Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom^−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom^−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5^−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5^−/− mice. In contrast to Apom^−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.     

Analysis of secondary structure predictions of dengue virus type 2 NS2B/NS3 against crystal structure to evaluate the predictive power of the in silico methods

Multiple sequence alignment was performed against eight proteases from the Flaviviridae family using ClustalW to illustrate conserved domains. Two sets of prediction approaches were applied and the results compared. Firstly, secondary structure prediction was performed using available structure prediction servers. The second approach made use of the information on the secondary structures extracted from structure prediction servers, threading techniques and DSSP database of some of the templates used in the threading techniques. Consensus on the one-dimensional secondary structure of Den2 protease was obtained from each approach and evaluated against data from the recently crystallised Den2 NS2B/NS3 obtained from the Protein Data Bank (PDB). Results indicated the second approach to show higher accuracy compared to the use of prediction servers only. Thus, it is plausible that this approach is applicable to the initial stage of structural studies of proteins with low amino acid sequence homology against other available proteins in the PDB.     

Protective effect of aminoguanidine against nephrotoxicity induced by cisplatin in normal rats

The effect of aminoguanidine (AG) on nephrotoxicity induced by cisplatin (CDDP) was investigated. A single dose of CDDP (7.5 mg/kg i.p.) induced nephrotoxicity, manifested biochemically by a significant elevation in serum urea, creatinine and a severe decrease in serum albumin. Moreover, marked increases in kidney weight, urine volume and urinary excretion of albumin were observed. Nephrotoxicity was further confirmed by a significant decrease in glutathione-S-transferase (GST, E.C. 2.5.1.18), glutathione peroxidase (GSH-Px, E.C. 1.11.1.9) and catalase (E.C. 1.11.1.6) and a significant increase in lipid peroxides measured as malondialdhyde (MDA) in kidney homogenates. Administration of AG (100 mg/kg per day p.o.) in drinking water 5 days before and 5 days after CDDP injection produced a significant protection against nephrotoxicity induced by CDDP. The amelioration of nephrotoxicity was evidenced by significant reductions in serum urea and creatinine concentrations. In addition, AG tended to normalize decreased levels of serum albumin. Urine volume, urinary excretions of albumin and GST and kidney weight were significantly decreased. Moreover, AG prevented the rise of MDA and the reduction of GST and GSH-Px activities in the kidney. These results suggest that AG has a protective effect on nephrotoxicity induced by CDDP and it may therefore improve the therapeutic index of CDDP.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.     

Phylogeny and infrageneric classification of Correa Andrews (Rutaceae) on the basis of nuclear and chloroplast DNA

This paper presents phylogenies of the small but ecologically and horticulturally important Australian genus Correa (Rutaceae). Consensus phylogenies generated using parsimony were congruent with their counterparts generated by Bayesian analysis, although usually less well resolved. The phylogeny generated from the second internal transcribed spacer region of the nuclear ribosomal DNA supported the monophyly of Correa and identified two well supported clades (one comprising C. lawrenceana and C. baeuerlenii and the other containing all other species of the genus). Phylogenetic reconstructions based on the combined trnL-trnF spacer and the trnK intron (including the matK gene) regions of chloroplast DNA also supported the monophyly of Correa and of the C. lawrenceana/C. baeuerlenii clade, but the topology among the other species differed markedly from that in the ITS-based phylogeny. The major clades identified in the chloroplast phylogenies seemed to follow geographic patterns rather than species boundaries, with different samples of C. glabra bearing chloroplast genotypes from different clades. These patterns are likely to be because of independent evolution of the chloroplast and nuclear genomes, and are typical of cases of introgressive hybridisation among species or incomplete lineage sorting of chloroplast genomes leading to incongruence between chloroplast and nuclear phylogenies. Thus, the phylogenies based on nuclear DNA should reflect species relations better than the chloroplast phylogeny in Correa, and we propose a new subgeneric classification of the genus on the basis of the ITS-based phylogeny and morphology. Correa subgenus Persistens Othman, Duretto and G.J. Jord., containing C. lawrenceana and C. baeuerlenii, is formally described.