Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

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.     

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.     

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.