Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.     

Airborne hyperspectral imaging for estimating acorn yield based on the PLS B-matrix calibration technique

Alternate bearing of acorn is a well-marked yield variability phenomenon in forest production. In Japan, this phenomenon is also related to wildlife management (e.g. of animals such as wild pigs, that rely on acorn as their major feed source). Effective management of animals dependent on acorn will require accurate estimation of acorn yield at an early stage. In this paper, we proposed a way to estimate acorn yield from the canopy reflectance values of individual trees. Using an Airborne Imaging Spectrometer for Application (AISA) Eagle System, hyperspectral images in 72 visible and near-infrared wavelengths (407–898 nm) were acquired over an acorn forest in Japan 10 times over three consecutive years (2003–2005) during the early acorn growing season. The canopy spectral reflectance values for individual trees at each wavelength were extracted from the images, and important wavelengths were determined as estimating factors by the B-matrix technique based on partial least squares (PLS) analysis. Yield-estimating models were then developed by multiple linear regression (MLR). Three models obtained from images acquired on June 27 in 2003, July 13 in 2004 and June 21 in 2005 estimated acorn yield well in comparison with ground truth, indicating that the procedure has considerable potential. The study also demonstrated the B-matrix technique based on PLS analysis to be reliable and efficient in identifying important wavelengths for determining suitable estimating factors that best contribute to the estimation model.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

How and why do plant nuclei move in response to light?

We recently found that nuclei take different intracellular positions depending upon dark and light conditions in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in both epidermal and mesophyll cells are distributed baso-centrally within the cell (dark position). Under light conditions, in contrast, nuclei are distributed along the anticlinal walls (light position). Nuclear repositioning from the dark to light positions is induced specifically by blue light at >50 µmol m⁻² s⁻¹ in a reversible manner. Using analysis of mutant plants, it was demonstrated that the response is mediated by the blue-light photoreceptor phototropin2. Intriguingly, phototropin2 also seems to play an important role in the proper positioning of nuclei and chloroplasts under dark conditions. Light-dependent nuclear positioning is one of the organelle movements regulated by phototropin2. However, the mechanisms of organelle motility, physiological significance, and generality of the phenomenon are poorly understood. In this addendum, we discussed how and why nuclei move depending on light, together with future perspectives.Key words: actin, Arabidopsis, blue light, cytoskeleton, nuclear positioning, nucleus, phototropin     

A dual resistance gene system prevents infection by three distinct pathogens

Colletotrichum higginsianum causes typical anthracnose lesions on the leaves, petioles, and stems of cruciferous plants. Inoculation of Arabidopsis thaliana ecotype Columbia leaves with C. higginsianum results in fungal growth and disease symptoms reminiscent of those induced in other cruciferous plants. We performed map-based cloning and natural variation analysis of 19 A. thaliana ecotypes to identify a dominant resistance locus against C. higginsianum. We found that the A. thaliana RCH2 (for recognition of C. higginsianum) locus encodes two NB-LRR proteins, both of which are required for resistance to C. higginsianum in the A. thaliana ecotype Ws-0. Both proteins are well-characterized R proteins involved in resistance against bacterial pathogens; RRS1 (resistance to Ralstonia solanacearum 1) confers resistance to strain Rs1000 of R. solanacearum and RPS4 to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). Furthermore, we found that both RRS1-Ws and RPS4-Ws genes are required for resistance to Pst-avrRps4 and to Rs1002 R. solanacearum. We therefore demonstrate that a pair of neighboring genes, RRS1-Ws and RPS4-Ws, function cooperatively as a dual R-gene system against at least three distinct pathogens.Key words: R gene, RPS4, RRS1, Colletotrichum higginsianum, Pseudomonas syringae, Ralstonia solanacearumPlants are exposed to various types of potentially invasive organisms, including viruses, bacteria, fungi, nematodes and protozoa, but are able to defend themselves by activating multiple defense mechanisms. The gene-for-gene hypothesis¹ provides a mechanism for specific recognition of the pathogen by the plant. This recognition is mediated by direct or indirect interactions between the product of a plant resistance (R) gene and the corresponding effectors encoded by avirulence genes in the pathogen.² Most R-genes encode non-membrane proteins that contain a conserved nucleotide-binding (NB) site and a carboxy-terminal leucine-rich repeat (LRR) domain.The A. thaliana genome contains about 150 genes coding for NB-LRR-containing proteins.³ This is far less than the number of genes that would be required to respond individually and specifically to all of its potential pathogens. However, plants may have been able to limit the number of required NB-LRR-encoding genes if host proteins perceive sets of distinct pathogens.⁴Colletotrichum species cause devastating anthracnose diseases in a large number of agronomically important crops. These diseases can often be controlled by introduction of genetic resistance traits, but the molecular components of resistance remain unknown. Inoculation of A. thaliana ecotype Columbia (Col-0) leaves with Colletotrichum higginsianum results in fungal growth and disease symptoms reminiscent of those induced in other cruciferous plants.^5,6 Inoculation of a large number of ecotypes with isolates of C. higginsianum showed that A. thaliana has at least two dominant resistance gene loci, designated RCH1 and RCH2 (for recognition of C. higginsianum), indicating that A. thaliana resistance to C. higginsianum is controlled by a “gene-for-gene” interaction.⁵ In a previous study, we identified a single putative R locus, RCH1 on the top of chromosome 4, in the C. higginsianum-resistant A. thaliana ecotype Eil-0.⁵In the present study, the locus named RCH2 maps in an extensive cluster of disease-resistance loci known as MRC-J in the A. thaliana ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1–2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21), which has a 5-bp deletion in the neighboring gene, RPS4-Ws, a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). To assess if RRS1-Ws and RPS4-Ws function in concert, we generated an rps4-21/rrs1-1 double mutant by crossing rps4-21 and rrs1-1 mutants. The susceptibility levels of rps4-21/rrs1-1 double mutant to C. higginsianum were similar to that exhibited by the single mutants, suggesting that RRS1-Ws and RPS-4-Ws function cooperatively. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, these two adjacent R genes confer resistance, in tandem or individually, to three distinct pathogens with very different infection strategies and virulence mechanisms (Fig. 1).Open in a separate windowFigure 1RPS4 and RRS1 function as a dual resistance gene system that prevents infection by three distinct pathogens (Colletotrichum higginsianum, Ralstonia solanacearum and Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4).Several examples of two NB-LRR genes acting cooperatively to confer resistance against a pathogen have been reported. For example, A. thaliana RPP2A and RPP2B reside adjacently in the RPP2 locus.⁷ Blast resistance in Pikm-containing rice is conferred by a combination of two NB-LRR encoding genes, Pikm1-TS and Pikm2-TS.⁸ Pi5-mediated resistance against rice blast requires two NB-LRR-encoding genes.⁹ It is not known whether these NB-LRR genes function cooperatively or independently. Because of structural similarity with RRS1/RPS4 genes, it is possible that resistance to the pathogens is conferred by cooperation between the two NB-LRR genes.Several reports have shown that a single R gene/locus can confer resistance to multiple pathogens. For instance, tomato Mi mediates resistance against three distinct types of pests, including root-knot nematodes, potato aphids and sweet potato whitefly.¹⁰ In the present study, we suggest that two distinct R-genes located in a conserved head-to-head organization confer resistance to three distinct pathogen species by acting cooperatively.The tandem function of RRS1-Ws and its neighboring gene RPS4-Ws is also supported by the evolutionary conservation of the gene pair. Close homologs of RPS4 are often physically paired with homologs of RRS1 in a head-to-head (inverted) tandem arrangement.¹¹ The evolutionary conservation of homologous gene pairs in a head-to-head arrangement also supports the idea that cooperative function of two R genes could be a common mechanism of defense against pathogens. Since the two open reading frames are only 264 bp apart, the promoter regions of the gene pairs possibly overlap, leading to co-regulation of the genes. The head-to-head configuration may assure balanced levels of the protein pair to meet a strict stoichiometric requirement to act together, possibly in a complex. As a practical application, this finding may provide a new strategy for creating transgenic plants that express R genes from other plants. Introduction of two R genes in a head-to-head orientation may be necessary for effective pathogen resistance.     

Effect of compost temperature on oxygen uptake rate, specific growth rate and enzymatic activity of microorganisms in dairy cattle manure

Investigations were carried out to find out the relationship between temperature and microbial activity in dairy cattle manure composting using oxygen uptake rate, specific growth rate and enzymatic activities during autothermal and isothermal composting experiments. In autothermal composting, oxygen uptake rate and specific growth rate were found to be most intensive in order of 43 degrees C, 60 degrees C and 54 degrees C. Isothermal composting at 54 degrees C resulted highest levels of enzymatic activity and promoted the volatile solids reduction. Based on the maximum enzymatic activity, specific growth rate appeared to be more closely linked with microbial activity in compost than with oxygen uptake rate. The enhancement of specific growth rate, enzymatic activity and volatile solids reduction were induced at 54 degrees C in cattle manure composting.     

Highly potent fibrinolytic serine protease from Streptomyces

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.