Search Results
Interaction of globular proteins with mixed lipid vesicles
A thermodynamic study of the lipid lateral phase separation
Vesicles of charged (Phosphatidic Acid) and neutral (Phosphatidylcholine) lipids were used as membranes model to examine the lateral phase separation induced by a globular protein, namely lysozyme.
This short report describes a case of tricuspid valvular metastasis of canine disseminated histiocytic sarcoma in a 9-year-old female Rottweiler. Immunohistochemically the malignant neoplastic cells gave a strong reaction for vimentin and lysozyme, and showed negativity for serotonin, CD3, CD79a and cytokeratin. The intratumoural microvessels were detected by immunohistochemistry using CD31 and claudin-5. This appears to be the first report of a valvular metastasis of canine malignant histiocytosis.
Selection of adsorbent for the development of purification process for biomolecules is crucial due to the requirement of large number of binding sites and adsorption area. Considering this, porous structure with high charge density is selected as an adsorbent for macromolecule purification. Such selection may provide high static binding capacity but causes loss of separation performance due to improper porosity of adsorbent in comparison to solute sizes involved. To address this problem for the screening of adsorbent, this work reports adsorbent selection procedure on the basis of adsorbent pore diameter (d p), solute hydrodynamic dimensions (RH), and flow velocity in support of binding capacity. Towards that end, this study evaluated the pore accessibility performance of varying characteristics adsorbents using tracers like acetone, lysozyme, and bovine serum albumin (BSA) by designing nonbinding conditions. All screened adsorbents showed certain loss of total surface area depending on the solute dimensions and pore size. Sepharose type adsorbents showed accessible area loss up to 25% for lysozyme and 50% for BSA. Sepabeads type showed 30% loss, while macroporous UNO type showed only 7% loss of surface area for lysozyme. The study correlates accessibility with size ratio β (d p/RH). The value of β > 38 is found to be required for the accessibility of total pore area and optimum separation performance of ion exchangers investigated. Accessibility and β provide useful information for the selection of suitable adsorbent for the purification of macromolecules.
This report describes a case of a canine cutaneous grade I mast cell tumour which developed within a lipoma in the right axillar region of an 8-year-old male Boxer. Immunohistologically, the neoplastic mast cells were positive for serotonin, CD45 vimentin and p53, and negative for lysozyme, CD3 and CD79a expression. The proliferation index of the mast cell tumour based on the Ki-67 antigen was 6.1%. Between the benign neoplastic lipocytes and mastocytoma tumour cells intratumoural microvessels were detected by immunohistochemical staining using CD31 and claudin-5 as markers for vascular endothelium.
The effect of pH on thermal stability of globular proteins
A critical insight
Abstract
In this study we try to re-analyze thepH dependence of thermal stability of small globular proteins. From the thermodynamic point of view a long series of calorimetric and spectroscopic investigations has shown that the decreased stability in very acidic conditions can be ascribed to entropic effects. The same conclusion is reached, from a microscopic point of view, by assuming that a binding of protons on equal and noninteracting sites takes place as a consequence of unfolding process. By linking the conformational unfolding equilibrium to the proton binding equilibrium, a model is developed that is able to describe the dependence on thepH of the thermal denaturation processes of small globular protiens. The application of the model to hen lysozyme and T4 lysozyme correctly accounts for the experimental results.
Abstract
A new technique for in vitro studies of biomacromolecules interactions, their adsorption at aqueous/organic liquid interfaces and distribution in the bulk of liquid/liquid systems was developed. The method includes (1) tritium labeling of biomolecules by tritium thermal activation method and (2) scintillation phase step with organic phase, which can be concerned as a model of cellular membrane. Two globular proteins lysozyme and human serum albumin tested. We have determined the conditions of tritium labeling when labeled by-products can be easy separated by means of dialysis and size-exclusion chromatography. Scintillation phase experiments were conducted for three types of organic liquids. Thus, the influences of the nature of organic phase on proteins adsorption and its distribution in the bulk of aqueous/organic liquid system were determined. It was found that proteins possess high surface activity at aqueous/organic liquid interface. Furthermore, values of hydrophobicity of globular proteins were found by the experiment.
Neutron and ion beams in biological research
On the scattering length density of proteins in H2O/D2O: Determination of H-D exchange using ES+I-MS
Summary
Neutron reflectometry is an excellent technique to determine the structure of a protein layer adsorbed at a solid interface. The use of contrast-variation makes it possible to highlight the adsorbed protein layer. The neutron scattering lengths of D and H have opposite signs. By choosing the H2O/D2O ratio, the scattering length density can be made equal to that of the adsorbing substrate. To determine the scattering length density of protein, both its volume and its scattering length in solution is needed. To calculate the scattering length, the H-D exchange of the labile protons of the protein should be taken into account. For monitoring the H-D exchange, electrospray ionization mass spectroscopy (ES+I-MS) was applied. We show that ES+I-MS is the appropriate technique for quantitative analysis of the H-D exchange. We compare the experimental results for the exchange in lysozyme and &-casein with theoretical calculations.
Abstract
Apparent molar volume and enthalpy changes for mixing NaCl (aq.) with albumin from human serum (aq.) are experimentally determined (25°C). Calorimetric experiments were carried out in an LKB 10700-2 calorimeter, whereas volumetric measurements were realized using an Anton Paar 60/602 densimeter. The density measurements were made after 1 and 24 h of the dissolution in the buffer (pH 4.2). The relation between the changes of the enthalpy and apparent molar volumes vs. molality of NaCl were determined. The obtained data are discussed together with data obtained previously for bovine albumin and hen egg lysozyme solutions with NaCl, Li2SO4 and (NH4)2SO4 salts of various concentration. As results correlations between the changes of enthalpy of salting and apparent molar volumes vs. molality of salts were made.
At clinical examination, a 5-year-old male domestic short-haired cat exhibited painful swelling and erythema of the pinnae of both ears. Microscopically, the lesions on both pinnae were composed of diffuse granulomatous chondritis with degeneration and necrosis of the pinnal cartilage. Numerous mast cells were also observed within and surrounding the inflammatory lesion. Immunohistochemistry showed a mixed inflammatory infiltrate characterised by the predominance of macrophages (CD68+, MAC 387+ and Lysozyme+), T lymphocytes (CD3+), some B lymphocytes (CD79α+) and neutrophils. Immunopathological characterisation of the lesion showed a granulomatous inflammation profile and suggests that the morphological changes and immunopathogenesis of auricular chondritis in cats presents a similarity with relapsing polychondritis in humans.
Abstract
A reliable and complete inactivation is an indispensable premise for any concentration of rickettsiae or for the development of diagnostic strategies based on their antigens. This study deals with the testing of methods to inactivate rickettsiae.
Rickettsia honei was used as a model organism. The inactivating potency of formalin, Qiagen® antiviral lysozyme (AVL) buffer, heating to 56 °C, and β-propiolactone was analyzed in cell culture.
The inactivation limits for rickettsiae were 0.1% formalin about 10 min, Qiagen AVL buffer about 5 min, 56 °C about 5 min, 0.125% β-propiolactone about 1 h, and 0.0125% β-propiolactone overnight. The interpretation was limited by cytotoxic effects of the inactivation procedures and by the culturally achievable rickettsial density in the cell culture supernatants that were used for the inactivation experiments.
Reliable modes of inactivation were identified, allowing for the secure handling of rickettsial antigens for diagnostic purposes.