CERT mediates intermembrane transfer of various molecular species of ceramides

Ceramide produced at the endoplasmic reticulum is transported to the Golgi apparatus for conversion to sphingomyelin. The main pathway of endoplasmic reticulum-to-Golgi transport of ceramide is mediated by CERT, a cytosolic 68-kDa protein, in a nonvesicular manner. CERT contains a domain that catalyzes the intermembrane transfer of natural C(16)-ceramide. In this study, we examined the ligand specificity of CERT in detail by using a cell-free assay system for intermembrane transfer of lipids. CERT did not mediate the transfer of sphingosine or sphingomyelin at all. The activity of CERT to transfer saturated and unsaturated diacylglycerols, which structurally resemble ceramide, was 5-10% of the activity toward C(16)-ceramide. Among four stereoisomers of C(16)-ceramide, CERT specifically recognized the natural d-erythro isomer. CERT efficiently transferred ceramides having C(14), C(16), C(18), and C(20) chains, but not longer acyl chains, and also mediated efficient transfer of C(16)-dihydroceramide and C(16)-phyto-ceramide. Binding assays showed that CERT also recognizes short chain fluorescent analogs of ceramide with a stoichiometry of 1:1. Moreover, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide, which inhibited the CERT-dependent pathway of ceramide trafficking in intact cells, was found to be an antagonist of the CERT protein. These results indicate that CERT can mediate transfer of various types of ceramides that naturally exist and their close relatives.     

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.     

Genetic mapping reveals a dominant awn-inhibiting gene related to differentiation of the variety anathera in the wild diploid wheat <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Aegilops tauschii, a wild wheat relative, is the D-genome donor of common wheat. Subspecies and varieties of Ae. tauschii are traditionally classified based on differences in their inflorescence architecture. However, the genetic information for their diversification has been quite limited in the wild wheat relatives. The variety anathera has no awn on the lemma, but the genetic basis for this diagnostic character is unknown. Wide variations in awn length traits at the top and middle spikes were found in the Ae. tauschii core collection, and the awn length at the middle spike was significantly smaller in the eastward-dispersed sublineage than in those in other sublineages. To clarify loci controlling the awnless phenotype of var. anathera, we measured awn length of an intervariety F₂ mapping population, and found that the F₂ individuals could be divided into two groups mainly based on the awn length at the middle of spike, namely short and long awn groups, significantly fitting a 3:1 segregation ratio, which indicated that a single locus controls the awnless phenotype. The awnless locus, Anathera (Antr), was assigned to the distal region of the short arm of chromosome 5D. Quantitative trait locus analysis using the awn length data of each F₂ individual showed that only one major locus was at the same chromosomal position as Antr. These results suggest that a single dominant allele determines the awnless diagnostic character in the variety anathera. The Antr dominant allele is a novel gene inhibiting awn elongation in wheat and its relatives.     

CD8+ suppressor T cell clone capable of inhibiting the antigen- and anti-T cell receptor-induced proliferation of Th clones without cytolytic activity

A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.     

Enhanced nodulation of soybean by Bradyrhizobium in the presence ofPseudomonas fluorescens

Experiments were undertaken to determine the effect ofPseudomonas fluorescens on nodulation of soybean by two strains ofBradyrhizobium japonicum, USDA I-110 and 61A76.Pseudomonas fluorescens can enhance the nodulation ability ofB. japonicum. Preincubation ofB. japonicum withP. fluorescens before inoculation further increased the level of nodulation.     

pH-dependent Ca2+ interaction with phospholipids related to phosphatidylethanolamine N-methylation

The interactions of PE and its N-methylated derivatives (PME, PDE AND PC) WITH Ca2+ were examined. PE and the intermediate phospholipids of PE N-methylation (PME and PDE) interacted with Ca2+ in a pH-dependent and reversible manner. When these phospholipids were present in the heptane phase, Ca2+ in the aqueous phase was translocated into the heptane phase at alkaline pH but not at acidic pH. PDE was also effective for the translocation even at around neutral pH, while PC hardly translocated Ca2+ at pH 6.0-9.2. The amounts of Ca2+ interacting with these phospholipids were in the following order: PDE is greater than PME is greater than PE is much greater than PC. P1, phosphatidic acid and PS interacted with Ca2+ in the whole pH range examined. The Ca2+ interactions with P1 and phosphatidic acid were independent of pH, while PS interacted with more Ca2+ at alkaline pH. These phospholipids interacted with Ca2+ most strongly among the cations studied. Liposomes containing PDE also bound the highest amounts Ca2+ among PE and its N-methylated derivatives. Furthermore, mammalian cultured cell membranes, which contain increased amounts of PDE by in vivo modification with N,N'-dimethylethanolamine, bound more Ca2+ than those prepared from choline-treated control cells.     

Synthesis of acyl phosphatidylglycerol from phosphatidylglycerol in Escherichia coli K-12. Evidence for the participation of detergent-resistant phospholipase A and heat-labile membrane-bound factor(s).

The conversion of phosphatidylglycerol to acyl phosphatidylglycerol by extracts of Escherichia coli K-12 strains was examined under various conditions. The maximum rate of conversion was observed at pH 7.2 in the presence of 50% (v/v) diethyl ether and 10 mM CaCl2. This conversion was found to involve two sequential reactions: (1) The formation of 2-acyl glycerophosphoglycerol and 2-acyl glycerophosphoethanolamine from phosphatidylglycerol and phosphatidylethanolamine, respectively, by detergent-resistant phospholipase A in the presence of Ca2+ and (2) transfer of the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol by a heat-labile factor(s) in the presence of diethyl ether. Neither fatty, acid acyl-CoA nor 1-acyl lysophospholipid could act as an acyl donor for phosphatidylglycerol. The heat-labile factor(s) was found in both the inner membrane and supernatant fractions.