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.     

A novel inhibitor of ceramide trafficking from the endoplasmic reticulum to the site of sphingomyelin synthesis

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3R)-HPA-12, which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3R)-HPA-12 did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3R)-HPA-12 inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3R)-HPA-12 inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.     

Role of PKC and TGF-beta receptor in glucose-induced proliferation of smooth muscle cells

The role of protein kinase C (PKC) and transforming growth factor (TGF)-beta in the proliferation of vascular smooth muscle cells (SMCs) under a high glucose condition was investigated. [3H]-thymidine incorporation under 20 mM glucose was significantly accelerated compared with that under 5.5 mM glucose, and this increase was inhibited by an anti-TGF-beta antibody or a PKC-beta specific inhibitor, LY333531. The amount of active and total TGF-beta1 in the conditioned media did not differ between 5.5 and 20 mM glucose. However, the expression of TGF-beta receptor type II under 20 mM glucose was significantly increased, but that of the TGF-beta receptor type I was not. This increased expression of the TGF-beta receptor type II was prevented by LY333531. These observations suggest that the increased expression of the TGF-beta receptor type II via PKC-beta plays an important role in the accelerated proliferation of SMCs under a high glucose condition, leading to the development of diabetic macroangiopathy.     

Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.     

Separate cis-acting DNA elements control cell type- and tissue-specific expression of collagen binding molecular chaperone HSP47

HSP47 is a collagen-binding heat shock protein and is assumed to act as a molecular chaperone in the biosynthesis and secretion of procollagen. As the synthesis of HSP47 is closely correlated with that of collagen in various cell lines and tissues, we performed a promoter/reporter assay using HSP47-producing and nonproducing cells. 280 base pairs (bp(s)) of upstream promoter were shown to be necessary for the basal expression but not to be enough for the cell type-specific expression. When the first and the second introns were introduced downstream of this 280-bp region, marked up-regulation of the reporter activity was observed in HSP47-producing cells but not in nonproducing cells. This was confirmed in transgenic mice by staining the lacZ gene product under the control of the 280-bp upstream promoter and the introns. Staining was observed in skin, chondrocytes, precursor of bone, and other HSP47/collagen-producing tissues. A putative Sp1-binding site at -210 bp in the promoter, to which Sp3 and an unidentified protein bind, was shown to be responsible for this up-regulation when combined with the introns. However no difference in the binding to this probe was observed between HSP47-producing and nonproducing cells. The responsible region for cell type-specific up-regulation was found to be located in a 500-bp segment in the first intron. On electrophoresis mobility shift assay using this 500-bp probe, specific DNA-protein complexes were only observed in HSP47-producing cell extracts. These results suggest that two separate elements are necessary for the cell type-specific expression of the hsp47 gene; one is a putative Sp1-binding site at -210 bp necessary for basal expression, and the other is a 500-bp region within the first intron, required for cell type-specific expression.     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

The Gln27Glu beta2-adrenergic receptor variant is associated with obesity due to subcutaneous fat accumulation in Japanese men

The Trp64Arg beta3-adrenergic receptor (AR) variant is associated with visceral obesity probably due to decreased lipolysis in visceral fat (H. Kim-Motoyama et al., Diabetologia 40, 469-472, 1997). Functional alteration of beta2AR may also change fat distribution. We investigated the influence of the Gln27Glu beta2AR variant upon obesity and fat distribution. We screened 278 unrelated Japanese men and detected 249 wild-type Gln27 homozygotes, 28 Gln27/Glu27 heterozygotes, and one mutant Glu27 homozygote. The frequency of mutant Glu27 allele was significantly higher in obese subjects than in nonobese/intermediate subjects (0.11 vs 0.04, P = 0. 004). The Gln27/Glu27 heterozygotes had a significantly higher mean age-adjusted body-mass index (BMI) and mean age-adjusted subcutaneous fat area assessed by CT scan than the wild-type homozygotes but not the mean age-adjusted visceral fat areas. In summary, we have found that in Japanese men the Gln27Glu beta2AR variant is associated with obesity due to subcutaneous fat accumulation.     

Chloroplast phylogeny indicates that bryophytes are monophyletic

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.