Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).     

Characterization of WiDr: A human colon carcinoma cell line

Summary We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Thalidomide and its analogues as cyclooxygenase inhibitors

Thalidomide showed cyclooxygenase (COX)-1/2 inhibitory activity with a potency comparable to that of aspirin. Structural development studies of thalidomide resulted in potent COX-1/2 inhibitors, and COX-1-selective and COX-2-selective inhibitors.     

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.     

Lamp-1 is upregulated in human glioblastoma cell lines induced to undergo apoptosis

Lysosome-associated membrane protein (LAMP)-1, one of the major protein components of the lysosomal membrane, is upregulated in the human glioblastoma cell lines, U-373 MG and LN-Z308, which undergo cisplatin-induced apoptosis. These human brain tumor cell lines demonstrated apoptosis in response to cisplatin/nifedipine treatment. Both cell lines demonstrated an apoptotic response by more than one criterion. Apoptosis was demonstrated by DNA fragmentation techniques such as DNA laddering, ApopTag in situ labeling, and an ELISA-based method of detecting liberated oligosomes. These cells also had characteristic morphologic changes and upregulation of bax consistent with apoptosis. LAMP-1 expression at the protein and mRNA level was examined and found to increase with cisplatin/nifedipine treatment. LAMP-1 expression was examined using indirect immunofluorescent staining, Northern blot analysis and Western blot analysis. The finding of an augmentation of LAMP-1 in these cells induced to die is enigmatic. These findings raise the possibility of LAMP-1 involvement in the apoptotic process.     

Involvement of the endocannabinoid system in periodontal healing

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.     

Different regulation of leaf respiration between Spinacia oleracea, a sun species, and Alocasia odora, a shade species

Mature leaves of shade species exhibit lower respiratory rates than those of sun species. To elucidate the mechanism underlying different respiratory rates between sun and shade species, we examined respiratory properties of leaves in Spinacia oleracea L., a sun species, and Alocasia odora (Lodd.) Spach, a shade species, with special reference to changes in the respiratory rate throughout the night. In S. oleracea , rates of both CO₂ efflux and O₂ uptake decreased with time during the night, whereas in A. odora both rates were virtually constant at lower levels. The rates of O₂ uptake in S . oleracea increased upon addition of sucrose, and the rates attained were virtually identical throughout the night. However, the addition of an uncoupler [carbonyl cyanide p -(trifluoromethoxy)-phenylhydrazone; FCCP] did not alter the rates. In contrast, the rates of O₂ uptake in A. odora were enhanced by the addition of FCCP, but not by sucrose. The concentrations of carbohydrates in the tissue decreased throughout the night in both species and the ATP/ADP ratio was always greater in A. odora. These results indicate that, in S. oleracea , the availability of respiratory substrate determines the respiratory rate, while the low respiratory rate in A. odora is ascribed to its low demand for ATP.     

Aspergillus melleus protease-catalyzed peptide synthesis using the carbamoylmethyl ester as an acyl donor in 1,1,1,3,3,3-hexafluoro- 2-propanol/N,N-dimethylformamide

The coupling between the carbamoylmethyl ester of an N-protected amino acid or dipeptide (at 25 mM) and an amino acid amide (at 100 mM) was achieved using Aspergillus melleus protease in 1,1,1,3,3,3-hexafluoro-2-propanol/N,N-dimethylformamide (1:1, v/v); the coupling efficiencies were dependent largely on the combination of amino acid residues: e.g. the dipeptide yields after 48 h were for l-Ala + Gly, 100% and for l-Leu + l-Leu, 16%.