Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export

Phosphorylation of the cyclin-dependent kinase inhibitor p27(Kip1) has been thought to regulate its stability. Ser(10) is the major phosphorylation site of p27(Kip1), and phosphorylation of this residue affects protein stability. Phosphorylation of p27(Kip1) on Ser(10) has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27(Kip1) protein was translocated from the nucleus to the cytoplasm at the G(0)-G(1) transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27(Kip1) at the G(0)-G(1) transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) export, whereas substitution of Ser(10) with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27(Kip1) on Ser(10) is required for its binding to CRM1 and for its subsequent nuclear export.     

Roles of galactose and sulfate residues in sulfatides for their antagonistic functions in the blood coagulation system

We previously reported that the sulfatide (galactosylceramide I³-sulfate) may have contradictory functions, namely both coagulant and anticoagulant roles in vivo: sulfatide induced giant thrombi formation when injected into rats with vein ligation, whereas no thrombi were formed when sulfatide was injected into rats without vein ligation. Rather it prolonged bleeding time. To investigate the structural features of sulfatide for both functions, a synthetic sulfatide (galactosylceramide I⁶-sulfate) which does not occur naturally, cholesterol 3-sulfate and ganglioside GM4 were examined together with naturally occurring sulfatide. Both sulfatides and cholesterol 3-sulfate induced giant thrombi in the rats with vein ligation within ten minutes of injection, although cholesterol 3-sulfate exhibited weaker coagulant activity than the sulfatides. On the contrary, both sulfatides significantly prolonged bleeding time but cholesterol 3-sulfate barely prolonged it when injected without vein ligation. GM4 exhibited neither coagulant nor anticoagulant activity. These results suggested that sulfate moiety in the sulfatides is essential for coagulant activity and that galactose residue enhances the activity, whereas both galactose and sulfate residues seem to be important for anticoagulant activity. This is because the sulfatides possess both residues but GM4 possesses galactose without sulfate and cholesterol 3-sulfate possesses sulfate without galactose. We previously reported that the possible mechanism of anticoagulation by sulfatide was due to its binding to fibrinogen, thereby inhibiting the conversion to fibrin. In this paper we reveal that both sulfatides inhibited thrombin activity independent of heparin cofactor II, thus providing evidence of another anticoagulation mechanism for the sulfatides.     

Investigation of varicella-zoster virus DNA in lymphocyte subpopulations by quantitative PCR assay

To investigate the nature of viremia during the acute phase of varicella, we studied the viral load in nine otherwise healthy children with varicella. Plasma and peripheral blood mononuclear cells (PBMC) were obtained, then PBMC were divided into CD4+T, CD8+T, and B lymphocytes and monocyte/macrophage fractions. The viral DNA in each component was quantified using a real-time quantitative polymerase chain reaction assay. Varicella-zoster virus (VZV) DNA was detected in plasma, PBMC and all subpopulations. The amount of viral DNA was similar in each PBMC subpopulation, suggesting that each lymphocyte fraction and monocytes carry similar amounts of VZV DNA during viremia.     

Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis

Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases.     

Inheritance Characteristics of Microsatellite DNA Loci in Experimental Families of Japanese Flounder Paralichthys olivaceus

Allele segregating patterns of microsatellite DNA loci in 5 experimental families of Japanese flounder Paralichthys olivaceus and genotype frequencies for deviation from Hardy-Weinberg expectations (HWE) in natural P. olivaceus populations were studied to assess inheritability. Of the 12 microsatellite loci examined, 1 locus had a possibility of scoring errors of heterozygous individuals caused by unreproducible polymerase chain reaction amplifications of a particular allele. At the remaining 11 loci, almost all of alleles were segregated according to Mendelian transmission, and observed genotype frequencies in natural populations were consistent with HWE. The results demonstrated here would provide useful information supporting the suitability of these microsatellite loci as inheritable P. olivaceus genetic markers. Received September 25, 2000; accepted March 1, 2001     

Analysis of Microsatellite DNA Polymorphisms in Rockfish Sebastes thompsoni and Application to Population Genetics Studies

Population differentiation and relationships among 6 natural rockfish populations collected from northern coastal seas around Japan were assayed using microsatellite DNA loci. Seven loci examined were polymorphic in all populations. The number of alleles per locus ranged from 6.7 to 9.3, and the average of observed and expected heterozygosity ranged from 0.63 to 0.68, and from 0.66 to 0.69, respectively. The observed genotype frequencies at each locus were almost in agreement with Hardy-Weinberg expectations with two exceptions (P < .05). The allele frequencies of 16 population pairs were significantly different (P < .05). Genetic distance (D _A ) between 6 populations ranged from 0.03 to 0.08. According to a neighbor-joining tree generated from the D _A values, the 6 populations fell into 3 clusters. These clusters were correlated with the geographical positions of each population; larval dispersions due to water current were also found to have an effect on these results. Received May 2, 2000; accepted July 17, 2000.     

FXIIIA subtypes in Indonesians, Bangladeshis, Tibetans, South African blacks and South African whites

The polymorphism of FXIIIA was investigated in 105 Indonesians, 141 Bangladeshis, 186 Tibetans, 101 South African Blacks and 100 South African Whites using isoelectric focusing followed by immunoblotting. These population data conformed to the Hardy-Weinberg equilibrium. A circum-pan-Pacific cline in the FXIII*1A (or FXIII*1B) allele frequency appeared to exist among the Mongoloids.     

Endogenous cholecystokinin plays a role in down-regulation of pancreatic amylase independent of dietary carbohydrate in rats

The role of cholecystokinin (CCK) in the regulation of pancreatic amylase has not been fully clarified. We examined the effects of hyperCCKemia with chronic pancreatico-biliary diversion (PBD) and blockade of CCK(A)-receptor on rat pancreatic amylase activity and mRNA abundance. Also, we examined the relationship between diet and CCK in terms of regulation of pancreatic amylase. PBD was produced by transposition of the duodenal segment containing the ampulla of Vater to the upper ileum. A potent CCK(A)-receptor antagonist, devazepide, was injected (6 mg/kg body weight per day for 5 days) in the PBD rats fed with diets containing normal or low level of carbohydrate (695 or 345 g sucrose/kg diet). The specific activity and mRNA abundance of the pancreatic amylase were constantly lower 4, 10 and 28 days after PBD than those after the sham operation. Devazepide treatment completely restored the amylase activity lowered by PBD without any increases in amylase mRNA. Feeding a high-protein low-carbohydrate diet suppressed the pancreatic amylase activity and mRNA abundance in PBD rats to a similar degree in those treated, and those untreated, with devazepide. We conclude that endogenous CCK suppresses pancreatic amylase production, and we speculate that CCK reduced translational efficiency of amylase mRNA. The effect of CCK on amylase production is independent of regulation by dietary carbohydrate.