An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Differential pulse anodic voltammetric determination of pantoprazole in pharmaceutical dosage forms and human plasma using glassy carbon electrode

Pantoprazole is used as an anti-ulcer drug through inhibition of H(+), K(+)-adenosine 5(')-triphosphatase in gastric parietal cells. It reduces the gastric acid secretion regardless of the nature of stimulation. The use of differential pulse voltammetry for the determination of pantoprazole in pharmaceutical dosage forms and human plasma using a glassy carbon electrode has been examined. The best voltammetric response was reached for a glassy carbon electrode in Britton-Robinson buffer solution of pH 5.0 submitted to a scan rate of 20.0 mVs(-1) and a pulse amplitude of 50.0 mV. This electroanalytical procedure was able to determine pantoprazole in the concentration range 6.0 x 10(-6)-8.0 x 10(-4)M. Precision and accuracy of the developed method was checked with recovery studies. The limit of detection and limit of quantitation were found to be 4.0 x 10(-7) and 9.0 x 10(-7)M, respectively. Rapidity, precision, and good selectivity were also found for the determination of pantoprazole in pharmaceutical dosage forms and human plasma. For comparative purposes high-performance liquid chromatography with a diode array and UV/VIS detection at 290.0 nm determination also was developed.     

Madin–Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen‐coated surfaces rather than in physiological 3‐D cultures

We investigate the influence of the dimensionality and the biochemistry of the culture system on the cellular functionality by analyzing the protein expression levels in Madin–Darby canine kidney (MDCK) cells grown in 3‐D and 2‐D substrates. We cultured MDCK cells on a hard and flat 2‐D uncoated plastic surface, on a 2‐D collagen‐coated plastic surface and in 3‐D collagen gel and employed 2‐D gel electrophoresis, MALDI‐TOF‐MS, and LC‐MS/MS analysis to identify the differentially regulated proteins. We found significant differences in the expression of antioxidant proteins, actin‐binding proteins, glycolytic enzymes, and heat‐shock proteins/chaperons among the three types of cultures. While MDCK cells cultured in 3‐D collagen up‐regulate antioxidant proteins and proteins involved in the dynamic remodeling of the actin cytoskeleton, 2‐D collagen‐coated plastic surfaces induce the up‐regulation of glycolytic enzymes. Our data shows that the culture conditions have profound effects on the physiology of the cell. Culture in 3‐D collagen induces a differentiated polarized phenotype. In contrast, collagen‐coated 2‐D substrates favor a tumor‐like phenotype with increased glycolysis. Thus, the suitability of 2‐D cultures to study the physiological behavior of cells, especially in drug discovery, bioprocessing, and toxicology, should be carefully reconsidered.     

Global Versus Local Centrality in Evolution of Yeast Protein Network

It is shown here that in the yeast protein interaction network the global centrality measure (betweenness) depends on the protein evolutionary age (i.e., on historic contingency) more weakly than the local centrality measure (degree). This phenomenon is not observed in mutational duplication-and-divergence models. The network domains responsible for this difference deal with DNA/RNA information processing, regulation, and cell cycle. A selection vector can operate in these domains, which integrates the network activity and thus compensates for the process of mutational divergence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.     

CT对于肝脏良性占位性病变及肝癌的鉴别诊断价值分析

目的:探讨CT对于肝脏良性占位性病变及肝癌的鉴别诊断价值。方法:收取2013年3月至2016年3月我院收治的肝脏良性占位性病变及肝癌患者101例作为研究对象,按照病变类型将其分为A、B、C三组。其中A组包含原发性肝癌患者32例,B组包含肝转移癌患者28例,C组包含肝血管瘤患者41例。采用CT全肝灌注扫描模式对三组患者占位病灶组织、病灶周围组织及正常肝脏组织灌注参数进行比较。结果:三组占位病灶组织,B组患者肝动脉灌注量(HAP)最低,C组患者HAP最高;A组患者门静脉灌注量(PVP)最低,C组患者PVP最高,三组两两比较均有显著差异(P0.05)。C组总肝灌注量(TLP)明显高于A组和B组(P0.05),A、B组间无统计学差异(P0.05)。三组肝动脉灌注指数(HPI)无明显差异(P0.05);B组病灶周围组织HAP及HPI明显高于A、C组(P0.05),A、C组间无统计学差异(P0.05);三组PVP及TLP差异不显著(P0.05);三组正常肝脏组织CT灌注参数均无显著差异(P0.05)。结论:CT灌注成像对于原发性肝癌、肝转移癌及肝血管瘤具有一定的鉴别诊断价值,但明确诊断仍需结合其他检测方法进行。