Multivariate map representation of phylogenetic relationships: application to tilapiine fish

Distance matrices obtained from allozyme studies on tilapiine fish are analysed by a multivarite approach. A hierarchical clustering procedure facilitated comparison with tree representations. A map-like representation provided information additional to the tree representation. The current belief that Sarotherodon is closer to Oreochromis than to Tilapia is strengthened. But while it may be the link between these genera at the species level, it is not entirely distinct from Oreochromis at the molecular level. Further, Sarotherodon and Oreochromis species may have arisen from Tilapia in several speciation events. Some of the species interrelations agreed with inferences from morphological data, and disagreed with those from a consensus maximum parsimony (MP) tree. It is suggested that both Chromidotilapia guntheri and Tylochromis jentinki are ancestral to diVerent sub-groups of Tilapia , so that inferences from morphological studies and the consensus MP method are both partially correct. The graphical representation also suggests that the Nile tilapia strains in Asia may be derived from Egypt rather than from Ghana. It is advantageous to use the map-like and tree representations together for maximum visual informativeness and inference from the data.     

Characterization of non-planar peptide groups in protein crystal structures

Peptide groups are generally assumed to be planar in protein structure, due to 'rigid' partial double bond character of peptide bonds, thus the value of peptide torsion angle omega should be restricted to 180 degrees for the usual trans form of peptide unit. However, on analyzing the ultra-high resolution protein crystal database, we find that in some cases, omega deviates >10 degrees from its usual value of 180 degrees, indicating significant non-planarity of peptide groups. Moreover, the non-planarity for most of the amino acids is found to be 'biased' towards values of omega smaller than 180 degrees. Similar trend for to is confirmed by the neutron diffraction data for proteins. The neutron diffraction database also reveals that non-planar peptide groups are generally correlated to 'pyramidal' structure of the peptide-nitrogen bonds. Consequently, the hydrogen atom of peptide group deviates from its planar position, as measured by the 'improper' torsion angle theta. Thus, we find that both the angles omega and theta point towards a significant amount of non-planarity of peptide groups, which cannot be ignored. The role of peptide nonplanarity in protein function is, however, not yet clear.     

Isolation, characterization, and chondrogenic potential of human bone marrow-derived multipotential stromal cells

Multipotential bone marrow stromal cells have the ability to differentiate along multiple connective tissue lineages including cartilage. In this study, we developed an efficient and reproducible procedure for the isolation of stromal cells from bone marrow aspirates of normal human donors based on the expression of endoglin, a type III receptor of the transforming growth factor-beta (TGF-beta) receptor family. We demonstrate that these cells have the ability of multiple lineage differentiation. Stromal cells represented 2-3% of the total mononuclear cells of the marrow. The cells displayed a fibroblastic colony formation in monolayer culture and maintained similar morphology with passage. Expression of cell surface molecules by flow cytometry displayed a stable phenotype with culture expansion. When cocultured with hematopoietic CD34(+) progenitor cells, stromal cells were able to maintain their ability to support hematopoiesis in vitro. Culture expanded stromal cells were placed in a 3-dimensional matrix of alginate beads and cultured in serum-free media in the presence of TGFbeta-3 for chondrogenic lineage progression. Increased expression of type II collagen messenger RNA was observed in the TGFbeta3 treated cultures. Immunohistochemistry performed on sections of alginate beads detected the presence of type II collagen protein. This isolation procedure for stromal cells and the establishment of the alginate culture system for chondrogenic progression will contribute to the understanding of chondrogenesis and cartilage repair.     

Identification and characterization of a spontaneously aggregating amyloid-forming variant of human PrP((90-231)) through phage-display screening of variants randomized between residues 101 and 112

The N-terminal 'unstructured' region of the human prion protein [PrP((90-231))] is believed to play a role in its aggregation because mutations in this region are associated with seeding-independent deposition disorders like Gerstmann-Straussler-Scheinker disease (GSS). One way of examining the effects of such mutations is to search combinatorially derived libraries for sequence variants showing a propensity to aggregate and/or the ability to interact with prion molecules folded into a beta-sheet-based conformation (i.e., beta-PrP or PrP(Sc)). We created a library of 1.8x10(7) variants randomized between positions 101 and 112, displayed it on filamentous bacteriophage, and 'spiked' it with a approximately 25% population of phages-bearing wild-type prion (wt-PrP). Screening was performed through four rounds of biopanning and amplification against immobilized beta-PrP, and yielded three beta-PrP-binding populations: wt-PrP (26% representation) and two non-wt-PrP variants ( approximately 10% and approximately 64% representation, respectively). The remarkable enrichment of one non-wt-PrP variant (MutPrP) incorporating residues KPSKPKTNMKHM in place of KGVLTWFSPLWQ, despite its initial representation at a 5 million-fold lower level than wt-PrP, caused us to produce it and discover: (i) that it readily aggregates into thioflavin-T-binding amyloids between pH 6.0 and 9.0, (ii) that it adopts a soluble beta-sheet based monomeric structure at pH 10.0, (iii) that it is less thermally stable and more compact than wt-PrP, and (iv) that it displays significantly greater resistance to proteolysis than wt-PrP. Our results suggest that sequence variations in the 101-112 region can indeed predispose the prion for aggregation.     

Endogenous cAbl regulates receptor endocytosis

There are two key processes underlying ligand-induced receptor endocytosis: receptor ubiquitylation and remodeling of the actin cytoskeleton. Tyrosine kinases play critical roles in both receptor endocytosis and actin reorganization. Interestingly, members of the Abl family are the only known tyrosine kinases that possess an actin-binding domain and thus have the potential to directly regulate the actin cytoskeleton. However, the role of non-transforming cAbl in receptor endocytosis remains undefined. We report that cAbl promotes ligand-induced antigen receptor endocytosis in B lymphocytes. We show that pharmacologic inhibition or genetic deletion of cAbl causes a defect in tyrosine phosphorylation of the cytoskeletal adapter CrkII. cAbl inhibition or ablation also impairs Rac activation downstream of CrkII, as well as antigen receptor capping and endocytosis. Although phosphorylation of CrkII has been suggested to maintain it in a closed inactive conformation, we demonstrate that it is in fact essential for the activation of Rac. On the other hand, association of CrkII with cCbl, a key mediator of receptor ubiquitylation, does not require CrkII phosphorylation and is cAbl-independent. Phosphorylation of cCbl itself is also cAbl-independent. Our results thus indicate that CrkII links receptor engagement to cytoskeletal remodeling by coupling cCbl- and cAbl-mediated signaling pathways that cooperatively regulate ligand-induced receptor endocytosis.     

pH dependence of conformational fluctuations of the protein backbone

Proton binding equilibria (pK_a values) of ionizable groups in proteins are exquisitely sensitive to their microenvironments. Apparent pK_a values measured for individual ionizable residues with NMR spectroscopy are actually population‐weighted averages of the pK_a in different conformational microstates. NMR spectroscopy experiments with staphylococcal nuclease were used to test the hypothesis that pK_a values of surface Glu and Asp residues are affected by pH‐sensitive fluctuations of the backbone between folded and locally unfolded conformations. ¹⁵N spin relaxation studies showed that as the pH decreases from the neutral into the acidic range the amplitudes of backbone fluctuations in the ps‐ns timescale increase near carboxylic residues. Hydrogen exchange experiments suggested that backbone conformational fluctuations promoted by decreasing pH also reflect slower local or sub‐global unfolding near carboxylic groups. This study has implications for structure‐based pK_a calculations: (1) The timescale of the backbone's response to ionization events in proteins can range from ps to ms, and even longer; (2) pH‐sensitive fluctuations of the backbone can be localized to both the segment the ionizable residue is attached to or the one that occludes the ionizable group; (3) Structural perturbations are not necessarily propagated through Coulomb interactions; instead, local fluctuations appear to be coupled through the co‐operativity inherent to elements of secondary structure and to networks of hydrogen bonds. These results are consistent with the idea that local conformational fluctuations and stabilities are important determinants of apparent pK_a values of ionizable residues in proteins. Proteins 2014; 82:3132–3143. © 2014 Wiley Periodicals, Inc.     

Putrescine overproduction does not affect the catabolism of spermidine and spermine in poplar and Arabidopsis

The effect of up-regulation of putrescine (Put) production by genetic manipulation on the turnover of spermidine (Spd) and spermine (Spm) was investigated in transgenic cells of poplar (Populus nigra × maximowiczii) and seedlings of Arabidopsis thaliana. Several-fold increase in Put production was achieved by expressing a mouse ornithine decarboxylase cDNA either under the control of a constitutive (in poplar) or an inducible (in Arabidopsis) promoter. The transgenic poplar cells produced and accumulated 8–10 times higher amounts of Put than the non-transgenic cells, whereas the Arabidopsis seedlings accumulated up to 40-fold higher amounts of Put; however, in neither case the cellular Spd or Spm increased consistently. The rate of Spd and Spm catabolism and the half-life of cellular Spd and Spm were measured by pulse-chase experiments using [¹⁴C]Spd or [¹⁴C]Spm. Spermidine half-life was calculated to be about 22–32 h in poplar and 52–56 h in Arabidopsis. The half-life of cellular Spm was calculated to be approximately 24 h in Arabidopsis and 36–48 h in poplar. Both species were able to convert Spd to Spm and Put, and Spm to Spd and Put. The rates of Spd and Spm catabolism in both species were several-fold slower than those of Put, and the overproduction of Put had only a small effect on the overall rates of turnover of Spd or Spm. There was little effect on the rates of Spd to Spm conversion as well as the conversion of Spm into lower polyamines. While Spm was mainly converted back to Spd and not terminally degraded, Spd was removed from the cells largely through terminal catabolism in both species.     

Regulation of PKC Mediated Signaling by Calcium during Visceral Leishmaniasis

Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca²⁺ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca²⁺ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca²⁺ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca²⁺-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca²⁺. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca²⁺ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca²⁺-ATPases play prominent roles during visceral leishmaniasis.     

Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles.     

Changes in lipid profiles of brain of albino rats following envenomation

Effect of high doses of cobra venom (150 micrograms/120 +/- 20 g body weight) and viper venom (300 micrograms/120 +/- 20 g body weight) on total lipid, triglyceride, phospholipid, cholesterol, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) of brain of albino rats was studied. Total lipid (TL) triglyceride (TG) and phospholipid (PL) are decreased in both viper and cobra venom treated groups while cholesterol (C), and LDL-C are increased in both the groups in relation to controlled ones. HDL-C content was almost unaltered. Decrease in triglyceride and phospholipid may be due to effect of lipases and phospholipases whereas increased cholesterol and LDL-C may be attributed to lysis of cell membrane.