Biomedical subjects

H Cai

Publications and source records attributed to H Cai.

At least 55 records · Page 3Linked to original sources

PubMed2001

High-throughput techniques for compound characterization and purification.

A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.

Chromatography↗

PubMed2001

[Cloning and sequencing of 16S rRNA gene of Phytoplasma CWB1 strain associated with cactus witches' broom].

A 1.5 kb DNA fragment was amplified in DNA samples extracted from Opuntia salmiana porm showed witches'-broom symptom. The result indicates the existence of phytoplasma associated with this disease and this phytoplasma was designated as CWB1. The amplified fragment was ligated to pGEM-T easy vector and then transformed into JM109 strain of E. coli. Cloned DNA fragments were verified by PCR, restriction endonuclease (EcoRI) digestion and sequence analysis. The result revealed that the 16S rRNA gene of CWB1 consists of 1489 bp and shared 99.7% homology with Faba bean phyllody which belongs to phytoplasma 16S rII-C subgroup. So we can classify this strain into phytoplasma 16S rII-C subgroup.

Cloning, Molecular↗

PubMed2001

[Determination of cobalt in solidified resin promoter by successive extraction and FAAS].

A new method had been investigated for the analysis of micro-amount of cobalt in solidified resin promoter. Methyl isobutyl ketone (MIBK) was used in 1 mol.L-1 NH4SCN medium followed by 4 mol.L-1 nitric acid back extraction step, then analysis by flame atomic absorption spectrometry. The results indicated that the method was accurate and the interference with concomitant in solidified resin promoter was negligible. The method had a recovery of 96.2%-104.6%.

Carboxylic Acids↗

PubMed2000

Oxidation of indole by cytochrome P450 enzymes.

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.

Animals↗

PubMed2000

Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress.

Accumulating evidence suggests that oxidant stress alters many functions of the endothelium, including modulation of vasomotor tone. Inactivation of nitric oxide (NO(.)) by superoxide and other reactive oxygen species (ROS) seems to occur in conditions such as hypertension, hypercholesterolemia, diabetes, and cigarette smoking. Loss of NO(.) associated with these traditional risk factors may in part explain why they predispose to atherosclerosis. Among many enzymatic systems that are capable of producing ROS, xanthine oxidase, NADH/NADPH oxidase, and uncoupled endothelial nitric oxide synthase have been extensively studied in vascular cells. As the role of these various enzyme sources of ROS become clear, it will perhaps be possible to use more specific therapies to prevent their production and ultimately correct endothelial dysfunction.

Animals↗

PubMed2000

Rapid DNA fingerprinting of pathogens by flow cytometry.

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.

DNA Fingerprinting↗

PubMed2000

Analysis of Proteins During Recovery from Lens Opacity. Analysis of Selenite Cataract Model Using Sprague- Dawley and Wistar Rat.

Background and Purpose: The cataract in Sprague-Dawley rats injected with selenite is a dense nuclear opacity that appears by 4 or 5 days after selenite injection and becomes irreversible by 7 days. Injection of Wistar rats with selenite resulted in a similar nuclear opacity by 4 or 5 days that began to recover transparency by 7 days. In this report, the cytoplasmic proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in lenses from Sprague-Dawley and Wistar rats at 4 and 7 days after injection.Results: In the opaque lens cells, degradation of the 31 kDa protein and cytoskeletal proteins (vimentin, spectrin, and actin) was observed during cataract development using SDS-PAGE and Western blot analysis. During recovery from opacity, the decreased 31 kDa protein and the vimentin increased.Conclusion: The results suggest that the 31 kDa protein and the vimentin may be important for recovery of transparency in a reversible model of cataract formation.

Journal Article↗

PubMed2000

SMARCAD1, a novel human helicase family-defining member associated with genetic instability: cloning, expression, and mapping to 4q22-q23, a band rich in breakpoints and deletion mutants involved in several human diseases.

Members of the DEAD/H box-containing helicase superfamily include proteins essential to genome replication, repair, and expression. We report here the cloning and initial characterization of a novel human member of this protein family, designated hHel1 (human helicase 1), now designated SMARCAD1 by HUGO. This DEAD/H box-containing molecule has seven highly conserved sequence regions that allow us to place it in the SNF2 family of the helicase superfamily. Uniquely, though, hHel1 contains two DEAD/H box motifs, a property not reported to be shared by any other SNF2 family members. This defines a new subfamily consisting of hHel1 and its homologues. In addition to these DEAD/H box/ATP-binding motifs, hHel1 has a putative nuclear localization signal and several regions that may mediate protein-protein interactions. Expression analysis indicates that hHel1 transcripts are ubiquitous, with particularly high levels in endocrine tissue. We have mapped the gene for hHel1 to human chromosome 4q22-q23; this region is rich in breakpoints and deletion mutants of genes involved in several human diseases, notably soft tissue leiomyosarcoma, hepatocellular carcinoma, and hematologic malignancies. Our observation that human Hel1 gene overexpression is present in an E1A-expressing cell line with increased capacity for gene reactivation events by genomic rearrangement suggests that human Hel1 may play a role in genetic instability development.

Adenovirus E1A Proteins↗

PubMed2000

Detection of antitumor and antimicrobial activities in marine organism associated actinomycetes isolated from the Taiwan Strait, China.

The purpose of this work was to screen the actinomycetes having antitumor or antimicrobial activity, which were isolated from the surface, epidermis and intestines of sea plants and animals collected from the Taiwan Strait, China. Antitumor activity was studied by the MTT assay and DNA target activity was studied by the biochemical induction assay while antimicrobial activity was determined by observing bacterial and fungal growth inhibition. 20. 6% of marine actinomycete cultures displayed cytotoxic activity on P388 cells at dilutions at and below 1:320 and 18.6% on KB cells. 2. 96% of marine actinomycete cultures displayed inducing activity. Among all marine actinomycetes isolated, the genus Micromonospora has the highest positive rate of inducing activity. However, most antimicrobial activity was found in the genus Streptomyces. These results indicate that marine organism associated actinomycetes could be a promising source for antitumor and antimicrobial bioactive agents.

Actinomycetales↗

PubMed2000

Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring.

Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. We have developed a sensitive and rapid flow cytometry-based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The new method involves subnanomolar concentrations of sample in small volumes ( approximately 10 microl) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexing microsphere arrays (GAMMArrays), enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. We have tested the new method by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles using the multiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and global-scale SNP analysis.

Base Sequence↗

PubMed2000

Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide.

Diverse stimuli, including shear stress, cyclic strain, oxidized LDL, hyperglycemia, and cell growth, modulate endothelial nitric oxide synthase (eNOS) expression. Although seemingly unrelated, these may all alter cellular redox state, suggesting that reactive oxygen intermediates might modulate eNOS expression. The present study was designed to test this hypothesis. Exposure of bovine aortic endothelial cells for 24 hours to paraquat, a superoxide (O(2)(-*))-generating compound, did not affect eNOS mRNA levels. However, cotreatment with paraquat and either Cu(2+)/Zn(2+) superoxide dismutase or the superoxide dismutase mimetic tetrakis(4-benzoic acid)porphyrin chloride increased eNOS mRNA by 2.3- and 2.2-fold, respectively, implicating a role for H(2)O(2). Direct addition of 100 and 150 micromol/L H(2)O(2) caused increases in bovine aortic endothelial cell eNOS mRNA that were dependent on concentration (ie, 3.1- and 5.2-fold increases) and time, and elevated eNOS protein expression and enzyme activity, accordingly. Nuclear run-on and 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-chase studies showed that H(2)O(2) caused a 3.0-fold increase in eNOS gene transcription and a 2.8-fold increase in eNOS mRNA half-life. Induction of eNOS by H(2)O(2) was not affected by the hydroxyl radical scavenger DMSO, mannitol, or N-tert-butyl-alpha-phenylnitrone, but it was inhibited by the antioxidants N-acetylcysteine, ebselen, and exogenously added catalase. Unlike H(2)O(2), the 4.0-fold induction of eNOS by shear stress (15 dyne/cm(2) for 6 hours) was not inhibited by N-acetylcysteine or exogenous catalase. In conclusion, H(2)O(2) increases eNOS expression through transcriptional and post-transcriptional mechanisms. Although H(2)O(2) does not mediate shear-dependent eNOS regulation, it is likely to be involved in regulation of eNOS expression in response to other physiological and/or pathophysiological stimuli.

Animals↗

PubMed2000

The alpha-synuclein gene and Parkinson disease in a Chinese population.

OBJECTIVE: To study the Ala53Thr and Ala30Pro mutations of the alpha-synuclein gene in a large number of Chinese patients with Parkinson disease (PD) as well as controls. METHODS: We recruited 183 Chinese patients with sporadic PD, 17 with younger-onset PD (onset age <50 years), and 7 with PD and a positive family history as well as 227 unaffected Chinese control subjects from the outpatient departments of 2 major hospitals in Hong Kong. All subjects were assessed for the the diagnosis of PD by a consultant neurologist or geriatrician. Subjects were interviewed with a standard questionnaire that also questioned for family history. Venous blood samples were obtained from the subjects and genomic DNA was extracted and studied for the presence of Ala53Thr mutation in exon 4 and Ala30Pro mutation in exon 3 of the alpha-synuclein gene using a polymerase chain reaction restriction fragment length polymorphism method. RESULTS: None of the Chinese PD patients or controls had either the Ala53Thr (exon 4) or Ala30Pro (exon 3) mutation of the alpha-synuclein gene. CONCLUSION: We failed to discover Ala53Thr or Ala30Pro mutations in a large number of Chinese patients with PD and control subjects, adding to the emerging consensus that variations in the alpha-synuclein gene are associated with PD in few families worldwide.

Alleles↗

PubMed2000

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection.

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.

Animals↗

PubMed2000

Genetic polymorphism of heparan sulfate proteoglycan (perlecan, HSPG2), lipid profiles and coronary artery disease in the Australian population.

Perlecan is one of the three major classes of heparan sulfate proteoglycans (HSPGs) within the cardiovascular system; it interacts with lipid metabolism by binding to lipoprotein lipase (LpL) and apolipoprotein B (apo B) and may be related to vascular disease. We explored interactions between an HSPG2 polymorphism (BamHI marker), and apo B and coronary artery disease (CAD) in patients undergoing coronary angiography. The frequencies of the HSPG2 BamHI +/+, +/-, and -/- genotypes were 4.7, 31.7 and 63.6%, respectively, with a '+' allele frequency of 20.6%. The genotype distribution was in Hardy-Weinberg equilibrium (chi(2)=0.669, P0.05). The +/+homozygotes had the lowest apo B levels (0.74+/-0.06 g/l, n=36) compared to +/- (0.89+/-0.03 g/l, n=241) and -/- (0.93+/-0.02 g/l, n=480) genotypes. Although plasma apo B concentration was the strongest lipid risk factor for significant CAD, the HSPG2 genotypes were not independently associated with the presence of CAD (P=0.640 in males; P=0.224 in females), with significant CAD (P=0.764; P=0.110) or with the number of significantly stenosed coronary arteries (P=0.945; P=0. 335). In Australian Caucasians undergoing coronary angiography the HSPG2 BamHI polymorphism is associated with lower circulating apo B but not with the occurrence or severity of CAD. This may be due to HSPG2-mediated alterations in the HSPG2-apo B-LpL system and requires further exploration.

Australia↗

PubMed2000

Acylation of protein lysines by trichloroethylene oxide.

Stable lysine adducts were formed in proteins following reaction with trichloroethylene (TCE) oxide, the major reactive compound generated by the metabolism of TCE. The order of formation of these adducts is N(6)-formyllysine > N(6)-(dichloroacetyl)lysine >> N(6)-glyoxyllysine, with the ratio being influenced by the particular protein. Protein lysine adducts were also analyzed following the enzymatic oxidation of TCE with several different cytochrome P450 (P450) enzyme systems. The ratio of formyl/dichloroacetyl lysine adducts was influenced by the enzyme system that was used. Chloral and TCE oxide formation was more extensive with rat liver microsomes isolated from phenobarbital-treated rats than with rat microsomes in which P450 2E1 was induced by treatment with isoniazid or in human P450 2E1 systems. Glutathione (GSH) and GSH transferase had inhibitory effects on the reaction of TCE oxide with albumin, with formylation being atteunated much more than the formation of dichloroacetyllysine. GSH is likely to react with the reactive acyl chloride intermediates formed from TCE oxide hydrolysis, instead of direct reaction with TCE oxide, as judged by the lack of an effect of GSH on the rate of decomposition of TCE oxide. Studies with the model enzymes aldolase and glucose-6-phosphate dehydrogenase, both known to have sensitive lysine groups, indicate that TCE oxide has effects similar to known acylating agents that form the same adducts; concentrations of TCE oxide (or the model acylating agents) in the low-millimolar range were needed for inhibition. The characterization of TCE-derived protein adducts can be used as a basis for consideration of the exposure and risk of TCE to humans. Human P450 2E1 was less likely to oxidize TCE to form TCE oxide and protein lysine adducts than rat P450 2B1, and the difference is rationalized in terms of the influence of the protein on chloride migration in an enzyme reaction intermediate.

Acetylation↗

PubMed2000

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates.

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.

Chromatography, High Pressure Liquid↗

PubMed2000

Effects of lipid composition on the transcorneal penetration of liposomes containing disulfiram, a potential anti-cataract agent, in the rabbit.

We previously demonstrated that topical treatment with disulfiram (DSF) prevented the development of cataracts in sodium selenite-injected rat pups. In biological systems, DSF is rapidly reduced to diethyldithiocarbamate (DDC), a potent antioxidant. In this study, we investigated the effect of altering the lipid composition of liposomes containing DSF on the transcorneal transit of DDC. Liposomes containing DSF were prepared with various molar ratios of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and cetylpyridinum chloride (CPC) by reverse-phase evaporation. Liposomes with a DMPC to DPPC molar ratio of 5:5, examined by differential scanning calorimetry, had the highest enthalpy of transition and the presence of one molar ratio of CPC further enhanced the enthalpy value. The addition of bovine serum albumin or a homogenate of rabbit cornea to the incubation buffer resulted in the release of DDC, but not DSF from the liposomes. The amount of DDC present in the aqueous humor of rabbit eyes following topical administration increased with increase in DMPC to DPPC ratios and was also enhanced by the addition of CPC to the liposomes. The results of this study suggest that liposome formulations are effective for transcorneal drug delivery of anticataract agents such as DSF. DSF in liposomes consisting of DMPC, DPPC, and CPC with a molar ratio of 8:2:1 may be a potential drug formulation for the prevention and/or treatment of cataracts.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Preparation and in vivo ocular absorption studies of disulfiram solid dispersion.

Disulfiram, a dimer of diethyldithiocarbamate (DDC) which is a strong radical scavenger, is known to prevent cataract development. However, disulfiram is hardly absorbed from the cornea and its bioavailability is extremely low. In this study, we attempted to prepare disulfiram solid dispersion for the improvement of ocular bioavailability. Solid dispersions of disulfiram were prepared by either an evaporation method or a spray-drying method, using polyvinylpyrrolidone (PVP) as a carrier. Preparations were analyzed by scanning electron microscopy, powder X-ray diffractometry and differential scanning calorimetry, and confirmed to be a solid dispersion. The particle size of the solid dispersion prepared by the spray-drying method was smaller than the preparation by the evaporation method (spray-drying: 3.3+/-0.04 microm, evaporation: 34.3+/-18.0 microm). An in vivo ocular absorption experiment was conducted by instilling solid dispersions to rabbit eye and measuring the DDC in the aqueous humor. After instillation of disulfiram and PVP physical mixture, DDC was not detected in the aqueous humor. On the other hand, DDC appeared in the aqueous humor after the instillation of a solid dispersion. Maximal concentration and the area under the aqueous humor concentration-time curve were greater in the solid dispersion prepared by the spray-drying method than the preparation by the evaporation method. Disulfiram solid dispersion, especially prepared by the spray-drying method, improved ocular bioavailability.

Absorption↗