Mapping cyclosporine-induced changes in protein secretion by renal cells using stable isotope labeling with amino acids in cell culture (SILAC)

Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies.     

AIR: A batch-oriented web program package for construction of supermatrices ready for phylogenomic analyses

Background  

Large multigene sequence alignments have over recent years been increasingly employed for phylogenomic reconstruction of the eukaryote tree of life. Such supermatrices of sequence data are preferred over single gene alignments as they contain vastly more information about ancient sequence characteristics, and are thus more suitable for resolving deeply diverging relationships. However, as alignments are expanded, increasingly numbers of sites with misleading phylogenetic information are also added. Therefore, a major goal in phylogenomic analyses is to maximize the ratio of information to noise; this can be achieved by the reduction of fast evolving sites.     

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.     

Immunomodulatory effects of probiotics in the intestinal tract

The intestinal microbiota is the largest source of microbial stimulation that exerts both harmful and beneficial effects on human health. The interaction between probiotic and enterocytes is the initiating event in immunomodulation and merits particular attention. The effects of probiotic is strain dependent and for each new probiotic strain, profiles of cytokines secreted by lymphocytes, enterocytes or dendritic cells that come in contact with the strain should be systematically established. To evaluate the effects of probiotics on the immune system, models that mimic the mucosa, and thus the physiological reality, should be preferred whenever it is possible. Then, the in vitro observed effects should be backed up by properly conducted randomized double bind clinical studies. More detailed studies are needed to determine the precise action mode of probiotics on both mucosal and systemic immunity.     

Invasion status of Florida bass Micropterus floridanus (Lesueur, 1822) in South Africa

Largemouth bass Micropterus salmoides are a popular North American angling species that was introduced into South Africa in 1928. To enhance the largemouth bass fisheries, Florida bass Micropterus floridanus were introduced into KwaZulu Natal, South Africa, in 1980. Knowledge on the status of M. floridanus in South Africa is required, because it lives longer and reaches larger sizes than M. salmoides, which may result in heightened impacts on native biota. Because M. floridanus are morphologically similar, but genetically distinct from M. salmoides, the distribution of this species was assessed by genetically screening 185 Micropterus sp. individuals sampled from 20 localities across South Africa using the mitochondrial ND2 gene. Individuals with mitochondrial DNA matching M. salmoides were recovered from 16 localities, whereas M. floridanus mitochondrial DNA was recovered from 13 localities. At nine localities (45%), the mitochondrial DNA of both species was detected. These results demonstrate M. floridanus dispersal to multiple sites across South Africa.     

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.