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.     

Effect of Exogenous Fatty Acids on the Cellular Fatty Acid Composition and Neomycin Formation in a Mutant Strain of Streptomyces fradiae

A mutant of Streptomyces fradiae which requires oleic acid for neomycin formation was isolated and the effects of exogenous fatty acids and other additives on the formation of neomycin were studied. Palmitic acid and high concentration of sodium ions could replace oleic acid in neomycin formation. The fatty acid spectrum of the mutant strain ST–5B was quite different from that of the parent strain 3123. The major fatty acid components of the mutant and the parent were anteiso 15:0 and iso 16: 0, respectively. However the fatty acid composition of the mutant was changed from the anteiso 15: 0-type to the parental iso 16: 0-type by the supplement of oleic acid or high concentration of sodium ions in the medium. In the case of palmitic acid, the major fatty acid component of the mutant cells was changed from anteriso 15: 0 to normal 16:0. The role of these additives in neomycin formation by the mutant is discussed.     

Changes in 1-Aminocyclopropane-1-carboxylic Acid Content and Ethylene Production in Hiproly Barley Callus during Differentiation

During differentiation after auxin withdrawal, the change in the ethylene production of Hiproly barley callus paralleled the change in 1-aminocyclopropane-1-carboxylic acid (ACC) content. The levels of ACC and ethylene production decreased rapidly, and then increased in Hiproly barley callus.

Aminooxyacetic acid (AOA) prevented the ACC and ethylene production of the callus. Moreover, aminoisobutyric acid (AIB) also inhibited the ethylene production, but did not prevent the ACC synthesis of the callus. On the other hand, methylglyoxal-bis(guanylhydrazone) (MGBG) greatly enhanced the ACC and ethylene production. Formation of adventitious roots in Hiproly barley callus was enhanced by the cultivation in the medium containing AIB or AOA. However, differentiation of the callus was strongly inhibited by MGBG.

Thus, prevention of ethylene production may be significant for differentiation of Hiproly barley callus.     

l-Glutamic Acid Fermentation

A study was made on the differences between Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant D-248 in some physiological characteristics.

The most important difference of the characteristics was found in their intracellular fatty acid contents. Namely, the cellular oleic acid content of D-248 was scarcely affected by biotin but limited by the oleic acid which was added to the medium.

On the other hand, various enzyme activities and rates of oxygen uptake for several organic acids were found to be slightly different between the two strains.

These observations suggest that oleic acid has an important role for the production of l-glutamic acid.

The effect of biotin and oleic acid on the cellular fatty acid contents, and the relation between the cellular fatty acid contents and the productivity of l-glutamic acid were investigated using Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant, D-248.

While the synthesis of palmitic acid in D-248 was stimulated by biotin and competitively reversed by oleic acid added to the culture medium, the level of cellular oleic acid was scarcely affected by biotin but regulated by oleic acid in the medium.

For the productivity of L-glutamic acid, the most important factor was the level of cellular oleic acid, and the effect of cellular palmitic acid was considerably weak. This relation was subjected to a figuration and able to be expressed on the whole as one exponential-like curve. An amount of over 70 per cent of cellular fatty acids was distributed in the phospholipid fraction and its fatty acid composition was almost the same as that of whole cells.     

“Nojirimycin” as a Potent Inhibitor of Glucosidase

Glucose-6-phosphate dehydrogenase in a yeast, Hansenula mrakii IFO 0895 is induced when the cells are cultured in a medium containing lipid hydroperoxide. The enzyme was purified from H. mrakii to the homogeneous state on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be approximately 52kDa by SDS-PAGE and 130 kDa by Sephadex G-150column chromatography, respectively. The enzyme was specific to glucose-6-phosphate and NADP⁺, and K_mvalues for glucose-6-phosphate and NADP⁺ were 293µM and 24.1 µM, respectively. The enzyme activity was inhibited by diethylpyrocarbonate and 2, 4, 6-trinitrobenzene sulfonate, and by metal ions such as Zn^{2 +}, Cd^{2 +}, Cu^{2 +}, and Al^{3 +} . tert-Butyl hydroperoxide, a kind of lipid hydroperoxide, slightly(approximately 20%) increased the enzyme activity.     

Antitumor Lipopolysaccharide from Heptoseless Mutant of Proteus mirabilis

Studies on lipopolysaccharide (LPS) from the cells of Proteus mirabilis RMS-203 were focused upon reduction of lethal toxicity and of pyrogenicity by biological and chemical modification. A heptoseless mutant, strain N-434, was isolated by the use of phage resistancy as a tool. LPS from that heptoseless mutant was completely deficient in neutral sugars and mainly composed of 2-keto-deoxy-octonic acid (KDO), glucosamine and fatty acids. It revealed almost the same antitumor activity as LPS of the wild type but it was less toxic and less pyrogenic.

Hydroxylaminolysis and reduction with LiAlH₄ resulted in removal of fatty acids from LPS accompanied with decrease in lethal toxicity and antitumor acitivity but not in pyrogenicity.

Lipid A fractions showed almost the same antitumor activity as intact LPS but less lethality and less pyrogenicity.     

Isolation and Characterization of Antitumor Lipopolysaccharide from Proteus mirabilis

Systematic isolation of the cell constituents of Proteus mirabilis RMS–203 was performed to find out localization of antitumor principle only in the lipopolysaccharide (LPS) layer of the cell wall fraction.

LPS with strong antitumor activity was extracted from P. mirabilis RMS–203 by phenol-water method followed by purification on DEAE-Sephadex A–50 column chromatography.

The main components of purified LPS were galactose, hexosamine, 2-keto-deoxy-octonic acid (KDO), myristic acid, β-hydroxymyristic acid and α,ε-diaminopimelic acid.

The minimal effective dose of LPS against Ehrlich solid carcinoma in mice was 0.1~1.0 μg/mouse. LD₅₀ in mice and pyrogenicity in rabbits were 28 mg/kg and 10^?3–10^?5 μg/rabbit, respectively.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.     

Relationship between G1287A of the NET Gene Polymorphisms and Brain Volume in Major Depressive Disorder: A Voxel-Based MRI Study

Background

Earlier studies implicated norepinephrine transporter (NET) gene (SLC6A2) polymorphisms in the etiology of major depressive disorder (MDD). Recently, two single nucleotide SLC6A2 polymorphisms, G1287A in exon 9 and T-182C in the promoter region, were found to be associated with MDD in different populations. We investigated the relationship between the brain volume and these two polymorphisms of the SLC6A2 in MDD patients.

Methods

We obtained 3D high-resolution T1-weighted images of 30 first-episode MDD patients and 48 age- and sex-matched healthy subjects (HS). All were divided into 4 groups based on polymorphism of either the G1287A or the T-182C genotype. VBM analysis examined the effects of diagnosis, genotype, and genotype-diagnosis interactions.

Results

Diagnosis effects on the brain morphology were found in the left superior temporal cortex. No significant genotype effects were found in the T-182C and the G1287A. A significant genotype (G1287A)–diagnosis interaction was found in the left dorsolateral prefrontal cortex. No significant genotype (T-182C)–diagnosis interaction effects were observed in any brain region.

Conclusions

In MDD patients there seems to be a relationship between the volume of the dorsolateral prefrontal cortex and polymorphism of the SLC6A2 G1287A gene.