Chemical synthesis and structure elucidation of bovine kappa-casein (1-44)

The caseins (alpha(s1), alpha(s2), beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1-44) of bovine K-casein, the protein which maintains the micellar structure of the caseins. K-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro(8) to Arg(34). This is the first report which demonstrates extensive secondary structure within the casein class of proteins. (c) 2006 Elsevier Inc. All rights reserved.

The effect of box shape on the dynamic properties of proteins simulated under periodic boundary conditions

The effect of the box shape on the dynamic behavior of proteins simulated under periodic boundary conditions is evaluated. In particular, the influence of simulation boxes defined by the near-densest lattice packing (NDLP) in conjunction with rotational constraints is compared to that of standard box types without these constraints. Three different proteins of varying size, shape, and secondary structure content were examined in the study. The statistical significance of differences in RMSD, radius of gyration, solvent-accessible surface, number of hydrogen bonds, and secondary structure content between proteins, box types, and the application or not of rotational constraints has been assessed. Furthermore, the differences in the collective modes for each protein between different boxes and the application or not of rotational constraints have been examined. In total 105 simulations were performed, and the results compared using a three-way multivariate analysis of variance (MANOVA) for properties derived from the trajectories and a three-way univariate analysis of variance (ANOVA) for collective modes. It is shown that application of roto-translational constraints does not have a statistically significant effect on the results obtained from the different simulations. However, the choice of simulation box was found to have a small (5-10%), but statistically significant effect on the behavior of two of the three proteins included in the study. (c) 2005 Wiley Periodicals, Inc.

Responsive hydrophobically associating polymers:a review of structure and properties

Responsive hydrophobically associating polymers can in many ways be considered to be analogous to proteins in their ability to form compact molecules with a defined secondary structure, and hence, functionality. These molecules are characterized by the presence of alternating charged and hydrophobic groups. The balance between charge repulsion and hydrophobic interactions is sensitive to environmental pH and therefore changes in pH produce controllable conformational changes. The change from a charged extended chain to a collapsed uncharged coil structure is sometimes referred to as hypercoiling behaviour and enables the polymer to act as a simple switch between an 'on' and 'off' state. The purpose of this review is to illustrate the structure and behaviour of polymers that exhibit hypercoiling behaviour and to highlight their potential pharmaceutical applications, which in terms of drug delivery is likely to be related to their surface behaviour and solubilizing activity. © 2001 Elsevier Science B.V. All rights reserved.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A(2) (sPLA2) from Both tops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the a-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of 5PLA2 and have a direct effect on the Ca2+-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this 5PLA2. (C) 2011 Elsevier Ltd. All rights reserved.

The Putative Leishmania Telomerase Rna (leishter) Undergoes Trans-splicing And Contains A Conserved Template Sequence.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

Phylogenetic position of the acariform mites: sensitivity to homology assessment under total evidence

Background: Mites (Acari) have traditionally been treated as monophyletic, albeit composed of two major lineages: Acariformes and Parasitiformes. Yet recent studies based on morphology, molecular data, or combinations thereof, have increasingly drawn their monophyly into question. Furthermore, the usually basal (molecular) position of one or both mite lineages among the chelicerates is in conflict to their morphology, and to the widely accepted view that mites are close relatives of Ricinulei. Results: The phylogenetic position of the acariform mites is examined through employing SSU, partial LSU sequences, and morphology from 91 chelicerate extant terminals (forty Acariformes). In a static homology framework, molecular sequences were aligned using their secondary structure as guide, whereby regions of ambiguous alignment were discarded, and pre-aligned sequences analyzed under parsimony and different mixed models in a Bayesian inference. Parsimony and Bayesian analyses led to trees largely congruent concerning infraordinal, well-supported branches, but with low support for inter-ordinal relationships. An exception is Solifugae + Acariformes (P. P = 100%, J. = 0.91). In a dynamic homology framework, two analyses were run: a standard POY analysis and an analysis constrained by secondary structure. Both analyses led to largely congruent trees; supporting a (Palpigradi (Solifugae Acariformes)) clade and Ricinulei as sister group of Tetrapulmonata with the topology (Ricinulei (Amblypygi (Uropygi Araneae))). Combined analysis with two different morphological data matrices were run in order to evaluate the impact of constraining the analysis on the recovered topology when employing secondary structure as a guide for homology establishment. The constrained combined analysis yielded two topologies similar to the exclusively molecular analysis for both morphological matrices, except for the recovery of Pedipalpi instead of the (Uropygi Araneae) clade. The standard (direct optimization) POY analysis, however, led to the recovery of trees differing in the absence of the otherwise well-supported group Solifugae + Acariformes. Conclusions: Previous studies combining ribosomal sequences and morphology often recovered topologies similar to purely morphological analyses of Chelicerata. The apparent stability of certain clades not recovered here, like Haplocnemata and Acari, is regarded as a byproduct of the way the molecular homology was previously established using the instrumentalist approach implemented in POY. Constraining the analysis by a priori homology assessment is defended here as a way of maintaining the severity of the test when adding new data to the analysis. Although the strength of the method advocated here is keeping phylogenetic information from regions usually discarded in an exclusively static homology framework; it still has the inconvenience of being uninformative on the effect of alignment ambiguity on resampling methods of clade support estimation. Finally, putative morphological apomorphies of Solifugae + Acariformes are the reduction of the proximal cheliceral podomere, medial abutting of the leg coxae, loss of sperm nuclear membrane, and presence of differentiated germinative and secretory regions in the testis delivering their products into a common lumen.

Diversity of free-living marine nematodes (Enoplida) from Baja California assessed by integrative taxonomy

We used morphological and molecular approaches to evaluate the diversity of free-living marine nematodes (order Enoplida) at four coastal sites in the Gulf of California and three on the Pacific coast of Baja California, Mexico. We identified 22 morphological species belonging to six families, of which Thoracostomopsidae and Oncholaimidae were the most diverse. The genus Mesacanthion (Thoracostomopsidae) was the most widespread and diverse. Five allopatric species, genetically and morphologically differentiated, were found in two localities in the Gulf of California (M. sp1 and M. sp2) and three in the Pacific coast (M. sp3, M. sp4 and M. sp5). Overall, we produced 19 and 20 sequences for the 18S and 28S genes, respectively. Neither gene displayed intraspecific polymorphisms, which allowed us to establish that some morphological variation was likely either ontogenetic or due to phenotypic plasticity. Although 18S and 28S phylogenies were topologically congruent (incongruence length difference test, P > 0.05), divergences between species were much higher in the 28S gene. Moreover, this gene possessed a stronger phylogenetic signal to resolve relationships involving Rhabdodemania and Bathylaimus. On the other hand, the close relationship of Pareurystomina (Enchilidiidae) with oncholaimids warrants further study. The 28S sequences (D2D3 domain) may be better suited for DNA barcoding of marine nematodes than those from the 18S rDNA, particularly for differentiating closely related or cryptic species. Finally, our results underline the relevance of adopting an integrative approach encompassing morphological and molecular analyses to improve the assessment of marine nematode diversity and advance their taxonomy.

Biophysical characterization of the recombinant merozoite surface protein-3 of Plasmodium vivax

Plasmodium vivax Merozoite Surface Protein-3 alpha and 3 beta are members of a family of related merozoite surface proteins that contain a central alanine-rich domain with heptad repeats that is predicted to form alpha-helical secondary and coiled-coil tertiary structures. Seven recombinant proteins representing different regions of MSP-3 alpha and MSP-3 beta of P. vivax were generated to investigate their structure. Circular dichroism spectra analysis revealed that some proteins are folded with a high degree of alpha-helices as secondary structure, whereas other products contain a high content of random coil. Using size exclusion chromatography, we found that the two smaller fragments of the MSP-3 alpha, named CC4 and CC5, predicted to form coiled-coil (CC) structures, eluted at volumes corresponding to molecular weights larger than their monomeric masses. This result suggests that both proteins are oligomeric molecules. Analytical ultracentrifugation experiments showed that the CC5 oligomers are elongated molecules. Together, these data may help to understand important aspects of P. vivax biology. (C) 2008 Elsevier B.V. All rights reserved.

Preparation and characterization of ethanol-treated silk fibroin dense membranes for biomaterials application using waste silk fibers as raw material

The possibility of producing valued devices from low cost natural resources is a subject of broad interest. The present study explores the preparation and characterization of silk fibroin dense membranes using waste silk fibers from textile processing. Morphology, crystallinity, thermal resistance and cytotoxicity of membranes as well as the changes on the secondary structure of silk fibroin were analyzed after undergoing treatment with ethanol. Membranes presented amorphous patterns as determined via X-ray diffraction. The secondary structure of silk fibroin on dense membranes was either random coil (silk I) or p-sheet (silk II), before and after ethanol treatment, respectively. The sterilized membranes presented no cytotoxicity to endothelial cells during in vitro assays. This fact stresses the material potential to be used in the fabrication of biomaterials, as coatings of cardiovascular devices and as membranes for wound dressing or drug delivery systems. (C) 2010 Elsevier Ltd. All rights reserved.

Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer- dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer- dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca(2+) flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca(2+) flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

A novel mutation in the L12 domain of keratin 5 in the Kobner variant of epidermolysis bullosa simplex

We have identified a novel mutation within the linker L12 region of keratin 5 (K5) in a family with the Kobner variant of epidermolysis bullosa simplex. The pattern of inheritance of the disorder in this family is consistent with an autosomal dominant mode of transmission. Affected individuals develop extensive and generalized blistering at birth or early infancy but in later years clinical manifestations are largely confined to palmo-plantar surfaces. Direct sequencing of polymerase chain reaction products revealed a T to C transition within codon 323 of K5 in affected individuals, resulting in a valine to alanine substitution of the seventh residue within the L12 linker domain. This mutation was not observed in unaffected family members or in 100 K5 alleles of unrelated individuals with normal skin. The other critical regions of K5 and K14 were unremarkable in this family except for common polymorphisms that have been previously described. The valine at position 7 of the L12 domain is absolutely conserved in all type II keratins, and in other intermediate filament subunits as well, which suggests that this residue makes an important contribution to filament integrity. Secondary structure analysis revealed that alanine at this position markedly reduces both the hydrophobicity and the beta-sheet nature of the L12 domain. This is the first report of a mutation at this position in an intermediate filament subunit and reinforces the importance of this region to filament biology.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.