The forkhead-associated domain of Ki-67 antigen interacts with the novel kinesin-like protein Hklp2

The Ki-67 antigen (pKi-67) is widely used as a cell proliferation marker protein. Its actual role in the cell cycle progression, however, is presently unclear. Using a two-hybrid screening in yeast, a novel protein, termed Hklp2 (human kinesin-like protein 2), was identified and shown to interact with the forkhead-associated (FHA) domain of pKi-67. Hklp2 has 1388 amino acids and shows a striking similarity (a 53% identity in amino acids) to Xklp2, a plus-end directed kinesin-like motor found in Xenopus. The interaction domain of Hklp2 was mapped to the portion that comprised residues 1017-1237 and that was phosphorylated in vitro by incubating with mitotic but not interphasic HeLa cell extracts. That the interaction was striking in the mitotic extract was also verified. In addition, immunofluorescence using specific antibodies revealed an association between pKi-67 and Hklp2 at the periphery of mitotic chromosomes, largely in close proximity to the centromeres. These findings suggest that pKi-67 is involved in the progression of mitosis via its interaction with Hklp2.     

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments

By affinity chromatography utilizing alpha-cobrotoxin from digitonin-solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin-dystrophin-associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802-807), alpha-actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X-100 solubilization released dystrophin-DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5-4 microM) significantly activated Mg(2+)-ATPase activity of digitonin-solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg(2+)-ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system.     

Heteroplasmic conjugates formed by the fusion of starfish oocyte pairs with a 12 minute time difference in the maturation phase

Two starfish oocytes with a 12 min time difference in the maturation phase were fused together with electric pulses to make a heteroplasmic conjugate. The starfish used were Asterina pectinifera. The emergence of the first meiotic spindle and the extrusion of the polar bodies in the conjugate were timed. Under polarization microscopy two meiotic spindles emerged with a time difference of 10-11 min, which is close to the time difference in the maturation phase between the original oocytes before fusion. In contrast, subsequent formation of the first two polar bodies occurred successively with a short time lag of 1-3 min between them. Times for the formation of both polar bodies were midway between the anticipated times for polar body formation in respective non-fused control oocytes. Thus, in one nucleus the meiotic division was delayed, while in another nucleus it was accelerated, in a single heteroplasmic conjugate. These two sets of observations indicate the presence of a certain control system that regulates progression of the cell cycle at a point during the period from the entry into metaphase through to late anaphase of meiosis I in starfish oocytes. This type of cell cycle control in starfish oocytes is obviously distinct from the currently accepted view of the cell cycle control by the spindle assembly checkpoint that monitors unattached kinetochores of mitotic chromosomes.     

Dietary troglitazone decreases oxidative stress in early stage type II diabetic rats

Oxidative stress is involved in the initiation and development of atherosclerosis in diabetes. We tested the hypothesis that oxidative stress is already increased in early stage type II diabetes, and that troglitazone may prevent the increase. Three groups of 20 week old rats were studied: untreated Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as an animal model of type II diabetes, OLETF rats treated with troglitazone, and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma lipid hydroperoxides (LOOH) concentration, as an indication of lipid peroxidation, and superoxide dismutase (SOD) activity in the thoracic aorta were measured. Plasma LOOH concentration was significantly higher in non-treated OLETF rats compared to LETO rats and treatment with troglitazone completely prevented this increase. SOD activity was significantly decreased in non-treated OLETF rats compared to LETO rats and troglitazone attenuated the diminution of it. These observations demonstrate oxidative stress is already increased in the early stage of type II diabetes and we confirmed troglitazone has the effect of an antioxidant in vivo.     

Reproductive cycle and sexual maturity of the anglerfish Lophiomus setigerus in the East China Sea with a note on specialized spermatogenesis

The reproductive cycle and sexual maturity of the anglerfish Lophiomus setigerus were examined. Spermatids were released from the germinal cysts into the lumina of the seminal lobules, and both spermatids and spermatozoa were present in the lumina of the seminal lobules and sperm ducts. Spermatogenesis and vitellogenesis occurred throughout most of the year. The testes of males were full of spermatozoa throughout the year, with spawning from May to November. Males and females reached sexual maturity at a mean total length and age of 178 mm, 3.3 years and 303 mm, 6.1 years, respectively. There were clear seasonal cycles in the gonadosomatic index (I_G) and hepatosomatic index ( I _H) in females. The mean I_G of females increased rapidly with ovarian development while the mean I _H decreased from the middle of vitellogenesis to final maturation. Mean values of I _G and I _H in males increased with testicular development.     

Leptin Is Required for Glucose Homeostasis after Roux-en-Y Gastric Bypass in Mice

MethodsTo investigate this hypothesis, we performed RYGB or sham operations on leptin-deficient ob/ob mice maintained on regular chow. To investigate whether leptin is involved in post-RYGB weight maintenance, we challenged post-surgical mice with high fat diet.ResultsRYGB reduced total body weight, fat and lean mass and caused reduction in calorie intake in ob/ob mice. However, it failed to improve glucose tolerance, glucose-stimulated plasma insulin, insulin tolerance, and fasting plasma insulin. High fat diet eliminated the reduction in calorie intake observed after RYGB in ob/ob mice and promoted weight regain, although not to the same extent as in sham-operated mice. We conclude that leptin is required for the effects of RYGB on glucose homeostasis but not body weight or composition in mice. Our data also suggest that leptin may play a role in post-RYGB weight maintenance.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer

Ovarian cancer remains the most lethal gynecologic cancer and new targeted molecular therapies against this miserable disease continue to be challenging. In this study, we analyzed the expressional patterns of Interleukin-6 (IL-6) and its receptor (IL-6R) expression in ovarian cancer tissues, evaluated the impact of these expressions on clinical outcomes of patients, and found that a high-level of IL-6R expression but not IL-6 expression in cancer cells is an independent prognostic factor. In in vitro analyses using ovarian cell lines, while six (RMUG-S, RMG-1, OVISE, A2780, SKOV3ip1 and OVCAR-3) of seven overexpressed IL-6R compared with a primary normal ovarian surface epithelium, only two (RMG-1, OVISE) of seven cell lines overexpressed IL-6, suggesting that IL-6/IL-6R signaling exerts in a paracrine manner in certain types of ovarian cancer cells. Ovarian cancer ascites were collected from patients, and we found that primary CD11b⁺CD14⁺ cells, which were predominantly M2-polarized macrophages, are the major source of IL-6 production in an ovarian cancer microenvironment. When CD11b⁺CD14⁺ cells were co-cultured with cancer cells, both the invasion and the proliferation of cancer cells were robustly promoted and these promotions were almost completely inhibited by pretreatment with anti-IL-6R antibody (tocilizumab). The data presented herein suggest a rationale for anti-IL-6/IL-6R therapy to suppress the peritoneal spread of ovarian cancer, and represent evidence of the therapeutic potential of anti-IL-6R therapy for ovarian cancer treatment.