Oomycetes and fungi: similar weaponry to attack plants

Fungi and Oomycetes are the two most important groups of eukaryotic plant pathogens. Fungi form a separate kingdom and are evolutionarily related to animals. Oomycetes are classified in the kingdom Protoctista and are related to heterokont, biflagellate, golden-brown algae. Fundamental differences in physiology, biochemistry and genetics between fungi and Oomycetes have been described previously. These differences are also reflected in the large variations observed in sensitivity to conventional fungicides. Recently, more pronounced differences have been revealed by genomics approaches. However, in this review we compare the mode of colonization of the two taxonomically distinct groups and show that their strategies have much in common.     

Differential expression of G protein alpha and beta subunit genes during development of Phytophthora infestans

A G protein alpha subunit gene (pigpa1) and a G protein beta subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of alpha,beta, and gamma subunits and participate in diverse signal transduction pathways. The deduced amino acid sequence of both pigpa1 and pigpb1, showed the typical conserved motifs present in Galpha or Gbeta proteins from other eukaryotes. Southern blot analysis revealed no additional copies of Galpha or Gbeta subunit genes in P. infestans, suggesting that pigpa1 and pigpb1 are single copy genes. By cross-hybridization homologues of gpa1 and gpb1 were detected in other Phythophthora species. Expression analyses revealed that both genes are differentially expressed during asexual development, with the highest mRNA levels in sporangia. In mycelium, no pigpa1 mRNA was detected. Western blot analysis using a polyclonal GPA1 antibody confirmed the differential expression of pigpa1. These expression patterns suggest a role for G-protein-mediated signaling during formation and germination of asexual spores of P. infestans, developmental stages representing the initial steps of the infection process.     

Amino acid sequences of the tryptic peptides from carboxymethylated L-asparaginase from Escherichia coli.

S-Carboxymethylated L-asparaginase was digested with trypsin and the resulting peptides were isolated by using gel filtration, ion exchange column chromatography and paper chromatography. Among the peptides thus isolated, 27 peptides were considered not to overlap and the sum of the amino acids from these 27 peptides is in good agreement with amino acid composition of the enzyme. The amino acid sequences of the peptides were determined by fragmentation with various enzymes and subtractive Edman degradation.     

Development of a monoclonal antibody against Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) that can detect EBNA-LP expressed in P3HR1 cells

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) fused to glutathione-S-transferase. The antibody detected endogenous and exogenous EBNA-LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA-LP. While another monoclonal antibody to EBNA-LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA-LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA-LP, especially investigations into the phenotypes of mutant EBNA-LP expressed in P3HR1 cells.     

Evidence for the association between two myosin heads in rigor acto-smooth muscle heavy meromyosin

The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and 60K were generated. These products were not obtained by EDC treatment of HMM in the absence of F-actin. The HMM fragments that participated in cross-linking were identified by fluorescent labeling and amino acid composition studies. The 115K peptide was determined to be a covalently cross-linked complex that formed between actin and the COOH-terminal 68K fragment of the HMM heavy chain. Our results are in agreement with a previous study which proposed that the site of cross-linking between HMM and F-actin resides within the COOH-terminal 22K fragment of the myosin subfragment 1 heavy chain [Marianne-Pépin, T., Mornet, D., Bertrand, R., Labbé, J.-P., & Kassab, R. (1985) Biochemistry 24, 3024-3029]. The 60K peptide, however, was not a product of cross-linking between HMM and F-actin. On the basis of its amino acid composition, we concluded that this 60K peptide was a cross-linked dimer of the NH2-terminal 24K fragments of the HMM heavy chain. The cross-linking of acto-gizzard HMM significantly increased the Mg-ATPase activity of gizzard HMM without any observable phosphorylation of the regulatory (20K) light chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lys-65 and Glu-168 are the residues for carbodiimide-catalyzed cross-linking between the two heads of rigor smooth muscle heavy meromyosin

We have previously demonstrated that the two heads of chicken gizzard heavy meromyosin (HMM) in a rigor complex with rabbit skeletal F-actin could be cross-linked by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Here, we report the location of the cross-linked sites in the amino acid sequence of the HMM heavy chain. One of the cross-linked residues was identified as Glu-168 by sequencing the CN1.CN6 cross-linked peptide containing residues 1-77 (CN1) and 164-203 (CN6). This site is located close to the ATP-binding site of HMM. Since the other site was further into the amino acid sequence of CN1, another cross-linked peptide corresponding to residues 53-66 and 145-182 was isolated from the 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-treated acto-tryptic gizzard HMM digested further by other proteolytic enzymes. The amino acid sequence of this peptide and its cyanogen bromide fragment indicated that the cross-linking occurred between Glu-168 and Lys-65. Our results suggests that these two amino acid side chains are in contact with each other in the acto-gizzard HMM rigor complex and participate in the electrostatic interaction between the two HMM heads bound to F-actin. Based on the head-to-head contact, we propose a three-dimensional model for the attachment of gizzard HMM heads to F-actin.     

Antibody neutralization of TGF-beta enhances the deterioration of collagen fascicles in a tissue-cultured tendon matrix with ex vivo fibroblast infiltration

A tissue-cultured tendon matrix infiltrated with cultured fibroblasts can be regarded as an ideal tissue-engineered tendon model. To clarify the role of TGF-beta in a tissue-cultured tendon matrix during ex vivo cellular infiltration, the present ex vivo study was conducted to test the following hypothesis that antibody neutralization of TGF-beta enhances weakening of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. In skeletally mature female rabbits, fibroblasts were isolated from the right patellar tendons using an explant culture technique, and the left patellar tendons underwent multiple freeze/thaw treatment with liquid nitrogen to obtain an acellular tendon matrix. Each acellular tendon was placed in a collagen gel containing cultured fibroblasts and then incubated with or without anti-TGF-beta1 antibody for 6 weeks. We found that antibody neutralization of TGF-beta enhanced the decrease in the tensile strength and tensile modulus of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. The present study indicates a possibility that TGF-beta may have a role in suppressing the material deterioration of the fascicles in the tendon during ex vivo cellular infiltration.