C-reactive protein suppresses insulin signaling in endothelial cells: role of spleen tyrosine kinase

Although few epidemiological studies have demonstrated that C-reactive protein (CRP) is related to insulin resistance, no study to date has examined the molecular mechanism. Here, we show that recombinant CRP attenuates insulin signaling through the regulation of spleen tyrosine kinase (Syk) on small G-protein RhoA, jun N-terminal kinase (JNK) MAPK, insulin receptor substrate-1 (IRS-1), and endothelial nitric oxide synthase in vascular endothelial cells. Recombinant CRP suppressed insulin-induced NO production, inhibited the phosphorylation of Akt and endothelial nitric oxide synthase, and stimulated the phosphorylation of IRS-1 at the Ser307 site in a dose-dependent manner. These events were blocked by treatment with an inhibitor of RhoA-dependent kinase Y27632, or an inhibitor of JNK SP600125, or the transfection of dominant negative RhoA cDNA. Next, anti-CD64 Fcgamma phagocytic receptor I (FcgammaRI), but not anti-CD16 (FcgammaRIIIa) or anti-CD32 (FcgammaRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway.     

A preliminary genetic association study of GAD1 and GABAB receptor genes in patients with treatment-resistant schizophrenia

Molecular Biology Reports - GABAergic system dysfunction has been implicated in the etiology of schizophrenia and of cognitive impairments in particular. Patients with treatment-resistant...     

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.     

eNOS and Hsp90 interaction directly correlates with cord formation in human lymphatic endothelial cells

Endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90) have been reported to contribute to angiogenesis and lymphangiogenesis. However, the functions of these proteins during lymphangiogenesis are unclear. In the present study, we first observed the cord formation pattern of human dermal microvascular lymphatic endothelial cells (HMVEC-dLy) on Matrigel over 2 to 8?h. The length of cord formation increased, peaked at 4?h, and then started to decline after 6 to 8?h of incubation. siRNA-targeted NOS3 significantly reduced the cord formation ability of HMVEC-dLy cells by 27% relative to control. This result confirmed the importance of eNOS in cord formation by human lymphatic endothelial cells. In addition, immunoprecipitation and Western blotting indicated that the interaction between eNOS and Hsp90 was maximal at 4?h, and then the proteins dissociated. This interaction correlated with the observation of cord formation of human lymphatic endothelial cells on Matrigel. Moreover, we found that the eNOS level decreased as the eNOS and Hsp90 complex disassociated during the late stage of cord formation. An Hsp90 inhibitor, 17-DMAG, was able to inhibit the eNOS and Hsp90 interaction, decrease the level of eNOS, and significantly inhibit cord formation to 38% of the level observed in the control. For the first time, we report that the interaction between eNOS and Hsp90 plays an important role in determining eNOS levels and in regulating cord formation of human lymphatic endothelial cells in vitro.     

Vaccine efficacy of Mycobacterium bovis BCG against Mycobacterium sp. infection in amberjack Seriola dumerili

Mycobacteriosis, caused by the intracellular parasitism Mycobacterium sp., causes economic damages to aquaculture production in Japan, particularly in seriola fish production. Antibiotics are not effective against Mycobacterium sp. and so a potent vaccine is needed. We previously reported that BCG vaccine (Mycobacterium bovis BCG) induces adaptive immunity against Mycobacterium sp. in Japanese flounder, Paralichthys olivaceus. In a phylogenetic tree, the genes for a major antigen, the Ag85 complex, in Mycobacterium sp. TUMSAT-Msp001 are closely related to homologues in Mycobacterium ulcerans. M. bovis BCG was detected until 7 days post-injection at the injection site (muscle) and 28 days post-vaccination in spleen. Cumulative mortality of amberjack, Seriola dumerili vaccinated intramuscularly (i.m.) and intraperitoneally (i.p.) with M. bovis BCG was 32.3% and 59.5% respectively, at 24 days post-infection of Mycobacterium sp., compared to 97.8% in PBS-injected fish. The bacterial counts of Mycobacterium sp. in spleen of both i.m.-and i.p.-vaccinated fish (6.2 × 10³ and 1.3 × 10⁴ CFU/mg tissue, respectively) at 20 days post-infection were significantly lower (P < 0.01) than those of PBS-injected fish (8.0 × 10⁶ CFU/mg). Furthermore, Immersion challenge with Mycobacterium sp. TUMSAT Msp-001 showed 50% RPS value in BCG i.m.-vaccinated fish at the end of the experiment. These results support our previous study using Japanese flounder and suggest that BCG vaccine is also effective against Mycobacterium sp. infection in amberjack.     

Uncovering the Mechanisms of Shrimp Innate Immune Response by RNA Interference

Because of the importance of shrimp in world aquaculture, there is much interest in understanding their immune system in order to improve their resistance to pathogenic microorganisms. An effective tool in studying genes involved in the immune response in shrimp is RNA interference (RNAi). RNAi, first recognized as an antiviral response against RNA viruses, is a cellular mechanism that is triggered by double-stranded RNAs and results in the degradation of homologous genes. In this review, we describe the current studies of genes in shrimp that employed RNAi technology to elucidate or confirm their functions. We also review the potential of RNAi to elicit antiviral response in shrimp.     

Domain movement within a gene: a novel evolutionary mechanism for protein diversification

A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.