Evaluation of two Japanese regulatory actions using medical information databases: a 'Dear Doctor' letter to restrict oseltamivir use in teenagers, and label change caution against co-administration of omeprazole with clopidogrel.

WHAT IS KNOWN AND OBJECTIVE: The implementation of appropriate epidemiological methodology using medical information databases (MIDs) to evaluate the effects of regulatory actions has been highly anticipated. To assess scientific methods for active pharmacovigilance using MIDs, we conducted a quantitative assessment of the impact of two regulatory actions by the Japanese government: (i) restriction of use of oseltamivir in teenagers in March 2007 and (ii) caution against the co-administration of omeprazole (OPZ) with clopidogrel (CPG) in April 2010. METHODS: Data were obtained from four hub hospitals in Japan. We measured the seasonal proportion of patients prescribed oseltamivir to those prescribed neuraminidase inhibitors for the 2002/2003 to 2010/2011 seasons. The monthly proportion of patients co-administered OPZ and CPG (OPZ+CPG) to those prescribed CPG was measured from May 2009 to April 2011. We evaluated the changes observed with implementation of the regulatory actions. To estimate the impact of the actions, we conducted segmented regression analysis using interrupted time series data. The impact was assessed by two parameter estimates of the regression model: the change in level for short-term effects and change in trend for long-term effects. RESULTS AND DISCUSSION: The use of oseltamivir in the target 10-19 years age group showed a significant and large decline (63·16%) immediately after the intervention (P = 0·0008). No change was observed in OPZ+CPG, although there was a relative inhibitory trend for OPZ+CPG compared with co-administration of lansoprazole or rabeprazole with CPG as the control group. When restricted to new users of CPG, the stratified results were consistent with the overall results. WHAT IS NEW AND CONCLUSION: The current analysis demonstrates the effectiveness of two regulatory actions. The results of the current study indicate that MID research can contribute to assessing and improving pharmacovigilance activities.

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

UNLABELLED: Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-ß-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. SIGNIFICANCE AND IMPACT OF THE STUDY: A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Development of a broadband telemedical network based on internet protocol in the Asia-Pacific region.

OBJECTIVES: To promote the exchange of knowledge and standardization of medical procedures and medical systems in the Asia-Pacific region, we established a medical network with high-quality moving images over broadband Internet lines in February 2003. METHODS: Real-time teleconferences and live demonstrations with medical-quality videos, broadcast via the Digital Video Transport System, have been used to teach surgical techniques and other medical procedures across national borders. The Asia-Pacific Advanced Network (APAN) committee in August 2005 formally approved our proposal to establish a medical working group within APAN. The network was expanded by the launch of the Trans-Eurasia Information Network 2 in 2006. By the end of 2006, we had conducted 82 events, in 10 countries in the Asia-Pacific region. The multi-station event has increased every year. RESULTS: There have been no serious transmission problems or ethical conflicts so far. With these experiences and current achievements, we hope to extend this advanced network system to the entire Asia-Pacific. CONCLUSION: This system is a promising and very useful tool for the standardization of medical system and procedures across national borders. Drawing upon these experiences and current achievements, we hope to extend this advanced network system to the entire Asia-Pacific region.

International transmission of uncompressed endoscopic surgery images via superfast broadband Internet connections.

BACKGROUND: Although telecommunication is increasing in popularity, poor-quality images sent through a narrowband network limit its use in the medical field. METHODS: Kyushu University Hospital in Japan and four hospitals in Korea were linked via superfast broadband Internet connection. The digital video transfer system, which can transmit digital videos without loss of image quality, was used, and the bandwidth was 30 Mbps per line. RESULTS: Of the 16 teleconferences conducted, 6 demonstrated real-time endoscopic surgery. In addition to the surgical images, preoperative diagnostic images, images of the operating room, and images of the staff in the conference room were transmitted to facilitate discussion. The network remained stable, and the sound delay was restricted to less than 0.3 s. In the other 10 teleconferences, recorded video images were used for discussion. CONCLUSIONS: The authors have established a high-quality, practical teleconference system that is economical and easy to use in clinical practice. This system shows promise for remote education beyond geographic borders.

Three novel mouse monoclonal antibodies, OM-A, OM-B, and OM-C, reactive with mucinous type ovarian tumors.

Three mouse IgG1 monoclonal antibodies (MoAbs) reactive predominantly with cytoplasmic antigens of mucinous type ovarian tumors were produced. OM-A MoAb was reactive with 11 of 14 mucinous type and two of three endometrioid type ovarian tumors, although only a minor population of the tumor cells was positive in the latter type. This MoAb was not reactive with serous type, clear cell type, or other types of ovarian tumors, nor with various types of uterine carcinoma. Normal adult and fetal tissues of female genital organs were not positive with this MoAb. Among nongynecological carcinomas, three of six metastatic tumors to the ovary from the gastrointestinal tract, one of five gastric carcinomas, and one of eight lung adenocarcinomas were positive. As for normal adult and fetal tissues of nongynecological origin, epithelium of the normal stomach, small bowel, and bronchus as well as epithelium of fetal small and large bowel and secretory products were weakly positive. Thus, this MoAb showed a selected specificity against mucinous and endometrioid types of ovarian tumors. OM-B MoAb showed a broader specificity than OM-A, reacting with all mucinous type, two of three endometrioid type, and three of 16 serous type ovarian tumors, but not with clear cell type tumors. Adenoma type, but not squamous type, cervical carcinomas and one-half of endometrial carcinomas were positive. This antigen is present in cervical mucosa, but not in ovary or endometrium. OM-C MoAb showed a specificity similar to, but broader than that of, OM-B; i.e., 11 of 14 mucinous type, two of three endometrioid type, nine of 16 serous type, and one of nine clear cell type ovarian tumors were positive. It is reactive with adenoma type uterine carcinoma and normal mucosa of the uterine cervix and with normal surface epithelium of the oviduct. Among nongynecological tumors, OM-B antigen was present in metastatic tumors to the ovary as well as in gastric and pancreatic carcinomas, while OM-C was in metastatic tumors to the ovary and gastric and colonic carcinomas. Thus, the serological analysis showed that these three MoAbs showed selective specificities to mucinous and endometrioid types of ovarian tumors. Preliminary characterization of these three OM antigens suggested that these are distinct from carcinoembryonic antigen or ABH blood group-related antigens.

Isolation and characterization of proteoglycans from human nonepithelial tumors.

Proteoglycans (PGs) and glycosaminoglycans (GAGs) were identified in myogenic and fibrogenic tumors. More PGs containing mainly chondroitin sulfate could be detected in malignant tumors (leiomyosarcomas) than in benign tumors (leiomyomas and fibromas). Two groups of PGs were detected in the malignant tumors by ion-exchange chromatography and gel chromatography. One group was a large molecule with chondroitin sulfate side chains, seemingly composed of two or more subpopulations that were eluted from Sepharose CL-4B with a kav of 0.45. After removal of GAG side chains from the PG by chondroitinase AC digestion, core molecules with molecular weights greater than 200,000 were obtained. Another PG detected was a fraction of small PG eluted from Sepharose CL-4B with a kav of 0.45. It consisted of a core molecule with a molecular weight approximately equal to 48,000 and GAG side chains containing chondroitin sulfate-dermatan sulfate hybrids. The mixed sequence of L-iduronic acid with D-glucuronic acid in the same GAG chain was demonstrated by the formation of a small proportion of tetrasaccharide after chondroitinase AC digestion. In the benign tumors, the large PG was found only in very small amounts, and PG detected was composed mainly of the small one eluted from Sepharose CL-4B with a kav of 0.45. Its core protein had a molecular weight of approximately equal to 46,000, which was similar to that of small PG obtained from leiomyosarcomas, but its GAG side chains were composed mainly of dermatan sulfate containing small amounts of glucuronic acid. The results suggest that the core molecules of small PGs from both benign and malignant tumors are the products of the same gene but that they are processed in a different manner to form proteoglycans with different types of GAG chains.

Health-Enabling and Ambient Assistive Technologies: Past, Present, Future.

BACKGROUND: During the last decades, health-enabling and ambient assistive technologies became of considerable relevance for new informatics-based forms of diagnosis, prevention, and therapy. OBJECTIVES: To describe the state of the art of health-enabling and ambient assistive technologies in 1992 and today, and its evolution over the last 25 years as well as to project where the field is expected to be in the next 25 years. In the context of this review, we define health-enabling and ambient assistive technologies as ambiently used sensor-based information and communication technologies, aiming at contributing to a person's health and health care as well as to her or his quality of life. METHODS: Systematic review of all original articles with research focus in all volumes of the IMIA Yearbook of Medical Informatics. Surveying authors independently on key projects and visions as well as on their lessons learned in the context of health-enabling and ambient assistive technologies and summarizing their answers. Surveying authors independently on their expectations for the future and summarizing their answers. RESULTS: IMIA Yearbook papers containing statements on health-enabling and ambient assistive technologies appear first in 2002. These papers form a minor part of published research articles in medical informatics. However, during recent years the number of articles published has increased significantly. Key projects were identified. There was a clear progress on the use of technologies. However proof of diagnostic relevance and therapeutic efficacy remains still limited. Reforming health care processes and focussing more on patient needs are required. CONCLUSIONS: Health-enabling and ambient assistive technologies remain an important field for future health care and for interdisciplinary research. More and more publications assume that a person's home and their interaction therein, are becoming important components in health care provision, assessment, and management.

Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide.

We isolated vitronectins from the plasma or sera of 14 animal species including mouse and rat by heparin affinity chromatography. They cross-reacted with anti-vitronectin antibody and their amino terminal sequences showed strong homology. They also promoted spreading of BHK cells and were bound to heparin and collagen in the same way. Therefore, these properties appear to be essential for vitronectin function. However, the apparent molecular weights of these vitronectins varied considerable from 59 to 78 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the number of bands also varied from 1 to 3. To search for the uniformity of vitronectin polypeptide, vitronectins were deglycosylated and examined by Ferguson plot analysis. The size of the polypeptide portion of vitronectins was estimated to range from 40 to 57 kDa which was 19-26 kDa smaller than original values. Supposing a possible cleavage site at 5-13 kDa far from the carboxyl terminus, all vitronectin polypeptides were speculated to be synthesized de novo in the size range of 50-57 kDa. Proteins reacting with anti-vitronectin antibody were also detected on the immunoblot of 13 more species including Drosophila and Physarum. Almost all of these vitronectin-like proteins showed marked species-specific variations in their apparent molecular weights from 51 to 96 kDa in SDS-PAGE.

Association of a T29-->C polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to myocardial infarction in Japanese.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is an important regulator of vascular remodeling and is involved in the pathogenesis of atherosclerosis. A T-->C transition at nucleotide 29 of the TGF-beta1 gene results in a Leu-->Pro substitution at amino acid 10 of the signal peptide. We have now examined a possible association of TGF-beta1 genotype with myocardial infarction (MI) in a Japanese population. METHODS AND RESULTS: TGF-beta1 genotype was determined in 315 Japanese patients (234 men and 81 women) with MI and 591 control subjects (289 men and 302 women). We found that age, body mass index, and incidence of habitual smoking, hypertension, diabetes mellitus, and hypercholesterolemia did not differ between the 2 groups for either men or women. Multivariable logistic regression analysis, however, demonstrated the frequency of the T allele to be significantly higher in male subjects with MI than in controls (TT + TC versus CC; P<0.0001, odds ratio 3.5, 95% CI 2.0 to 6.3). In contrast, the T allele was not associated with the prevalence of MI in women. In both male MI patients and controls, the serum concentration of TGF-beta1 was significantly higher in individuals with the CC genotype than in subjects with the TT or TC genotype. CONCLUSIONS: Findings suggest that the T allele at nucleotide 29 in the TGF-beta1 gene is a risk factor for genetic susceptibility to MI, at least in middle-aged Japanese men.

Saccharomyces cerevisiae putative G protein, Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p.

Prp20p and Rna1p are GDP/GTP exchanging and GTPase-activating factors of Gsp1p, respectively, and their mutations, prp20-1 and rna1-1, can both be suppressed by Saccharomyces cerevisiae gtr1-11. We found that gtr1-11 caused a single amino acid substitution in Gtr1p, forming S20L, which is a putative GDP-bound mutant protein, while Gtr1p has been reported to bind to GTP alone. Consistently, gtr1-S20N, another putative GDP-bound mutant, suppressed both prp20-1 and rna1-1. On the other hand, gtr1-Q65L, a putative GTP-bound mutant, was inhibitory to prp20-1 and rna1-1. Thus, the role that Gtr1p plays in vivo appears to depend upon the nucleotide bound to it. Our data suggested that the GTP-bound Gtr1p, but not the GDP-bound Gtr1p, interacts with itself through its C-terminal tail. S. cerevisiae possesses a novel gene, GTR2, which is homologous to GTR1. Gtr2p interacts with itself in the presence of Gtr1p. The disruption of GTR2 suppressed prp20-1 and abolished the inhibitory effect of gtr1-Q65L on prp20-1. This finding, taken together with the fact that Gtr1p-S20L is a putative, inactive GDP-bound mutant, implies that Gtr1p negatively regulates the Ran/Gsp1p GTPase cycle through Gtr2p.

The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome.

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

The p53-Mdm2 association in epithelial cells in idiopathic pulmonary fibrosis and non-specific interstitial pneumonia.

BACKGROUND: Wild-type p53 is increased during cellular responses to various stresses. Mdm2, which is induced by p53, regulates p53 protein concentrations through the ubiquitin-proteasome pathway. AIM: To investigate whether the Mdm2 mediated ubiquitination of p53 is associated with epithelial cell apoptosis in idiopathic pulmonary fibrosis (IPF). METHODS: Immunohistochemistry and western blot analysis were carried out on lung samples obtained by lung biopsy from patients with IPF and non-specific interstitial pneumonia (NSIP). RESULTS: The expression of p53, phosphorylated p53, Mdm2, p21, and Bax was upregulated in epithelial cells from patients with IPF and NSIP compared with normal lung parenchyma. Except for p21, there was a significant increase in the expression of these factors in IPF compared with NSIP. In addition, the number of apoptotic cells and the number of p53 and Bax positive cells was increased compared with controls. p53 conjugated with Mdm2 was decreased in IPF compared with NSIP and controls. Ubiquitinated p53 was increased in both IPF and NSIP compared with controls. CONCLUSIONS: Signalling molecules associated with p53 mediated apoptosis may participate in epithelial cell apoptosis, and the attenuation of p53-Mdm2 conjugation and of p53 degradation may be involved in the epithelial cell apoptosis seen in IPF. Augmented epithelial apoptosis in IPF may lead to the poor prognosis compared with NSIP.

Effects of beta-D-xylosides on proliferation and matrix formation of adherent fibroblastic cells in mouse bone marrow culture.

Mouse bone marrow cells were seeded on small pieces of cover glasses placed in culture dishes, and after 3 days, the pieces of glass to which only small spindle cells adhered were transferred to another dish containing fresh medium. The adherent small spindle cells proliferated and some of them became large polygonal cells with abundant cytoplasm. When phenyl beta-D-thioxyloside (0.5 mM), an artificial initiator of chondroitin sulfate chain synthesis, was added to the culture medium, the cells showed a marked increase in number as compared to controls, with conversion of about 35% of the cells to large size cells. Immunohistochemical staining with monoclonal antibodies showed that the intercellular matrix of adherent cells consists mainly of a large proteglylcan with chondroitin 6-sulfate side chains. By histochemical analysis, the amount of chondroitin sulfate was shown to be greater in the intercellular matrices of xyloside-treated groups than those of control cultures. The amount of chondroitin sulfate in the growth medium of the adherent cells, as measured by uronic acid analysis, was also significantly increased by treatment with phenyl beta-D-thioxyloside compared with controls.

Betaine prevents homocysteine-induced memory impairment via matrix metalloproteinase-9 in the frontal cortex.

Betaine plays important roles that include acting as a methyl donor and converting homocysteine (Hcy) to methionine. Elevated plasma Hcy levels are known as hyperhomocysteinemia (HHcy) and contribute to impairments of learning and memory. Although it is commonly known that betaine plays an important role in Hcy metabolism, the effects of betaine on Hcy-induced memory impairment have not been investigated. Previously, we demonstrated the beneficial effects of betaine on acute stress and lipopolysaccharide-induced memory impairment. In the present study, we investigated whether betaine ameliorates Hcy-induced memory impairment and the underlying mechanisms of this putative effect. Mice were treated with Hcy (0.162mg/kg, s.c.) twice a day for nine days, and betaine (25mg/kg, s.c.) was administered 30min before the Hcy injections. The memory functions were evaluated using a spontaneous alternation performance test (Y-maze) at seven days and a step-down type passive avoidance test (SD) at nine and ten days after Hcy injection. We found that betaine suppressed the memory impairment induced by repeated Hcy injections. However, the blood concentrations of Hcy were significantly increased in the Hcy-treated mice immediately after the passive avoidance test, and betaine did not prevent this increase. Furthermore, Hcy induces redox stress in part by activating matrix metalloproteinase-9 (MMP-9), which leads to BBB dysfunction. Therefore, we tested whether betaine affected MMP-9 activity. Interestingly, treatment with betaine significantly inhibited Hcy-induced MMP-9 activity in the frontal cortex but not in the hippocampus after acute Hcy injection. These results suggest that the changes in MMP-9 activity after betaine treatment might have been partially responsible for the amelioration of the memory deficits and that MMP-9 might be a candidate therapeutic target for HHcy.

Effects of general receptor for phosphoinositides 1 on insulin and insulin-like growth factor I-induced cytoskeletal rearrangement, glucose transporter-4 translocation, and deoxyribonucleic acid synthesis.

We investigated the effects of general receptor for phosphoinositides-1 (GRP1), a recently cloned protein that binds 3,4,5-phosphatidylinositol [PtdIns(3,4,5)P3] with high affinity, but not PtdIns(3,4)P2 nor PtdIns(3)P, on insulin and insulin-like growth factor I (IGF-I)-induced cytoskeletal rearrangement, glucose transporter-4 (GLUT4) translocation, and DNA synthesis. GRP1 consists of an NH2-terminally located coiled coil domain followed by a Sec7 domain and a COOH-terminal pleckstrin homology (PH) domain that is required for PtdIns binding. We used microinjection of glutathione-S-transferase fusion proteins containing residues 239-399 (PH domain), residues 52-260 (Sec7 domain), residues 5-71 (N-terminal domain), full-length GRP1, and an antibody (AB) raised against full-length GRP1 coupled with immunofluorescent detection of actin filament rearrangement, GLUT4 translocation, and 3'-bromo-5'-deoxyuridine incorporation. Microinjection of these constructs and the AB had no effect on insulin-induced GLUT4 translocation or DNA synthesis. However, microinjection of the GRP1-PH and the GRP1-Sec7 domain as well as the alpha-GRP1-AB significantly inhibited insulin- and IGF-I-stimulated actin rearrangement in an insulin receptor-overexpressing cell line (HIRcB) compared with that in control experiments. Coinjection of GRP1-Sec7 along with constitutively active Rac (Q67L) did not inhibit Rac-induced actin rearrangement. Furthermore, GRP1 is not able to bind and act as a nucleotide exchange factor for the small GTP-binding proteins of the Rho family. As GRP1 acts as a guanine nucleotide exchange factor for ARF6 proteins, we propose a signaling pathway distinct from the small GTP-binding protein Rac, connecting PtdIns(3,4,5)P3 via GRP1 to ARF6, leading to insulin- and IGF-I-induced actin rearrangement.

Persistent activation of phosphatidylinositol 3-kinase causes insulin resistance due to accelerated insulin-induced insulin receptor substrate-1 degradation in 3T3-L1 adipocytes.

Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.

Insulin resistance associated with substitution of histidine for arginine 252 in the alpha-subunit of the human insulin receptor: trial of insulin-like growth factor I injection therapy to enhance insulin sensitivity.

Mutation of the insulin receptor gene can compromise the ability of the receptor to mediate insulin action. A homozygous point mutation that results in the substitution of histidine for arginine 252 in the insulin receptor alpha-subunit has now been identified by polymerase chain reaction and single stranded conformational polymorphism analysis in a 20-yr-old Japanese woman with type A syndrome and severe insulin resistance. The proband's consanguineous parents (diabetic mother and normal father) and her sister (impaired glucose tolerance), each of whom showed an exaggerated insulin response to an oral glucose load, were heterozygous for this mutation. Her brother showed a normal insulin response and lacked the mutation, as did 50 healthy Japanese control subjects. The chronic sc administration of insulin-like growth factor I (IGF-I) improved the patient's hyperglycemia and corrected certain metabolic abnormalities over a 9-month period, even though the binding of 125I-labeled IGF-I to her cultured fibroblasts was decreased by 40% relative to that to cells from healthy controls. Studies of the binding of 125I-labeled insulin to the proband's cultured fibroblasts, to COS-I cells transfected with complementary DNA encoding the mutant insulin receptor, and to partially purified mutant receptors revealed that the Arg252-->His mutation decreased both cell surface expression and the affinity for insulin for the receptor. These observations suggest that the homozygous Arg252-->His mutation is responsible for the type A insulin resistance of the proband, whereas in the heterozygous state, the mutation results in mild insulin resistance indistinguishable from that observed in noninsulin-dependent diabetes mellitus.

Point mutation in a family with hyperproinsulinemia detected by single stranded conformational polymorphism.

We previously described a case of familial hyperproinsulinemia, the fifth to be reported. In the present study we characterized the genetic defect carried by this family and demonstrated that it could be detected by polymerase chain reaction-single stranded conformational polymorphism. Since the serum proinsulin molecule from the propositus, a 63-yr-old Japanese man, was eluted on the same fraction of human proinsulin intermediate cleaved only at the B-C junction, we sequenced exon 3 of his insulin gene, including the C-A junction. A point mutation was discovered that changed codon 65 from arginine (CGT) to histidine (CAT) in one allele. This was the same point mutation as that described previously in three unrelated kindreds representing two races, consistent with the hypothesis that the dinucleotide sequence CpG may be a "hot spot" for mutations. Recently, developed polymerase chain reaction-single stranded conformational polymorphism proved useful in detecting this mutation in the family members. The daughter of the propositus and one of his two grandsons were also demonstrated to be heterozygous for this point mutation by this method.