The effect of hen-egg antibodies on Clostridium perfringens colonization in the gastrointestinal tract of broiler chickens.

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n=10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n=15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.

The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis.

Flowering plants display a remarkable range of inflorescence architecture, and pedicel characteristics are one of the key contributors to this diversity. However, very little is known about the genes or the pathways that regulate pedicel development. The brevipedicellus (bp) mutant of Arabidopsis thaliana displays a unique phenotype with defects in pedicel development causing downward-pointing flowers and a compact inflorescence architecture. Cloning and molecular analysis of two independent mutant alleles revealed that BP encodes the homeodomain protein KNAT1, a member of the KNOX family. bp-1 is a null allele with deletion of the entire locus, whereas bp-2 has a point mutation that is predicted to result in a truncated protein. In both bp alleles, the pedicels and internodes were compact because of fewer cell divisions; in addition, defects in epidermal and cortical cell differentiation and elongation were found in the affected regions. The downward-pointing pedicels were produced by an asymmetric effect of the bp mutation on the abaxial vs. adaxial sides. Cell differentiation, elongation, and growth were affected more severely on the abaxial than adaxial side, causing the change in the pedicel growth angle. In addition, bp plants displayed defects in cell differentiation and radial growth of the style. Our results show that BP plays a key regulatory role in defining important aspects of the growth and cell differentiation of the inflorescence stem, pedicel, and style in Arabidopsis.

Genetic transformation of Trametes versicolor to phleomycin resistance with the dominant selectable marker shble.

We have developed a stable, DNA-mediated transformation system for the white-rot basidiomycete Trametes versicolor based on the dominant selectable marker shble (phleomycin resistance). We employed a vector containing the selectable marker under control of expression sequences from the basidiomycete Schizophyllum commune and a polyethylene glycol/ CaCl2 protoplast-fusion technique to introduce the transforming DNA. This transformation system generated stable phleomycin-resistant transformants at a frequency of four to seven transformants/microg of transforming DNA.

Isogloboside biosynthesis in metastatic R3230AC cells results from a decreased GM3 synthase activity.

We have determined that the production of a metastasis-associated neutral glycosphingolipid, isogloboside (iGb(4)Cer, GalNAcbeta1-3Galalpha1-3Galbeta1-4Glcbeta1-O-ceramide) is associated with the loss of G(M3) synthase activity. Assays for neutral glycosphingolipid-forming glycosyltransferases in cells producing various levels of iGb(4)Cer revealed no consistent differences that could account for the difference in iGb(4)Cer biosynthesis. However, comparison of the activity of G(M3) synthase in homogenates of these two cell types revealed that cells that did not synthesize iGb(4)Cer had activity significantly greater than that of cells possessing this antigen. Furthermore, somatic cell hybrids generated using clones of the iGb(4)Cer -producing and nonproducing cell lines lacked iGb(4)Cer while possessing high levels of G(M3) synthase activity. When iGb(4)Cer-producing cells were transfected with a G(M3) synthase expression vector, all of the resultant clones were negative for iGb(4)Cer production. The results of these studies clearly show that the presence of G(M3) synthase prevents the formation of iGb(4)Cer in these cells.

Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase.

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus (Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.

Cloning and sequencing of a gene encoding cellobiose dehydrogenase from Trametes versicolor.

Cellobiose dehydrogenase (CDH) is an enzyme produced under lignocellulose-degrading conditions by Trametes versicolor strain 52J (Tv) and several other wood-degrading fungi, including Phanerochaete chrysosporium (Pc). In order to understand better the nature and properties of this enzyme, we isolated a genomic clone of Tv cdh using heterologous probes derived from the sequence of Pc cdh. DNA sequence analysis revealed that Tv cdh consists of 3091 bp of coding sequence interrupted by 14 introns. Southern blotting showed that the gene was present in a single copy in the strain of Tv analyzed. Tv cdh was shown by Northern blot analysis to be expressed as a single transcript under cellulolytic conditions. RT-PCR of poly(A)+ RNA isolated under cellulolytic conditions was used to generate a near full-length cDNA copy of the cdh mRNA. The deduced protein encoded by Tv cdh consists of 768 amino acids (aa), including a predicted 19 aa signal peptide. The protein had 73% identity to the corresponding protein from Pc, which is the only other CDH-encoding gene that has been cloned. Based upon its deduced primary structure and alignment to similar sequences, Tv CDH shares a general structural organization with Pc CDH and other hemoflavoenzymes. Amino acid residues H-109 and M-61 in the N-terminal heme domain are hypothesized to function in heme binding; the C-terminal flavin domain contained a consensus sequence for flavin binding between residues 217-222. Although the protein is known to bind to cellulose, no obvious homology to bacterial or fungal cellulose binding domains was observed. However, a strong homology was observed to a region of Pc CDH that is hypothesized to be involved in cellulose binding.

Purification and Characterization of Cellobiose Dehydrogenases from the White Rot Fungus Trametes versicolor.

The white rot fungus Trametes versicolor degrades lignocellulosic material at least in part by oxidizing the lignin via a number of secreted oxidative and peroxidative enzymes. An extracellular reductive enzyme, cellobiose dehydrogenase (CDH), oxidizes cellobiose and reduces insoluble Mn(IV)O(inf2), commonly found as dark deposits in decaying wood, to form Mn(III), a powerful lignin-oxidizing agent. CDH also reduces ortho-quinones and produces sugar acids which can promote manganese peroxidase and therefore ligninolytic activity. To better understand the role of CDH in lignin degradation, proteins exhibiting cellobiose-dependent quinone-reducing activity were isolated and purified from cultures of T. versicolor. Two distinct proteins were isolated; the proteins had apparent molecular weights of 97,000 and 81,000 and isoelectric points of 4.2 and 6.4, respectively. The larger CDH (CDH 4.2) contained both flavin and heme cofactors, whereas the smaller contained only a flavin (CDH 6.4). These CDH enzymes were rapidly reduced by cellobiose and lactose and somewhat more slowly by cellulose and certain cello-oligosaccharides. Both glycoproteins were able to reduce a very wide range of quinones and organic radical species but differed in their ability to reduce metal ion complexes. Temperature and pH optima for CDH 4.2 were affected by the reduced substrate. Although CDH 4.2 showed rather high substrate specificity among the ortho-quinones, it could also rapidly reduce a structurally very diverse collection of other species, from negatively charged triiodide ions to positively charged hexaquo ferric ions. CDH 6.4 showed a higher K(infm) and a lower V(infmax) and turnover number than did CDH 4.2 for all substrates tested. Furthermore, CDH 6.4 did not reduce the transition metals Fe(III), Cu(II), and Mn(III) at concentrations likely to be physiologically relevant, while CDH 4.2 was able to rapidly reduce even very low concentrations of these ions. The reduction of Fe(III) and Cu(II) by CDH 4.2 may be important in sustaining a Fenton's-type reaction, which produces hydroxyl radicals that can cleave both lignin and cellulose. Unlike the CDH proteins from Phanerochaete chrysosporium, CDH 4.2 and CDH 6.4 are unable to produce hydrogen peroxide.

Production and Characterization of Trametes versicolor Mutants Unable To Bleach Hardwood Kraft Pulp.

Protoplasts of the monokaryotic strain 52J of Trametes versicolor were treated with UV light and screened for the inability to produce a colored precipitate on guaiacol-containing agar plates. Mutants unable to oxidize guaiacol had absent or very low secretion of laccase and manganese peroxidase (MnP) proteins. All isolates unable to secrete MnP were also unable to bleach or delignify kraft pulp. One mutant strain, M49, which grew normally but did not oxidize guaiacol, was tested further with a number of other substrates whose degradation has been associated with delignification by white rot fungi. Compared with the parent, 52J, mutant M49, secreting no MnP and low laccase, could not brighten or delignify kraft pulp, produced less ethylene from 2-keto methiolbutyric acid, released much less (sup14)CO(inf2) from [(sup14)C]DHP (a synthetic lignin-like polymerizate), and produced much less methanol from pulp. This mutant also displayed decreased abilities to oxidize the dyes poly B-411, poly R-478, and phenol red compared with the wild-type strain and was also unable to decolorize kraft bleachery effluent or mineralize its organochlorine. Addition of purified MnP in conjunction with H(inf2)O(inf2), MnSO(inf4), and an Mn(III) chelator to M49 cultures partially restored methanol production, pulp delignification, and biobleaching in some cases.