Deregulation of allosteric response of Lactococcus lactis prolidase and its effects on enzyme activity

The allosteric behaviour of Lactococcus lactis prolidase (Xaa-Pro dipeptidase) of this proline-specific peptidase was investigated where it was hypothesized that intersubunit interactions between a loop structure and three residues near the active site contributed to this behaviour. Seven mutant prolidases were constructed, and it was observed that the loopless mutant and His303 substitution inactivated the enzyme. Ser307 substitution revealed that this residue influenced the substrate binding, as judged from its kinetic constants and substrate specificity; however, this residue did not contribute to allostery of prolidase. R293S mutation resulted in the disappearance of the allosteric behaviour yielding a Hill constant of 0.98 while the wild type had a constant of 1.58. In addition, the R293S mutation suppressed the substrate inhibition that was observed in other mutants and wild type. The K_m value of R293S was 2.9-fold larger and V_max was approximately 50% less as compared to the wild type. The results indicated that Arg293 increased the affinity for substrates while introducing allosteric behaviour and substrate inhibition. Computer modelling suggested that negative charges on the loop structure interacted with Arg293 and Ser307 to maintain these characteristics. It was, therefore, concluded that Arg293, His303, Ser307 and the loop contributed to the enzyme's allosteric characteristics.     

Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle–eye–brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of α-DG, and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^?/? muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1^?/? satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^?/? myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of α-DG is important for maintenance of the proliferative activity of satellite cells in vivo.     

Systematic study of the genus Compsilura Bouché in Southeast and East Asia with morphological and molecular data (Diptera,Tachinidae)

Compsilura concinnata (Meigen) is one of the most famous, most polyphagous and most widely distributed tachinid flies (Diptera, Tachinidae) in the world. This species is well known as a biocontrol agent of some injurious pests of cultural and wild plants and has been introduced from Europe to the United States to control mainly the gypsy moth. Recently we found three new species very closely resembling C. concinnata from Southeast and East Asia: C. lobata sp. nov. (Japan and Thailand), C. malayana sp. nov. (Malaysia) and C. pauciseta sp. nov. (Japan and Taiwan). Additionally, C. samoaensis Malloch is treated as a junior synonym of C. concinnata based on the examination of the type specimen. The genetic differences in the mitochondrial COI gene data are examined to assess the accuracy of species delimitation of Compsilura. The male postabdominal characters of these species are illustrated. The piercing female postabdomen of C. concinnata is illustrated and compared to those of other members belonging to the Blondelia group including Blondelia Robineau-Desvoidy, Celatoria Coquillett, Eucelatoria Townsend and Vibrissina Rondani.     

Monitoring Escape and Feeding Behaviours of Cruiser Fish by Inertial and Magnetic Sensors

A method was developed and applied for monitoring two types of fast-start locomotion (feeding and escape) of a cruiser fish, Japanese amberjacks Seriola quinqueradiata. A data logger, which incorporated a 3-axis gyroscope, a 3-axis accelerometer and a 3-axis magnetometer, was attached to the five fish. The escape, feeding and routine movements of the fish, which were triggered in tank experiments, were then recorded by the data logger and video cameras. The locomotor variables, calculated based on the high resolution measurements by the data logger (500 Hz), were investigated to accurately detect and classify the types of fast-track behaviour. The results show that fast-start locomotion can be detected with a high precision (0.97) and recall rate (0.96) from the routine movements. Two types of fast-start movements were classified with high accuracy (0.84). Accuracy was greater if the data were obtained from the data logger, which combined an accelerometer, a gyroscope and a magnetometer, than if only an accelerometer (0.80) or a gyroscope (0.66) was used.     

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases

Background

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.

Methods and results

In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at ²⁹⁶SNLD²⁹⁹ and ³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified ³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at ³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the ³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.

Conclusion

CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore, ³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.

General significance

CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.     

Effect of osmolarity and viscosity on the motility of pathogenic and saprophytic Leptospira

The motility of bacteria is an important factor in their infectivity. In this study, the motility of Leptospira, a member of the spirochete family that causes a zoonotic disease known as leptospirosis, was analyzed in different viscous or osmotic conditions. Motility assays revealed that both pathogenic and saprophytic strains increase their swimming speeds with increasing viscosity. However, only pathogenic Leptospira interrogans maintained vigorous motility near physiological osmotic conditions. This suggests that active motility in physiological conditions is advantageous when Leptospira enters hosts and when it migrates toward target tissues.     

Identification of a Novel Subgroup of Koala Retrovirus from Koalas in Japanese Zoos

We identified a new subgroup of koala retrovirus (KoRV), named KoRV-J, which utilizes thiamine transport protein 1 as a receptor instead of the Pit-1 receptor used by KoRV (KoRV-A). By subgroup-specific PCR, KoRV-J and KoRV-A were detected in 67.5 and 100% of koalas originating from koalas from northern Australia, respectively. Altogether, our results indicate that the invasion of the koala population by KoRV-J may have occurred more recently than invasion by KoRV-A.     

ER stress response induced by the production of human IL-7 in rice endosperm cells

Rice seed has been used as a production platform for high value recombinant proteins. When mature human interleukin 7 (hIL-7) was expressed as a secretory protein in rice endosperm by ligating the N terminal glutelin signal peptide and the C terminal KDEL endoplasmic reticulum (ER) retention signal to the hIL-7 cytokine to improve production yield, this protein accumulated at levels visible by Coomassie Brilliant Blue staining. However, the production of this protein led not only to a severe reduction of endogenous seed storage proteins but also to a deterioration in grain quality. The appearance of aberrant grain phenotypes (such as floury and shrunken) was attributed to ER stress induced by the retention of highly aggregated unfolded hIL-7 in the ER lumen, and the expression levels of chaperones such as BiPs and PDIs were enhanced in parallel with the increase in hIL-7 levels. The activation of this ER stress response was shown to be mainly mediated by the OsIRE1-OsbZIP50 signal cascade, based on the appearance of unconventional splicing of OsbZIP50 mRNA and the induction of OsBiP4&5. Interestingly, the ER stress response could be induced by lower concentrations of hIL-7 versus other types of cytokines such as IL-1b, IL-4, IL-10, and IL-18. Furthermore, several ubiquitin 26S proteasome-related genes implicated in ER-associated degradation were upregulated by hIL-7 production. These results suggest that severe detrimental effects on grain properties were caused by proteo-toxicity induced by unfolded hIL-7 aggregates in the ER, resulting in the triggering of ER stress.     

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.