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.     

Infrared spectroscopy of proteins

This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.     

Reactivity and binding of benzo(a)pyrene diol epoxide to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms

The physical and covalent binding of the carcinogen benzo(a)pyrene-7,8-diol-9,10-oxide (BaPDE) to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms were studied utilizing absorbance, fluorescence and linear dichroism techniques. In the case of poly(dG-dC).(dG-dC) the decrease in the covalent binding of BaPDE with increasing NaCl concentration (0.1-4 M) as the B form is transformed to the Z form is attributed to the effects of high ionic strengths on the reactivity and physical binding of BaPDE to the polynucleotides; these effects tend to obscure differences in reactivities with the B and Z forms of the nucleic acids. In the case of poly(dG-m5dC).(dG-m5dC) the B-to-Z transition is induced at low ionic strength (2 mM NaCl + 10 microM Co(NH3)6Cl3) and the covalent binding is found to be 2-3-times lower to the Z form than to the B form. Physical binding of BaPDE by intercalation, which precedes the covalent binding reaction, is significantly lower in the Z form than in the B form, thus accounting, in part, for the lower covalent binding. The linear dichroism characteristics of BaPDE covalently bound to the Z and B forms of poly(dG-m5dC).(dG-m5dC) are consistent with nonintercalative, probably external conformations of the aromatic pyrenyl residues.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

An automated technique for monitoring carbon dioxide respired by insects*

ABSTRACT. A non-dispersive infrared gas analyser equipped with a Luft-type sonic detector and flow-through reference cell was automated to monitor the total volume of carbon dioxide (CO₂) respired by single insects or groups of insects. The infrared analyser was interfaced with an integrator for quantification, a microprocessor to control intermittent air flow through the insect respiration chambers, and a microcomputer for data storage and reduction. This technique has been used to monitor the CO₂ Output of diapausing and non-diapausing mature fifth instar larvae and of developing pupae of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). The resulting data were accurate, quantitative and reproducible.     

Thermographic visualization of cell death in tobacco and Arabidopsis

Pending cell death was visualized by thermographic imaging in bacterio‐opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) ( Chaerle et al. 1999 Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction ( Chaerle et al. 1999 ), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.     

Vacuum ultraviolet circular dichroism of poly(galacturonic acid), sodium polygalacturonate and calcium polygalacturonate

Vacuum ultraviolet circular dichroism spectra are reported for poly(galacturonic acid) solution and film, sodium polygalacturonate solution and film, and calcium polygalacturonate gel. In addition to the positive c.d. band near 208 nm previously observed, we find a pair of higher energy bands at 170 180 nm (negative) and 145 nm (positive). The low energy band, assigned to an $\text{n-π}{}^1$ carboxyl transition, is blue-shifted upon gelation or film formation.     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.     

Zoning eco-environmental vulnerability for environmental management and protection

Eco-environmental vulnerability assessment is crucial for environmental and resource management. However, evaluation of eco-environmental vulnerability over large areas is a difficult and complex process because it is affected by many variables including hydro-meteorology, topography, land resources, and human activities. The Thua Thien – Hue Province and its largest river system, the Perfume River, are vital to the social-economic development of the north central coastal region of Vietnam, but there is no zoning system for environmental protection in this region. An assessment framework is proposed to evaluate the vulnerable eco-environment in association with 16 variables with 6 of them constructed from Landsat 8 satellite image products. The remaining variables were extracted from digital maps. Each variable was evaluated and spatially mapped with the aid of an analytical hierarchy process (AHP) and geographical information system (GIS). An eco-environmental vulnerability map is assorted into six vulnerability levels consisting of potential, slight, light, medium, heavy, and very heavy vulnerabilities, representing 14%, 27%, 17%, 26%, 13%, 3% of the study area, respectively. It is found that heavy and very heavy vulnerable areas appear mainly in the low and medium lands where social-economic activities have been developing rapidly. Tiny percentages of medium and heavy vulnerable levels occur in high land areas probably caused by agricultural practices in highlands, slash and burn cultivation and removal of natural forests with new plantation forests. Based on our results, three ecological zones requiring different development and protection solutions are proposed to restore local eco-environment toward sustainable development. The proposed integrated method of remote sensing (RS), GIS, and AHP to evaluate the eco-environmental vulnerability is useful for environmental protection and proper planning for land use and construction in the future.