Central and peripheral cardiovascular actions of apelin in conscious rats

APJ was cloned as an orphan G protein-coupled receptor and shares a close identity with angiotensin II type 1 receptor (AT1R). Apelin is a peptide that has recently been identified as an endogenous ligand of the APJ. Apelin and APJ mRNA are expressed in peripheral tissue and the central nervous system. However, little is known about the effects of apelin in cardiovascular regulation. To examine the central and peripheral role of apelin, we injected the active fragment of apelin [(Pyr1)apelin-13] intracerebroventricularly (ICV, 5 and 20 nmol, n=6) or intravenously (IV, 20 and 50 nmol, n=4 or 5) in conscious rats. ICV injection of (Pyr1)apelin-13 dose-dependently increased mean arterial pressure (MAP) and heart rate (HR) (19+/-3 mm Hg and 162+/-26 bpm at 20 nmol). Pretreatment with ICV injection of the AT1R antagonist (CV-11974, 20 nmol) did not alter the apelin-induced increase in MAP and HR. IV injection of (Pyr1)apelin-13 also dose-dependently increased MAP and HR (13+/-2 mm Hg and 103+/-18 bpm at 50 nmol); however, the peripheral effects of apelin were relatively weak compared to its central effects. Expression of c-fos in the paraventricular nucleus (PVN) of hypothalamus was increased in the rat that received ICV injection of (Pyr1)apelin-13 but not in the rat that received IV injection of (Pyr1)apelin-13. These results suggest that apelin plays a role in both central and peripheral cardiovascular regulation in conscious rats, and that the cardiovascular effects of apelin are not mediated by the AT1R.     

Differential gene expression profiling between wild-type and ALAS2-null erythroblasts: identification of novel heme-regulated genes

To identify erythroid-specific heme-regulated genes, we performed differential expression analysis between wild-type and heme-deficient erythroblasts, which had been prepared from wild-type and erythroid-specific delta-aminolevulinate synthase-null mouse ES cells, respectively. Among 8737 clones on cDNA array, 40 cDNA clones, including 34 unknown ESTs, were first selected by their high expression profiles in wild-type erythroblasts, and evaluated further for their erythroid-lineage specificity, expression in hematopoietic tissues in vivo, and heme-dependent expression, which yielded 11, 4, and 4 genes, respectively. Because of the selection strategy employed, the final 4 were considered as the newly identified erythroid-specific heme-regulated genes. These 4 genes were uncoupling protein 2, nucleolar spindle-associated protein, cellular nucleic acid-binding protein, and a novel acetyltransferase-like protein. These findings thus suggest that heme may regulate a wide variety of hitherto unrecognized genes, and further analysis of these genes may clarify their role in erythroid cell differentiation.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non-allelic temperature-sensitive virescent mutants of rice. Temperature-shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast-encoded genes but do not affect the subsequent morphological development of the leaf or the photo-regulation of the expression of nuclear genes encoding chloroplast proteins.     

Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families

Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan.     

Tissue-Protective Effects of NKG2A in Immune-Mediated Clearance of Virus Infection

Virus infection triggers a CD8⁺ T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8⁺ T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1^b, which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8⁺ T cells. We have previously shown that Qa1^b-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8⁺ T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8⁺ effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8⁺ T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8⁺ T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8⁺ NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during virus clearance.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.