MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Comparative morphology of the foliage leaf epidermis, with emphasis on papillae characters, in key taxa of woody bamboos of the Asian tropics (Poaceae: Bambusoideae)

The foliage leaf epidermis of 35 species representing 12 key genera of woody bamboos of the Asian tropics was investigated using light and scanning electron microscopy. The results indicated that papillae forms and distributional patterns around the stomatal apparatus of the abaxial foliage leaf epidermis were usually constant and were of great taxonomic significance at the specific and generic levels. However, papillae characters were not suitable for dividing subtribes within woody bamboos of the Asian tropics. On the basis of papillae characters, Schizostachyum s.s. and Cephalostachyum were confirmed, but their delimitations should be modified. The transfer of Leptocanna chinensis and Schizostachyum sanguineum into Cephalostachyum was supported, and Cephalostachyum virgatum and C .  pergracile were confirmed to be members of Schizostachyum s.s. The subtribe Racemobambosinae did not obtain support and Racemobambos appeared to be better placed in subtribe Bambusinae. Neomicrocalamus was supported as a close relative and better treated as a synonym of Racemobambos . Gigantochloa was closely related to Dendrocalamus .  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 411–423.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Muscle fine structure reflects ecotype in two nototheniids

The fine structure of swimming (pectoral) and myotomal (axial) skeletal muscle and myocardium of two species of Antarctic nototheniid fishes were studied by electron microscopy, comparing the cryopelagic Pagothenia borchgrevinki and the benthic Trematomus bernacchii . Mean fibre size varied by a factor of four among muscles within each species and may have reflected the locomotory power available, being larger in pectoral oxidative (red) and axial glycolytic (white) muscle of P. borchgrevinki . Both species use labriform locomotion, and the more active P. borchgrevinki had a greater capillary supply, expressed as a capillary to fibre ratio, than T. bernacchii to both red (3·48 ± 0·36 v . 1·63 ± 0·14, mean ±  s . e .; P  < 0·01) and white (2·70 ± 0·20 v . 1·53 ± 0·18, mean ±  s . e .; P  < 0·01) regions of the pectoral musculature. The greater aerobic scope of P. borchgrevinki was strikingly demonstrated in the higher mitochondrial content of all skeletal muscle types sampled, and the ventricular myocardium (0·269 ± 0·011 v . 0·255 ± 0·012 mean ±  s . e .; P  < 0·05). Minor differences were found in other elements of fibre composition, with the exception of a five‐fold greater lipid content in pectoral red fibres of P. borchgrevinki (0·074 ± 0·014 mean ±  s . e .) v . T. bernacchii (0·010 ± 0·003; P  < 0·05). Differences in muscle fine structure among species clearly reflected differences in their ecotype.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

An anatomical study of anther development in Acorus L.: Phylogenetic implications

 Critical morphological synapomorphies have not been found in support of the Acoranan hypothesis, the molecular phylogenetic discovery that Acoranae are the basal monocots. The previously undetermined pattern of anther wall development in Acorus has been suggested to be one such character. Two main types of anther wall development have been recognized: 1) the “monocotyledonous” type, which characterizes both monocots and dicots, and 2) the “dicotyledonous” type, which is almost exclusively found among dicots. An anatomical study of anther wall development in Acorus was here undertaken using the electron microscope. Development of the anther wall in Acorus was found to be somewhat irregular or perhaps even intermediate between the two types although largely consistent with the “monocotyledonous” type. The presumed significance of anther wall development and other critical morphological characters to the Acoranan hypothesis in the absence of knowledge about the sister group to the monocots is evaluated. Received August 28, 2000 Accepted February 19, 2001     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Water and solute transport by the Malpighian tubules of the stick insect,Carausius morosus

Summary Structural features of the principal, urine-secreting cells (type 1 cells) of the Malpighian tubules of Carausius are de scribedquantitatively and discussed in relation to possible mechanisms of water and solute transport. Mitochondria are arranged in two bands of about equal volume near to the basal and apical surfaces, suggesting active processes occur at both surfaces. Basal infoldings and apical microvilli which greatly amplify the cell surface are probably primarily devices to increase the passive permeability of the tissue to solutes. They do not provide functionally significant standing-osmotic-gradients. The extensive endoplasmic reticulum is locally differentiated into several components and ramifies between the infoldings and along microvilli but probably is not an intracellular conduit for the majority of urinary constituents. Vesicles and stages in their formation or liberation are observed both basally and apically although they probably do not contribute significantly to transcellular transport. At present it remains a problem to satisfactorily account for observations that the urine of Carausius can be hypotonic.This investigation formed part of a dissertation for the degree of Ph. D. in the University of Newcastle upon Tyne. It is a pleasure to thank Prof. J. Shaw for his advice and encouragement and the Science Research Council for financial support.