Immunological roles of Pasteurella multocida toxin (PMT) using a PMT mutant strain

The immunological role of the Pasteurella multocida toxin (PMT) in mice was examined using a PMT mutant strain. After a nasal inoculation, the mutant strain failed to induce interstitial pneumonia. Moreover, PMT had no significant effect on the populations of CD4+, CD8+, CD3+, and CD19+ immunocytes in blood or on the populations of CD4+ and CD8+ splenocytes (P<0.01). However, there was a significant increase in the total number of cells in the BAL samples obtained from the wild-type P. multocida-inoculated mice. On the other hand, the level of IL-1 expression decreased when the macrophages from the bronchio-alveolar lavage were stimulated with PMT. Overall, PMT appears to play some role (stimulating and/or inhibiting) in the immunological responses but further studies will be required to confirm this.     

Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3

Summary Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of ³⁵⁹serine and ⁸³⁵serine of 14-3-3β binding sites to alanine, which slightly reduces the interaction with 14-3-3β, does not influence the PTPN3 effect. In contrast, mutation of the invariant ⁸⁴²cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3β, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3β, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression. En-Chi Hsu, Yen-Cheng Lin have equal contributions to this work.     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

Association of the FADS2 gene with omega-6 and omega-3 PUFA Concentration in the egg yolk of Japanese quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low omega-6:omega-3 PUFA ratio (h2 = 0.36-0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the omega-6 and omega-3 PUFAs (P < 0.05). No effects of the other SNPs were found - indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to omega-6 and omega-3 PUFA concentration in the egg yolk.     

Combined mitochondrial and nuclear sequences support the monophyly of forcipulatacean sea stars

Previous molecular phylogenetic analyses of forcipulatacean sea stars (Echinodermata: Asteroidea) have reconstructed a non-monophyletic order Forcipulatida, provided that two or more forcipulate families are included. This result could mean that one or more assumptions of the reconstruction method was violated, or else the traditional classification could be erroneous. The present molecular phylogenetic analysis included 12 non-forcipulatacean and 39 forcipulatacean sea stars, with multiple representatives of all but one of the forcipulate families and/or subfamilies. Bayesian analysis of approximately 4.2kb of sequence data representing seven partitions (nuclear 18S rRNA and 28S rRNA, mitochondrial 12S rRNA, 16S rRNA, 5 tRNAs and cytochrome oxidase I with first and second codon positions analyzed separately from third codon positions) recovered a consensus tree with three well-supported clades (78%-100% bootstrap support) that corresponded at least approximately to traditional taxonomic ranks: the superorder Forcipulatacea (Forcipulatida + Brisingida) + Pteraster, the Brisingida/Brisingidae and Asteriidae + Rathbunaster + Pycnopodia. When a molecular clock was enforced, the partitioned Bayesian analysis recovered the traditional Forcipulatacea. Five of six genera represented by two or more species were monophyletic with 100% bootstrap support. Most of the traditional subfamilial and familial groupings within the Forcipulatida were either unresolved or non-monophyletic. The separate partitions differed considerably in estimates of model parameters, mainly between nuclear sequences (with high GC content, low rates of sequence substitution and high transition/transversion rate ratios) and mitochondrial sequences.     

Endemic freshwater finfish of Asia: distribution and conservation status

Freshwater finfish species richness and level of endemism in East, and South and South‐East Asia that included 17 nations were studied using available databases, and included nation‐wise distribution, habitat types, and conservation status. The number of endemic finfish species in the region was 559, belonging to 47 families. Families Cyprinidae and Balitoridae accounted for 43.5% and 16.2% of the total number of endemic species in the region, respectively, followed by Sisoridae (25), Gobiidae (20), Melanotaeniidae (19), and Bagridae (16), and the other 41 families had at least one endemic species. Nation‐wise the most number of endemic freshwater finfish species occur in India (191), followed by China (88), Indonesia (84), and Myanmar (60). In India, the endemic species accounted for 26.4% of the native freshwater fish fauna, followed by South Korea (16.9%), the Philippines, (16.3%) and Myanmar (15.7%). Statistically significant relationships discerned between the number of indigenous and endemic species richness to land area (X_la in 10³ km²) of the nations in the region were, Y_in = 218.961 Ln(X_la) – 843.1 (R² = 0.735; P < 0.001) and Y_e = 28.445 Ln X_la?134.47 (R² = 0.534; P < 0.01), respectively, and between indigenous and endemic species richness was Y_e = 0.079X_n? 1.558 (R² = 0.235; P < 0.05). The overall conservation status of endemic finfish in Asia was satisfactory in that only 92 species were in some state of vulnerability, of which 37 species (6.6%) are endangered or critically endangered. However, the bulk of these species (83.7%) were cave‐ and or lake‐dwelling fish. However, nation‐wise, the endemic freshwater finfish fauna of the Philippines and Sri Lanka, based on the imperilment index, were found to be in a highly vulnerable state. Among river basins, the Mekong Basin had the highest number of endemic species (31.3%). The discrepancies between databases are highlighted and the need to consolidate information among databases is discussed. It is suggested that the Mekong Basin be considered as a biodiversity hotspot, and appropriate management strategies be introduced in this regard.     

Minimizing the overlap problem in protein NMR: a computational framework for precision amino acid labeling

MOTIVATION: Recent advances in cell-free protein expression systems allow specific labeling of proteins with amino acids containing stable isotopes ((15)N, (13) C and (2)H), an important feature for protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. Given this labeling ability, we present a mathematical optimization framework for designing a set of protein isotopomers, or labeling schedules, to reduce the congestion in the NMR spectra. The labeling schedules, which are derived by the optimization of a cost function, are tailored to a specific protein and NMR experiment. RESULTS: For 2D (15)N-(1)H HSQC experiments, we can produce an exact solution using a dynamic programming algorithm in under 2 h on a standard desktop machine. Applying the method to a standard benchmark protein, calmodulin, we are able to reduce the number of overlaps in the 500 MHz HSQC spectrum from 10 to 1 using four samples with a true cost function, and 10 to 4 if the cost function is derived from statistical estimates. On a set of 448 curated proteins from the BMRB database, we are able to reduce the relative percent congestion by 84.9% in their HSQC spectra using only four samples. Our method can be applied in a high-throughput manner on a proteomic scale using the server we developed. On a 100-node cluster, optimal schedules can be computed for every protein coded for in the human genome in less than a month. AVAILABILITY: A server for creating labeling schedules for (15)N-(1)H HSQC experiments as well as results for each of the individual 448 proteins used in the test set is available at http://nmr.proteomics.ics.uci.edu.