Simultaneous assay for protease activities of hepatitis C virus and human immunodeficiency virus based on fluorescence detection.

Hepatitis C virus protease (HCV-PR) and human immunodeficiency virus protease (HIV-PR) are important for virus maturation, and thus can be used as potential target molecules for the development of antiviral drugs for the treatment of viral infections. In this study, a novel assay was developed to determine HCV-PR activity. This assay is based on a fluorogenic reaction, in which peptide fragments generated from an acetyl peptide substrate by HCV-PR can be selectively converted into a fluorescent derivative, and quantified by high-performance liquid chromatography (HPLC) with fluorescent detection. Herein, several acetyl-peptides can be used as substrates for HPLC. The application of this assay was further validated by simultaneous detection of HCV-PR and HIV-PR in a reaction mixture. The proposed method can differentiate the enzyme activities of HCV-PR and HIV-PR in a sample using their corresponding substrates. The results suggest that this assay can detect various proteases by employing set of substrate peptides under the same reaction conditions.

Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker.

We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic acid substrate, followed by fluorescence (FL) detection specific for orotic acid using the 4-trifluoromethyl-benzamidoxime fluorogenic reagent. The DHODH activities in the biologically complex samples were readily measured by the assay method. Our data indicate significantly higher DHODH activity in HeLa cells (340 ± 25.9 pmol/105 cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/105 cells/h), and in malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) than in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). This is the first report that DHODH activity may be a diagnostic biomarker for cancer.

DNA Aptamers in the Diagnosis and Treatment of Human Diseases.

Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility of generation and synthesis. To better understand the specific potential of DNA aptamers, an overview of the progress in the generation and application of DNA aptamers in human disease diagnosis and therapy are presented in this review. Special attention is given to researches that are relatively close to practical application. DNA aptamers are expected to have great potential in the diagnosis and treatment of human diseases.

Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction.

Orotic acid is an intermediate in the synthesis pathway of uridine-5'-monophosphate, and increases in body fluids of patients suffering from hereditary disorders such as orotic aciduria and hyperammonemia. In this study, we developed a spectrofluorometric method with or without high-performance liquid chromatography for the selective and sensitive quantification of orotic acid in human biological specimens, using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. This reagent provided intensive fluorescence for only orotic acid amongst 62 compounds including structurally related bio-substances such as nucleic acid bases, nucleosides, nucleotides, amino acids, vitamins, bilirubin, uric acid, urea, creatine, creatinine and sugars. Under optimized reaction conditions, orotic acid was reacted with 4-TFMBAO, K3[Fe(CN)6] and K2CO3 in an aqueous solution. The fluorescence produced from the orotic acid derivative was measured at an excitation of 340 nm and an emission of 460 nm. A concentration of 1.2 µM orotic acid per 1.0 mM creatinine in normal urine and 0.64 nmol orotic acid per 5.0 × 10(5) HeLa cells were determined by this method. The present method permitted the facile quantification of orotic acid in healthy human urine and cultured HeLa cells by spectrofluorometry and/or high-performance liquid chromatography.

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants.

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance.

Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe.

Carbon nanofiber-based luminol-biotin probe for sensitive chemiluminescence detection of protein.

A carbon nanofiber-based luminol-biotin probe was synthesized for the sensitive chemiluminescence (CL) detection of a target protein by grafting luminol and biotin onto an oxidized carbon nanofiber. This carbon nanofiber was prepared by chemical vapor-deposition with methane in the presence of the Ni-Cu-MgO catalyst, which was followed by oxidization with HNO3-H2SO4 to produce a carboxyl group on the surface of the nanofiber. The material was grafted with luminol and biotin by means of a standard carbodiimide activation of COOH groups to produce corresponding amides. The substance was water-soluble and thus could be utilized as a sensitive CL probe for a protein assay. The probe showed highly specific affinity towards the biotin-labeled antibody via a streptavidin-biotin interaction. The detection limit for this model assay was approximately 0.2 pmol of the biotinized IgG spotted on a polyvinylidene fluoride (PVDF) membrane. Nonspecific binding to other proteins was not observed. Therefore, the synthesized carbon nanofiber-based CL probe may be useful for a sensitive and specific analysis of the target protein.

Amplified and selective assay of collagens by enzymatic and fluorescent reactions.

Sensitive and selective assay of collagen is of substantial importance to the diagnostic study of health- and aging-related failures. In this paper, we describe a highly specific and sensitive method for the assay of whole collagens in biological samples using a novel fluorogenic reagent, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA reagent can selectively detect N-terminal Gly-containing peptides (NGPs) in the presence of sodium borate and NaIO4. Under conditions optimized, this assay format for collagen, termed 3,4-DHPAA assay method showed a good linear relationship between the amplified FL signals and the collagen concentrations from 0.18 to 12 µg/ml. Therefore the sensitive determination of intracellular collagens in cheek tissue and HeLa cells was individually possible without any separation protocol. The dual recognitions of the collagens in the samples could be performed by the enzymatic digestion and the FL reaction. The proposed assay method enables the determination facile, specific, sensitive and quantitative for biogenic collagens.

Selective, sensitive and fluorometric determination of urinary cytosine with 4-trifluoromethylbenzamidoxime and N,N-dimethylformamide.

BACKGROUND: Cytosine in urine is one of the biomarkers for the diagnosis of metabolic immunodeficiency. It has been mentioned that a high level of cytosine is found in urine of children having immunodeficiency. In this study, we have developed a fluorescence (fluorescence) derivatization reaction of cytosine using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. METHODS: In this reaction, cytosine was mixed with 4-TFMBAO, K3[Fe(CN)6], N,N-dimethylformamide (DMF) and KOH in an aqueous solution. The mixture was heated at 100°C for 20 min. The fluorescence intensity of the mixture was measured with a spectrofluorometer. RESULTS: Under the optimized reaction conditions, a strong fluorescence was produced only from cytosine amongst 62 compounds including structurally related bio-substances. The selectivity and sensitivity of this method were compared with a conventional fluorescence one using 2-bromoacetophenone that reacts with cytosine, adenine and their related substances. The present method was sufficiently selective toward cytosine, and approximately 50 times more sensitive than the conventional one. CONCLUSIONS: Our method permitted the quantitative determination of cytosine in human urines without any pretreatment for a primary screening test of inborn disorder in pyrimidine metabolism with immunodeficiency, and indicated the lower detection limit of 0.1 µmol/l cytosine which gave 3 times greater fluorescence intensity than that observed for the reagent blank.

Dendrimer-like polymeric DNAs as chemiluminescence probes for amplified detection of telomere DNA on a solid-phase membrane.

For the first time, an amplified chemiluminescence (CL) detection of the telomere DNA spotted on a nylon membrane is described here, based on the direct hybridization with the CL probe of dendrimer-like polymeric DNAs possessing a large number of guanine moieties. This probe was synthesized by sense and antisense hybridization between Y-shaped DNAs and then could hybridize with the target DNA.

Selective FL quenching or enhancing of diimine ligands by guanine.

Diimine ligand (DL) 1 significantly exhibited the fluorescence quenching upon binding to guanine. Changing at the para-substituent of the phenyl ring from the hydroxyl to bromo groups reversely enhanced the fluorescence in the presence of guanine. The reverse in the fluorescence selectivity indicated the profound effect of the substituent at the para-position of the phenyl ring. The simple synthesis of DL 1 and DL 2 with good selectivity for guanine offers these DLs as promising compounds for chemosensors of other guanine derivatives.

Hybridization chain reaction-based instantaneous derivatization technology for chemiluminescence detection of specific DNA sequences.

We propose here a new amplifying strategy that uses hybridization chain reaction (HCR) to detect specific sequences of DNA, where stable DNA monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe that this simple technique is promising for improving medical diagnosis and treatment.

A novel fluorescence reaction for N-terminal Ser-containing peptides and its application to assay caspase activity.

Caspases are the key regulatory factors of apoptosis and are also found to be involved in inflammatory cytokinesis. Sensitive and selective determination of caspases has significant importance in evaluation of apoptosis, disease diagnosis, and drug development. Here, we developed an assay method for the determination of caspase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal Ser-containing peptides. FL derivatization of peptides requires heating in the presence of catechol, HEPES buffer (pH 7.5), and sodium periodate. Under optimized conditions, the reaction showed a unique sequence preference for N-terminal Ser-containing peptides, and a lower detection limit (signal/noise [S/N] = 3) of approximately 0.1 µM was obtained for SKTS and SSNSF. Acetylated substrates were enzymatically cleaved to produce N-terminal Ser-containing peptides, which were selectively converted to FL compounds. The enzyme activities were simultaneously determined as low as 2 U (4.3 nM) caspase-3 and 2.5 U (3.3 nM) caspase-8 by high-performance liquid chromatography (HPLC) with FL detection. The proposed assay method does not require any labeled substrates and can be applied to evaluate cell-based apoptosis and also to study apoptosis inhibitors or inducers.

Inhibition of HIV-1 protease expression in T cells owing to DNA aptamer-mediated specific delivery of siRNA.

Targeted delivery is a promising way to improve the safety and efficiency of siRNA delivery. We show that a DNA aptamer could be used to deliver siRNA into CD4(+) T cells specifically. The DNA aptamer was obtained from the conversion of a reported RNA aptamer that binds to CD4 protein on the surface of T cells. It was covalently conjugated to the sense strand of the siRNA targeting HIV-1 protease (HIV-PR). The resulting DNA aptamer-siRNA chimera could specifically enter into CD4(+) T cells and efficiently knock down the expression of exogenous HIV-PR gene. This study provides the first evidence that the DNA aptamer with intrinsic stability has a greater potential to be used for siRNA delivery.

Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons.

The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 µM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 µM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 µM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.

Selective and sensitive determination of peptides using 3,4-dihydroxyphenylacetic acid as a fluorogenic reagent.

A novel fluorescence (FL) reaction for N-terminal Gly-containing peptides has been developed using 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The reaction of the peptides with 3,4-DHPAA was carried out in borate buffer (pH 8.0) in the presence of sodium periodate at 37°C for 10 min, and the FL was measured with a spectrofluorimeter at the excitation and emission wavelengths of 370 nm and 465 nm, respectively. The 3,4-DHPAA reagent generated particularly strong FL for peptides containing Gly at their N-termini. When various other bio-substances, such as amino acids, sugars, nucleic bases, nucleotides, and proteins, were reacted with 3,4-DHPAA, no FL was observed. Under optimized reaction conditions, the lower detection limit of 0.25 µmol L(-1) was obtained for the N-terminal Gly-containing peptides of Gly-Pro (GP) and Gly-Pro-Pro (GPP), which gave 3 times greater FL intensity than that observed for the reagent blank. The proposed reaction with 3,4-DHPAA as a fluorogenic reagent is selective and sensitive for the detection of N-terminal Gly-containing peptides, and therefore, this method could be a useful tool for the determination of these particular oligopeptides.

Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane.

In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27-31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14-20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays.

Sensitive chemiluminescence detection of prion protein on a membrane by using a peroxidase-labeled dextran probe.

We synthesized dextran-based macromolecular probes carrying multiple molecules of horseradish peroxidase (HRP) as a signal-trigger enzyme and of biotin as an assembly mediator without losing the enzymatic activity. Multiple attachments of HRP and biotin to the dextran backbone were readily accomplished after the formation of aldehyde groups into the dextran macromolecule by periodate oxidation. The synthesized macromolecular probe was successfully used for sensitive chemiluminescence (CL)-imaging detection of mouse recombinant prion protein on a nylon membrane. The prion protein at a small amount of 20 fmol blotted on a nylon membrane was specifically detected, indicating at least a 10-times higher sensitivity than that of a conventional biotinylated HRP probe. Therefore, the synthesized dextran-based probes containing HRP and biotin should be used for the sensitive high-throughput analysis of various proteins on a solid-phase membrane.

A manual sequence method of peptides and phosphopeptides using 4-(1'-cyanoisoindolyl)phenylisothiocyanate.

A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1'-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.

Chemiluminescence detection of telomere DNA in human cells on a membrane by using fluorescein-5-isothiocyanate-labeled primers.

Telomere DNA is related to cell aging and cancer genesis because the telomeric region of DNA sequences at chromosome ends are shortened with cell divisions. Therefore, a sensitive and specific detection method is required for the telomere DNA. Here we propose a chemiluminescence (CL)-based method for the sensitive detection of telomere DNA in human cells. In this study, the telomere DNA was amplified by polymerase chain reaction (PCR) using special forward and reverse primers labeled with fluorescein-5-isothiocyanate (FITC) at the 5' end, and then the FITC-containing PCR products were detected by CL reaction with 3,4,5-trimethoxyphenylglyoxal (TMPG) after electrophoresis followed by Southern blot onto a nylon membrane. The TMPG reagent specifically reacted with guanine moiety in DNA at room temperature and provided CL intensities. The CL intensities from the PCR products could be enhanced approximately 10-fold using FITC-labeled primers as compared with those using nonlabeled primers. The detection limit of the PCR products with the proposed method was 0.3 ng on the membrane. The developed CL method could quantitatively determine the telomere DNA in a small number of human cells (∼350) and gave approximately 10 times higher sensitivity than a conventional fluorescence-based method.