Changes in intracellular location of DNA polymerase-α during oocyte maturation of the toad,bufo bufo japonicus

Intracellular location of DNA polymerase-α during oocyte maturation of the toad was studied. Quantitative and qualitative changes in the activity of DNA polymerase-α were not observed during the maturational process. Nearly all activity was found in isolated germinal vesicles from full grown oocytes and in enucleated mature oocytes. The cytoplasmic DNA polymerase-α of mature oocytes was recovered at buoyant densities equivalent to microsome by isopycnic centrifugation. These findings indicate that DNA polymerase-α in the germinal vesicle is released into the cytoplasm and binds to the endoplasmic reticulum when the germinal vesicle breaks down.     

Structure of Ovarian Asterosaponin-1 in the Starfish Asterias amurensis

Cultured crown gall cells of Catharanthus roseus Don (Vinca rosea L.) was found to contain brassinosteroids. These were identified as brassinolide and castasterone by GC/MS. This is the first conclusive identification of endogenous brassinosteroids in cultured cells.     

Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes

Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.     

Effect of insulin-mimetic vanadyl sulfate on cytochrome P450 2E1-dependent p-nitrophenol hydroxylation in the liver microsomes of streptozotocin-induced type 1 diabetic rats

CYP2E1 is known to be induced in streptozotocin (STZ)-treated diabetic rats (STZ rats), and its induction is improved by insulin. We have examined the age-dependent changes of CYP2E1 in the liver microsomes of type 1 diabetic STZ rats, the effects of VOSO4 on the contents of total P450 and CYP2E1, and the activities of CYP2E1 in terms of p-nitrophenol hydroxylation. The contents of P450 and CYP2E1 and CYP2E1 activity were enhanced with the development of diabetes. When the hyperglycemia of STZ rats was improved by daily intraperitoneal injections of VOSO4 for 10 days at the doses of 7 mg/kg body weight for 5 days, 5 mg/kg for the following 3 days, and then 2.5 mg/kg for 2 days, the P450 and CYP2E1 levels and CYP2E1 activity were lowered than those in the untreated STZ rats. To understand the mechanism underlying CYP2E1-dependent hydroxylation activity, the production of reactive oxygen species was examined in the NADPH-liver microsomal systems by ESR spin-trapping. Singlet oxygen (1O2) was detected in all microsomal systems, while superoxide anion radical(*O2-) and hydroxyl radical (*OH) were not. On the basis of these results, we conclude that (1) CYP2E1 level and activity are enhanced in the diabetic state, however, they are improved by VOSO4 treatment, and (2) 1O2 is generated during CYP2E1-dependent substrate oxygenation.     

Osteoblastic activity and estrogenic response in the regenerating scale of goldfish, a good model of osteogenesis

Osteogenesis in the teleost was morphologically observed using regenerating scales of goldfish. Histological observations indicated that osteoblasts around the regenerating scales on days 7 to 10 were greater in size and number than those at other stages. Therefore, further experiments were carried out to examine the activity of osteoblasts in the regenerating period. To quantify their osteoblastic activities, scales on the left side of the body were taken, and the regenerating scales were then used to measure the activities of alkaline phosphatase (ALP), a marker of osteoblasts, on days 7, 10, and 15. The ontogenic scales on the right side of the body were also collected and used to measure ALP activity on the same days. Osteoblasts at all stages of regenerating scales were more active than those in the remaining ontogenic scales. The regenerating scales on day 10 had the highest activity. Furthermore, we found that estrogen receptor (ER) mRNA was expressed in the regenerating scales because estrogen participates in osteoblastic growth and differentiation in mammals. Therefore, using a scale culture system reported previously, the estrogenic response was examined in the ontogenic and regenerating scales on day 10. The reactivity was much higher in regenerating scales, although estrogen treatment significantly activated the osteoblastic activities in both scales. We are the first to demonstrate that ER is expressed in regenerating scales and that estrogen participates in osteogenesis as it does in mammalian bone. Our findings strongly suggest that regenerating scales can be used as a model of osteogenesis in vertebrates.     

Requirement of hCenexin for proper mitotic functions of polo-like kinase 1 at the centrosomes

Outer dense fiber 2 (Odf2) was initially identified as a major component of sperm tail cytoskeleton and later was suggested to be a widespread component of centrosomal scaffold that preferentially associates with the appendages of the mother centrioles in somatic cells. Here we report the identification of two Odf2-related centrosomal components, hCenexin1 and hCenexin1 variant 1, that possess a unique C-terminal extension. Our results showed that hCenexin1 is the major isoform expressed in HeLa cells, whereas hOdf2 is not detectably expressed. Mammalian polo-like kinase 1 (Plk1) is critical for proper mitotic progression, and its association with the centrosome is important for microtubule nucleation and function. Interestingly, depletion of hCenexin1 by RNA interference (RNAi) delocalized Plk1 from the centrosomes and the C-terminal extension of hCenexin1 was crucial to recruit Plk1 to the centrosomes through a direct interaction with the polo-box domain of Plk1. Consistent with these findings, the hCenexin1 RNAi cells exhibited weakened gamma-tubulin localization and chromosome segregation defects. We propose that hCenexin1 is a critical centrosomal component whose C-terminal extension is required for proper recruitment of Plk1 and other components crucial for normal mitosis. Our results further suggest that the anti-Odf2 immunoreactive centrosomal antigen previously detected in non-germ line cells is likely hCenexin1.     

Dissecting the role of Rho-mediated signaling in contractile ring formation

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation.     

Metamorphosis in Balanomorphan, Pedunculated, and Parasitic Barnacles: A Video-Based Analysis

Cypris metamorphosis was followed using video microscopy in four species of cirripeds representing the suspension-feeding pedunculated and sessile Thoracica and the parasitic Rhizocephala. Cirripede metamorphosis involves one or more highly complex molts that mark the change from a free cypris larva to an attached suspension feeder (Thoracica) or an endoparasite (Rhizocephala). The cyprids and juveniles are so different in morphology that they are functionally incompatible. The drastic reorganization of the body implicated in the process can therefore only commence after the cyprid has irreversibly cemented itself to a substratum. In both Megabalanus rosa and Lepas, the settled cyprid first passes through a quiescent period of tissue reorganization, in which the body is raised into a position vertical to the substratum. In Lepas, this is followed by extension of the peduncle. In both Lepas and M. rosa, the juvenile must free itself from the cypris cuticle by an active process before it can extend the cirri for suspension feeding. In M. rosa, the juvenile performs intensely pulsating movements that result in shedding of the cypris carapace ～8?h after settlement. Lepas sp. sheds the cypris cuticle ～2 days after settlement due to contractile movements of the peduncle. In Lepas anserifera, the juvenile actively breaks through the cypris carapace, which can thereafter remain for several days without impeding cirral feeding. Formation of the shell plates begins after 1-2 days under the cyprid carapace in Lepas. In M. rosa, the free juvenile retains its very thin cuticle and flexible shape for some time, and shell plates do not appear until sometime after shedding of the cypris cuticles. In Sacculina carcini, the cypris settles at the base of a seta on the host crab and remains quiescent and aligned at an angle of ～60° to the crab's cuticle. The metamorphosis involves two molts, resulting in the formation of an elongated kentrogon stage with a hollow injection stylet. Due to the orientation of the cyprid, the stylet points directly towards the base of the crab's seta. Approximately 60?h after settlement the stylet penetrates down one of the cyprid antennules and into the crab. Almost immediately afterwards the unsegmented vermigon stage, preformed in the kentrogon, passes down through the hollow stylet and into the crab's hemocoel in a process lasting only 30?s. In S. carcini, the carapace can remain around the metamorphosing individual without impeding the process.     

Visualization of biodistribution of Zn complex with antidiabetic activity using semiconductor Compton camera GREI

Various types of zinc (Zn) complexes have been developed as promising antidiabetic agents in recent years. However, the pharmacological action of Zn complex is not elucidated because the biodistribution of the complex in a living organism has not been studied. Nuclear medicine imaging is superior technology for the noninvasive analysis of the temporal distribution of drug candidates in living organisms. Gamma-ray emission imaging (GREI), which was developed by our laboratory as a novel molecular imaging modality, was adopted to visualize various γ-ray–emitting radionuclides that are not detected by conventional imaging techniques such as positron emission tomography and single-photon emission computed tomography. Therefore, we applied GREI to a biodistribution assay of Zn complexes. In the present study, ⁶⁵Zn was produced in the ^natCu(p,n) reaction in an azimuthal varying field cyclotron for the GREI experiment. The distribution was then noninvasively visualized using GREI after the intravenous administration of a ⁶⁵Zn-labeled di(1-oxy-2-pyridinethiolato)zinc [Zn(opt)₂], ZnCl₂, and di(l-histidinato)zinc. The GREI images were validated using conventional invasive assays. This novel study showed that GREI is a powerful tool for the biodistribution analysis of antidiabetic Zn complexes in a living organism. In addition, accumulation of ⁶⁵Zn in the cardiac blood pool was observed for [Zn(opt)₂], which exhibits potent antidiabetic activity. These results suggest that the slow elimination of Zn from the blood is correlated to the antidiabetic activity of [Zn(opt)₂].