Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.     

Long-term Administration of Probiotics to Asymptomatic Pre-school Children for Either the Eradication or the Prevention of Helicobacter pylori Infection

Background:  The role of probiotics in the armamentarium remains to be defined. The aims of this study were to investigate whether the long-time administration of Lactobacillus gasseri OLL2716 (LG21) strain can eradicate H. pylori in asymptomatic pre-school children and/or prevent H. pylori infection.
Methods: A total of 440 children, from 5–7 years of age, attending a kindergarten in Thailand were screened by the Helicobacter pylori stool antigen (HpSA) test. Thereafter 132 H. pylori positive and 308 H. pylori negative children were recruited to eradication and randomized prevention arms, respectively. Children in the active and placebo treatment groups received Lactobacillus gasseri OLL2716 (LG21) containing cheese and ordinary cheese, respectively, for 12 months. Eradication was defined as reversion by HpSA at 12 months. Prevention was defined as persistently HpSA negative at 12 months.
Results:  Eighty-two of 132 H. pylori positive (62%) completed the eradication arm, of which 24 (29.3%) were negative at 12 months according to the HpSA test. In the randomized prevention arm, 123 of 156 (79%) and 99 of 122 (81%) completed active and placebo arms, respectively, of which 4.1% and 8.1%, respectively, were HpSA positive at 12 months based on a per-protocol analysis ( p  = .21).
Conclusion: Further trials are needed.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.     

Characterization of a HMT2-like enzyme for sulfide oxidation from Pseudomonas putida

The open reading frame pp0053, which has a high homology with the sequence of mitochondrial sulfide dehydrogenase (HMT2) conferring cadmium tolerance in fission yeast, was amplified from Pseudomonas putida KT2440 and expressed in Escherichia coli JM109(DE3). The isolated and purified PP0053-His showed absorption spectra typical of a flavin adenine dinucleotide (FAD)--binding protein. The PP0053-His catalyzed a transfer of sulfide-sulfur to the thiophilic acceptor, cyanide, which decreased the Km value of the enzyme for sulfide oxidation and elevated the sulfide-dependent quinone reduction. Reaction of the enzyme with cyanide elicited a dose-dependent formation of a charge transfer band, and the FAD-cyanide adduct was supposed to work for a sulfur transfer. The pp0053 deletion from P. putida KT2440 led to activity declines of the intracellular catalase and ubiquinone-H2 oxidase. The sulfide-quinone oxidoreductase activity in P. putida KT2440 was attributable to the presence of pp0053, and the activity showed a close relevance to enzymatic activities related to sulfur assimilation.     

Contribution of physical fitness component to health status in middle-aged and elderly females

This study determined the physical fitness component that contributes to improving and maintaining health status for each age group as well as quantifying the degree of the relationship between health status and physical fitness in middle-aged and elderly females. The participants were 2,371 females aged 30 to 69 years. Ten physical fitness tests and medical checkups were performed. The participants were divided into a healthy group and an unhealthy group according to health status. Multiple discriminant analysis was applied to the multivariate data. Correct discriminant probabilities of the multiple discriminant function to discriminate the healthy and unhealthy groups for females ranged from 63.0% to 77.5%. These results suggest that there is a relatively high relationship between health status and physical fitness level for middle-aged and elderly females. With each individual's discriminant score calculated by the obtained multiple discriminant function as the index of the degree of health, the Pearson's correlation coefficient of the discriminant score and the performance in each physical fitness test were calculated. The aging change from 30 to 69 years old was classified into four patterns according to the contribution. The result of this study is considered to be useful as objective data to prepare an exercise program considering the contribution of the physical fitness component of health status.