Formation of Threonine Aldolase by Bacteria and Yeasts

Threonine aldolase was found to be formed in various strains of bacteria and yeasts when they were grown in media containing l-threonine as a sole source of carbon. As the other sources of carbon, d, l-allothreonine, l-serine and glycine were effective but glucose and sucrose were inert for the formation of the enzyme.

The maximal formation of the enzyme was observed in the initial of stationary phase of growth and, thereafter, the enzyme disappeared with the consumption of l-threonine. It seems that the enzyme is adaptive in nature and that it is responsible for the growth in threonine as the carbon source.     

Adipose tissue-organotypic culture system as a promising model for studying adipose tissue biology and regeneration

Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed “adipose tissue-organotypic culture system” that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. Our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro.Key words: adipose tissue-organotypic culture, three-dimensional, tissue fragments, peripheral zone, central zone, mature adipocytes, preadipocytes (immature adipocytes), mesenchymal stem cells, adipokines, tissue regeneration     

A single amino acid in the PSPG-box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site-directed mutagenesis was carried out. Exchange of the PSPG-box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG-box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site-directed mutagenesis revealed that the size of the amino acid side-chain in that particular site is critical to the catalytic activity of CaUGT2.     

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.     

Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF

We recently reported that mast cells stimulated via FcepsilonRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3(+) T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.     

Floral fragrances in two closely related fruit fly orchids,Bulbophyllum hortorum and B. macranthoides (Orchidaceae): assortments of phenylbutanoids to attract tephritid fruit fly males

Floral chemical components are important cues used by plants to attract pollinators. One outstanding case is “fruit fly orchids” in the genus of Bulbophyllum to attract their pollinators by releasing characteristic fragrances. Dacini fruit flies are main or exclusive pollinators which are strongly attracted to certain natural chemicals, either methyl eugenol (ME: a phenylpropanoid) or raspberry ketone (RK: a phenylbutanoid). Furthermore, zingerone (ZN: a phenylbutanoid) has been characterized as the attractant for both ME- and RK-sensitive fruit fly species. In the present study, we examined chemical profiles of two closely related Bulbophyllum orchids—B. hortorum, and B. macranthoides subsp. tollenoniferum—distributed in Papua New Guinea and the Southeast Asian countries, respectively. We first observed that RK-sensitive flies were attracted to these orchids by ex situ cultivation in Penang, Malaysia. These Bulbophyllum orchids contained RK and/or ZN as their main floral components. Other than these attractants, multiple phenylbutanoids including potential attractants for RK-sensitive species were identified from these orchids. Therefore, we examined attractiveness of potential phenylbutanoid attractants to an RK-sensitive melon fly, Zeugodacus cucurbitae, using laboratory-reared flies. Furthermore, we analyzed molecular phylogenetic relationships among phenylpropanoid- or phenylbutanoid-producing orchids to see a relation between chemical profiles and phylogenetic classification in the related species.     

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.