Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.     

Flavor Constituents of Pouchong Tea and a Comparison of the Aroma Pattern with Jasmine Tea

The aroma constituents of the highest quality pouchong tea were characterized by GC–MS. Forty-eight components, including five newly identified compounds were characterized. GC peak area percentages of the main components in pouchong tea were compared with those of jasmine tea to differentiate compounds contributing to the aroma characteristics of pouchong tea with superior floral elegant flavor. Nerolidol, jasmine lactone, methyl jasmonate, indole, benzyl cyanide and linalool oxides were found in much higher concentration in pouchong tea than in jasmine tea. These compounds seemed to contribute to the aroma characteristics of the highest quality pouchong tea.     

Glutamyl Oligopeptides as Factors Responsible for Tastes of a Proteinase-modified Soybean Protein

AK-toxin I, a host-specific toxin to Japanese pear (Pyrus serotina), was synthesized as its methyl ester from three precursor fragments: conjugated diene-carboxylic acid, chiral epoxyalcohol and β-methylphenylalanine. The epoxyalcohol fragment was derived from D-fructose, in which effective homologation of the hemiacetal carbon to alkyne by using dimethyl 1-diazo-2-oxopropylphosphonate was the key reaction. The diene-carboxylic acid fragment was prepared by repeated Wittig reactions, and was combined with the epoxyalcohol fragment by the Stille reaction. Esterification of the combined product with the stereochemically-pure β-methylphenylalanine fragment afforded the target compound. This method was used to prepare the methyl ester of tritium-labeled AK-toxin I with a specific radioactivity of 213 GBq/mmol.     

Production and Isolation of a New Antifungal Antibiotic,Prumycin and Taxonomic Studies of Streptomyces sp., Strain No. F-1028

A newly described species of Streptomyces (named Sm. kagawaensis ATCC No. 21811) isolated from soil was found to produce a new antifungal antibiotic, prumycin, which belongs to amino sugar group. Prumycin was isolated from the fermentation broth by ion-exchange adsorption and gel-filtration methods. This antibiotic inhibited specifically the growth of Sclerotinia sp. and Botrytis sp. on flower pot test with kidney bean leaves and also was effective on the field test with various plants.     

Studies on the Flavor of Green Tea

By continuing flavor analysis of green tea from a previous paper, further twenty seven compounds were newly identified. These compounds are limonene, α-cubebene, α-copaene, caryophyllene, α-humulene, α- and γ-muurolene, β-sesquiphellandrene, δ-cadinene, calamenene, cubenol, α-cadinol, α-terpineol, n-heptanol, n-nonanol, furfurylalcohol, n-nonanal, N-ethylformylpyrrole, pyrrylmethylketone, 6-methyl-trans-3,5-heptadien-2-one, 2′,2″-dihydro-α-ionone, 6,10,14-trimethyl-2-pentadecanone, cis-3-hexenylcaproate, cis-3-hexenylbenzoate, α-terpinylacetate, coumarin and diphenylamine.

Relative quantities of known compounds in intermediate- and high-boiling fraction were determined.     

Applying Proteolytic Enzymes on Soybean

An indole derivative having blue fluorescence was produced in cooked soybean digested at 37°C for 24 hr with an acid proteinase Molsin (optimum pH: 2.8) from Aspergillus saitoi or a usual acid proteinase pepsin (optimum pH: 1.6) from beef stomach. This indole derivative was identical with a condensation product from l-tryptophan and n-hexanal. Based on MS, NMR, IR and UV spectrometry, the condensation product was identified as l-pentyl-2, 3, 4, 9-tetrahydro-lH-pyrido [3, 4-b]-indole-3-carboxylic acid [trivial name: 1-pentyl-l, 2, 3, 4-tetrahydro-2-carboline carboxylic acid-(3)].

Data were presented of the formation of the above indole derivative and of the resulting consumption of l-tryptophan and n-hexanal.

The possible ocurrence of the formation of Harmala alkaloids, i.e. 2-carboline derivatives, through in vitro digestion of soybean with acid proteinases was discussed.

A carbonyl-trapping ability of l-tryptophan was suggested.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Applying Proteolytic Enzymes on Soybean

Soy proteins were incubated with a microbial acid protease (Molsin) under the following condition: substrate concentration, 1%; enzyme-substrate ratio (by weight), 1/100; pH, 2.8; and temperature, 40°C—flavor components and related impurities are removable from crude soy-protein concentrates by their incubation for 2 hr under the above condition. The acid-precipitated fraction of soy protein incubated for 2 hr with Molsin (i.e. 2 hr-proteolyzate) showed the following composition: 10% trichloroacetic acid (TCA) insoluble fraction, 47.52%; 10% TCA soluble peptide fraction, 52.02%; and free amino acid fraction, 0.46%. Gel filtration of the 2 hr-proteolyzate gave an elution pattern showing its molecular weight distribution.

In the process of the incubation of the acid-precipitated protein, the 10% TCA insoluble fraction showed increase in amino nitrogen content, its solubility in a phosphate buffer increased to change at 6 hr, and a hydrophobic amino acid share in this fraction increased gradually.

In vitro digestibility of the acid-precipitated fraction were improved and the lipoxygenase activity in this fraction decreased through the Molsin treatment.

Ultracentrifugal analysis showed a decreasing tendency of the cold-insoluble fraction of soy protein during its incubation with Molsin. Optical rotatory dispersion and circular dichroism study elucidated conformational changes in this fraction during its incubation either with or without Molsin.     

Condensation Reaction Occurring during Plastein Formation by α-Chymotrypsin

An investigation was conducted on myosin and actin-activated heavy meromyosin (HMM) ATPase activities in normal porcine muscle stored for varying periods of time after death. Studies were also made on temperature dependent myosin ATPase, initial burst of ATPase and actin-activated HMM ATPase in normal and in pale, soft and exudative (PSE) porcine muscle. The maximum velocity of acto-HMM ATPase of normal muscle decreased considerably with postmortem time, while the apparent dissociation constant decreased slightly. The maximum velocity of acto-HMM ATPase of postmortem normal muscle was approximately two-times larger than that of the corresponding PSE muscle. However, almost no difference was found in the apparent dissociation constant. The size of the initial burst of phosphate-liberation of myosin prepared from normal muscle was approximately 1.2 mol/mol of myosin and from PSE muscle 0. It is assumed that the lack of contractility of PSE muscle was brought about by two basic myosin malfunctions: one, the irreversible binding of myosin to actin filament and the other, the functional damage of myosin ATPase, responsible for the formation of phosphorylated complex, even when dissociable.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.