Interaction between C18 fatty acids and DOPE PEG2000 in Langmuir monolayers: effect of degree of unsaturation

In this study, we address the effect of the cis-double bond in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamide-N-[methoxy(polyethylene glycol)-2000, DOPE PEG2000 (DP), on the Langmuir monolayer of C18 fatty acids—namely, stearic acid (SA), oleic acid (L1), linoleic acid (L2), and linolenic acid (L3)—with the same head group but different degrees of saturation on their hydrocarbon chains. Negative values of Gibbs free energy of mixing (ΔG _mix) were obtained throughout the investigated ranges of the unsaturated C18 fatty-acid (L1, L2 and L3) mixed systems, indicating that very strong attractions occurred between molecules in the monolayers. The bend and kink effects from the cis-double bond(s) in the hydrocarbon chain affected the membrane fluidity and molecular packing in the monolayers, which resulted in a greater interaction between unsaturated C18 fatty acids and DP. The most thermodynamically stable mole composition of unsaturated C18 fatty acids to DP was observed at 50:1; this ratio is suggested to be the best mole ratio and will be subsequently used to prepare DP–C18 fatty-acid nanoliposomes. The presence of cis-double bonds in both hydrocarbon chains of DOPE in DP also created an imperfection in the membrane structure of lipid-drug delivery systems, which is expected to enhance lipid-based systems for antibody conjugation and drug encapsulation.     

The peptide PIN changes the timing of transitory burst activation of timer-ATPase TIME in accordance with diapause development in eggs of the silkworm, Bombyx mori

TIME is an ATPase that measures a time interval by exhibiting transitory burst activation in eggs of the silkworm, Bombyx mori L. PIN is a peptide that regulates the time measurement of TIME. To address the mode of action of PIN, interactions between TIME and PIN were investigated. First, TIME was mixed with PIN for various periods (days) at 25 degrees C. The longer TIME was mixed with PIN, the later the transitory burst activation of TIME ATPase activity occurred, while no such delay occurred at 5 degrees C. Second, the capacity of PIN to bind with TIME was measured at the two temperatures by fluorescence polarization. The binding interaction was much tighter (nearly 1000 times) at 25 degrees C than that at 4 degrees C. Because the log EC50 (in nM) at 4 degrees C was about 7, PIN must dissociate from TIME at low temperatures at the physiological concentration of TIME in eggs. Thus, TIME appears to be restructured into a time-measuring conformation by PIN at the high temperatures of summer, whereas the TIME-PIN complex would dissociate at the low temperatures of winter. This dissociation acts as the preliminary cue for the ATPase activity burst of TIME, which in turn causes the completion of diapause development and initiates new developmental programs.     

Mitochondrial and cytosolic expression of human peroxiredoxin 5 in Saccharomyces cerevisiae protect yeast cells from oxidative stress induced by paraquat

Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides. To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat. The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species. Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat.     

The Saccharomyces cerevisiae RNase mitochondrial RNA processing is critical for cell cycle progression at the end of mitosis

We have identified a cell cycle delay in Saccharomyces cerevisiae RNase MRP mutants. Mutants delay with large budded cells, dumbbell-shaped nuclei, and extended spindles characteristic of "exit from mitosis" mutants. In accord with this, a RNase MRP mutation can be suppressed by overexpressing the polo-like kinase CDC5 or by deleting the B-type cyclin CLB1, without restoring the MRP-dependent rRNA-processing step. In addition, we identified a series of genetic interactions between RNase MRP mutations and mutations in CDC5, CDC14, CDC15, CLB2, and CLB5. As in most "exit from mitosis" mutants, levels of the Clb2 cyclin were increased. The buildup of Clb2 protein is not the result of a defect in the release of the Cdc14 phosphatase from the nucleolus, but rather the result of an increase in CLB2 mRNA levels. These results indicate a clear role of RNase MRP in cell cycle progression at the end of mitosis. Conservation of this function in humans may explain many of the pleiotropic phenotypes of cartilage hair hypoplasia.     

Demarcation of diapause development by cold and its relation to time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori

We investigated the mode of action of winter cold in the termination of diapause by investigating Time-Interval-Measuring Enzyme (TIME). First, we determined the period of cold required for the completion of diapause development. Synchronously developing egg batches of a pure strain (C108 Bombyx mori silkworm) were used to minimize variations in hatching time. Hatching occurred with only 18 days chilling at 5 degrees C when the incubation at 25 degrees C after the chilling was elongated. The 18-day period was much shorter than we expected; diapause in B. mori is known to terminate completely with about 100 days of chilling. Even in such a short period of chilling, no sporadic hatching occurred. Moreover, we determined that a temperature-insensitive stage, which we called "Neboke", followed the short cold-requiring stage. Thus, the stage of diapause development was demarcated from other stages of diapause. While the length of diapause development was elongated when chilling was delayed after oviposition, the Neboke stage length was invariant. Cold evidently exerts its effect only on diapause development. When TIME was purified from eggs and chilled in test tubes, a transitory burst of its ATPase activity occurred at a time equivalent to shortly before the completion of diapause development; this was an interval-timer activation. The mechanism by which cold activates TIME to measure the time interval may help explain in biochemical terms the insect's adaptation to its seasonal environments.     

Novel cellulase profile of Trichoderma reesei strains constructed by cbh1 gene replacement with eg3 gene expression cassette

To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbhl gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transforments denoted as L 13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbhl promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.     

Prostaglandin F2α facilitates collagen synthesis in cardiac fibroblasts via an F-prostanoid receptor/protein kinase C/Rho kinase pathway independent of transforming growth factor β1

Accumulation of collagen I and III in the myocardium is a prominent feature of interstitial fibrosis. Prostaglandin F(2α) (PGF(2α)) facilitates fibrosis by increasing collagen synthesis. However, the underlying mechanisms mediating the effect of PGF(2α) on collagen expression in cardiac fibroblasts are not yet fully elucidated. We measured the mRNA and protein levels of collagen I and III by quantitative real-time PCR and ELISA, respectively. Activation of signaling pathways was determined by western blot analysis. In primary rat cardiac fibroblasts, treatment with PGF(2α) stimulated both the mRNA and protein levels of collagen I and III, and pretreatment with the F-prostanoid (FP) receptor antagonist AL-8810, protein kinase C inhibitor LY-333531, and Rho kinase inhibitor Y-27632 significantly inhibited PGF(2α)-induced collagen I and III expression. FP receptor, protein kinase C, and Rho kinase were activated with PGF(2α) treatment. PGF(2α) may be an important regulator in the synthesis of collagen I and III via an FP receptor/protein kinase C/Rho kinase cascade in cardiac fibroblasts, which might be a new therapeutic target for myocardial fibrosis.     

Replication Study in Chinese Population and Meta-Analysis Supports Association of the 11q23 Locus with Colorectal Cancer

Background

A common single nucleotide polymorphism (SNP), rs3802842, located at 11q23, was identified by genome-wide association studies (GWAS) to be significantly associated with the risk of colorectal cancer (CRC); however, the results of following replication studies were not always concordant. Thus, a case-control study and a meta-analysis were performed to clearly discern the effect of this variant in CRC.

Method and Findings

We determined the genotypes of rs3802842 in 641 unrelated Chinese patients with CRC and 1037 cancer-free controls. Additionally, a meta-analysis comprising current and previously published studies was conducted. In our case-control study, significant associations between the polymorphism and CRC risk were observed in all genetic models, with an additive OR being 1.45 (95% CI = 1.26–1.67). The meta-analysis of 38534 cases and 39446 controls further confirmed the significant associations in all genetic models but with obvious between-study heterogeneity. Nevertheless, ethnicity, study type and whether subjects affected by Lynch syndrome could synthetically accounted for the heterogeneity. Besides, the cumulative and sensitivity analyses indicated the robust stability of the results.

Conclusion

The results from our case-control study and meta-analysis provided convincing evidence that rs3802842 significantly contributed to CRC risk.     

The mouse Mageb18 gene encodes a ubiquitously expressed type I MAGE protein and regulates cell proliferation and apoptosis in melanoma B16-F0 cells

Although many cancer vaccines have been developed against type?I MAGE (melanoma antigen) genes owing to their shared tumour-specific expression properties, studies about their expression and functions are relatively limited. In the present study, we first identify a non-testis-specific type?I MAGE gene, Mageb18 (melanoma antigen family B 18). Mouse Mageb18 is also expressed in digestion- and immune-related tissues as well as testis, and its expression in testis is age-dependent. Mageb18 is expressed in many mouse-derived cell lines, and DNA demethylation and histone acetylation mediate the reactivation of Mageb18 in Mageb18-negtive H22 and C6 cells. We also show that mouse Mageb18 encodes a 46?kDa protein which is predominantly localized in the cytoplasm. In testis, the endogenous MAGEB18 protein is mainly expressed in proliferative spermatogonia and primary and secondary spermatocytes, but less so in spermatids. Finally, we demonstrate that knockdown of MAGEB18 inhibits the growth of B16-F0 cells and induces apoptosis, which correlates with increased levels of TP53 (tumour protein 53), p21, Bax and caspase 3. The results of the present study thus uncover an important phenomenon that the expression of certain type?I MAGE genes, at least for Mageb18, is non-testis-specific. Although they can regulate various malignant phenotypes of cancer cells, it is necessary to study further their expression pattern in normal tissues before using them to develop more effective and safer cancer vaccines.     

A protein microarray prepared with phage-displayed antibody clones

Using a large phage antibody library, a protein microarray spotted directly with phage-displayed antibody clones was created to discriminate between recognition profiles of samples from healthy donors and leukemia patients. The protocol for preparing antibody-displaying phage chips was presented. Some conditions such as substrates and blocking buffers were compared and optimized. The major improvements of this microarray are higher throughput and lower cost compared to previous antibody chips. Due to its convenience and sensitivity, it can be extensively used for rapid and high throughput detection of protein profiles of experimental and clinical samples.