Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase,that needs product of upstream gene,sldB, for activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

Heat-inducible transgenic expression in the silkmoth Bombyx mori

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.     

Use of a biomimetic chromatographic stationary phase for study of the interactions occurring between inorganic anions and phosphatidylcholine membranes

A liquid chromatographic method for the study of ion-membrane interactions is reported. A phosphatidylcholine biomimetic stationary phase was established by loading dimyristoylphosphatidylcholine (DMPC) onto a reversed-phase octadecylsilica packed column. This column was then used to study the interaction of some inorganic anions with the stationary phase by UV and conductivity detection. Ten inorganic anions were selected as model ions and were analyzed with the proposed chromatographic system. Anion-DMPC interactions of differing magnitudes were observed for all of the model anions. Perchlorate-DMPC interactions were strongest, followed by thiocyanate-DMPC, iodide-DMPC, chlorate-DMPC, nitrate-DMPC, bromide-DMPC, chloride-DMPC, fluoride-DMPC, and then sulfate-DMPC. Cations in the eluent, especially H(+) ions and divalent cations such as Ca(2+), showed strong effects on anion-DMPC interactions. The chromatographic data suggest that DMPC interacts with both the anions and the cations. Anion-DMPC interactions were dependent on the surface potential of the stationary phase: at low surface potentials anion-DMPC interactions were predominantly solvation dependent in nature whereas at more positive surface potentials anion-DMPC interactions were predominantly electrostatic in nature. Cation-DMPC interactions served to raise the surface potential, causing the anion-DMPC interactions to vary from solvation dependent to electrostatic. The chromatographic data were used to provide quantitative estimates of the enthalpies of the anion-DMPC interactions.     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

(+)-Strigol,a witchweed seed germination stimulant,from Menispermum dauricum root culture

(+)-Strigol was isolated from Menispermum dauricum root culture filtrate. Its identity was confirmed by HPLC, 1H NMR, UV and MS, and on the basis of its CD spectrum. This is the first report on isolation of strigolactone from aseptic plant culture.     

Damage to the gills,skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts,Tanichthys albonubes and Oreochromis mossambicus

Lysenin is a 33-kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 microg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM-liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine.     

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s⁻¹), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s⁻¹), and high tolerance to product inhibition (K_i for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.     

Development of galactomannan-hydrolyzing activity in the micropylar endosperm tip of tomato seed prior to germination

Development of galactomannan-hydrolyzing activity, that is involved in the weakening of the mechanical restraint of the endosperm, was followed at pre-germinative stages in tomato ( Lycopersicon esculentum ) seed. Prior to germination the activity developed exclusively in the endosperm portion just adjacent to the radicle tip. In other parts of the endosperm, the activity developed only after germination occurred. Under the conditions where germination was suppressed (far-red light- or ABA-treatment). no activity was detected in the endosperm at the pre-germinative stages. Under the conditions where the inhibition of germination was alleviated (far-red + red or ABA + GA₃), the activity developed prior to germination in the endosperm part in front of the radicle tip. Thus, a clear parallel relationship was observed between germinability of the seed and the pre-germinative development of activity in the part of the endosperm portion adjacent to the radicle tip.