Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.     

Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely. The estimated dimer dissociation constant (K(1,2)) for CheA(T) (3.1 microM) was weaker than K(1,2) for CheA(L) (0.49 microM), which was attributed to the P domain-catalytic domain interactions that were present in CheA(L) but not CheA(T). In contrast, CheA(S) dimerization was unexpectedly stronger (K(1,2) approximately 20 nM), which arose through interactions between two P domain remnants in the CheA(S) dimer. This conclusion was supported by the results of sedimentation equilibrium experiments conducted with P domains and P domain remnants expressed in the absence of the dimerization/catalytic/regulatory domains. The P domain remnant had a measurable tendency to self-associate; the full-length P domain did not. Hydrophobic forces probably drive this interaction, since hydrophobic amino acids buried in the intact P domain are solvent-exposed in CheA(S). Also, the nascent N-terminus of CheA(S) bound to the phosphatase (CheZ) more effectively, a conclusion based on the demonstrably greater ability of the P domain remnant to co-sediment CheZ, compared to the intact P domain.     

Bioorthogonal noncovalent chemistry: Fluorous phases in chemical biology

Chemical entities designed to noncovalently interact with predetermined partners have fashioned a new paradigm in chemical biology. Fluorocarbons are extremely promising as supramolecular synthons toward these objectives. Bioorthogonal noncovalent interactions provide a way to modulate self-assembled systems in environments where such control has hitherto not been possible. Fluorocarbons have now found applications in self-assembly as well as proteomics, biomolecule purification and in the creation of microarray platforms. Other self-assembly motifs with similar attributes might be exploited using the same general approach.     

Nuclear localisation of endogenous SUMO-1-modified PDGF-C in human thyroid tissue and cell lines

We investigated post-translational modification and subcellular localisation of endogenous platelet-derived growth factor-C (PDGF-C) in human thyroid papillary carcinomas (PTC), non-neoplastic thyroid tissues, and a selection of cultured cell lines. PDGF-C expressed nuclear localisation in 95% of all tested cell types in culture and in 10% of the thyrocytes from both PTC and non-neoplastic tissue. The cell lines expressed two forms of full-length PDGF-C, approximately 39 and approximately 55 kDa, in cell membrane and cytosol, while the approximately 55 kDa form dominated in the nucleus where it was partly chromatin-associated. The approximately 55 kDa form was post-translationally modified by SUMO-1. The putative PDGF-C SUMOylation site is the surface exposed (314)lysine part of a positively charged loop ((312)RPKTGVRGLHK(322)) with characteristics of a nuclear localisation signal. The tissue thyrocytes expressed a non-SUMOylated approximately 43 kDa and the 55 kDa PDGF-C. The SUMO-1 modified approximately 55 kDa PDGF-C expression was low in PTC where the approximately 43 kDa PDGF-C dominated. This is in contrast to non-neoplastic tissue and cultured cells where the SUMOylated approximately 55 kDa PDGF-C was strongly expressed. Our data provide novel evidence for nuclear localisation of PDGF-C, post-translational modification by SUMOylation and the expression of a novel form of PDGF-C in human papillary thyroid carcinomas.     

Postrelease dive ability in rehabilitated harbor seals

The efficacy of seal rehabilitation is examined in a postrelease study of dive ability in harbor seal pups (Phoca vitulina) in the Wash, United Kingdom. Six rehabilitated seals were fitted with Sea Mammal Research Unit (SMRU) Argos Satellite Relay Data Logger tags and their individual dive behavior was monitored for an average of 122 d. The upper 90 percentile edge of dive behavior (dive duration [DD₉₀] and percentage of time at‐sea spent in a dive [PD₉₀]), in 7 d bins, was used as a proxy for physiological dive ability. The results are compared with data from five wild adult harbor seals. There was no statistically significant difference between (1) the mean track duration of rehabilitated seals (126.20 ± 27.48 [SD] d) and adult seals (150.2 ± 24.62 d) (P= 0.108), indicating no evidence that short‐term survival was less in the rehabilitated group; (2) the mean mass‐scaled DD₉₀ of rehabilitated seals (3.95 ± 0.37 min) and adult seals (4.09 ± 0.55 min) (P= 0.632); and (3) the mean PD₉₀ of rehabilitated seals (81.62 ± 1.21%) and adult seals (81.48 ± 3.93%) (P= 0.943). These three results all suggest the success of the rehabilitation program in terms of short‐term survival and dive ability.     

Dos especies nuevas de Borreria (Rubiaceae) y sinopsis de las especies de Bolivia

Two new species are described, Borreria cerradoana and B. velascoana, from Bolivia and Brazil, and a new combination is made, B. spinosa var. minima. Through an identification key, all 28 species of Borreria for Bolivia are recorded, and comments on geographical distribution and habitat are provided. Borreria brachystemonoides, B. poaya, and B. tenera are confirmed as new records for the country.     

Nonlinearity between action potential alternans and restitution, which both predict ventricular arrhythmic properties in Scn5a+/- and wild-type murine hearts

Electrocardiographic QT- and T-wave alternans, presaging ventricular arrhythmia, reflects compromised adaptation of action potential (AP) duration (APD) to altered heart rate, classically attributed to incomplete Na(v)1.5 channel recovery prior to subsequent stimulation. The restitution hypothesis suggests a function whose slope directly relates to APD alternans magnitude, predicting a critical instability condition, potentially generating arrhythmia. The present experiments directly test for such correlations among arrhythmia, APD alternans and restitution. Mice haploinsufficient in the Scn5a, cardiac Na(+) channel gene (Scn5a(+/-)), previously used to replicate Brugada syndrome, were used, owing to their established arrhythmic properties increased by flecainide and decreased by quinidine, particularly in right ventricular (RV) epicardium. Monophasic APs, obtained during pacing with progressively decrementing cycle lengths, were systematically compared at RV and left ventricular epicardial and endocardial recording sites in Langendorff-perfused Scn5a(+/-) and wild-type hearts before and following flecainide (10 μM) or quinidine (5 μM) application. The extent of alternans was assessed using a novel algorithm. Scn5a(+/-) hearts showed greater frequencies of arrhythmic endpoints with increased incidences of ventricular tachycardia, diminished by quinidine, and earlier onsets of ventricular fibrillation, particularly following flecainide challenge. These features correlated directly with increased refractory periods, specifically in the RV, and abnormal restitution and alternans properties in the RV epicardium. The latter variables were related by a unique, continuous higher-order function, rather than a linear relationship with an unstable threshold. These findings demonstrate a specific relationship between alternans and restitution, as well as confirming their capacity to predict arrhythmia, but implicate mechanisms additional to the voltage feedback suggested in the restitution hypothesis.     

A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos

To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.     

Venomics and antivenomics profiles of North African Cerastes cerastes and C. vipera populations reveals a potentially important therapeutic weakness

We report the proteomic analysis of the venom of the medically relevant snake, Cerastes cerastes, from Morocco, and the immunoreactivity profile of an experimental monospecific (CcMo_AV against Moroccan C. cerastes venom) and a commercial (Gamma-VIP against Tunisian C. cerastes and M. lebetina venoms) F(ab')(2) antivenoms towards geographic variants of C. cerastes and C. vipera venoms. The venom of C. cerastes is a low-complexity proteome composed of 25-30 toxins belonging to 6 protein families, mainly targetting the hemostatic system. This toxin arsenal explains the clinical picture observed in C. cerastes envenomings. Despite geographic compositional variation, the monospecific CcMo_AV and the Gamma-VIP divalent antivenom produced at Institut Pasteur de Tunis, showed similar immunocapturing capability towards Moroccan, Tunisian, and Egyptian C. cerastes venom proteins. Proteins partially escaping immunorecognition were all identified as PLA(2) molecules. Antivenomic analysis showed low degree of cross-reactivity of Moroccan CcMo_AV and Tunisian Gamma-VIP antivenoms towards C. vipera venom toxins. This study indicates that a more complete therapeutic cover could be achieved by including C. vipera venom in the formulation of venom immunization mixtures, thereby generating a pan-Cerastes antivenom.