A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets

X-ray and light scattering study of the structure of large protein aggregates at neutral pH

The structure of large ovalbumin and ß-lactoglobulin aggregates formed after heat-denaturation at neutral pH was studied using a combination of light and small-angle X-ray scattering. The effect of the electrostatic interactions was investigated by varying the ionic strength. The results were compared with images obtained using cryo-TEM. The structure of ovalbumin aggregates is compatible with that of semi-flexible strings of monomers that are more flexible and increasingly branched with increasing ionic strength. The persistence length increases with decreasing ionic strength. ß-lactoglobulin aggregates consist of clusters of primary aggregates that are formed in the first step of the aggregation process. At low ionic strength the association of primary aggregates is mostly head to tail, while with increasing ionic strength denser clustering of the primary aggregates is observed

Iso-scattering points during heat-induced aggregation and gelation of globular proteins indicating micro-phase separation

Small-angle X-ray scattering was done in situ during heat-induced aggregation and gelation of concentrated solutions of two globular proteins (β-lactoglobulin and ovalbumin) over a wide range of ionic strengths at pH 7. A well-defined iso-scattering point independent of heating time was observed in all cases, which is interpreted in terms of micro-phase separation between native proteins and aggregates. At high scattering wave vectors the structure factor is simply the weighted average of the initial state (native protein) and the final state (gel). In this q-range the relative variation of the scattering intensity is controlled by the depletion rate of native proteins

Hemorrhagic, Hemostatic, and Thromboelastometric Disorders in 35 Dogs with a Clinical Diagnosis of Leptospirosis: A Prospective Study

International audienceBackgroundLeptospirosis in dogs is occasionally associated with a hemorrhagic syndrome, the pathophysiology of which is not fully understood. Hypothesis/ObjectivesTo characterize hematologic, hemostatic, and thromboelastometric abnormalities in dogs with leptospirosis and to study their association with hemorrhagic diatheses and outcomes. AnimalsThirty-five client-owned dogs. MethodsA prospective observational single cohort study was conducted. Results from the CBC, coagulation tests (prothrombin, activated partial thromboplastin and thrombin times, fibrinogen, fibrin(ogen) degradation products, and D-dimer concentrations), rotational thromboelastometry (TEM), signalment, hemorrhagic diatheses, occurrence of disseminated intravascular coagulation (DIC) at admission, and survival to discharge were recorded. ResultsThe most common hematologic and hemostatic abnormalities were anemia (30/35), thrombocytopenia (21/35), and hyperfibrinogenemia (15/35). Eight dogs were diagnosed with DIC. A normal TEM profile was found in 14 dogs, a hypercoagulable profile in 14 dogs, and a hypocoagulable profile in 7 dogs. The 8 dogs with hemorrhagic diatheses at admission had significantly decreased platelet counts (P = .037) and increased D-dimer concentrations (P = .015) compared with other dogs. Dogs with a hypocoagulable profile exhibited more hemorrhagic diatheses compared with the dogs that had normal and hypercoagulable profiles (P = .049). The mortality rate was lower in dogs with a hypercoagulable profile than in those with a hypocoagulable profile (21% vs 57%; P = .043). Disseminated intravascular coagulation was not a significant prognostic factor. Conclusions and Clinical ImportanceThromboelastometric parameters were altered in dogs with both hypercoagulable and hypocoagulable profiles. A hypocoagulable profile was significantly correlated with hemorrhagic diathesis and higher mortality rate

Reliability of the nitrogen washout-washin technique to assess end-expiratory lung volume at variable PEEP and tidal volumes

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