Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells

Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic β cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic β cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic β cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.     

Identification and biocontrol efficacy of Streptomyces miharaensis producing filipin III against Fusarium wilt

A number of bacterial strains were isolated from the internal tissue of Trapa japonica. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of Streptomyces miharaensis showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP-1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH-20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP-1 inhibited the mycelial growth of plant pathogenic fungi such as Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum at 1-10 μg ml(-1) and also markedly inhibited the development of Fusarium wilt caused by F. oxysporum f.sp. lycopersici in tomato plants by treatment with 10 μg ml(-1) under greenhouse conditions. The efficacy of FP-1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An egfp -tagged strain of KPE62302H confirmed its ability to colonize tomato plants.     

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.     

Remodeling of the glycosylation pathway in the methylotrophic yeast Hansenula polymorpha to produce human hybrid-type N-glycans

As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc₁Man₅GlcNAc₂ and GlcNAc₁Man₃GlcNAc₂, respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.     

Increased Expression of ATG10 in Colorectal Cancer Is Associated with Lymphovascular Invasion and Lymph Node Metastasis

Background

Autophagy has paradoxical and complex functions in cancer development, and autophagy-related genes (ATG) are key regulators in autophagy. Until now, more than 30 different ATG proteins have been identified in yeast, and their mammalian counterparts also have been reported. Although the roles of a few ATG proteins in cancer have been characterized, the role of ATG10 is almost completely unknown.

Methodology/Principal Findings

To investigate the clinicopathological role of ATG10 in colorectal cancer, we analyzed ATG10 expression in colorectal cancer tissues and cell lines. Protein expression analysis showed that ATG10 is highly increased in colorectal cancer (tissue - 18/37 cases, 48%; cell line –8/12 cell lines, 66%). Immunohistochemical analysis with clinicopathological features indicated a strong association of the up-regulation of ATG10 with tumor lymph node metastasis (p = 0.005) and invasion (p<0.001). Moreover, both 5-year disease free survival and overall survival rates of patients bearing tumors that did not express ATG10 were significantly higher than those of patients bearing ATG10-expressing tumors (p = 0.012).

Conclusion/Significance

Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis indicating that ATG10 may be a potential prognostic maker in colorectal cancer.     

Non-labeled detection of waterborne pathogen Cryptosporidium parvum using a polydiacetylene-based fluorescence chip

A non-labeling fluorescence sensor system was developed using polydiacetylene (PDA) liposomes composed of 10,12-pentacosadiynoic acid (PCDA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a 8:2 molar ratio. The PDA liposomes were immobilized onto an amine-coated glass surface using peptide bonding between the carboxyl group of the liposome and the amine group of the glass surface. The optimum ratio of the cross linker (NHS/EDC) to PDA liposome was determined to be 50% for strong immobilization of the liposomes. Residual carboxyl groups of the PDA liposomes were selectively biotinylated, followed by sequential binding of streptavidin and biotin-antibody (bioreceptor). Finally, the performance of the PDA liposome chip was tested for detecting Cryptosporidium parvum, and yielded a detection limit of 1 x 10(3) oocysts/mL. From these results, it is expected that the PDA liposome chip will have high application potential for the detection of waterborne pathogens including C. parvum.     

Increased expression of the F(1)F(o) ATP synthase in response to iron in heart mitochondria

The objective of the present study was to identify mitochondrial components associated with the damage caused by iron to the rat heart. Decreased cell viability was assessed by increased presence of lactate dehydrogenase (LDH) in serum. To assess the functional integrity of mitochondria, Reactive Oxygen Species (ROS), the Respiratory Control Ratio (RCR), ATP and chelatable iron content were measured in the heart. Chelatable iron increased 15-fold in the mitochondria and ROS increased by 59%. Deterioration of mitochondrial function in the presence of iron was demonstrated by low RCR (46% decrease) and low ATP content (96% decrease). Using two dimensional gel electrophoresis (2DE), we identified alterations in 21 mitochondrial proteins triggered by iron overload. Significantly, expression of the alpha, beta, and d subunits of F(1)F(o) ATP synthase increased along with the loss of ATP. This suggests that the F(1)F(o) ATP synthase participates in iron metabolism.     

Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H–15N multidimensional NMR spectroscopy

We used xenon-perturbed ¹H–¹⁵N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the β3-, β5- and β6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.