D-glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Differential expression of phospholipases D1 and D2 in mouse tissues

The differential expression of phospholipase D (PLD) isozymes, which include PLD1 and PLD2, was examined in various murine tissues, including the cerebrum, cerebellum, heart, lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus. In Western blot analysis, only PLD1 was detected in the heart and ovary, while only PLD2 was detected in the pancreas and ileum. Both PLD1 and PLD2 were strongly expressed in the cerebrum, cerebellum, and lung, and both were also expressed in the liver, spleen, stomach, colon, kidney, testes, and uterus. Immunohistochemistry showed intense PLD immunostaining in the cerebrum, cerebellum, lungs, intestines, and testis, and weak PLD immunostaining in the liver, kidneys, spleen, and heart. These findings suggest that PLD1 and PLD2 are differentially expressed in the various organs of mice, and that each PLD isozyme plays a distinct role in each organ.     

Solution structure of termite-derived antimicrobial peptide,spinigerin, as determined in SDS micelle by NMR spectroscopy

Spinigerin is a linear antibacterial peptide derived from a termite insect. It consists of 25 amino acids and is devoid of cysteines. Spinigerin displays good lytic activities against Gram-positive and Gram-negative bacteria, but has no hemolytic activities against human erythrocytes. In this study, we present a three-dimensional solution structure of spinigerin in SDS micelles. According to CD data spinigerin has an alpha-helical conformation in the presence of TFE, DPC micelles, and SDS micelles. The three-dimensional structure of spinigerin as determined by NMR spectroscopy contains a stable alpha-helix from Lys4 to Thr23. Spinigerin (4-21), an 18-residue fragment from Lys4 to Leu21, contains a similar content of alpha-helical structure compared to native spinigerin and was found to retain antibacterial activity, too. Therefore, this alpha-helical structure and the strong electrostatic attraction between four Lys and three Arg residues in spinigerin and the negatively charged polar head groups of the phospholipids on the membrane surface play important roles in disrupting membrane and subsequent cell death.     

Expression of vascular endothelial growth factor in the progression of cervical neoplasia and its relation to angiogenesis and p53 status

OBJECTIVE: To evaluate vascular endothelial growth factor (VEGF) expression in the successive steps of cervical neoplasia and to determine its correlation with angiogenesis and p53 status. STUDY DESIGN: Immunohistochemical staining with a VEGF monoclonal antibody was performed on a total of 161 cervical specimens representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 30 CIN 3 and 86 squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody to CD34. Computerized image analysis was used to evaluate microvessel density (MVD). p53 Status was determined by immunohistochemistry and direct sequencing of exons 5-8 of the p53 gene. RESULTS: VEGF expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < .05). MVD increased significantly with cervical neoplasia progression, from normal epithelium, through CIN, to squamous cell carcinoma (P < .001). A strong correlation was observed between VEGF expression and MVD (P < .001). p53 Protein expression was not detected in the normal epithelium or in CIN 1, while 3 (10%) of 30 CIN 3 and 28 (33%) of 86 squamous cell carcinomas were positive for p53. VEGF expression correlated statistically with p53 protein expression (P < .001). In double VEGF- and p53-stained sections, the 2 markers were generally expressed in the same tumor cells. Of the 4 p53 gene mutations, 3 exhibited strong VEGF expression, and 1 exhibited moderate VEGF expression. VEGF expression did not correlate significantly with outcome variables in patients with squamous cell carcinoma. CONCLUSION: Our results suggest that VEGF expression is involved in the promotion of angiogenesis in cervical neoplasia and that p53 is likely to be involved in the regulation of VEGF expression.     

Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress

Previous studies have shown that dermal fibroblast cell lines derived from young adult mice of the long-lived Snell dwarf mutant stock are resistant, in vitro, to the cytotoxic effects of H(2)O(2), cadmium, UV light, paraquat, and heat. We show here that similar resistance profiles are seen in fibroblast cells derived from a related mutant, the Ames dwarf mouse, and that cells from growth hormone receptor-null mice are resistant to H(2)O(2), paraquat, and UV but not to cadmium. Resistance to UV light, cadmium, and H(2)O(2) are similar in cells derived from 1-wk-old Snell dwarf or normal mice, and thus the resistance of cell lines derived from young adult donors reflects developmental processes, presumably hormone dependent, that take place in the first few months of life. The resistance of cells from Snell dwarf mice to these stresses does not reflect merely antioxidant defenses: dwarf-derived cells are also resistant to the DNA-alkylating agent methyl methanesulfonate. Furthermore, inhibitor studies show that fibroblast resistance to UV light is unaffected by the antioxidants ascorbic acid and N-acetyl-L-cysteine. These data suggest that postnatal exposure to altered levels of pituitary hormones leads to development of cellular resistance to oxidative and nonoxidative stressors, which are stable through many rounds of in vitro cell division and could contribute to the remarkable disease resistance of long-lived mutant mice.     

Protective effect of the phosphodiesterase III inhibitor cilostazol on amyloid β-induced cognitive deficits associated with decreased amyloid β accumulation

Alzheimer’s disease (AD), which is characterized by progressive cognitive impairment, is the most common neurodegenerative disease. Here, we investigated the preventive effect of a phosphodiesterase III inhibitor, cilostazol against cognitive decline in AD mouse model. In vitro studies using N2a cells stably expressing human amyloid precursor protein Swedish mutation (N2aSwe) showed that cilostazol decreased the amyloid β (Aβ) levels in the conditioned medium and cell lysates. Cilostazol attenuated the expression of ApoE, which is responsible for Aβ aggregation, in N2aSwe. Intracerebroventricular injection of Aβ_25–35 in C57BL/6J mice resulted in increased immunoreactivity of Aβ and p-Tau, and microglia activation in the brain. Oral administration of cilostazol for 2 weeks before Aβ administration and once a day for 4 weeks post-surgery almost completely prevented the Aβ-induced increases of Aβ and p-Tau immunoreactivity, as well as CD11b immunoreactivity. However, post-treatment with cilostazol 4 weeks after Aβ administration, when Aβ was already accumulated, did not prevent the Aβ-induced neuropathological responses. Furthermore, cilostazol did not affect the neprilysin and insulin degrading enzymes involved in the degradation of the Aβ peptide, but decreased ApoE levels in Aβ-injected brain. In addition, cilostazol significantly improved spatial learning and memory in Aβ-injected mice. The findings suggest that a phosphodiesterase III inhibitor, cilostazol significantly decreased Aβ accumulation and improved memory impairment induced by Aβ_25–35. The beneficial effects of cilostazol might be explained by the reduction of Aβ accumulation and tau phosphorylation, not through an increase in Aβ degradation but via a significant decrease in ApoE-mediated Aβ aggregation. Cilostazol may be the basis of a novel strategy for the therapy of AD.     

Brap2 functions as a cytoplasmic retention protein for p21 during monocyte differentiation

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.     

Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.