Mutation in insulin receptor attenuates oxidative stress and apoptosis in pancreatic beta-cells induced by nutrition excess: reduced insulin signaling and ROS.

Type 2 diabetes results from the failure of beta-cells to adequately compensate for insulin resistance. Although the reduction of beta-cell mass is because of increased cell death and/or inadequate replication or neogenesis, the mechanism underlying beta-cell mass reduction is not fully understood. Here, we clarify the role of insulin signaling pathway in the beta-cell apoptosis using insulin resistant model mice. Wild-type mice and those carrying a mutation in the insulin receptor (mIR) were fed either regular chow or a high-fat diet for 6 weeks and subsequently investigated for beta-cell apoptosis, endoplasmic reticulum stress, and oxidative stress. Insulin tolerance tests revealed that mIR mice fed a high-fat diet (mIRHF) had higher insulin resistance. Beta-cell apoptosis was increased 2-fold in the wild-type mice fed a high-fat diet (wHF) compared with control mice, whereas beta-cell apoptosis in mIRHF mice did not increase compared with that in mIR mice. The expression of endoplasmic reticulum stress markers in isolated islets did not differ between the groups. Staining of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal in islets of wHF mice significantly increased, but the staining in mIRHF mice was not different from that in control group. Gene expression of the antioxidant enzyme MnSOD was significantly higher in mIRHF mice than those in the other 3 groups. A mutation in the insulin receptor attenuated the oxidative stress and apoptosis in beta-cells even though high caloric nutrient was loaded. Our results suggest that reduced insulin signaling protects beta-cells thorough decline of oxidative stress.

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.

Polymyxin-B hemoperfusion for acute exacerbation of idiopathic pulmonary fibrosis: serum IL-7 as a prognostic marker.

BACKGROUND: Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) has an extremely poor prognosis. Direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) has been used to improve oxygenation for acute respiratory distress syndrome. The study aim was to retrospectively determine the predictive factors affecting the prognosis of AE of IPF treated with PMX-DHP. METHODS: We studied patients suffering from AE of IPF, treated with PMX-DHP combined with high-dose corticosteroid therapy. Stored serum taken before and after PMX-DHP therapy was analyzed for 27 cytokines and chemokines. RESULTS: Nineteen patients with AE of IPF were studied. The median survival time after diagnosis of AE was 22 days. Survival rates after diagnosis of AE were 47.4% at 30 days, 31.6% at 60 days, and 26.3% at 90 days. Serum levels of Interleukin (IL)-7, an anti-fibrotic cytokine, in survivors at day 30 following PMX-DHP therapy ('Survivors') significantly increased after the treatment, compared to serum levels of non-survivors at day 30 after the therapy ('Nonsurvivors'), which did not demonstrate a significant change. Serum levels of IL-1beta, interferon-y and chemokine ligand (CCL) 2 levels were not significantly altered in 'Survivors', but were significantly changed in 'Nonsurvivors.' Multivariate Cox proportional-hazards analysis showed that an increase in IL-7 levels after PMX-DHP therapy and treatment without intubation (other than invasive positive-pressure ventilation) were significantly better prognostic factors. CONCLUSION: The results suggest that serum IL-7 may be a useful prognostic factor for patients with AE of IPF treated with PMX-DHP, possibly reflecting underlying anti-fibrotic mechanisms.

[Subscapular elastofibroma].

Elastofibroma is a tumor that is localized mainly at the subscapular region. We report 2 cases of subscapular elastofibromas. Case 1, 75-year-old woman was seen at the hospital because of a left dorsal tumor. Computed tomography (CT) scan revealed the tumor of 6 cm in diameter in the inferior angle of left scapula. The patient underwent excision of the tumor. Case 2, 90-year-old man underwent excision a tumor of 5 cm in diameter in the inferior angle of right scapula simultaneously with the operation of right lung cancer. Histological examinations showed increased elastic fiber with elastica van Gieson staining. These specimens confirmed the diagnosis of elastofibroma There have been no signs of recurrence after surgery.

Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.

Structure and function of Cas-L, a 105-kD Crk-associated substrate-related protein that is involved in beta 1 integrin-mediated signaling in lymphocytes.

Integrin/ligand binding evokes tyrosine phosphorylation of various proteins. We reported previously that a 105 kD protein (pp105) was tyrosine phosphorylated by the engagement of beta 1 integrins in T lymphocytes. We show here that pp105 is a novel p130Cas (Crk-associated substrate)-related protein. Deduced amino acid sequence revealed that pp105 contains conserved motifs with p130Cas, and both pp105 and p130Cas bind to focal adhesion kinase (pp125FAK) and Crk. However, pp105 has a clearly distinct structure from p130Cas, and pp105 is preferentially expressed in lymphocytes, whereas p130Cas is expressed in adherent cells. With these findings, we designate pp105 as Cas-L, lymphocyte-type Cas. Furthermore, we demonstrate that integrin/ligand binding results in the recruitment of Crk, Nck, and SHPTP2 to pp105. These findings further define the roles of pp105/Cas-L and pp125FAK in the integrin-mediated signaling pathways.

CXCR4/fusin is not a species-specific barrier in murine cells for HIV-1 entry.

Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection.

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit human immunodeficiency virus type 1 (HIV-1) entry and infection. The CXC chemokine receptor CXCR4/fusin is a receptor for pre-B cell growth stimulating factor (PBSF)/stromal cell-derived factor 1 (SDF-1) and serves as a coreceptor for the entry of T cell line-tropic HIV-1 strains. Thus, the development of CXCR4 antagonists or agonists may be useful in the treatment of HIV-1 infection. T22 ([Tyr5,12,Lys7]-polyphemusin II) is a synthesized peptide that consists of 18 amino acid residues and an analogue of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus). T22 was found to specifically inhibit the ability of T cell line-tropic HIV-1 to induce cell fusion and infect the cell lines transfected with CXCR4 and CD4 or peripheral blood mononuclear cells. In addition, T22 inhibited Ca2+ mobilization induced by pre-B cell growth stimulating factor (PBSF)/SDF-1 stimulation through CXCR4. Thus, T22 is a small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 entry into target cells.

Initial triggering of M-phase in starfish oocytes: a possible novel component of maturation-promoting factor besides cdc2 kinase.

G2-phase-arrested immature starfish oocytes contain inactive cdc2 kinase and cdc25 phosphatase, and an inactivator for cdc2 kinase. In this system, we have studied how the regulatory balance is apped toward the initial activation of cdc2 kinase. During the hormone-dependent period (Guerrier, P., and M. Doree, 1975. Dev. Biol. 47:341-348), p34cdc2 and cdc25 protein are already converted, though not fully, to active forms, whereas the inactivators for cdc2 kinase and cdc25 phosphatase are able to exhibit their activities if the hormone were removed. We produced "triggered oocytes," in which due to a neutralizing anticdc25 antibody, the activation of cdc2 kinase is prevented out cdc25 protein is phosphorylated slightly after the maturation-inducing hormonal stimulus. In contrast to control immature oocytes, in triggered oocytes the injected cdc2 kinase is not inactivated, and accordingly the level of cdc2 kinase activity required for meiosis reinitiation is much less. These results imply the presence of a cdc2 kinase activity-independent process(es) that suppresses the inactivator for cdc2 kinase and initially phosphorylates cdc25 protein, although this process is reversible during the initial activation of cdc2 kinase. At the most initial triggering of M-phase, the cdc2 kinase activity-independent process might trip the switch leading to the initial activation of cdc2 kinase. Thereafter, in parallel, the cdc2 kinase-dependent feedback loops described by others may cause further increase in cdc2 kinase activity. We propose that a putative suppressor, which downregulates the inactivator for cdc2 kinase independently of nuclear components, might be a previously unrecognized component of maturation-promoting factor.

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins.

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

Pavoninins: shark-repelling ichthyotoxins from the defense secretion of the pacific sole.

A series of ichthyotoxic and hemolytic steroid aminoglycosides, pavoninins-1, to -6, has been isolated from the defense secretion of the sole Pardachirus pavoninus, and their respective chemical structures have been established by spectroscopic studies and chemical conversions. The pavoninins exert repellent activity against sharks and are considered to be the factors responsible for the predator-repelling property of the sole.

Melittin-Like Peptides from the Shark-Repelling Defense Secretion of the Sole Pardachirus pavoninus.

Three ichthyotoxic peptides, pardaxins P-I to P-3, have been isolated from the defense secretion of the sole Pardachirus pavoninus. Pavoninins, the steroid glycosides with shark-repelling ability, had previously been isolated therefrom. Each pardaxin consists of 33 amino acid residues having a distinctly hydrophilic carboxyl terminal region and a predominantly hydrophobic remainder; the pardaxin is thus strongly surfactant. These peptides show marked physical and pharmacological similarities to melittin, the major active constituent of bee venom, yet they lack sequence homology. They are probably also responsible for the predator-repelling property of the sole.

The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis.

The RASSF1A isoform of RASSF1 is frequently inactivated by epigenetic alterations in human cancers, but it remains unclear if and how it acts as a tumor suppressor. RASSF1A overexpression reduces in vitro colony formation and the tumorigenicity of cancer cell lines in vivo. Conversely, RASSF1A knockdown causes multiple mitotic defects that may promote genomic instability. Here, we have used a genetic approach to address the function of RASSF1A as a tumor suppressor in vivo by targeted deletion of Rassf1A in the mouse. Rassf1A null mice were viable and fertile and displayed no pathological abnormalities. Rassf1A null embryonic fibroblasts displayed an increased sensitivity to microtubule depolymerizing agents. No overtly altered cell cycle parameters or aberrations in centrosome number were detected in Rassf1A null fibroblasts. Rassf1A null fibroblasts did not show increased sensitivity to microtubule poisons or DNA-damaging agents and showed no evidence of gross genomic instability, suggesting that cellular responses to genotoxins were unaffected. Rassf1A null mice showed an increased incidence of spontaneous tumorigenesis and decreased survival rate compared with wild-type mice. Irradiated Rassf1A null mice also showed increased tumor susceptibility, particularly to tumors associated with the gastrointestinal tract, compared with wild-type mice. Thus, our results demonstrate that Rassf1A acts as a tumor suppressor gene.

Prolonged impairment of very late activating antigen-mediated T cell proliferation via the CD3 pathway after T cell-depleted allogeneic bone marrow transplantation.

One of the major obstacles in allogeneic bone marrow transplantation (allo-BMT) is prolonged T cell dysfunction resulting in a variety of infectious complications in the months to years after hematologic engraftment. We previously showed that immobilized extracellular matrix (ECM) proteins such as fibronectin (FN), the CS-1 domain of FN, or collagen (CO) acted synergistically with immobilized anti-CD3 to induce T cell proliferation. In addition, the comitogenic effect of ECMs could be mimicked by immobilized mAb reactive with a common beta 1 chain (CD29) of very late activating (VLA) antigens which include ECM receptors. Since the interaction of T cells with ECMs appears to play an important role in the process of T cell reconstitution following allo-BMT, we examined the expression of VLA antigens (alpha 1-alpha 6, beta 1) and their functional roles in CD3-mediated T cell proliferation at various times after T cell depleted allo-BMT. VLA beta 1 as well as VLA alpha 4, alpha 5, and alpha 6 expression was lower than normal controls during the first 3 mo after allo-BMT and auto-BMT, whereas these expressions returned to normal levels by 4 mo after allo-BMT and auto-BMT. Although alpha 1 and alpha 2 were not expressed on lymphocytes from normal controls, these antigens were expressed on lymphocytes at the detectable levels (5-15%) from patients after allo-BMT and auto-BMT. Both CD29 and CD3 were expressed at normal levels on lymphocytes from patients > 3 mo after allo-BMT, whereas T cell interaction with ECM through VLA proteins or crosslinking of VLA beta 1 expressed by T cells with anti-CD29 mAb results in poor induction of CD3-mediated T cell proliferation for a prolonged period (> 1 yr) after allo-BMT. In contrast, T cell proliferation induced by crosslinking of anti-CD2 or anti-CD26 with anti-CD3 was almost fully recovered by 1 yr post-allo-BMT. After autologous BMT, impaired VLA-mediated T cell proliferation via the CD3 pathway after auto-BMT returned to normal levels within 1 yr despite no significant difference in CD3 and CD29 expression following either allo- or auto-BMT. The adhesion of T cells from post-allo-BMT patients to FN-coated plate was normal or increased compared to that of normal controls. Moreover, the induction of the tyrosine phosphorylation of pp105 protein by the ligation of VLA molecules was not impaired in allo-BMT patients. These results suggest that there are some other defects in the process of VLA-mediated signal transduction in such patients. Our results imply that disturbance of VLA function could explain, at least in part, the persistent immunoincompetent state after allo-BMT and may be involved in susceptibility to opportunistic infections after allo-BMT.

Spontaneous puberty in 46,XX subjects with congenital lipoid adrenal hyperplasia. Ovarian steroidogenesis is spared to some extent despite inactivating mutations in the steroidogenic acute regulatory protein (StAR) gene.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. We report here the clinical, endocrinological, and molecular analyses of two unrelated Japanese kindreds of 46,XX subjects affected with lipoid CAH who manifested spontaneous puberty. Phenotypic female infants with 46,XX karyotypes were diagnosed with lipoid CAH as newborns based on a clinical history of failure to thrive, hyperpigmentation, hyponatremia, hyperkalemia, and low basal values of serum cortisol and urinary 17-hydroxycorticosteroid and 17-ketosteroid. These patients responded to treatment with glucocorticoid and 9alpha-fludrocortisone. Spontaneous thelarche occurred in association with increased serum estradiol levels at the age of 10 and 11 yr, respectively. Pubic hair developed at the age of 12 yr 11 mo in one subject and menarche was at the age of 12 yr in both cases. Both subjects reported periodic menstrual bleeding and subsequently developed polycystic ovaries. To investigate the molecular basis of the steroidogenic lesion in these patients, the StAR gene was characterized by PCR and direct DNA sequence analyses. DNA sequence analysis revealed that one patient is homozygous for the Gln 258 Stop mutation in exon 7 and that the other patient is a compound heterozygote with the Gln 258 Stop mutation and a single A deletion at codon 238 in the other allele causing a frame-shift, which renders the StAR protein nonfunctional. These findings demonstrate that ovarian steroidogenesis can be spared to some extent through puberty when the StAR gene product is inactive. This is in marked contrast to the early onset of severe defects in testicular and adrenocortical steroidogenesis which are characteristics of this disease.

Hic-5-reduced cell spreading on fibronectin: competitive effects between paxillin and Hic-5 through interaction with focal adhesion kinase.

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKbeta/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK(-/-) mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

Colony-stimulating factor 1 expression is down-regulated during the adipocyte differentiation of H-1/A marrow stromal cells and induced by cachectin/tumor necrosis factor.

We isolated clonal sublines of the established mouse marrow stromal cell line, H-1. These clonal sublines underwent differentiation into adipocytes in various degrees. One subline, H-1/A, underwent adipocyte differentiation after confluence, while another subline, H-1/D, did not differentiate. In H-1/A cells, the 4.5- and 2.5-kb major mRNA species of colony-stimulating factor 1 (CSF-1) were expressed before differentiation and were down-regulated at a posttranscriptional level during the differentiation of H-1/A cells. The down-regulation of the CSF-1 gene was not a result of arrested cellular growth, because no down-regulation was detected in the nondifferentiating sister line, H-1/D. This down-regulation appeared to be an early event in differentiation. Cachectin/tumor necrosis factor transiently induced the expression of CSF-1 and inhibited the differentiation of H-1/A cells into adipocytes. This induced expression of CSF-1 was due to an increased rate of transcription.