Hardy W. Chan - Academia.edu (original) (raw)

Papers by Hardy W. Chan

Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1977

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotid... more The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of Aplc5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p-, the INT promoter (pI), several sites in the b2 region, the mis promoter, the oop promoter (or po), and pro. Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the CIII-tL region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites.

Proceedings of the National Academy of Sciences of the United States of America, Feb 1, 1988

Transcription of the interleukin 1pf (IL-1,) gene was studied by mRNA hybridization with a cDNA p... more Transcription of the interleukin 1pf (IL-1,) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1i mRNA. Glucocorticoids markedly decreased IL-1(3 mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-113 mRNA, without affecting the stability of 3-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.

International Journal of Molecular Sciences, May 16, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Infection and Immunity, 1988

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] ha... more The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability w...

Biochemical Journal, 1996

Human dopamine β-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) ... more Human dopamine β-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are i...

Scientific Reports, Jul 30, 2019

Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule an... more Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and label-free detection. In nucleic acid detection, Gc-rich nucleic acid sequences form self-and cross-dimers and stem-loop structures, which can easily obtain data containing signals from nonspecific DNA binding. The features of GC-rich nucleic acid sequences cause inaccuracies in nucleic acid detection and hinder the development of precision medicine. To improve the inaccurate detection results, we used phosphate-methylated (neutral) nucleotides to synthesize the neutralized chimeric DNA oligomer probe. The probe fragment originated from a primer for the detection of hepatitis C virus (HCV) genotype 3b, and single-mismatched and perfect-matched targets were designed for single nucleotide polymorphisms (SNP) detection on the SiNW FET device. Experimental results revealed that the HCV-3b chimeric neutralized DNA (nDNA) probe exhibited better performance for SNP discrimination in 10 mM bis-tris propane buffer at 25 °C than a regular DNA probe. The SNP discrimination of the nDNA probe could be further improved at 40 °C on the FET device. Consequently, the neutralized chimeric DNA probe could successfully distinguish Snp in the detection of Gc-rich target sequences under optimal operating conditions on the SiNW FET device. Single nucleotide polymorphisms (SNPs) are the most common forms of genetic variations, which are important indicators to disclose individual susceptibility to disease and differences in treatment effect. To achieve the goals of precision medicine, there are strong demands to develop rapid, affordable, easy-to-use, sensitive, and specific techniques for SNP analysis. Researchers have devoted extensive efforts to improve the various techniques for SNP genotyping in the previous decade, such as mass spectroscopy 1 , polymerase chain reaction 2 , microarray 3 , and molecular beacon probes 4,5. However, most of the abovementioned methods require expensive instruments, complicated procedures, and radioactive/fluorescent labels to amplify the detection signals and sample numbers. Therefore, biosensing techniques have been adopted as platforms of SNP detection due to their high sensitivity, simplicity, short detection time, and good reproducibility. Electrochemical 6 , surface plasmon resonance (SPR) 7-9 and nanowire-based biosensors are the frequently used platforms for the SNP discrimination 10,11. In general, a biosensing platform comprises a recognizing element (the probe) and transducer. Therefore, to enhance the ability of SNP discrimination, the probe design must be enhanced for improved hybridization efficiency and specificity to the target gene molecule. Hence, some DNA analogs, such as peptide nucleic acid (PNA), locked nucleic acid (LNA), and phosphate-methylated (neutral) nucleotide, are applied in the probe design because these analogs have unique properties unfound in nature and can hybridize specifically with natural target DNA. By using LNA modifications on the probe, the melting temperature (T m) difference between matched and mismatched duplexes can be adjusted, and a large T m difference may lead to improved performance in mismatch discrimination 12. Ananthanawat et al. 13 reported that immobilized thiolated-PNA can discriminate

ACS Omega, 2019

Silicon nanowire field-effect transistors (SiNW-FETs) have been demonstrated as a highly sensitiv... more Silicon nanowire field-effect transistors (SiNW-FETs) have been demonstrated as a highly sensitive platform for label-free detection of a variety of biological and chemical entities. However, detecting signal from immunoassays by nano-FETs is severely hindered by the distribution of different charged groups of targeted entities, their binding orientation, and distances to the surface of the FET. Aptamers have been widely applied as a recognition element for plentiful biosensors because of small molecular sizes and moderate to high specific binding affinity with different types of molecules. In this study, we propose an effective approach to enhance the electrical responses of both direct (6×-histidine) and sandwich (amyloid β 1−42) immunoassays in SiNW-FETs with R18, a highly negative charged RNA aptamer against rabbit immunoglobulin G (IgG). Empirical results presented that the immunosensors targeted with R18 expressed a significantly stabilized and amplified signal compared to the ones without this aptamer. The research outcome provides applicability of the highly negative charged aptamer as a bioamplifier for immunoassays by FETs.

Sensors, 2021

Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire fi... more Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire field-effect transistors (SiNWFETs) is severely hindered due to the weakened signal, primarily caused by screening effects. In this study, aptamer as a signal amplifier, which has already been reported by our group, is integrated into SiNWFET immunosensors employing antigen-binding fragments (Fab) as the receptors to improve its detection limit for the first time. The Fab-SiNWFET immunosensors were developed by immobilizing Fab onto Si surfaces modified with either 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Fab/APTES-SiNWFETs), or mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) and GA (Fab/PEG-SiNWFETs), to detect the rabbit IgG at different concentrations in a high-ionic-strength environment (150 mM Bis-Tris Propane) followed by incubation with R18, an aptamer which can specifically target rabbit IgG, for signal enhancement. Empirical results reveal...

PROBLEM TO BE SOLVED: To obtain the subject new substance in a vaculovirus expression sys tem, co... more PROBLEM TO BE SOLVED: To obtain the subject new substance in a vaculovirus expression sys tem, consisting of biologically active humanNGF or a derivative therefrom with variation in the amino acrid sequence while retaining the same activity as that of biologically active humanNGF, and useful for e.g. treating neuropathy. SOLUTION: This new substance is a biologically active humanNGF or a derivative therefrom through addition, insertion, deletion or substitution of amino acid(s) to, into, from or with the humanNGF while retaining essentially the same activity as the humanNGF, being useful for e.g medicinal compositions for treating neuropathy such as Alzheimer's disease. This biologically active humanNGF is obtained by the following process: a human leukocyte genome library is subjected to Southern blotting analysis using a terminal-labeled synthetic oligonucleotide complementary to the fragment of a humanNGF gene as a hybridization probe to select positive clone, its DNA is then...

ES provided a method for the construction of DNA molecules RECOMBINANT FACTOR CAPABLE OF PRODUCIN... more ES provided a method for the construction of DNA molecules RECOMBINANT FACTOR CAPABLE OF PRODUCING A NERVE GROWTH OF HUMAN biologically active (hNGF) in insect cells. MATURE protein expression TRIGGERED USandA BACULOVIROSA expression system. Biologically active hNGF thus produced is a value POTENTIAL IN THE TREATMENT OF PATIENTS SUFFERING FROM DISEASE AND OTHER ALZHEIMAR Neurological Disorders.

Brain Research, 1993

In cerebral amyloid angiopathy, the amyloid-/3 (A/3) deposits lie primarily in the tunica media s... more In cerebral amyloid angiopathy, the amyloid-/3 (A/3) deposits lie primarily in the tunica media suggesting that smooth muscle cells play an important role in A/3 deposition. To define this role, we conducted an immunocytochemical study of brain tissue from cases of Alzheimer disease with extensive cerebral amyloid angiopathy and cerebral hemorrhage. Antibodies specific to recombinant /3 protein precursor (/3PP) and synthetic peptides homologous to various /3PP sequences from residue 18 to 689 of /3PP695 were used. Antibodies to actin, tropomyosin, a-actinin or desmin were used to label muscle cells. Antibodies to A/3 sequences intensely recognized the extracellular amyloid deposit. Antibodies raised against /3PP sequences other than the A/3 domain recognized smooth muscle cells. /3PP-immunoreactivity was reduced in regions of A/3 deposits, since no muscle cells were recognized by cytoskeletal markers or observed ultrastructurally. In order to assess why A/3 is deposited in the tunica media, we used biotin-labelled /3PP to determine if/3PP can be locally retained. We found /3PP bound to the tunica media of vessels but not other brain elements. These findings suggest A/3 in blood vessels derives from degenerating /3PP-containing smooth muscle cells.

Proceedings of the National Academy of Sciences, 1988

Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a... more Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1 beta mRNA. Glucocorticoids markedly decreased IL-1 beta mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1 beta mRNA, without affecting the stability of beta-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.

Proceedings of the National Academy of Sciences, 1987

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1... more A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

BMC Neuroscience, 2010

Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alz... more Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions...

L'invention decrit l'hydrolyse enantioselective d'esters de naproxene racemiques par ... more L'invention decrit l'hydrolyse enantioselective d'esters de naproxene racemiques par des esters hydrolases, les hydrolases etant derivees d'un groupe de micro-organismes dont le plus approprie etait le micro-organisme, Zopfiella latipes. Les esters hydrolases sont utilises dans une hydrolyse d'esters de naproxene racemiques dont le cout est faible et le rendement eleve.

PURPOSE: To obtain a novel biologically active human nerve growth factor(hNGF) and a derivative t... more PURPOSE: To obtain a novel biologically active human nerve growth factor(hNGF) and a derivative thereof for the treatment of human neurological disorders such as Alzheimer's disease or the like, which is obtained by expressing a recombinant DNA of hNGF in insect cells using a baculovirus expression system. CONSTITUTION: The novel biologically active hNGF and a derivative thereof useful as a therapeutic agent for human neurological disorders such as Alzheimer's disease or the like can be obtained by infecting insect cells such as Spodoptera-frugiperda(Sf9) with a baculovirus transfer vector having been integrated with the gene of a human βNGF(hNGF) comprising the prepro-portion of the DNA coding sequence of mouse nerve growth factor(NGF) and the DNA coding sequence of maturation human βNGF(hNGF) or the prepro-portion of the DNA coding sequence of human NGF and the DNA coding sequence of maturation human βNGF(hNGF) or a derivative thereof which has been bonded to the controlli...