Kazumi Sasamoto

A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability

A highly water soluble disulfonated tetrazolium salt, 4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt, was synthesized. The compound is reduced by NADH in good yields at neutral pHs in the presence of 1-methoxy PMS to produce the corresponding formazan dye that absorbs at 460 nm. The formazan is soluble to water at concentrations higher than 0.1 M. The tetrazolium salt thus proved to be useful as a sensitive chromogenic indicator for NADH. It is also applicable to cell proliferation assays as a cell viability indicator.

A water-soluble tetrazolium salt useful for colorimetric cell viability assay

The application of a tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt (WST-8), to cell viability assays and in vitro drug sensitivity tests is described. With a higher sensitivity as a chromogenic indicator for cell viability compared with conventional tetrazolium salts, WST-8 produced results of cell viability and IC50 values that were in good agreement, respectively, with the MTT method and [3H]thymidine uptake method.

Novel disulfonated tetrazolium salt that can be reduced to a water-soluble formazan and its application to the assay of lactate dehydrogenase

A new tetrazolium salt, 4-[3-(4-iodophenyl)-2-(2,4-dinitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, sodium salt, that produces a highly water-soluble formazan dye upon reduction by reduced nicotinamide adenine dinucleotide (NADH) was synthesized. The reduction of the compound by NADH at a neutral pH is fast owing to its small reduction potential. The applicability of the compound to the assay of lactate dehydrogenase is described in comparison with a prevalent tetrazolium salt.

2-(5-hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography and its application to the assay of fatty acids in human serum.

Abstract A unique acid hydrazide, 2-(5-hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole, that is characterized by the benzothiazole structure conjugated to an oxazoline moiety was synthesized, and its applicability as a precolumn derivation reagent for carboxylic acids in HPLC was examined in view of sensitivity and separability. The sensitivity of the hydrazide for carboxylic acids was determined using lauric acid to be 0.1 pmol (per 10-μ1 injection volume) at a signal-to-noise ratio of 3. The reagent allowed rapid assays of carboxylic acids within 20 min with satisfactory separability. The method was applied to the determination of fatty acids in human sera from healthy volunteers as well as from patients with diabetes or thyroid dysfunction.

Detection of nitric oxide and nitrite by using a Rhodamine-type fluorescent indicator

A unique, colorless Rhodamine derivative, Rhodamine B hydrazide (RBH), was shown to react with nitrite in acidic pH to produce an absorption at 561 nm. The sensitivity of RBH toward nitrite was higher than conventional methods. RBH also demonstrated to give a fluorescence at 581 nm when incubated with an NO donor at neutral pH, and the reaction was enhanced by using an NO scavenger, Carboxy-PTIO.

Benzothiazole derivatives as substrates for alkaline phosphatase assay with fluorescence and chemiluminescence detection

Unique fluorogenic and chemiluminescence substrates for alkaline phosphatase (ALP), 2-(N-phenylphthalimidyl)benzothiazolyl-5-phosphate and 2-(2′,3′-dihydro-1′,4′-dionephthalazyl)benzothiazolyl-5-phosphate, respectively, that contain a highly fluorescent benzothiazole moiety as the fluorophore were synthesized and their applicabilities to the assay of ALP were examined. The chemiluminogenic substrate exhibited a linear chemiluminescence response to the amount of ALP in the range 0–20 pmol.

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujitas organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N′-bis[N″, N‴-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.

2-(5-hydrazinocarbonyl-2-thienyl)-5,6-methylenedioxybenzofuran and 2-(5-hydrazinocarbonyl-2-furyl)-5,6-methylenedioxybenzofuran as novel fluorescence derivatisation reagents for carboxylic acids in liquid chromatography

Abstract The new acid hydrazides, 2-(5-hydrazinocarbonyl-2-thienyl)-5,6-methylenedioxybenzofuran (TMBH) and 2-(5-hydrazin-ocarbonyl-2-furyl)-5, 6-methylenedioxybenzofuran (FMBH), were synthesized as precolumn fluorescence derivatisation reagents for carboxylic acids in liquid chromatography. These compounds readily react with carboxylic acids in the presence of a coupling reagent under mild conditions in aqueous solution to give fluorescent derivatives. The detection limits of lauric acid were both 0.1 pmol per 10 μl injection volume at a signal-to-noise ratio of 3. The methods in which these compounds are used were applied to the assay of a standard mixture of prostaglandins (25 μM) and prostaglandins in a human seminal fluid.

Live Cell Imaging of Mitochondrial Autophagy with a Novel Fluorescent Small Molecule

There has been a growing interest in mitophagy, mitochondria-selective autophagy, which plays an essential role in maintaining intracellular homeostasis. We have developed a small-molecule fluorescent probe, Mtphagy Dye, for visualizing mitophagy, which was readily synthesized from a known perylene derivative, perylene-3,4-dicarboxylic anhydride. Mtphagy Dye has suitable fluorescent properties for detecting mitochondrial acidification during mitophagy in the long-wavelength region that does not damage mitochondria. Using Mtphagy Dye, we were able to visualize mitophagy with both cases of Parkin-dependent and -independent HeLa cells.

Small fluorescent molecules for monitoring autophagic flux

We have developed two types of fluorescent probes, DALGreen and DAPGreen, for monitoring autophagy, that exhibit fluorescence upon being incorporated into autophagosomes. DALGreen enhances its fluorescence at acidic pH, which is favorable for monitoring late‐phase autophagy, whereas DAPGreen remains fluorescent with almost constant brightness during the autophagic process. With these probes that stain autophagosomes as they are being formed, the real‐time change of autophagic phenomena of live cells may be traced, which is an advantage over conventional approaches with small molecules that stain mature autophagosomes. The use of both dyes allows monitoring of the membrane dynamics of autophagy in any type of cell without the need for genetic engineering, and therefore, will be useful as a tool to study autophagic phenomena.