Comparison of dihydrodiazepam enantiomers: Metabolism,serum binding and brain receptor binding

Summary Dihydrodiazepam is a diazepam prodrug, as shown by its in vitro metabolism by rat and mouse liver and brain microsomal fractions, and its displacing activity on brain diazepam binding. The mechanism of bioactivation is discussed. Stereoselectivity of metabolism and of binding to specific benzodiazepine binding sites in brain synpatosomes and serum albumin were studied.Acknowledgment. The authors are grateful to Dr. J. Wolford for the synthesis of³H-diazepam.     

Immunohistochemical localization of glutamine synthetase in human liver

Glutamine synthetase (GS) of human liver was recognized with a polyclonal antibody to pig brain GS, but failed to stain with an antibody against rat liver GS. Using the latter antibody GS of human liver was shown to be localized within small rings of 1 to 3 hepatocytes surrounding the terminal hepatic venules. This pattern was analogous to that seen in rat and mouse liver.     

Immunohistochemical localization of glutamine synthetase in human liver

Glutamine synthetase (GS) of human liver was recognized with a polyclonal antibody to pig brain GS, but failed to stain with an antibody against rat liver GS. Using the latter antibody GS of human liver was shown to be localized within small rings of 1 to 3 hepatocytes surrounding the terminal hepatic venules. This pattern was analogous to that seen in rat and mouse liver.     

The multi-KH protein vigilin associates with free and membrane-bound ribosomes

The-multi-KH domain protein vigilin has been identified by ex vivo experiments as both a tRNA- and/or mRNA-binding protein. We show here that in vitro under conditions previously shown to allow tRNA binding, recombinant vigilin also binds to selected mRNA species and ribosomal RNA. An in vivo link of vigilin to mRNA and rRNA was elucidated by several approaches. (i) Coexpression/costimulation of vigilin was found with many other proteins independently of whether their mRNA was translated on free or membrane-bound ribosomes. (ii) A close codistribution of vigilin with free ribosomes was seen in the cytoplasm while nucleoli were a major organelle of vigilin accumulation in the nucleus. (iii) Furthermore, free and membrane-bound ribosomes can be enriched for vigilin which suggests that this binding does not depend on the class of mRNA translated. Therefore, we suggest that vigilin does not distinguish between free or membrane-bound ribosomes but is generally necessary for the localization of mRNAs to actively translating ribosomes.Received 20 June 2003; received after revision 25 July 2003; accepted 29 July 2003     

Endothelial dysfunction in the klotho mouse and downregulation of klotho gene expression in various animal models of vascular and metabolic diseases

The human aging process is associated with vascular endothelial dysfunction. However, humoral factors which might protect against endothelial dysfunction during aging have not yet been identified. We recently identified the klotho gene as a possible regulator of human aging. In the present study using the klotho-deficient heterozygous mouse, we examined whether the Klotho protein is a humoral factor protecting against endothelial dysfunction. We further cloned rat klotho cDNA and investigated whether klotho mRNA expression in rat kidney is altered under pathological conditions such as hypertension, hyperlipidemia, renal failure, and inflammatory stress. The Klotho protein itself, or its metabolites, promotes endothelial NO production in aorta as well as arterioles, and klotho mRNA in kidney is downregulated under sustained circulatory stress.     

Alterations in bone-marrow cellularity following thymectomy in rats.

The number of nucleated marrow cells was decreased following neonatal thymectomy in rats, and was corrected by administration of syngeneic lymphoid cells, or by implantation of a syngeneic testis. These results suggest that, in the rat, as has been shown previously in the mouse, lymphoid cells exert parital control over bone marrow cellularity and this effect may be further modulated by sex steroids.     

Binding property of rat andLimulus C-reactive proteins (CRP) to mercury

Summary The C-reactive proteins (CRP) from both rat andLimulus were found to bind mercury (Hg) in both in vivo and in vitro conditions. CRP has high-affinity binding sites for Hg as evidenced by the loss of free sulfhydryl groups, arrested mobility in polyacrylamide gel electrophoresis, and the consumption of CRP in the serum after Hg administration. The binding was tight as it could not be inhibited either by the addition of cysteine or EDTA. By using a direct titration method it was shown that binding of Hg to CRP was saturable at a molar ratio of Hg/CRP=13.11. The possibility that CRP may act as a scavenger for Hg is discussed.     

Alterations in bone-marrow cellularity following thymectomy in rats

Summary The number of nucleated marrow cells was decreased following neonatal thymectomy in rats, and was corrected by administration of syngeneic lymphoid cells, or by implantation of a syngeneic testis. These results suggest that, in the rat, as has been shown previously in the mouse, lymphoid cells exert parital control over bone marrow cellularity and this effect may be further modulated by sex steroids.Supported in part by NSF-RIAS 77-06922, NIH AM21137 and the Morseman Foundation.     

Alternative exon usage selectively determines both tissue distribution and subcellular localization of the acyl-CoA thioesterase 7 gene products

Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of acyl-CoAs to free fatty acids and coenzyme A. Recent studies have demonstrated that one gene named Acot7, reported to be mainly expressed in brain and testis, is transcribed in several different isoforms by alternative usage of first exons. Strongly decreased levels of ACOT7 activity and protein in both mitochondria and cytosol was reported in patients diagnosed with fatty acid oxidation defects, linking ACOT7 function to regulation of fatty acid oxidation in other tissues. In this study, we have identified five possible first exons in mouse Acot7 (Acot7a–e) and show that all five first exons are transcribed in a tissue-specific manner. Taken together, these data show that the Acot7 gene is expressed as multiple isoforms in a tissue-specific manner, and that expression in tissues other than brain and testis is likely to play important roles in fatty acid metabolism. Received 5 February 2007: received after revision 3 April 2007; accepted 19 April 2007     

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11)

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.Received 8 December 2004; received after revision 27 January 2005; accepted 8 March 2004The nucleotide sequence for the cDNA to mouse TFIP11 (previously known as TIP39 and TIP39kDa) has been submitted to Gen- Bank^TM/ EBI Data Bank with accession numbers AF290474 and NM_018783. The accession number for the human TFIP11 homologueis NM_012143.     

Über die Beeinträchtigung der Wirkung von Spindelgiften in der Zellkultur durch menschliches Serum sowie durch Seren verschiedener Tierspezies

Summary Chick embryo fibroblasts, cultivatedin vitro, require higher concentrations of podophyllotoxin derivatives for complete mitotic arrest when the medium contains human adult or cord serum than when it contains horse, bovine, guinea pig, rat, mouse, or cock serum. This difference is due to a higher binding capacity of human serum for these compounds. No such difference was found with a colchicine and a peltatin derivative.     

Upregulation of rat P23 (a member of the YjgF protein family) by fasting, glucose diet and fatty acid feeding

In a previous study, we identified and purified a 99-amino-acid rat liver-kidney perchloric-acid-soluble 23-kDa protein (P23) which displays 30% identity with a highly conserved domain of heat shock proteins (HSPs), as well as an AT-rich 3 untranslated region, which has also been described to play a role in H70 mRNA life span and protein expression. An identical perchloric-acid-soluble protein inhibiting protein synthesis in a rabbit reticulocyte lysate system was also found 2 years later by another group. More recently, the novel, the YjgF, protein family has been described, comprising, 24 full-length homologues, including P23, highly conserved through evolution, and consisting of approximately 130 residues each and sharing a common ternary structure. Independent studies from different laboratories have provided various hypothetical functions for each of these proteins. The high degree of evolutionary conservation may suggest that these proteins play an important role in cellular regulation. Although the function of none of these proteins is known precisely, we present experimental evidence which, combined with the relationship to glucose-regulating protein revealed here, and the relationship to fatty-acid-binding protein revealed by others, allow us to propose a role for P23. In rat liver, P23 expression is developmentally regulated and modulated by dietary glucose, and its mRNA is induced by starvation, in the presence of fatty-acids and in 3-MeDAB-induced hepatomas. The mRNA encoding mouse liver P23 is also hormonally modulated in a mouse line AT1F8. These data indicate that P23 protein might be a key controller of intermediary metabolism during fasting.Received 7 June 2003; received after revision 8 September 2004; accepted 10 October 2004     

Recombinant expression of perchloric acid-soluble protein reduces cell proliferation

Perchloric acid-soluble protein (PSP) may play an important role in the regulation of cellular physiological functions because it has been highly conserved throughout evolution; however, this role has not been well elucidated. In previous reports, we suggested that PSP regulates cell proliferation. In this study, we examined the effect of PSP expression on proliferation of the normal rat kidney cell line NRK-52E, the rat hepatocyte cell line RLN-10, and the rat hepatoma cell line dRLh-84. Cells transfected with pcDNA-sense-PSP (pcDNA-S-PSP) over-expressed PSP mRNA and protein, and cell proliferation of the transfected cells was suppressed compared with that of cells transfected with pcDNA-empty (pcDNA-E). Cell viability of pcDNA-S-PSP-transfected cells was similar to that of pcDNA-E-transfected cells. Thus, over-expression of PSP suppresses cell proliferation without any influence on cell viability. These findings are the first to report an inhibitory activity of PSP on cell proliferation. Received 27 April 2001; received after revision 8 June 2001; accepted 8 June 2001     

Differential expression of glucose transporters during chick embryogenesis

The patterns of Glut1 and Glut3 glucose transporter protein and mRNA expression were assessed during embryogenesis of chicken brain and skeletal muscle, Glut4 protein levels were also evaluated in skeletal muscle and heart, and Glut1 was examined in the developing heart and liver. Glut1 protein expression was detectable throughout brain ontogeny but was highest during early development. Glut1 mRNA levels in the brain remained very high throughout development. Glut3 protein was highest very early and very late and mRNA was highest during the last half of development. In embryonic skeletal muscle, the levels of Glut1and Glut3 proteins and mRNA were highest very early, and declined severely by mid-development. Glut1 protein and mRNA in the heart also peaked early and then decreased steadily. Although Glut1 mRNA levels were consistently high in the embryonic liver, Glut1 protein expression was not detected. These results suggest that (1) Glut1 is developmentally regulated in chick brain, skeletal muscle, and heart, (2) Glut1 mRNA is present in liver but does not appear to be translated, (3) Glut3 in brain increases developmentally but is virtually absent in muscle, and (4) Glut4 protein and mRNA appear to be absent from chick heart and skeletal muscle. Received 11 January 2001; accepted 14 February 2001     

Interaction of DISC1 with the PTB domain of Tensin2

The gene for Disrupted-in-Schizophrenia 1 (DISC1) is amongst the most significant risk genes for schizophrenia. The DISC1 protein is an intracellular scaffolding molecule thought to act an important hub for protein interactions involved in signalling for neural cell differentiation and function. Tensin2 is an intracellular actin-binding protein that bridges the intracellular portion of transmembrane receptors to the cytoskeleton, thereby regulating signalling for cell shape and motility. In this study, we probed in molecular detail a novel interaction between DISC1 and Tensin2. Western blot and confocal microscopic analyses revealed widespread expression of both DISC1 and Tensin2 proteins throughout the mouse brain. Furthermore, we have developed novel anti-DISC1 antibodies that verified the predominant expression of a 105-kDa isoform of DISC1 in the rodent brain as well as in human cells. In the mouse brain, both proteins showed region-specific expression patterns, including strong expression in the pyramidal cell layer of the hippocampus and dentate gyrus. DISC1–Tensin2 colocalisation was most clearly observed in the Purkinje cells of the mouse cerebellum. Biochemical coimmunoprecipitation experiments revealed an interaction between endogenous DISC1 and Tensin2 proteins in the mouse brain. Further pulldown studies in human cells using Myc-tagged Tensin2 constructs revealed that DISC1 specifically interacts with the C-terminal PTB domain of Tensin2 in a phosphorylation-independent manner. This new knowledge on the DISC1–Tensin2 interaction, as part of the wider DISC1 interactome, should further elucidate the signalling pathways that are perturbed in schizophrenia and other mental disorders.     

Biosynthesis of phosphatidylethanolamine from CDP-ethanolamine by the Golgi complex of rat liver in vitro

Summary A Golgi-rich fraction from rat liver has been shown to synthesize phosphatidylethanolamine from CDP-ethanolamine in vitro. The implications of the existence of such a pathway for the membrane flow hypothesis are discussed.Acknowledgments. This study was supported by the Medical Research Council of Canada.     

Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets

Introduction

Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets.

Materials and methods

GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay.

Results

We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion.

Discussion

The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.