Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

Isolation of human β-defensin-4 in lung tissue and its increase in lower respiratory tract infection

Background

Human β-defensin-4 (hBD-4), a new member of the β-defensin family, was discovered by an analysis of the genomic sequence. The objective of this study was to clarify hBD-4 expression in human lung tissue, along with the inducible expression in response to infectious stimuli, localization, and antimicrobial activities of hBD-4 peptides. We also investigated the participation of hBD-4 in chronic lower respiratory tract infections (LRTI) by measuring the concentrations of hBD-4 peptides in human bronchial epithelial lining fluid (ELF).

Methods

The antimicrobial activity of synthetic hBD-4 peptides against E. coli and P. aeruginosa was measured by radial diffusion and colony count assays. We identified hBD-4 in homogenated human lung tissue by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay (RIA). Localization of hBD-4 was studied through immunohistochemical analysis (IHC). We investigated the effects of lipopolysaccharide (LPS) on hBD-4 expression and its release from small airway epithelial cells (SAEC). We collected ELF from patients with chronic LRTI using bronchoscopic microsampling to measure hBD-4 concentrations by RIA.

Results

hBD-4 exhibited salt-sensitive antimicrobial activity against P. aeruginosa. We detected the presence of hBD-4 peptides in human lung tissue. IHC demonstrated the localization of hBD-4-producing cells in bronchial and bronchiolar epithelium. The levels of hBD-4 peptides released from LPS-treated SAECs were higher than those of untreated control cells. ELF hBD-4 was detectable in 4 of 6 patients with chronic LRTI, while the amounts in controls were all below the detectable level.

Conclusion

This study suggested that hBD-4 plays a significant role in the innate immunity of the lower respiratory tract.     

Development of simultaneous gas chromatography-mass spectrometric and liquid chromatography-electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine

To prove the intake of recently controlled designer drugs, N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a simple, sensitive and reliable method which allows us to simultaneously detect BZP, TFMPP and their major metabolite in human urine has been established by coupling gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). GC-MS accompanied by trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed after the enzymatic hydrolysis and the solid phase extraction with OASIS HLB, and BZP, TFMPP and their major metabolites, 4'-hydroxy-BZP (p-OH-BZP), 3'-hydroxy-BZP (m-OH-BZP) and 4'-hydroxy-TFMPP (p-OH-TFMPP), have found to be satisfactorily separated on a semi-micro SCX column with acetonitrile-40 mM ammonium acetate buffer (pH 4) (75:25, v/v) as the eluent. The detection limits produced by GC-MS were estimated to be from 50 ng/ml to 1 microg/ml in the scan mode, and from 200 to 500 ng/ml in the selected ion monitoring (SIM) mode. Upon applying the LC-ESI-MS technique, the linear calibration curves were obtained by using the SIM mode for all analytes in the concentration range from 10 ng/ml to 10 microg/ml. The detection limits ranged from 5 to 40 ng/ml in the scan mode, and from 0.2 to 1 ng/ml in the SIM mode. These results indicate the high reliability and sensitivity of the present procedure, and this procedure will be applicable for proof of intake of BZP and TFMPP in forensic toxicology.     

Human M-ficolin is a secretory protein that activates the lectin complement pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a co-immunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio)

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.     

The First Finding of a Rickettsia Bacterium Associated with Parthenogenesis Induction Among Insects

The larval endoparasitoid, Neochrysocharis formosa (Westwood), is an important natural enemy of the leafminer Liriomyza trifolii (Burgess) in Japan. The thelytokous strain of N. formosa mostly produces female progeny. Male progeny were produced by females treated with tetracycline, suggesting that microorganisms induce thelytoky in N. formosa. The result of nucleotide sequencing of the 16S rRNA gene indicated that the parasitoid is infected with a Rickettsia bacterium, which appears to be causative of the thelytoky. Although Rickettsia-bellii-like bacteria have been found to be associated with various reproductive disorders, this is the first finding of a parthenogenesis-inducing Rickettsia among insects.     

Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO₂Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO₂Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO₂Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO₂Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO₂Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.