Macrophage colony-stimulating factor is produced by human T lymphoblastoid cell line, CEM-ON: identification by amino-terminal amino acid sequence analysis

A subclone, designated CEM-ON, derived from an azaguanine-resistant human leukemic T cell line, CEM-AG(R), constitutively secretes a colony-stimulating factor (CSF) which stimulates the production of macrophages from murine bone marrow progenitor cells. This CSF has been purified from serum-free conditioned medium. Highly purified CEM-ON CSF with a specific activity of 4.7 X 10(7) units/mg protein was obtained. Amino-terminal sequence analysis showed that the first 27 amino acids were identical to the amino-terminal sequence of the M-CSF (CSF-1) based on the cDNAs for human M-CSF. On SDS-PAGE analysis, CEM-ON CSF had an apparent molecular weight of 33,000-43,000; following reduction with 2-mercaptoethanol, this migrated as a 20,000-24,000 subunit, suggesting a homodimer structure. These results show that a human T cell line, CEM-ON, secretes M-CSF into its medium.     

Mouse coq7/clk-1 orthologue rescued slowed rhythmic behavior and extended life span of clk-1 longevity mutant in Caenorhabditis elegans

The coq7/clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a regulatory role in biological rhythm and longevity. The mouse COQ7 is homologous to Saccharomyces cerevisiae COQ7/CAT5 that is required for the biosynthesis of coenzyme Q (ubiquinone), an essential messenger in mitochondrial respiration. In the present study, we characterized the expression and processing of mouse COQ7. We found that COQ7 is highly expressed in tissues with high energy demand such as heart, muscle, liver, and kidney in mice. Biochemical analysis revealed that COQ7 is targeted to mitochondria where it is processed to mature form. Transgenic expression of mouse coq7 completely rescued the slowed rhythmic behaviors of clk-1 such as defecation. In life-span analysis, transgenic expression reverted the extended life span of clk-1 to the comparable level with wild-type control. These data strongly suggested that coq7 plays a pivotal role in the regulation of biological rhythms and the determination of life span in mammalian species.     

Nuclear localization of yeast Nfs1p is required for cell survival

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Detection of Oxidant Producing-sites in Glutaraldehyde-fixed Human Neutrophils and Eosinophils Stimulated with Phorbol Myristate Acetate

The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2– generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10sec followed by O2– production. The maximal rate reached was 3.18±0.07nmol/min/1×106 cells (mean±S.D.; n=4) after 30sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2– without a lag period at a rate of 0.35±0.05nmol/min/1×106 cells (mean±S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20sec. The fact that the pre-fixed PMNs stimulated for 30sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.     

Regulation of natural killer cell-mediated swine endothelial cell lysis through genetic remodeling of a glycoantigen

The effect of remodeling of a glycoantigen such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for beta1,4-N-acetylglucosaminyltransferase III, alpha2, 3-sialyltransferase and alpha1,2-fucosyltransferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the alpha-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-alpha-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with alpha-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the alpha-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the alpha-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts.     

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.     

Ecto-ATPase activity in the rat cardiac muscle: biochemical characteristics and histocytochemical localization

We used a combined biochemical and histocytochemical approach to study ecto-ATPase in the rat cardiac muscle. The reaction medium employed for histocytochemical detection was optimized in biochemical assays to achieve the highest enzyme activity and lowest inhibition by the capture agent used for visualization of the reaction product. Approximately 70% of the enzyme activity was retained in samples after the fixation procedure. Divalent cations stimulated ecto-ATPase. High activity was detectable within a wide pH range. Histocytochemical reaction was observed at sites at which extracellular ATP can potentially exert its actions on the cardiac muscle: nerve endings, plasma membranes of cardiac myocytes and capillary endothelial cells, and T-tubules. Product of the reaction was found exclusively at the outer surface of the cells. In controls, enzyme activity was abolished by diethyl pyrocarbonate and slightly stimulated by digitonin and concanavalin A, whereas sodium orthovanadate, N-ethylmaleimide, and sodium azide yielded no effect. Our results support the view that cardiac ecto-ATPase is involved in important physiological functions and suggest that its activity may be regulated by the release of ATP from nerve endings.