Phylogeny and biogeography of the freshwater crayfish Euastacus (Decapoda: Parastacidae) based on nuclear and mitochondrial DNA

Euastacus crayfish are endemic to freshwater ecosystems of the eastern coast of Australia. While recent evolutionary studies have focused on a few of these species, here we provide a comprehensive phylogenetic estimate of relationships among the species within the genus. We sequenced three mitochondrial gene regions (COI, 16S, and 12S) and one nuclear region (28S) from 40 species of the genus Euastacus, as well as one undescribed species. Using these data, we estimated the phylogenetic relationships within the genus using maximum-likelihood, parsimony, and Bayesian Markov Chain Monte Carlo analyses. Using Bayes factors to test different model hypotheses, we found that the best phylogeny supports monophyletic groupings of all but two recognized species and suggests a widespread ancestor that diverged by vicariance. We also show that Euastacus and Astacopsis are most likely monophyletic sister genera. We use the resulting phylogeny as a framework to test biogeographic hypotheses relating to the diversification of the genus.     

N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.     

The levels of nicotinamide nucleotides in liver microsomes and their possible significance to the function of hexose phosphate dehydrogenase.

The concentrations of NAD and NADP have been determined in detergent extracts of washed rat liver microsomes. Precautions were taken during the preparation of the microsomes to remove nicotinamide nucleotides from their external surface both by hydrolysis by nucleotide pyrophosphatase (EC 3.6.1.9) and by washing them three times in 0.15 M-Tris/HCl, pH 8.0, to remove soluble proteins which bind these nucleotides. The mannose phosphatase was essentially completely latent, indicating that the microsomes were intact. Assuming these nucleotides are in the cisternae of the microsomes, the concentrations in the cisternae are 240 +/- 25 microM-NAD and 55 +/- 12 microM-NADP. These levels of nucleotides are compatible with both the glucose:NAD+ and the glucose 6-phosphate:NADP+ oxidoreductase activities of hexose phosphate dehydrogenase (EC 1.1.1.47). Since the organ and subcellular distributions of this dehydrogenase and glucose-6-phosphatase are similar, and Pi stimulates the glucose:NAD+ oxidoreductase activity, it is proposed that the combined action of these two enzymes leads to the reduction of both coenzymes in the lumen of the endoplasmic reticulum. A modification of the colorimetric method of Nisselbaum & Green [(1969) Anal. Biochem. 27, 212-217] for the determination of NADP+ is described. Colour formation is linear with the concentration of NADP+ and is sensitive to less than 0.3 nmol of NADP+.     

Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation.

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several alpha(v)beta(3) integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of alpha(v)beta(3) integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of alpha(v)beta(3) integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the alpha(v)beta(3) integrin.     

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires

Background

Plasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine.

Methods

Isolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp -1 block 2, and msp -2 block 3, to analyze both parasite population polymorphism and clone fluctuations.

Results

Twenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence.

Conclusion

P. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity.     

The crystal structure of the heparin-binding reelin-N domain of f-spondin

The extracellular matrix protein F-spondin mediates axon guidance during neuronal development. Its N-terminal domain, termed the reelin-N domain, is conserved in F-spondins, reelins, and other extracellular matrix proteins. In this study, a recombinant human reelin-N domain has been expressed, purified, and shown to bind heparin. The crystal structure of the reelin-N domain resolved to 2.0 Å reveals a variant immunoglobulin-like fold and potential heparin-binding sites. Substantial conformational variations even in secondary structure are observed between the two chemically identical reelin-N domains in one crystallographic asymmetric unit. The variations may result from extensive, highly specific interactions across the interface of the two reelin-N domains. The calculated values of buried surface area and the interface's shape complementarity are consistent with the formation of a weak dimer. The homophilic asymmetric dimer can potentially offer advantages in binding to ligands such as glycosaminoglycans, which may, in turn, bridge the two reelin-N domains and stabilize the dimer.     

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro.

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.     

C-mannosylation and O-fucosylation of the thrombospondin type 1 module

Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 repeats (TSRs), modules of approximately 60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an alpha-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is well described in epidermal growth factor-like modules. Both C- and O-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.