Tomohito Iwasaki

Histone H1 null vertebrate cells exhibit altered nucleosome architecture

In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes, and chromatin fibers are thought to be stabilized by linker histones of the H1 type. Higher eukaryotes express multiple variants of histone H1; chickens possess six H1 variants. Here, we generated and analyzed the phenotype of a complete deletion of histone H1 genes in chicken cells. The H1-null cells showed decreased global nucleosome spacing, expanded nuclear volumes, and increased chromosome aberration rates, although proper mitotic chromatin structure appeared to be maintained. Expression array analysis revealed that the transcription of multiple genes was affected and was mostly downregulated in histone H1-deficient cells. This report describes the first histone H1 complete knockout cells in vertebrates and suggests that linker histone H1, while not required for mitotic chromatin condensation, plays important roles in nucleosome spacing and interphase chromatin compaction and acts as a global transcription regulator.

Hydrostatic pressure-induced solubilization and gelation of chicken myofibrils

Hydrostatic pressure effect on chicken myofibrils was studied. Some of myofibrillar proteins were solubilized by pressure application above 200 MPa. Most of them were derived from thin filament; namely, actin, tropomyosin, and troponin components. Myosin heavy chain was hardly solubilized in 0.1 M NaCl, while its solubilization occurred in 0.2 M NaCl at 200–300 MPa. Solubilization of myosin dependend on magnitude of applied pressure and duration of treatment, and also salt concentration and pH. The maximal solubilization of myosin occurred at 200 MPa in 0.2 M NaCl at pH 7. Myofibrils in 0.1–0.2 M NaCl with a protein concentration of 40 mg/ml formed a gel by pressure application. The gel strength at 0.1 M NaCl increased with pressure, while it remained almost at the same level in 0.2 M NaCl. The intrinsic structure of myofibril was retained after pressure treatment in 0.1 M NaCl; however, pressure-induced disruption of myofibrillar structure occurred in 0.2 M NaCl.

Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis

ABSTRACT Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolated Myoviridae or Podoviridae phages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused by P. aeruginosa in a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis. IMPORTANCE Antibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad range of P. aeruginosa strains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models of P. aeruginosa keratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Bacteriophage can lyse antibiotic-resistant Pseudomonas aeruginosa isolated from canine diseases

Pseudomonas aeruginosa is a pathogen frequently identified as the cause of diverse infections or chronic disease. This microbe has natural resistance to several kinds of antibiotics, because of the species’ outer membrane, efflux pumps and growth as a biofilm. This bacterium can acquire increased resistance with specific point mutations. Bacteriophage (phage), however, can lyse these bacteria. Therefore, in the present study, we assessed the host range of phages isolates and their ability to lyse antibiotic-resistant P. aeruginosa. Present phages could lyse many strains of P. aeruginosa (28/39), including strains with high resistance to fluoroquinolones (4/6). In conclusion, application of phages for antibiotic-resistant bacteria is greatly effective. To avoid pervasive antibiotic-resistant bacteria, further development of phage usage for disease treatment is required.

Structural changes in chicken myosin subfragment-1 induced by high hydrostatic pressure

ATP-induced fluorescence increment of pressurized S1 was almost the same as unpressurized one at least up to 150 MPa, whereas it decreased above 200 MPa. The binding of e-ADP to S1 decreased at 250–300 MPa. S1 pressurized at 100–250 MPa and unpressurized S1 bound to F-actin similarly, although binding of S1 to actin decreased when the pressure treatment was done above 250 MPa. S1 was easily cleaved by tryptic digestion into three domains. Tryptic fragments of S1 digested after pressure treatment were essentially the same as those of unpressurized one. On the other hand, additional fragments appeared when the digestion was performed under pressure at 300 MPa. It is concluded that pressure-induced structural changes of S1 begin to occur about 150 MPa, and ATPase and actin binding sites lose those intrinsic structures at 250–300 MPa.

Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl.

The actinobacterium Kribbella flavida NBRC 14399T produces cyclobis-(1→6)-α-nigerosyl (CNN), a cyclic glucotetraose with alternate α-(1→6)- and α-(1→3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-α-glucosyltransferase and 3-α-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the α-(1→3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s−1) was much higher than that of panose (0.242 s−1), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the α-(1→3)-isomaltosyl moiety and effectively hydrolyzes the α-(1→3)-glucosidic linkage, it should be termed 1,3-α-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of β-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The kcat/Km for isomaltose (4.81 ± 0.18 s−1 mm−1) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.

A novel method for monitoring troponin T fragment from rabbit skeletal muscle during aging using quartz crystal microbalance.

BACKGROUND Troponin T (TnT) is degraded during aging of meat. The proteolytic fragment of TnT, especially the 30 kDa fragment, is used as one of indices for estimating aging of meat. We have tried to use quartz crystal microbalance (QCM), which is widely used to analyze interaction among macromolecules, to detect proteolytic fragments of TnT during aging of meat. RESULT The frequency of the QCM sensor with immobilized anti-TnT antibody in high-salt solution extracts of both myofibrils and whole meat decreased with time of aging. The staining intensity of the bands, including a 30 kDa fragment bound to anti-TnT antibody, also increased with time of aging in western blotting. These results confirm that TnT is degraded during aging and released from thin filaments, and QCM analysis is sufficiently sensitive to detect the TnT fragments. CONCLUSION The QCM analysis of muscle and myofibrillar extracts using anti-TnT antibody-immobilized sensor can be used as a convenient tool for monitoring the extent of aging of meat.

Corticosterone biosynthesis in mouse clonal myoblastic C2C12 cells

Graphical abstract Figure. No Caption available. Highlights3ß‐HSD, cytochrome P450c21 and cytochrome P45011ß, exist in C2C12 cells.C2C12 cells do not reveal P450scc activity, but the cells show sulfatase activity.Cholesterol is not initial substrate for local corticosterone synthesis in C2C12 cells.Pregnenolone‐sulfate is deconjugated by C2C12 cells and functioned as an initial substrate. ABSTRACT Corticosterone (CORT), the major glucocorticoid in rodents, is secreted from the adrenal gland, affects various organs in the body and regulates energy metabolism as a stress response. Although local steroidogenesis of androgens and estrogens in skeletal muscles has been previously reported, local CORT synthesis in skeletal muscle remains unconfirmed. In the present study, we investigated steroidogenic activities in a clonal myoblastic cell line, C2C12 cells. Three enzymes involved in CORT synthesis, 3ß‐hydroxysteroid dehydrogenase (3ß‐HSD), cytochrome P450c21 and cytochrome P45011ß, were identified in C2C12 cells by detecting the enzymatic reaction products with LC–MS/MS analysis. Only one enzyme that mediates cholesterol cleavage was not detected in the cells. After the addition of pregnenolone‐sulfate conjugates to the cell culture medium, pregnenolone was detected and increased according to the incubation time. In conclusion, CORT synthesis occurs in C2C12 cells, and it is suggested that the initial steroidogenesis substrate is the pregnenolone‐sulfate conjugate.

Gelation Characteristics of Heat-Induced Gels Mixed Meat with Fish Proteins

The aim of this study was to make a comprehensive assessment of the quality of fish surimi (FS) from scale-eye plaice (SP) and surimi-like products from culled hen and FS from sand eel (SE) and Arabesque greenling (AG), which are regarded as low-valued food resources. The thermal gelation properties of a variety of the gels mixed meat with fish proteins were investigated by the use of the rheological and protein chemistry approaches, and the obtained results are as follows: (1) SP surimi has advantages such as high whiteness and suwari phenomenon at low temperature. (2) A variety of heat-induced gel products were able to produce with different mixing ratios of ground culled hen (GCH) and FS regardless of adding egg albumen powder (EAP). (3) Disulfide bond (S-S bond) was related to an improvement in gel properties of the directly heated gel mixed FS with GCH by addition of EAP. In particular, cleavage of myosin heavy chains in the gels, which have a high mixing ratio of SE surimi, was inhibited by addition of EAP. (4) AG can be used as ingredients of heat-induced gel products mixed fish with hen, while SP is inappropriate for the product because of syneresis phenomenon by use of microbial transglutaminase as a reagent for gelation. These findings revealed that adequate understanding of gel-forming ability of FS could be essential to improve gel properties of the heat-induced gels mixed meat with FS.