Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii

The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before. In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.     

Intraperitoneal immunization led to T cell hyporesponsiveness to Helicobacter pylori infection in mice

During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori-challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori-challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized-challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL-2, IFN-gamma, and IL-4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL-2 and IFN-gamma. CTLA-4 has been documented to be a negative regulator of IL-2 production and lymphoproliferation that induces peripheral tolerance and functions 24-72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized-challenged mice showed higher levels of CTLA-4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori-associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA-4 following challenge.     

Whole chloroplast genome comparison of rice,maize, and wheat: implications for chloroplast gene diversification and phylogeny of cereals

The fully sequenced chloroplast genomes of maize (subfamily Panicoideae), rice (subfamily Bambusoideae), and wheat (subfamily Pooideae) provide the unique opportunity to investigate the evolution of chloroplast genes and genomes in the grass family (Poaceae) by whole-genome comparison. Analyses of nucleotide sequence variations in 106 cereal chloroplast genes with tobacco sequences as the outgroup suggested that (1) most of the genic regions of the chloroplast genomes of maize, rice, and wheat have evolved at similar rates; (2) RNA genes have highly conservative evolutionary rates relative to the other genes; (3) photosynthetic genes have been under strong purifying selection; (4) between the three cereals, 14 genes which account for about 28% of the genic region have evolved with heterogeneous nucleotide substitution rates; and (5) rice genes tend to have evolved more slowly than the others at loci where rate heterogeneity exists. Although the mechanism that underlies chloroplast gene diversification is complex, our analyses identified variation in nonsynonymous substitution rates as a genetic force that generates heterogeneity, which is evidence of selection in chloroplast gene diversification at the intrafamilial level. Phylogenetic trees constructed with the variable nucleotide sites of the chloroplast genes place maize basal to the rice-wheat clade, revealing a close relationship between the Bambusoideae and Pooideae.     

Evolutionary distance estimation under heterogeneous substitution pattern among lineages

Most of the sophisticated methods to estimate evolutionary divergence between DNA sequences assume that the two sequences have evolved with the same pattern of nucleotide substitution after their divergence from their most recent common ancestor (homogeneity assumption). If this assumption is violated, the evolutionary distance estimated will be biased, which may result in biased estimates of divergence times and substitution rates, and may lead to erroneous branching patterns in the inferred phylogenies. Here we present a simple modification for existing distance estimation methods to relax the assumption of the substitution pattern homogeneity among lineages when analyzing DNA and protein sequences. Results from computer simulations and empirical data analyses for human and mouse genes are presented to demonstrate that the proposed modification reduces the estimation bias considerably and that the modified method performs much better than the LogDet methods, which do not require the homogeneity assumption in estimating the number of substitutions per site. We also discuss the relationship of the substitution and mutation rate estimates when the substitution pattern is not the same in the lineages leading to the two sequences compared.     

Cutting edge: a novel role for Fas ligand in facilitating antigen acquisition by dendritic cells

Fas ligand (FasL)-expressing tumor cells are found to effectively mediate rejection of the coinoculated FasL negative parental cells while having no effect on the growth of histologically distinct tumor cells. These observations indicate that FasL induces a specific immune response against Ag derived from FasL-bearing tumors and suggest a possible role for FasL in tumor Ag presentation. Indeed, tumor cells expressing FasL can efficiently interact with dendritic cells (DCs) and this interaction requires the expression of membrane-bound FasL on tumors and Fas on DCs. Moreover, DCs cocultured with FasL-expressing tumors are able to elicit a tumor-specific immune response in vivo, suggesting that DCs acquire tumor Ag during the Fas/FasL-mediated DC-tumor contact. These results identify a novel role for FasL in augmenting tumor-DC interactions and subsequent tumor Ag acquisition by DCs, and suggest that FasL-expressing tumor cells could be used to generate tumor-specific DC vaccines.     

Characterization of two noncellulosomal subunits,ArfA and BgaA,from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation

Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.     

Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid

In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). Here we cloned a cDNA for A. belladonna SAMT (AbSAMT1), which consisted of 357 aa residues. It was expressed in E. coli, and the recombinant AbSAMT1 showed SAMT activity. When A. belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.     

Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna

Phytosulfokine (PSK)- is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK- on growth and tropanealkaloid production in Atropa belladonna hairy rootstransformed with Agrobacterium rhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK- for 4 weeks did not show any differences, the productivityof tropane alkaloids, especially of hyoscyamine, was enhanced by10^–7 or 10^–8 M PSK-. Inaddition, the content of tropane alkaloids in transformed roots treated withPSK- was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and tropane alkaloid production inAtropa belladonna transformed roots suggested thatPSK- influenced the growth of transformed roots during the activegrowingphase, but not tropane alkaloid production.