Regulation of subcellular localization of alpha1-adrenoceptor subtypes.

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface.

The role of CTLA-4 in murine contact hypersensitivity.

CTLA-4 (CD152) shares its ligand with a costimulatory molecule, CD28, and functions as a negative regulator in T cell activation. We examined the role of CTLA-4 in both induction and effector phases of contact hypersensitivity induced by using two allergens. Treatment with anti-CTLA-4 monoclonal antibody at sensitization, but not at challenge, significantly enhanced Th1- and Th2-dominant contact hypersensitivity reactions elicited by dinitrofluorobenzene and fluorescein isothiocyanate, respectively.

Long-term graft acceptance in rat heart transplantation by CTLA4Ig gene transfection combined with FTY720 treatment.

CTLA4Ig strongly adheres to B7 molecules on antigen-presenting cells to block intracellular signal transduction via CD28 on helper T cells, which eventually inhibits immune responses. We have demonstrated that the administration to recipient animals of adenoviral vectors containing CTLA4Ig gene (adCTLA4Ig) prolonged graft survival, although the gene expression diminished in a time-dependent manner and the grafts were finally rejected. In addition, recipient animals treated with FTY720, a new immunosuppressant, exhibited a decrease in the number of peripheral lymphocytes due to apoptosis.

In vitro prevention of cell-mediated xeno-graft rejection via the Fas/FasL-pathway in CrmA-transducted porcine kidney cells.

Cell-mediated cytotoxicity may be involved in delayed and/or chronic xenograft rejection in which apoptosis is induced in the grafted cells via the Fas/Fas-ligand (FasL) and perforin/granzyme pathways. One barrier to the potential use of xeonogenic grafts for humans may be Fas/FasL-mediated apoptosis, which would be blocked by the gene expression of cytokine response modifier A (CrmA), a cowpox virus gene product. The purpose of this study is to explore whether crmA is an effective candidate gene for inhibiting apoptosis in an in vitro model of xenograft rejection, using Fas-expressing non-primate cells cultured with a soluble recombinant human FasL (sFasL).

Protein determination by high-performance gel-permeation chromatography: applications to human pancreatic juice, human bile and tissue homogenate.

A high-performance gel-permeation chromatography (HPGPC) method to determine the proteins of human pancreatic juice, bile, and tissue homogenate has been developed. A diol-type silica gel column (35 x 8 mm I.D.

CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system.

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells.

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4.

A role for lipid rafts in immune cell signaling.

Cross-linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src-family tyrosine kinase, rafts can function as centers of signal transduction by forming patches.

Comprehensive studies on subcellular localizations and cell death-inducing activities of eight GFP-tagged apoptosis-related caspases.

By using green fluorescent protein fusion, we investigated the subcellular localization of all the caspases that have been cloned from humans and implicated in the execution of apoptosis. We divided these caspases into three groups according to subcellular localization. The first group includes caspase-1, -3, -6, -7, and -9, which are expressed mainly in the cytoplasm with various levels of nuclear localization depending on the cell type.

Fulminant hepatitis by Fas-ligand expression in MRL-lpr/lpr mice grafted with Fas-positive livers and wild-type mice with Fas-mutant livers.

Background: Fulminant hepatitis in mice could be induced by gene-transfection of Fas ligand (FasL). However, the mechanisms of this event still remain controversial as to whether it is mediated by direct Fas/FasL interaction and/or neutrophil migration. To investigate the role of exogenous FasL-expression, we established a simple but clear mouse model on which we performed liver transplantation between Fas-mutant mice (MRL-lpr/lpr) and wild-type mice (MRL+/+).

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation.

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation.

Engraftment of genetically engineered amniotic epithelial cells corrects lysosomal storage in multiple areas of the brain in mucopolysaccharidosis type VII mice.

Cell-mediated gene therapy for visceral lesions of lysosomal storage diseases is promising; however, the treatment of central nervous system (CNS) lesions remains a challenge. In this study, we generated rat amniotic epithelial cells (AEC) that overexpress and secrete human beta-glucuronidase (GUSB) following transduction with an adenoviral vector encoding human GUSB. The AEC were used as donor cells for cell-mediated gene therapy of CNS lesions in mice with mucopolysaccharidosis type VII (MPSVII), a lysosomal storage disorder caused by an inherited deficiency of GUSB activity.

Japanese familial patients with male-limited precocious puberty.

Familial male-limited precocious puberty (FMPP) is a rare disease caused by constitutively activating mutations in the luteinizing hormone receptor (LH-R) gene. In the present study, we analyzed the LH-R gene in members of a Japanese FMPP family. Two males of the family were affected and had a heterozygous M398T mutation; one patient developed pubertal signs as early as 2 years of age, and the other at 6 years of age.

[A model for clinical study based on genome science--trials in disorders of the immune system and allergies].

Disorders of the immune system, such as allergies, have multi-factorial etiologies that include both genetic and environmental components. The recent advances in genome science have facilitated two strategies for studying the genetic basis of disease: (1) systematic analysis of gene expression profiles and (2) comprehensive analysis of gene variations, such as polymorphisms. Here, we describe a unique research institute, Genox Research Inc.

Effect of high-frequency oscillatory ventilation on the upper airways of kittens.

Background: High-frequency oscillatory ventilation (HFOV) has become the preferred method of ventilation for the fragile lungs of neonates and infants because its beneficial effects on lungs are well known; however, its benefits on upper airways are not yet known. We investigated the effects of HFOV and conventional mechanical ventilation (CMV) on the airways of kittens with normal lungs.

Methods: Ten healthy cross-bred kittens, 2-3-months-old, with a mean bodyweight of 0.

Activation of the caspase cascade during Stx1-induced apoptosis in Burkitt's lymphoma cells.

Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1-mediated cytotoxicity, we observed that multiple caspases, including caspase-3, -7, and -8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases.

Selective down-regulation of high-affinity IgE receptor (FcepsilonRI) alpha-chain messenger RNA among transcriptome in cord blood-derived versus adult peripheral blood-derived cultured human mast cells.

Substantial numbers of human mast cells (MCs) were generated from umbilical cord blood (CB) and from adult peripheral blood (PB). A single CB progenitor produced 15 436 MCs, whereas a single PB progenitor produced 807 MCs on average. However, PB-derived MCs were far more active than CB-derived MCs in terms of high-affinity IgE receptor (FcepsilonRI)-mediated reactions.

Breakage and fusion of the TEL (ETV6) gene in immature B lymphocytes induced by apoptogenic signals.

TEL-AML1 fusion resulting from the t(12;21)(p13;q22) is one of the most common genetic abnormalities in childhood acute lymphoblastic leukemia. Recent findings that site-specific cleavage of the MLL gene can be induced by chemotherapeutic agents such as topoisomerase-II inhibitors suggest that apoptogenic agents can cause chromosomal translocations in hematopoietic cells. This study demonstrates a possible relationship between exposure to apoptogenic stimuli, TEL breaks, and the formation of TEL-AML1 fusion in immature B lymphocytes.

Characterization of monoclonal antibodies against mouse and rat platelet glycoprotein V (CD42d).

The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes.

Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of a bioartificial liver.

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes.

Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer.

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human beta-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells.

Strong, long-term transgene expression in rat liver using chicken beta-actin promoter associated with cytomegalovirus immediate-early enhancer (CAG promoter).

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken beta-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucuronosyltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively.

Tumour rejection by gene transfer of 4-1BB ligand into a CD80(+) murine squamous cell carcinoma and the requirements of co-stimulatory molecules on tumour and host cells.

NRS1 is a murine squamous cell carcinoma that constitutively expresses the co-stimulatory molecule CD80 at a high level yet grows as a tumour in syngeneic C3H mice. We examined the effect of gene transfer of the 4-1BB ligand (4-1BBL) into NRS1 cells. Introduction of the 4-1BBL gene efficiently elicited anti-tumour immune responses in syngeneic mice which acquired specific immunity against wild-type tumour.