Characterization of AFLP Markers Associated with Growth in the Kuruma Prawn, <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>, and Identification of a Candidate Gene

Growth rate of the Kuruma prawn, Marsupenaeus japonicus is an important economic trait, with larger animals commanding higher market prices. To identify gene markers associated with growth, a genetic map of a full-sib F₂ intercross family of M. japonicus has previously been generated and quantitative trait loci (QTL) influencing weight, total length, and carapace length were identified. In this study, amplified fragment length polymorphism (AFLP) markers associated with the major QTL region, contributing 16% to phenotypic variation, were characterized. Flanking sequence has been obtained and allelic variants responsible for segregation patterns of these markers have been identified. The genomic sequence surrounding the AFLP band 7.21a, residing under the QTL peak, contains a gene sequence homologous to the elongation of very long chain fatty acids-like (ELOVL) protein family. A full-length mRNA (ELOVL-MJ) encoding this protein was isolated from M. japonicus, representing both the first ELOVL gene in crustacea and the first candidate gene identified via QTL studies in crustacea.     

The cytosolic protein talin induces an intermediate affinity integrin alphaLbeta2

The integrin alphaLbeta2 mediates leukocyte adhesion and migration that are required for a functional immune system. It is known that inside-out signaling triggers alphaLbeta2 conformational changes, which affect its ligand-binding affinity. At least three alphaLbeta2 affinity states (low, intermediate, and high) were described. The cytosolic protein talin connects alphaLbeta2 to the actin filament. The talin head domain is also known to activate alphaLbeta2 ligand binding. However, it remains to be determined whether talin promotes an intermediate or high affinity alphaLbeta2. In this study using transfectants and T cells, we showed that talin induced an intermediate affinity alphaLbeta2 that adhered constitutively to its ligand intercellular adhesion molecule (ICAM)-1 but not ICAM-3. Adhesion to ICAM-3 was induced when an additional exogenous activating agent was included. Similar profiles were observed with soluble ICAMs. In addition, the intermediate affinity alphaLbeta2 induced by talin allowed adhesion and migration of T cells on immobilized ICAMs.     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

Characterization and identification of Sox2+ radial glia cells derived from rat embryonic cerebral cortex

During the central nervous system (CNS) development, radial glia cells (RGCs) play at least two essential roles, they contribute to neuronal production and the subsequent guidance of neuronal migration, whereas its precise distribution and contribution to cerebral cortex remains less understood. In this research, we used Vimentin as an astroglial marker and Sox2 as a neural progenitor marker to identify and investigate RGCs in rat cerebral cortex at embryonic day (E) 16.5. We found that the Sox2+ progenitor cells localized in the germinal zone (GZ) of E16.5 cerebral cortex, ~95% Sox2+ cells co-localized with Vimentin+ or Nestin+ radial processes which extended to the pial surface across the cortical plate (CP). In vitro, we obtained RG-like cells from E16.5 cerebral cortex on adherent conditions, these Sox2+ Radial glia (RG)-like cells shared some properties with RGCs in vivo, and these Sox2+ RG-like cells could differentiate into astrocytes, oligodendrocytes and presented the radial glia—neuron lineage differentiation ability. Taken together, we identified and investigated some characterizations and properties of Sox2+ RGCs derived from E16.5 cerebral cortex, we suggested that the embryonic Sox2+ progenitor cells which located in the cortical GZ were mainly composed of Sox2+ RGCs, and the cortex-derived Sox2+ RG-like cells displayed the radial glia—neuron lineage differentiation ability as neuronal progenitors in vitro.     

Towards a global database of weed risk assessments: a test of transferability for the tropics

Worldwide spread and establishment of alien plant species continues to accelerate and damage ecological and agricultural systems. Early warning and prevention of high-risk introductions is the most cost-effective approach to minimise losses while maximising benefits, and the Australian Weed Risk Assessment (A-WRA) system has been the most well-developed and successful predictive scheme. However, any system would be limited if the results or scores were confined to the locality of assessment. We compiled A-WRA scores conducted in four tropical to sub-tropical regions and tested the accuracy of these scores for predicting naturalisations for a separate well-documented, equatorial, exotic flora where weed risk assessments have never been conducted. Receiver Operating Characteristic (ROC) curves reflect high accuracies of predictions, comparable to those in other studies. No significant differences in accuracy were found between each regional subset and the compiled set of scores. Our results show that A-WRA scores assessed at one locality can be used for others of similar climate, increasing the utility of every species’ assessment. A global database of A-WRA scores would enable rapid local decision-making in border controls on imported plant species. A growing record of species assessments would also facilitate monitoring evolutionary and ecological aspects of invasive species.     

Leucine nutrition in animals and humans: mTOR signaling and beyond

Macronutrients, such as protein or amino acid, not only supply calories but some components may also play as signaling molecules to affect feeding behavior, energy balance, and fuel efficiency. Leucine, a branched-chain amino acid is a good example. After structural roles are satisfied, the ability of leucine to function as signal and oxidative substrate is based on a sufficient intracellular concentration. Therefore, leucine level must be sufficiently high to play the signaling and metabolic roles. Leucine is not only a substrate for protein synthesis of skeletal muscle, but also plays more roles beyond that. Leucine activates signaling factor of mammalian target of rapamycin (mTOR) to promote protein synthesis in skeletal muscle and in adipose tissue. It is also a major regulator of the mTOR sensitive response of food intake to high protein diet. Meanwhile, leucine regulates blood glucose level by promoting gluconeogenesis and aids in the retention of lean mass in a hypocaloric state. It is beneficial to animal nutrition and clinical application and extrapolation to humans.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.