Determination of junction avidity of cytolytic T cell and target cell

A direct measurement of the avidity of the junction between a cytotoxic T lymphocyte and its target cell was achieved by using a biophysical approach. A micromanipulation technique was used to determine the force required to separate a cytotoxic T cell (human clone F1, with specificity for HLA-DRw6) from its specific target cell (JY: HLA-A2, -B7, -DR4, w6) prior to delivery of the lethal hit. The force required to separate the F1-JY pair is 1.5 X 10(4) dynes per square centimeter. This junction avidity for F1-JY pairs is 6 to 13 times greater than that for F1-F1 and JY-JY pairs; the F1-JY conjugate requires a stronger separating force and is more easily rejoined than the homologous cell pairs. This study provides an estimate of the avidity of cytotoxic T cells for their target cells and insights into the biophysical correlates of the molecular complexes formed in the interaction of cytotoxic T cells and their targets during the cytotoxic process.     

Efficacy of probiotics from anaerobic microflora with prebiotics on growth performance and noxious gas emission in growing pigs

We investigated the effect of probiotics from anaerobic microflora with prebiotics (synbiotics) on growth performance, noxious gas emission and fecal pathogenic bacteria populations in growing pigs. The basal diet, which contained approximately 25% corn, 24% whey, 12% wheat and 12% soybean meal, was supplemented alternatively with 0.15% antibiotics (US diet), prebiotics and 0.2% probiotics from anaerobic bacteria (BS diet), yeast (YS diet), mold (MS diet) or compounds (CS diet). One hundred and fifty pigs were fed an experimental diet for 15 days. Although the growth performance was not affected by supplemental synbiotics, the BS group showed higher dry matter and crude protein digestibility. The BS group decreased fecal ammonia and amine gas emissions, and increased fecal acetate gas emission compared with the US group. All synbiotics groups decreased in fecal propionate gas emission. Fecal Escherichia coli population was lower in the synbiotics groups than in the US group. Therefore, the results of the present study suggest that synbiotics exert similar effects with antibiotics on the nutrient digestibility and fecal microflora composition in growing pigs. Moreover, synbiotics can also decrease the fecal noxious gas emission in growing pigs.     

Occurrence of Honeysuckle Yellow Vein Virus (HYVV) containing a monopartite DNA-A genome in Korea

Two newly emerged begomoviruses were isolated from naturally infected tomato (Solanum lycopersicum) plants grown in greenhouses at Jeju Island and Dangjin in Korea and their genomes were characterized. These viruses-infected plants had very small leaves that curled upward, yellow margins and a leathery appearance, and a bushy and stunted appearance with short internodes. Nucleotide (nt) sequence analysis of their genomes showed that they have a DNA-A component of a monopartite begomovirus. Their genomes comprised 2763 and 2764 nucleotides with six open reading frames. The results of nt sequence similarity analysis of DNA-A genome between the two Korean isolates and isolates of Tobacco leaf curl Japan virus (TbLCJV), Honeysuckle yellow vein virus (HYVV), Honeysuckle yellow vein mosaic virus (HYVMV), and Eupatorium yellow vein virus in Japan (EpYVV) showed that they are likely similar to HYVV-[Masuda] (89.4–92.8% nt identity). Consequently, we tentatively propose the two isolates’ names as HYVV-Jeju and -DJ according to the ICTV geminivirus rules. Phylogenetic relationship analysis of 33 DNA-A genome sequences using PAUP* 4.0b10 and MrBayes revealed that HYVV-Jeju and -DJ belong to the Far East Asian begomovirus species complex. Within the Far East Asian begomovirus species complex, HYVV-Jeju and -DJ are distantly related to EpYVV, HYVMV, and TbLCJV groups. Based on the presence of a recombination fragment spanning the C3 ORF, a recombinant origin was suggested for both HYVV-Jeju and –DJ, with parents close to Japanese isolates HYVMV-[SP1:00] and Eupatorium yellow vein virus (EpYVV)-[Suya]. In addition, the presence of a further recombination fragment spanning the IR suggested the parents of HYVV-DJ were close to HYVV-Jeju and EpYVV-[Suya].     

Differences in polymer formation through disulfide bonding of recombinant light meromyosin between white croaker and walleye pollack and their possible relation to species specific differences in thermal unfolding

Fast skeletal light meromyosins (LMMs) of white croaker and walleye pollack were prepared in our expression system using Escherichia coli and determined for their polymer-forming ability and thermodynamic properties by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and differential scanning calorimetry (DSC), respectively. White croaker LMM formed dimer by heating at 80 degrees C and showed only a single peak at 32.1 degrees C of temperature transition in DSC. On the other hand, walleye pollack LMM hardly formed polymer and showed four peaks at 27.7, 30.5, 35.8, and 43.9 degrees C. When Cys525 of white croaker LMM was replaced by alanine, this point-mutated LMM showed no change in its DSC profile but formed no dimer upon heating, suggesting a possible role of Cys525 in dimer formation. On the other hand, walleye pollack LMM where Cys491 was substituted by alanine changed its DSC profile, showing four peaks at 27.9, 29.1, 38.4, and 43.9 degrees C. However, this point-mutated LMM formed no dimer upon heating as in the case of native LMM. These results suggest that cysteine residue(s) participates in thermal gel formation of LMM when it locates in a suitable position of the sequence.     

Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.     

Purification and characterization of cysteine proteinase inhibitors from crucian carp <Emphasis Type="Italic">Carassius auratus</Emphasis> eggs

Two cystatins (cst-I and cst-II) were purified from crucian carp eggs by acidification and subsequent ion exchange and molecular sieve chromatography. The molecular masses of cst-I and cst-II analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were 11.9 and 14.4 kDa, respectively, under reducing conditions and 13.5 and 12.7 kDa, respectively, under non-reducing conditions. The cst-I and cst-II molecules were stable after 30 min of incubation at 60 and 50°C, respectively. There was no significant loss in the inhibitory activity of either cst in the pH range 4–11. These two cystatins were able to affect the proteolysis of papain, cathepsin L, and bromelain, but they were unable to inhibit cathepsin B and trypsin. The partial N-terminal amino acid sequences of both cst inhibitors were homologous and that of cst-I was recognized as NH₂-AGIPGGLVDADINDADVQ. This latter fragment shared 88.9% identity to common carp cystatin and 44.4–55.6% to cystatins of other aquatic animals. Based on these results, we conclude that the two cst inhibitors are members of family II cystatin.     

Novel Caryophyllane-Related Sesquiterpenoids with Anti-Inflammatory Activity from Rumphella antipathes (Linnaeus, 1758)

Two previously undescribed caryophyllane-related sesquiterpenoids, antipacids A (1) and B (2), with a novel bicyclo[5.2.0] core skeleton, and known compound clovane-2β,9α-diol (3), along with rumphellolide L (4), an esterified product of 1 and 3, were isolated from the organic extract of octocoral Rumphella antipathes. Their structures, including the absolute configurations were elucidated by spectroscopic and chemical experiments. In vivo anti-inflammatory activity analysis indicated that antipacid B (2) inhibited the generation of superoxide anions and the release of elastase by human neutrophils, with IC₅₀ values of 11.22 and 23.53 μM, respectively, while rumphellolide L (4) suppressed the release of elastase with an IC₅₀ value of 7.63 μM.     

Suppression of pine wilt disease by an antibacterial agent,oxolinic acid

BACKGROUND: Pine wilt disease (PWD) is very complex and has been reported to be caused by pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and its accompanying bacteria. However, there is no report on the control of PWD by antibacterial agent. The present study was performed to investigate disease control efficacy of antibacterial agents against PWD. RESULTS: Among six antibacterial antibiotics tested, oxolinic acid (OA) showed the strongest antibacterial activity against five bacteria isolated from three strains of pine wood nematode. In in vivo assay, it effectively suppressed the development of PWD in three‐year‐old seedlings of Pinus densiflora Sieb. & Zucc.; it showed 71% control when injected at 3 mg per seedling. A mixture of OA and the nematicidal agent abamectin (Ab) showed higher disease control efficacy against PWD than either OA or Ab alone. In addition, OA alone and a mixture of OA and Ab also controlled PWD in approximately 20‐year‐old pine trees under field conditions. CONCLUSION: This is the first report on the suppression of PWD by OA. The result strongly indicates that PWD could be controlled by antibacterial antibiotic alone and a combination of antibacterial and nematicidal agents. Copyright © 2010 Society of Chemical Industry     

Exploring genetic diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development of SNP marker for Bph18

Brown planthopper (BPH) is the most devastating insect pest in rice‐growing areas. Information on availability of BPH resistance alleles and their sources enhances BPH‐resistant breeding programmes. In this study, 260 highly diversified rice cultivars or breeding lines were screened for the presence of five major BPH resistance genes (Bph10, Bph13, Bph18, Bph20 and Bph21) using gene‐specific markers. The analysis revealed that 137 of the 260 cultivars possess at least one BPH resistance gene. Bph10 was predominant while Bph20 was the least distributed. Moreover, two and three different resistance gene combinations were found in the cultivars. Molecular markers play an important role in molecular breeding programmes. A tightly linked PCR‐based co‐dominant Bph18 marker was developed, which is cost effective and time effective and simpler than available Bph18 CAPS marker (7312.T4A). We strongly believe that the identified BPH‐resistant cultivars can be used as alternative resistance gene sources and also as resource for novel BPH resistance genes. The developed Bph18 marker will be highly useful in molecular breeding applications of BPH‐resistant breeding programmes.