Structural Analysis of a Trimer of β2-Microgloblin Fragment by Molecular Dynamics Simulations

A peptide ${\beta }_2$-${\text{m}}_{21?31}$, which is a fragment from residue 21 to residue 31 of ${\beta }_2$-microgloblin, is experimentally known to self-assemble and form amyloid fibrils. In order to understand the mechanism of amyloid fibril formations, we applied the replica-exchange molecular dynamics method to the system consisting of three fragments of ${\beta }_2$-${\text{m}}_{21?31}$. From the analyses on the temperature dependence, we found that there is a clear phase transition temperature in which the peptides aggregate with each other. Moreover, we found by the free energy analyses that there are two major stable states: One of them is like amyloid fibrils and the other is amorphous aggregates.     

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.     

Predation risk and resource abundance mediate foraging behaviour and intraspecific resource partitioning among consumers in dominance hierarchies

Dominance hierarchies and the resulting unequal resource partitioning among individuals are key mechanisms of population regulation. The strength of dominance hierarchies can be influenced by size‐dependent tradeoffs between foraging and predator avoidance whereby competitively inferior subdominants can access a larger proportion of limiting resources by accepting higher predation risk. Foraging‐predation risk tradeoffs also depend on resource abundance. Yet, few studies have manipulated predation risk and resource abundance simultaneously; consequently, their joint effect on resource partitioning within dominance hierarchies are not well understood. We addressed this gap by measuring behavioural responses of masu salmon Oncorhynchus masou ishikawae to experimental manipulations of predation risk and resource abundance in a natural temperate forest stream. Responses to predation risk depended on body size and social status such that larger fish (often social dominants) exhibited more risk‐averse behaviour (e.g. lower foraging and appearance rates) than smaller subdominants after exposure to a simulated predator. The magnitude of this effect was lower when resources were elevated, indicating that dominant fish accepted a higher predation risk to forage on abundant resources. However, the influence of resource abundance did not extend to the population level, where predation risk altered the distribution of foraging attempts (a proxy for energy intake) from being skewed towards large individuals to being skewed towards small individuals after predator exposure. Our results imply that size‐dependent foraging–predation risk tradeoffs can weaken the strength of dominance hierarchies by allowing competitively inferior subdominants to access resources that would otherwise be monopolized.     

Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe²⁴⁶ (PsOYE)/Phe²⁵⁰ (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences.

Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE     

Strain- and age-dependent loss of sarcoglycan complex in cardiomyopathic hamster hearts and its re-expression by delta-sarcoglycan gene transfer in vivo

We found that 5-S-GAD, an insect-derived antibacterial peptide, inhibited murine osteoclast formation in vitro. We examined the specific time point of the inhibitory action of 5-S-GAD on osteoclast formation and found that it mainly suppressed differentiation of osteoclasts in the middle of the culture period. Using HL60 cells that are able to differentiate into multinucleated macrophage-like cells, we found that 5-S-GAD induced apoptosis of HL60 cells by producing H(2)O(2). Thus, the inhibition of osteoclast formation by 5-S-GAD could be, in part, due to apoptosis of the cells of an osteoclast lineage.     

Expression of DNase gamma during Fas-independent apoptotic DNA fragmentation in rodent hepatocytes

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase ) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). DNase immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase immunoreactivity was not detected during LPS-induced or CCl₄-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.     

Intergeneric conjugal transfer of plasmid DNA from<Emphasis Type="Italic"> Escherichia coli</Emphasis> to<Emphasis Type="Italic"> Kitasatospora setae</Emphasis>, a bafilomycin B<Subscript>1</Subscript> producer

An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1×10^-6) was achieved on ISP4 medium containing 30 mM MgCl₂ using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non-Streptomyces actinomycetes. K. setae contains a single C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.     

Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide–pulsed dendritic cells

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24–restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.