharide units in mucins, glycoproteins and other microbial polymers. Existing methods for determining these acids are long, tedious, and not specific. A simple, rapid, and sensitive method for determining the most commonly occurring acids, N-acetylneuraminic and N-glycolylneuraminic acid, using LC-MS is described. Standard solutions of the sialic acids with the internal standard, N-acetylneuraminic acid methyl ester, were quantitatively analyzed by positive ion electrospray ionization. Fetuin was used as a model glycoprotein and the hydrolysate was injected directly onto an ES Industries AquaSep 3 microm 150x4.6 mm column eluted with a 0.1% aqueous formic acid mobile phase at a flow-rate of 0.5 ml/min. Detection was achieved using the Finnigan Navigator MS system in the selected ion monitoring mode for the protonated molecular ions at m/z 310, 324, and 326. The linearity over the dynamic range 10 to 1000 ng of sialic acids on-column had a correlation coefficient greater than 0.999. The amount of sialic acids found in the fetuin hydrolysate was in agreement with values reported in the literature.

Chromatography, Liquid↗

PubMed2001

DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes:.

Two complexes of [Co(phen)2IP]3+ (IP=imidazo[4,5-f][l,10]phenanthroline) and [Co(phen)2PIP]3+ (PIP=2-phenylimidazo[4,5-f][1,10]phenanthroline) have been synthesized and characterized by UV/VIS, IR, EA and mass spectra. The binding of the two complexes with calf thymus DNA has been investigated by absorption spectroscopy, cyclic voltammetry, viscosity measurements and DNA cleavage assay. The spectroscopic studies together with cyclic voltammetry and viscosity experiments support that both of the complexes bind to CT DNA by intercalation via IP or PIP into the base pairs of DNA. [Co(phen)2PIP]3+ binds more avidly to CT DNA than [Co(phen)2IP]3+, which is consistent with the extended planar and pi system of PIP. Noticeably, the two complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA.

Cobalt↗

PubMed2001

Sustained and complete phenotype correction of hemophilia B mice following intramuscular injection of AAV1 serotype vectors.

We previously reported that direct intramuscular injection of non-serotype-2 AAV vectors, especially AAV serotype 1 (AAV1), resulted in expression of supranormal levels of canine F9 in immunodeficient mice. Here we test the ability of the AAV1-F9 vector to deliver sustained expression and correction of factor IX (FIX) deficiency in genetically engineered hemophilic mice. Intramuscular injection of AAV1-F9 resulted in 100-1000 times more canine F9 in plasma of recombinant AAV1-F9 mice compared with injection of AAV2-F9. Assessment of clotting activity by activated partial thromboplastin time confirmed that circulating canine FIX was indeed functional. Moreover, phenotypic correction assayed by tail clip challenge resulted in survival of all AAV1-F9 treated animals, in contrast to naive mice and 50% of AAV2-treated hemophilia B mice, which failed to survive. Administration of cyclophosphamide (CTX) was required to suppress formation of anti-canine FIX antibodies for AAV2-treated animals, whereas it was dispensable for those treated with AAV1-F9. This difference in immunogenicity further emphasizes the usefulness of serotype-specific vectors. Finally, we report that correction of the hemophilia phenotype using AAV1-F9 was complete and persistent (over 8 months), a result that underscores the value of continued exploration of alternative AAV serotype vectors.

Animals↗

PubMed2001

The design of new molecular "light switches" for DNA.

Two novel ruthenium(II) complexes, [Ru(pztp)2(phen)](ClO4)2 and [Ru(pztp)2(bpy)] (ClO4)2, have been synthesized and characterized by UV/Vis and 1H NMR spectroscopies and mass spectrometry. The MeCN solutions of both complexes display fluorescence that was found to be highly sensitive to the presence and concentration of water. The complexes behave like a "light switch" for DNA in that they do not luminesce in water but were "turned on" in the presence of DNA and show emission enhancement with the increase of DNA concentration. Their DNA binding behavior was also studied by absorption spectroscopy and viscosity measurements, which suggest that the DNA-complex interaction involves intercalation of the metal-bound pztp ligand into the base pairs of duplex DNA.

Animals↗

PubMed2001

Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.

Since its discovery three decades ago, sterol carrier protein-2 (SCP-2) has remained a fascinating protein whose physiological function in lipid metabolism remains an enigma. Its multiple proposed functions arise from its complex gene structure, post-translational processing, intracellular localization, and ligand specificity. The SCP-2 gene has two initiation sites coding for proteins that share a common 13 kDa SCP-2 C-terminus: (1) One site codes for 58 kDa SCP-x which is partially post-translationally cleaved to 13 kDa SCP-2 and a 45 kDa protein. (2) A second site codes for 15 kDa pro-SCP-2 which is completely post-translationally cleaved to 13 kDa SCP-2. Very little is yet known regarding how the relative proportions of the two transcripts are regulated. Although all three proteins contain a C-terminal SKL peroxisomal targeting sequence, it is unclear why all three proteins are not exclusively localized in peroxisomes. However, the recent demonstration that the SCP-2 N-terminal presequence in pro-SCP-2 dramatically modulated the intracellular targeting coded by the C-terminal peroxisomal targeting sequence may account for the observation that as much as half of total SCP-2 is localized outside the peroxisome. The tertiary and secondary structure of the 13 kDa SCP-2, but not that of 15 kDa pro-SCP-2 and 58 kDa SCP-x, are now resolved. Increasing evidence suggests that the 58 kDa SCP-x and 45 kDa proteins are peroxisomal 3-ketoacyl-CoA-thiolases involved in the oxidation of branched chain fatty acids. Since 15 kDa pro-SCP-2 is post-translationally completely cleaved to 13 kDa SCP-2, relatively little attention has been focused on this protein. Finally, although the 13 kDa SCP-2 is the most studied of these proteins, because it exhibits diversity of its ligand partners (fatty acids, fatty acyl CoAs, cholesterol, phospholipids), new potential physiological function(s) are still being proposed and questions regarding potential compensation by other proteins with overlapping specificity are only beginning to be resolved.

Acetyl-CoA C-Acetyltransferase↗

PubMed2001

Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking.

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).

Animals↗

PubMed2001

[Bax gene expression in endometrial carcinoma].

OBJECTIVE: To study the relationship between bax gene expression and endometrial carcinoma. METHODS: Bax mRNA expression was evaluated by RT-PCR in a series of 8 normal proliferative endometrium (NPE), 6 simple and complex hyperplasia (SCH), 6 atypical hyperplasia (AH) and 42 endometrial carcinoma (EC). RESULTS: All of the 8 NPE showed moderate bax mRNA expression. In 1 of 6 SCH, 2 of 6 AH and 27 of the 42 EC (64.3%), there was overexpression of bax mRNA. In endometrial carcinoma, with the progression of clinical stage, increase in histologic grade and in depth of invasion to muscle layer, and lymph node metastasis, the positive rate of bax overexpression was correspondingly increased. CONCLUSION: The results suggest that bax expression affects the balance between the endometrial cell proliferation and apoptosis. Bax might play an important role in carcinogenesis and tumor progression of endometrial carcinoma. As a high risk factor it can be used to evaluate the biological behavior of endometrial carcinoma.

Disease Progression↗

PubMed2000

Sustained expression of human factor VIII in mice using a parvovirus-based vector.

Persistent therapeutic levels of human factor VIII (hFVIII) would signify a major advance in the treatment of hemophilia A. Here we report sustained expression of hFVIII in immunocompetent mice using recombinant adeno-associated virus (rAAV) vectors. AAV can stably transduce liver cells, the target tissue for efficient hFVIII production. Because of rAAV packaging constraints, we tested 2 constructs using small regulatory elements designed for liver-specific transgene expression linked to B-domain-deleted hFVIII (BDD-hFVIII) cDNA. More than 10(12)/mL rAAV/BDD-hFVIII virion particles were generated using a transfection scheme that eliminates adenovirus. Coatest and APTT assays confirmed the production of functional BDD-hFVIII protein after transduction of 293 and HepG2 cells. In vivo experiments were performed in C57BL/6 and NOD/scid mice receiving 10(10-11) rAAV/hFVIII particles via portal vein injection. All C57BL/6 mice tested developed anti-hFVIII antibody. In contrast, NOD/scid mice expressed hFVIII reaching 27% of normal human plasma levels. As expected, we could not detect hFVIII antigen from plasma samples isolated from control animals receiving equivalent doses of rAAV expressing enhanced green fluorescent protein (EGFP). Transgene mRNA expression was detected primarily in the liver and histologic analysis of the liver revealed no pathologic abnormalities. These results demonstrate a promising approach for treatment of hemophilia A. (Blood. 2000;95:1594-1599)

Animals↗

PubMed2000

Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.

We previously demonstrated that rAAV vectors carrying human and canine factor IX (FIX) cDNA can infect, stably persist, and secrete functional human and canine FIX following direct intramuscular injection. In an attempt to improve FIX protein secretion for eventual therapeutic use, we set out to determine if alteration of the AAV capsid would affect skeletal muscle transduction and factor IX secretion. Two reasons to pursue this question were (1) the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) our previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expression cassette, we cross-packaged this genome into virions generated from each of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo. In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending on the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same viruses were tested in vivo using immunodeficient NOD/SCID animals, we obtained surprisingly different results. While the time to onset of detectable serum levels appeared the same for all serotypes, types 1, 3, and 5 produced 100- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, type 1 continued to express levels of cFIX on average at 80 microg/ml followed by type 5 (6.52 microg/ml), type 3 (3.27 microg/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT supported the circulating levels measured by ELISA demonstrating the secreted protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant differences in cFIX expression upon introducing various rAAV serotypes into mouse muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.

Animals↗

PubMed2000

Sterol carrier protein-2 suppresses microsomal acyl-CoA hydrolysis.

Although sterol carrier protein 2 (SCP-2) has long been regarded primarily as a sterol transfer protein, its actual physiological function is not known. The recent discovery that SCP-2 binds long chain fatty acyl-CoAs (LCFA-CoAs) with high affinity suggests additional roles for SCP-2 in cellular utilization of LCFA-CoAs for synthesis of glycerides and cholesterol esters. Concomitant to these anabolic pathways, LCFA-CoAs are also degraded by cellular hydrolases. The purpose of the work presented herein was to determine if SCP-2 altered the aqueous pool of LCFA-CoA by (i) extracting LCFA-CoA from microsomal membranes, and (ii) protecting LCFA-CoA from microsomal hydrolase activity. The data demonstrated for the first time that SCP-2 increases the aqueous pool of oleoyl-CoA by increasing the aqueous/membrane distribution oleoyl-CoA by 2.4-fold. In addition, SCP-2 inhibited the hydrolysis of oleoyl-CoA by microsomal acyl-CoA hydrolase 1.6-2.4 fold, depending on the concentration of oleoyl-CoA. By simultaneously extracting LCFA-CoA from membranes and inhibiting LCFA-CoA degradation SCP-2 may potentiate LCFA-CoA transacylation and modulate the role of LCFA-CoAs as intracellular signaling molecules.

Acyl Coenzyme A↗

PubMed2000

pRB-dependent, J domain-independent function of simian virus 40 large T antigen in override of p53 growth suppression.

Simian virus 40 (SV40) large T antigen (LT) can immortalize and transform many cell types. These activities are attributed in large part to the binding and functional inactivation by LT of two major tumor suppressors: p53 and the retinoblastoma protein, pRB. Most effects of LT on pRB have been shown to additionally require an intact J domain, which mediates binding to Hsc70. We show here that the J domain is not required for p53 override in full-length LT. Although LT binds p53, it was shown previously that overcoming a p53-induced cell cycle arrest requires binding to pRB family members (R. S. Quartin et al., J. Virol. 68:1334-1341). We demonstrate that an LT mutant defective for pRB family member binding (K1) can be complemented for efficient override of p53 arrest by a construct encoding the first 135 amino acids of LT with a J domain-inactivating mutation, H42Q. Hence, complementation does not require the J domain, and pRB binding by LT is important for more than dissociating pRB-E2F complexes, which is J dependent. In accordance with this notion, LT alleviates pRB small-pocket-mediated transcriptional repression independently of the J domain. The LT K1 mutant can also be complemented for p53 override by small t antigen (st) in a manner independent of its J domain. Our observations underscore the importance of multiple SV40 functions, two in LT and one in st, that act cooperatively to counteract p53 growth suppression.

Animals↗

PubMed2000

[Growth inhibition effect of 5-aza-CdR on endometrial carcinoma xenografted in nude mice by p16 gene demethylation].

OBJECTIVE: To study the antitumor effect and mechanism of 5-aza-CdR on endometrial carcinoma xenografted in nude mice. METHODS: The effects of 5-aza-CdR on tumor growth inhibition were observed on human endometrial carcinoma xenografted in nude mice. The methylation status of the 5' CpG island of the p16 gene was evaluated using methylation-sensitive restriction enzymes and polymerase chain reaction. p16 mRNA expression was determined by reverse transcription-polymerase chain reaction. RESULTS: The inhibition rates of the tumor were 79.10% in 5-aza-CdR group (P < 0.01), 90.32% in 5-aza-CdR + DDP group (P < 0.01), and 10.13% in DDP group (P > 0.05), respectively, in comparison with control group. The methylation level of p16 gene was gradually decreased and the p16 mRNA expression restored after exposure to 5-aza-CdR. CONCLUSION: Our data show that 5-aza-CdR may inhibit tumor growth by reactivating growth-regulatory gene silenced by de novo methylation.

Animals↗

PubMed2000

[Methylation and expression of the p16 gene in endometrial carcinoma].

OBJECTIVE: To analyse the relationship between methylation status of the 5' CpG island and mRNA expression of the p16 gene in endometrial carcinoma. METHODS: Methylation status of p16 gene was determined by methylation-sensitive restriction enzymes, PCR and p16 mRNA expression was evaluated by RT-PCR in a series of 8 specimens with normal endometrium(NE), 6 with simple and complex hyperplasia(SCH), 6 with atypical hyperplasia (AH) and 38 with endometrial carcinoma(EC). RESULTS: Eight specimens of NE displayed no methylation and showed normal expression of the p16 mRNA. In 1 of the 6 AH and 13 of the 38(34.21%) EC, there was methylation of p16 exon 1; 5 of the 6 SCH showed overexpression of p16 mRNA, 4 of the 6 AH and 27 of the 38 (71.05%) EC exhibited decrease or loss of p16 mRNA expression. CONCLUSION: Hypermethylation of p16 gene is an early event of endometrial carcinogenesis and is associated with the progression of endometrial carcinoma. Hypermethylation is highly correlated with inhibition of p16 gene transcription.

Cyclin-Dependent Kinase Inhibitor p16↗

PubMed1999

New ice-binding face for type I antifreeze protein.

Type I antifreeze protein (AFP) from winter flounder is an alanine-rich, 37 amino acid, single alpha-helix that contains three 11 amino acid repeats (Thr-X(2)-Asx-X(7)), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice-binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn-rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully alpha-helical and remained as monomers in solution. Wild-type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr-rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wild-type activity and demonstrated weak interactions with the ice surface. We propose a new ice-binding face for type I AFP that encompasses the conserved Ala-rich surface and adjacent Thr.

Alanine↗

PubMed1999

Microsomal long chain fatty acyl-CoA transacylation: differential effect of sterol carrier protein-2.

The recent discovery that sterol carrier protein-2 (SCP-2) binds long chain++ (LCFA-CoA) with high affinity (A. Frolov et al., J. Biol. Chem. 271 (1997) 31878-31884) suggests new possible functions of this protein in LCFA-CoA metabolism. The purpose of the present investigation was to determine whether SCP-2 differentially modulated microsomal LCFA-CoA transacylation to cholesteryl esters, triacylglycerols, and phospholipids in vitro. Microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity measured with liposomal membrane cholesterol donors depended on substrate LCFA-CoA level, mol% cholesterol in the liposomal membrane, and total amount of liposomal cholesterol. As compared to basal activity without liposomes, microsomal ACAT was inhibited 30-50% in the presence of cholesterol poor (1.4 mol%) liposomes. In contrast, cholesterol rich (>25 mol%) liposomes stimulated ACAT up to 6.4-fold compared to basal activity without liposomes and nearly 10-fold as compared to cholesterol pool (1.4 mol%) liposomes. Increasing oleoyl-CoA reversed the inhibition of microsomal ACAT by cholesterol poor (1.4 mol%) liposomes, but did not further stimulate ACAT in the presence of cholesterol rich (35 mol%) liposomes. In contrast, high (100 microM) oleoyl-CoA inhibited ACAT nearly 3-fold. This inhibition was reversed by LCFA-CoA binding proteins, bovine serum albumin (BSA) and SCP-2. SCP-2 was 10-fold more effective (mole for mole) than BSA in reversing LCFA-CoA inhibited microsomal ACAT. Concomitantly, under conditions in which SCP-2 stimulated ACAT it equally enhanced transacylation of oleoyl-CoA into phospholipids, and 5.2-fold enhanced oleoyl-CoA transacylation to triacylglycerols. In summary, SCP-2 appeared to exert its greatest effects on microsomal transacylation in vitro by reversing LCFA-CoA inhibition of ACAT and by differentially targeting LCFA-CoA to triacylglycerols. These data suggest that the high affinity interaction of SCP-2s with LCFA-CoA may be physiologically important in microsomal transacylation reactions.

Acyl Coenzyme A↗

PubMed1999

Alternative roles for putative ice-binding residues in type I antifreeze protein.

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.

Amino Acid Sequence↗