Stabilization of the fibrous structure of an alpha-helix-forming peptide by sequence reversal

The alpha3-peptide, which comprises three repeats of the sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala and forms an amphipathic alpha-helix, is unique among various alpha-helix-forming peptides in that it assembles into fibrous structures that can be observed by transmission electron microscopy. As part of our investigation of the structure-stability relationships of the alpha3-peptide, we synthesized the r3-peptide, whose amino acid sequence is the reverse of that of the alpha3-peptide, and we investigated the effects of sequence reversal on alpha-helix stability and the formation of fibrous structures. Unexpectedly, the r3-peptide formed a more-stable alpha-helix and longer fibers than did the alpha3-peptide. The stability of the r3-peptide helix decreased when the ionic strength of the buffer was increased and when the pH of the buffer was adjusted to 2 or 12. These results suggest that the r3-peptide underwent a "magnet-like" oligomerization and that an increase in the charge-distribution inequality may be the driving force for the formation of fibrous structures.     

Cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro

ER-60 is a PDI family protein that has protein thiol-disulfide oxidoreductase activity. It has been shown to associate with BiP in the endoplasmic reticulum. Here, we analyzed the cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro. ER-60 facilitated the refolding of 20 or 30% of denatured alpha-lactalbumin or ribonuclease B during incubation for 80 min, and these levels of nearly doubled on the addition of BiP to the reaction mixture. BiP alone could not refold denatured ribonuclease B, but could refold denatured alpha-lactalbumin a little. Enhancement of oxidative refolding of alpha-lactalbumin by ER-60 could be detected only when ER-60 was present from the start of refolding. On surface plasmon resonance analysis, ER-60 was shown to associate with BiP. The association was not influenced by ATP or ADP. Domains a and/or b' of ER-60 were implicated in the association with BiP.     

Application of a Combination of a Knowledge-Based Algorithm and 2-Stage Screening to Hypothesis-Free Genomic Data on Irinotecan-Treated Patients for Identification of a Candidate Single Nucleotide Polymorphism Related to an Adverse Effect

Interindividual variation in a drug response among patients is known to cause serious problems in medicine. Genomic information has been proposed as the basis for “personalized” health care. The genome-wide association study (GWAS) is a powerful technique for examining single nucleotide polymorphisms (SNPs) and their relationship with drug response variation; however, when using only GWAS, it often happens that no useful SNPs are identified due to multiple testing problems. Therefore, in a previous study, we proposed a combined method consisting of a knowledge-based algorithm, 2 stages of screening, and a permutation test for identifying SNPs. In the present study, we applied this method to a pharmacogenomics study where 109,365 SNPs were genotyped using Illumina Human-1 BeadChip in 168 cancer patients treated with irinotecan chemotherapy. We identified the SNP rs9351963 in potassium voltage-gated channel subfamily KQT member 5 (KCNQ5) as a candidate factor related to incidence of irinotecan-induced diarrhea. The p value for rs9351963 was 3.31×10⁻⁵ in Fisher''s exact test and 0.0289 in the permutation test (when multiple testing problems were corrected). Additionally, rs9351963 was clearly superior to the clinical parameters and the model involving rs9351963 showed sensitivity of 77.8% and specificity of 57.6% in the evaluation by means of logistic regression. Recent studies showed that KCNQ4 and KCNQ5 genes encode members of the M channel expressed in gastrointestinal smooth muscle and suggested that these genes are associated with irritable bowel syndrome and similar peristalsis diseases. These results suggest that rs9351963 in KCNQ5 is a possible predictive factor of incidence of diarrhea in cancer patients treated with irinotecan chemotherapy and for selecting chemotherapy regimens, such as irinotecan alone or a combination of irinotecan with a KCNQ5 opener. Nonetheless, clinical importance of rs9351963 should be further elucidated.     

Skeletal isotope microprofiles of growth perturbations in<Emphasis Type="Italic"> Porites</Emphasis> corals during the 1997–1998 mass bleaching event

Severe coral bleaching occurred throughout the tropics in 1997/98. We report high-resolution skeletal oxygen isotope (¹⁸O) and carbon isotope (¹³C) microprofiles for bleached corals from Pandora Reef, Great Barrier Reef, and Ishigaki Island, Japan, in order to examine the ability of Porites corals to record clear signals of bleaching. Analysis of the annual cycle in ¹⁸O revealed abrupt reductions in skeletal extension immediately after the 1997–98 summer temperature maximum, indicating that bleaching inhibits coral calcification. Skeletal ¹³C in the Ishigaki corals showed lower values during bleaching, indicating depressed coral metabolism associated with a reduction in calcification. In contrast, microprofiles of skeletal ¹³C from the shaded sides of Pandora Reef corals exhibited little change, possibly because algal photosynthesis was already slow prior to bleaching, thus subduing the ¹³C-response to bleaching. Comparison of ¹⁸O microprofiles from bleached corals with instrumental temperature records showed that Porites corals can recover following 5 months with little skeletogenesis. The results indicate that isotopic microprofiling may be the key to identifying gaps in coral growth that are diagnostic of past bleaching events. We have tested this hypothesis using blue UV fluorescent bands to guide us to coral skeleton where isotope microprofiling identifies bleaching events in 1986, 1989, and 1990. These events, detected by proxy, suggest that coral bleaching may have occurred more commonly on Ishigaki Island than previously recorded.     

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.     

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.     

Insulin-dependent phosphatidylinositol 3-kinase/Akt and ERK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis

TLR3, one of the TLRs involved in the recognition of infectious pathogens for innate and adaptive immunity, primarily recognizes viral-associated dsRNA. Recognition of dsRNA byproducts released from apoptotic and necrotic cells is a recently proposed mechanism for the amplification of toxicity, suggesting a pivotal participation of TLR3 in viral infection, as well as in lung diseases where apoptosis plays a critical role, such as asthma and chronic obstructive pulmonary disease. In addition to metabolic control, insulin signaling was postulated to be protective by inhibiting apoptosis. Therefore, we explored the role of insulin signaling in protecting against TLR3-mediated apoptosis of human bronchial epithelial cells. Significant TLR3-mediated apoptosis was induced by polyinosinic-polycytidylic acid, a dsRNA analog, via caspase-8-dependent mechanisms. However, insulin efficiently inhibited TLR3/polyinosinic-polycytidylic acid-induced human bronchial epithelial cell apoptosis via PI3K/Akt and ERK pathways, at least in part, via upregulation of cellular FLIPs and through protein synthesis-independent mechanisms. These results indicate the significance of TLR3-mediated dsRNA-induced apoptosis in the pathogenesis of apoptosis-driven lung disease and provide evidence for a novel protective role of insulin.     

Effect of vitamin B12-enriched thraustochytrids on the population growth of rotifers

Newly isolated thraustochytrids showed uptake of vitamin B12 from the culture into the cells. Cultivation of thraustochytrids in a medium containing 1 microg/ml of vitamin B12 greatly increased the contents of vitamin B12 in the cells. Similarly to Schizochytrium limacinum, odd numbered fatty acids decreased in the cells of new isolates cultivated with vitamin B12. Vitamin B12-enriched thraustochytrids, strain mh0186, enhanced the population growth of rotifers fed on the cells as sole feed.     

Effects of G-CSF on cardiac remodeling after acute myocardial infarction in swine

We examined whether granulocyte colony-stimulating factor (G-CSF) prevents cardiac dysfunction and remodeling after myocardial infarction (MI) in large animals. MI was produced by ligation of left anterior descending coronary artery in swine. G-CSF (10 microg/kg/day, once a day) was injected subcutaneously from 24h after ligation for 7 days. Echocardiographic examination revealed that the G-CSF treatment induced improvement of cardiac function and attenuation of cardiac remodeling at 4 weeks after MI. In the ischemic region, the number of apoptotic endothelial cells was smaller and the number of vessels was larger in the G-CSF treatment group than in control group. Moreover, vascular endothelial growth factor was more abundantly expressed and Akt was more strongly activated in the ischemic region of the G-CSF treatment group than of control group. These findings suggest that G-CSF prevents cardiac dysfunction and remodeling after MI in large animals.