Activation of ADP-ribosylation factors (ARFs), ϳ20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ϳ200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ϳ50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ϳ209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFAinhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ϳ74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ϳ202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His 6 fusion protein in Sf9 cells, accelerated guanosine 5-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ϳ50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ϳ200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFAsensitive ARF function in Golgi transport.

Mast cells play a critical role in the pathogenesis of allergic asthma. Histamine is a central mediator released from mast cells through allergic reactions. Histamine plays a role in airway obstruction via smooth muscle contraction, bronchial secretion, and airway mucosal edema. However, previous clinical trials of H1 receptor antagonists (H1RAs) as a treatment for asthma were not successful. In recent years, type 2 innate immunity has been demonstrated to be involved in allergic airway inflammation. Allergic asthma is defined by IgE antibody-mediated mast cell degranulation, while group 2 innate lymphoid cells (ILC2) induce eosinophilic inflammation in nonallergic asthma without allergen-specific IgE. Anti-IgE therapy has demonstrated prominent efficacy in the treatment of severe allergic asthmatics sensitized with specific perennial allergens. Furthermore, recent trials of specific cytokine antagonists indicated that these antagonists were effective in only some subtypes of asthma. Accordingly, H1RAs may show significant clinical efficacy for some subtypes of allergic asthma in which histamine is deeply associated with the pathophysiology.

Activation of ADP-ribosylation factors (ARFs), ϳ20-kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. A novel ARF GEP, designated cytohesin-4, was cloned from a human brain cDNA library. Deduced amino acid sequence of the 47-kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an ϳ200-amino acid Sec7 domain with an ϳ100-residue pleckstrin homology domain near the C terminus. The Sec7 domain sequence is 77% identical to those of other cytohesins. Structures of the cytohesin-4 and cytohesin-1 genes were remarkably similar, except for an extra 3-base pair (GAG) exon present in cytohesin-1. Two mRNAs with and without the 3-base pair sequence were found in brain in different ratios for cytohesin-1, -2, and -3 but not cytohesin-4. Recombinant cytohesin-4 stimulated guanosine 5-3-O-(thio)triphosphate binding by human ARF1 and ARF5 but not ARF6. Like other cytohesins and unlike the ϳ200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of ϳ3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, ϳ90% of CD33 ؉ (monocytes), 80% of CD2 ؉ (NK/T), and 10 -20% of CD19 ؉ (B) cells contained cytohesin-4 mRNA by in situ hybridization. Thus, in gene structure and brefeldin A-insensitive GEP activity, cytohesin-4 resembles other cytohesins, but its tissue distribution differs considerably, consistent with a different specific function. ADP-ribosylation factors (ARFs),1 initially identified as activators of cholera toxin-catalyzed ADP-ribosylation, are ϳ20-kDa guanine nucleotide-binding proteins involved in vesicular trafficking pathways, including endoplasmic reticulum and intra-Golgi transport (1, 2) and endocytosis (3). Effects of ARF on vesicular trafficking may result from its interaction with coatomer (4) and/or activation of phospholipase D (5-7). Mammalian ARFs are grouped into three classes (class I, ARF1, -2, and -3; class II, ARF4 and -5; and class III, ARF6) based on amino acid sequence, size, gene structure, and phylogenetic analysis (8). ARFs are GTPases that function as molecular switches by alternation between inactive GDP-bound and active GTP-bound states. Inactivation of ARF-GTP requires interaction with a GTPase-activating protein that accelerates hydrolysis of bound GTP (9 -12). Activation of ARF-GDP depends on the replacement of bound GDP by GTP, which is catalyzed by guanine nucleotide-exchange proteins or GEPs (13). Liu and Pohajdak (14) cloned a human cDNA named B2-1 using subtractive hybridization (natural killer cells minus Thelper Jurkat cells); the gene was localized to chromosome 17-qter (15). The B2-1 gene product cytohesin-1 is a 47-kDa protein found at high levels in natural killer and cytotoxic T-cells and at very low levels in monocytes and several cultured cell lines (16). Part of its deduced amino acid sequence resembles that of the so-called Sec7 domain of the Saccharomyces cerevisiae Sec...

Objective: To describe the relation between body composition and age measured by dual-energy X-ray absorptiometry (DXA) in healthy Japanese adults. Design: Cross-sectional study. Subjects and measurements: The subjects were 2411 healthy Japanese adults (males 625, females 1786, age 20 ± 79 y) who attended the Fukuoka Health Promotion Center, Fukuoka, Japan for health check-up. Body composition was determined by DXA (QDR-2000, Hologic) for the whole body and three anatomical regions of arms, legs and trunk. Results:The mean values of body mass index (BMI) and percentage fat mass (%FM) were 23.2 AE 3.1 (s.d.) kgam 2 and 21.8 AE 6.8% for males and 22.1 AE 3.3 kgam 2 and 32.0 AE 7.5% for females, respectively. For males, curvilinear relations with the peaks in their forties or ®fties were seen for the variables associated adiposity, ie BMI, waist and hip circumference, waist ± hip ratio, total or regional fat mass (FM), %FM and ratio of trunk FM to leg FM. For females, most of these variables increased linearly in older subjects. Lean mass (LM), bone mineral content (BMC) and bone mineral density (BMD) of the whole body and appendicular LM were relatively constant until the forties and then decreased in both sexes. The rates of decrease in the total or appendicular LM were larger for males than for females, whereas those in BMC or BMD were larger for females than for males. Conclusions: This study presents the ®rst detailed data on body composition in Japanese, which may be useful when comparing with populations of different racial and ethnic backgrounds and studying ill subjects.

Abstract. Histamine is inactivated by the histamine-metabolizing enzyme histamine N-methyltransferase (HNMT) in bronchus, kidney, and the central nervous system. HNMT seems to be localized in the cytoplasm, but histamine is unable to easily enter the intracellular space. Therefore, two hypotheses can be elicited: one is the plasma membrane hypothesis that HNMT can be translocated to the plasma membrane and function at the cell surface under growth factor stimulation and the other is the transporter hypothesis that organic cation transporter (OCT)-2 and -3 can function as a histamine transporter as well. To investigate the involvement of OCT2, HEK293 cells stably double transfected with C-terminal hemagglutinin (HA)-tagged HNMT cDNA and/ or C-terminal myc-tagged rat OCT2 were prepared for analysis of HNMT activity associated with OCT2 function. After 60-min incubation of these cells with PBS including HA (100 µM), N τ -methylhistamine (MHA) concentration of the supernatants was determined by the HPLC-fluorometry method. MHA from cells with HNMT plus OCT-2 was produced at about 3-fold higher level than that from cells with HNMT alone, suggesting that OCT-2 could function as a histamine transporter as well and that HNMT function could partly depend on OCT-2 transporter activity. Using OCT-3 knockout (OCT-3 −/ − ) mice, histamine content and survival rates were investigated in lipopolysaccharide (LPS)-induced endotoxemia model. Without LPS stimulation, histamine content was compared between OCT-3 −/ − and wild mice. Histamine content in the spleen of OCT-3 −/ − mice was higher than that of wild mice. With LPS stimulation, the survival rate of OCT-3 −/ − mice was significantly decreased 12 h after LPS (20 mg / kg) stimulation, suggesting that before immunological stimulation, a higher content of histamine in spleen could stimulate histamine receptors in mast cells, macrophages, dendritic cells, as well as T lymphocytes and explaining the decreased survival rate in OCT-3 −/ − mice possibly due to the functional changes of immunological cells.

Genetic defects in the methylmalonyl-CoA mutase (MCM) gene result in methylmalonic acidemia which is inherited as an autosomal recessive disease. We investigated fibroblast cultures obtained from two Japanese patients with MCM deficiency. MCM mRNA was not detected by Northern blot analysis, suggesting that MCM mRNA was markedly decreased. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of MCM mRNA in these fibroblasts was less than 1% of normal controls. This minute amount of MCM mRNA was successfully amplified by nested RT-PCR and subjected to primary structure analysis. Sequence analysis revealed two novel mutations: a G-to-T substitution at nucleotide position 425 and a 2 bp deletion at nucleotide positions 769 and 770. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The second mutation (769 delta CA) resulted in a frame shift which created a premature termination codon 508 amino acid upstream of the C-terminus of the protein. Patient 1 was homozygous for G425T and patient 2 was a compound heterozygote for G425T and 769 delta CA. Our report is the first to identify MCM mutations that affect the stability of MCM mRNA. An analysis of 16 Japanese patients revealed the presence of G425T in six patients, suggesting a relatively high incidence of the mutation among Japanese patients. This is in sharp contrast to a previous report describing diverse heterogeneity of MCM mutations among Caucasians.

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