Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process ofHypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH₂ were 0.9×10⁻³M (PE-1) and 1.2×10⁻³M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.     

Influence of long-latency reflex modulation on the performance of quick adjustment movements

The effect of long-latency reflex modulation on the performance of a quick adjustment movement following a muscle stretch was studied in 26 healthy male subjects. When the subjects felt a sudden angle displacement in the direction of a wrist extension they were required to make an adjustment movement by moving a handlebar, held in the hand, to align with a target position as quickly and as accurately as possible. The index of performance (adjustment time) was the time taken to move the handle to the target position from stretch onset. A DC torque motor was used to evoke electromyographic (EMG) reflex responses on a wrist flexor. Averaging of the rectified EMG, recorded from surface electrodes placed over the flexor, showed short- and long-latency reflexes (M1 and M2 components). For all subjects, the amplitudes of the reflex components decreased during the adjustment movement because the target position for this study was fixed to the extension side of the wrist joint. The decrease in the M2 component, which is considered to be a transcortical reflex, was significantly larger than the decrease in the M1 component, which is spinal reflex. The main finding was of a positive correlation between the length of adjustment time and the degree of reduction of M1 and M2 with the adjustment movement (r = 0.602 for M1, P < 0.01; r = 0.850 for M2, P < 0.001). Moreover, there were correlations between the consistency of the voluntary response onset and the degree of M2 decrease (r = 0.577, P < 0.01), and between the consistency of the voluntary response onset and the length of the adjustment time (r = 0.603, P < 0.01). Therefore, we have concluded that the subjects who were able to perform adjustment movements within a short time could modulate the long-latency reflex of the muscle involved in such movements in order to make the function of their voluntary muscle activity more effective, and thus were able to respond appropriately. Accepted: 19 February 1997     

Time course of recovery from nerve injury in skeletal muscle: energy state and local circulation

Hayashi, Yoshihiro, Takaaki Ikata, Hiroaki Takai, ShinjiroTakata, Takayuki Sogabe, and Keiko Koga. Time course of recoveryfrom nerve injury in skeletal muscle: energy state and localcirculation. J. Appl. Physiol. 82(3):732-737, 1997.This study examined the time course of recoveryfrom nerve injury on energy state assessed by phosphorus-31 magneticresonance spectroscopy and local circulation dynamics by fluorine-19magnetic resonance spectroscopy in skeletal muscles ofrats. The hindlimb muscles that had undergone unilateralsciatic nerve compression for 2 wk (CN) were compared withsham-operated (SO) muscles and with muscles that had the compressionremoved after 2 wk and were allowed to recover for 4 wk (R4) or for 6 wk (R6). The energy state and local circulation dynamics of CN muscleswere less than those of SO muscles (P < 0.01). The energy state of R4 muscles remained at levels similar toCN muscles, whereas the local circulation dynamics improved but notback to SO values. In R6 muscles, both parameters returned to SOvalues. These results showed that the recovery processes of circulationprecede those of energy state in skeletal muscles.

     

The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins

Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.     

“DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint

The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.     

Subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli induces stress granule formation

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)‐negative strains of Shiga‐toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase‐dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB‐induced SG formation is regulated by activation of double‐stranded RNA‐activated protein kinase (PKR)‐like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH₄Cl and chloroquine, suppressed SubAB‐induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death‐associated protein 1 (DAP1) knockdown increased basal phospho‐PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA‐treated cells. Our findings show that SubAB‐induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin‐induced SG formation.     

Protective effect of vitamin E on spinal cord injury by compression and concurrent lipid peroxidation

Studies were made on the influence of vitamin E on the effects of compression injury of the spinal cord associated with ischemia in rats. The motor disturbance induced by spinal cord injury was greatly reduced by vitamin E supplementation. After injury, the spinal cord evoked potentials showed greater recovery of both amplitude and latency in the vitamin E-supplemented group than in the control group. Spinal cord blood flow was promptly restored and remained normal after injury in the vitamin E-supplemented group, but was significantly decreased from 3 h after injury in the control group. Thiobarbituric acid (TBA)—reactive substances in the spinal cord was immediately increased by compression injury in both groups, and after injury it persisted at a high value for 24 h in the control group, but decreased within 1 h in the vitamin E-supplemented group. Pathological examination of the spinal cord showed less damage, such as bleeding and edema, in the vitamin E-supplemented group than in the control group. Vitamin E may have protective effects on the spinal cord by inhibiting damage induced by lipid peroxidation and/or by sustaining the blood flow by maintaining the normal metabolism of arachidonic acid.