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.     

Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.     

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.     

Polyacrylamide Gel Electrophoresis of Plasteins

A peptic hydrolysate of soybean protein was filtered with Sephadex G–25 and was separated approximately into four fractions (I, II, II, and IV in the order of mol. wt.). Fraction II (av. mol. wt: 1043) and III (av. mol. Wt.: 685) were more plastein-productive than others. When plastein produced from Fraction II with Nagarse was investigated by plate electrophoresis using 7.5% polyacrylamide-gel, the upper limit of the molecular weight was found to be about 25,000. A similar result was obtained also with Fraction III. The increase of molecular weight in the course of the plastein formation with the mixture (substrate) of Fractions II and III was shown that the final product lay mainly in a position between cytochrome c (mol. wt.: 11,700) and Nagarse (mol wt.: 27,600). In addition, the gel-electrophoretic experiments revealed that the most favorable condition for the plastein synthesis were pH 6.5 and 35% in substrate concentration.     

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.     

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Influenza and Other Respiratory Viruses Detected by Influenza-Like Illness Surveillance in Leyte Island,the Philippines, 2010–2013

This study aimed to determine the role of influenza-like illness (ILI) surveillance conducted on Leyte Island, the Philippines, including involvement of other respiratory viruses, from 2010 to 2013. ILI surveillance was conducted from January 2010 to March 2013 with 3 sentinel sites located in Tacloban city, Palo and Tanauan of Leyte Island. ILI was defined as fever ≥38°C or feverish feeling and either cough or running nose in a patient of any age. Influenza virus and other 5 respiratory viruses were searched. A total of 5,550 ILI cases visited the 3 sites and specimens were collected from 2,031 (36.6%) cases. Among the cases sampled, 1,637 (75.6%) were children aged <5 years. 874 (43.0%) cases were positive for at least one of the respiratory viruses tested. Influenza virus and respiratory syncytial virus (RSV) were predominantly detected (both were 25.7%) followed by human rhinovirus (HRV) (17.5%). The age distributions were significantly different between those who were positive for influenza, HRV, and RSV. ILI cases were reported throughout the year and influenza virus was co-detected with those viruses on approximately half of the weeks of study period (RSV in 60.5% and HRV 47.4%). In terms of clinical manifestations, only the rates of headache and sore throat were significantly higher in influenza positive cases than cases positive to other viruses. In conclusion, syndromic ILI surveillance in this area is difficult to detect the start of influenza epidemic without laboratory confirmation which requires huge resources. Age was an important factor that affected positive rates of influenza and other respiratory viruses. Involvement of older age children may be useful to detect influenza more effectively.     

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.