Optimized production of lignolytic manganese peroxidase in immobilized cultures of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Manganese peroxidase (MnP) is a key enzyme involved in the lignolysis of white-rot fungi. The purpose of this study is to investigate the effect of immobilization and culture conditions on MnP production in cultures of Phanerochaete chrysosporium grown on polyurethane foam. Higher concentrations of foam and lower levels of spore inoculums resulted in the formation of scattered mycelial pellets, increased autolysis of chlamydospore-like cells (a reservoir of MnP), and a higher activity of MnP. Even though MnP was a secondary metabolite, the addition of 5 times more glucose and diammonium tartrate, as carbon and nitrogen sources, resulted in a 4 fold increase in the dry cell mass. However, MnP activity decreased under these conditions to less than half, due to the formation of increasingly dense pellets and the inhibited lysis of chlamydospore-like cells.     

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.     

Comparative Analysis of Disease-Linked Single Nucleotide Polymorphic Markers from Brassica rapa for Their Applicability to Brassica oleracea

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KCTC 3014

Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected.     

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.     

A knee-specific finite element analysis of the human anterior cruciate ligament impingement against the femoral intercondylar notch

This work presents a finite element analysis of anterior cruciate ligament (ACL) impingement against the intercondylar notch during tibial external rotation and abduction, as a mechanism of noncontact ACL injuries. Experimentally, ACL impingement was measured in a cadaveric knee in terms of impingement contact pressure and six degrees-of-freedom tibiofemoral kinematics. Three-dimensional geometries of the ACL, femur and tibia were incorporated into the finite element model of the individual knee specimen. A fiber-reinforced model was adopted, which accounts for the anisotropy, large deformation, nonlinearity and incompressibility of the ACL. With boundary conditions specified based on the experimental tibiofemoral kinematics, the finite element analysis showed that impingement between the ligament and the lateral wall of intercondylar notch could occur when qthe knee at 45° was externally rotated at 29.1° and abducted at 10.0°. Strong contact pressure and tensile stress occurred at the impinging and nonimpinging sides of the ligament, respectively. The impingement force and contact area estimated from the model matched their counterparts from the corresponding cadaver experiment. The modeling and experimental approach provides a useful tool to characterize potential ACL impingement on a knee-specific basis, which may help elucidate the ACL injury mechanism and develop more effective treatments.     

Bacillus daqingensis sp. nov. isolated from near poultry farm soil

A novel bacterial strain, designated PFS-5^T, was isolated from the soil environment with feces of a live poultry farm located in Cheonan, Republic of Korea. Strain PFS-5^T was Gram-staining-positive, motile, strictly aerobic bacterium, rod-shaped, and endospore-forming. The strain contained meso-diaminopimelic acid in their peptidoglycan and MK-7 menaquinone. The major fatty acids were anteiso-C_15:0 (44.2%), C_16:0 (22.2%), and iso-C_15:0 (16.7%). The DNA G+C content was 40.1 mol%. Comparative 16S rRNA gene sequence analysis identified strain PFS-5^T in the genus Bacillus, exhibiting the highest level of sequence similarity with type strain of B. herbersteinensis D-1,5a^T (96.9%), B. humi LMG 22167^T (96.7%), B. alkalitelluris BA288^T (96.1%), B. litoralis SW-211^T (96.0%), and B. luteolus YIM93174^T (95.5%). The major polar lipids of PFS-5^T were diphosphatidylglycerol and phosphatidylglycerol. On the basis of result from poly-phasic data, strain PFS-5^T represents a novel species, for which the name Bacillus cheonanensis sp. nov. is proposed (Type strai PFS-5^T = KACC 17469^T = JCM19333^T).