Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control

It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76–94], des[64–80], and des [6–127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64–80] and des[6–127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5–45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHS^ox. When reduced HEL was reacted with 1–4 equivalents of DHS^ox, 1S, 2S, 3S, and 4S intermediate ensembles with 1–4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76–94] was populated in the largest amount, but the oxidation to N was slower than that of des[64–80] and des[6–127]. At 35 °C, on the other hand, des[64–80] and des[6–127] were no longer stable, and only des[76–94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control.     

Effect of Long-Term Supplementation with Acetic Acid on the Skeletal Muscle of Aging Sprague Dawley Rats

Mitochondrial function in skeletal muscle, which plays an essential role in oxidative capacity and physical activity, declines with aging. Acetic acid activates AMP-activated protein kinase (AMPK), which plays a key role in the regulation of whole-body energy by phosphorylating key metabolic enzymes in both biosynthetic and oxidative pathways and stimulates gene expression associated with slow-twitch fibers and mitochondria in skeletal muscle cells. In this study, we investigate whether long-term supplementation with acetic acid improves age-related changes in the skeletal muscle of aging rats in association with the activation of AMPK. Male Sprague Dawley (SD) rats were administered acetic acid orally from 37 to 56 weeks of age. Long-term supplementation with acetic acid decreased the expression of atrophy-related genes, such as atrogin-1, muscle RING-finger protein-1 (MuRF1), and transforming growth factor beta (TGF-β), activated AMPK, and affected the proliferation of mitochondria and type I fiber-related molecules in muscles. The findings suggest that acetic acid exhibits an anti-aging function in the skeletal muscles of aging rats.     

Improving discovery phase of reactive ad hoc routing protocols using Jaccard distance

Recently, more and more research focus has been directed towards optimum designs for Mobile Ad Hoc Networks (MANETs). In this paper, the authors explore novel broadcasting schemes to improve the discovery phase of reactive routing protocols in MANETs. The objective is to reduce the redundancy of routing packets during the discovery phase of the reactive routing protocols for MANETs. For this purpose, the Jaccard distance is used to select dissimilar nodes during the discovery phase in order to reduce redundancy. Furthermore, it is shown that the Jaccard distance is correlated to the Euclidean distance. The simulation results obtained indicate that the Jaccard distance is a suitable metric for improving the discovery phase of the reactive routing protocols in MANETs.     

CO2 Hydrogenation to Methanol Over Cu/ZnO/Al2O3 Catalyst: Kinetic Modeling Based on Either Single- or Dual-Active Site Mechanism

CO₂ hydrogenation to CH₃OH via heterogeneous catalysis is one of the most promising and available approaches for mitigation of anthropogenic CO₂ issues. In this work, thermodynamic equilibria of CO₂ to methanol were compared with experimental results at given conditions using a commercial Cu/ZnO/Al₂O₃ catalyst for CO hydrogenation to methanol. It was found that, the high pressure, low temperature, and high H₂/CO₂ ratio are favorable to methanol synthesis from CO₂. Furthermore, the kinetic data were measured with an isothermal integral reactor under temperature between 160 and 240 °C, lower than that for CO hydrogenation to methanol reaction. Based on the single-active site and dual-active site LH mechanisms, both kinetic models can achieve full illustration of the influence of the operating conditions and the mechanisms. According to comparative analysis of the error variances of model correlations and the adsorbate coverages on the active sites, the dual-site mechanism identified to be superior to the single-site one for methanol synthesis from CO₂ feedstock. Overall, this paper provides fundamental understanding of the thermodynamic and kinetic aspects of a central route for CO₂ Valorisation.

Graphical Abstract

     

Polymorphism of gamma-adducin gene in genetic hypertension and mapping of the gene to rat chromosome 1q55

Adducin (ADD) is a heterodimeric protein involved in cellular signal transduction. A mutation in the alpha subunit affects ion transport and blood pressure in primary hypertension of Milan rats (MHS) and humans. In rats this effect is modulated by another mutation in the beta subunit. The recently described gamma subunit is a new member of the ADD family that should take the place of beta subunit in cells and tissues expressing alpha but not beta-Add. A missense mutation (Q572K) has been found in the gamma subunit of the Milan rats. Nineteen normotensive and five hypertensive inbred rat strains were genotyped for the polymorphisms in alpha, beta and gamma-Add genes. A disequilibrium was evident in the distribution of MHS-like Add genotype, being more frequent between the hypertensive than the normotensive strains (Chi-Square = 13.03, p = 0.0003). In kidney, brain, spleen, liver and heart a cDNA differing from gamma subunit by an in-frame insertion of 96 nucleotides, was found by PCR amplification and confirmed by RNase protection analysis. The rat gamma-Add gene was localized to chromosome 1q55 by fluorescence in situ hybridization.     

The carboxy-terminal region of human lipoprotein lipase is necessary for its exit from the endoplasmic reticulum

Certain missense substitutions on the human lipase (hLPL) gene produce mutated proteins that are retained in different compartments along the secretory pathway. The purpose of the present study was to elucidate whether the C-terminal domain of the hLPL molecule could be important for secretion. We constructed by site-directed mutagenesis three carboxy-terminal mutated (F388-->Stop, K428-->Stop and K441-->Stop) hLPL cDNAs that were expressed in COS1 cells. Immunoblotting of cell extracts showed that all three constructs led to similar levels of protein. Both wild type (WT) hLPL and the truncated K441-->Stop hLPL were secreted to the extracellular medium, and presented a similar intracellular distribution pattern as shown by immunofluorescence. Neither F388-->Stop nor K428-->Stop hLPL protein was detected in cell medium. Immunofluorescence experiments showed that both truncated hLPL were retained within an intracellular compartment, which became larger. Double immunofluorescence analysis using antibodies against LPL and antiprotein disulfide isomerase as a marker showed that the truncated K428-->Stop hLPL was retained within the rough endoplasmic reticulum. This truncated protein was not found in other compartments in the secretory pathway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal region of the LPL molecular model showed both that F388-->Stop and K428-->Stop hLPL truncated proteins are highly hydrophobic. As retention of secretory proteins in the rough endoplasmic reticulum is a quality control mechanism of the secretory pathway, we conclude that the C-terminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.     

Electron microscopy of the lesions produced in the human dura mater by Quincke beveled and Whitacre needles

INTRODUCTION: Comparisons of Quincke needles and non traumatic "pencil point" needles in recent years have reported lower rates of post dural puncture headache using the later type. Our new understanding of the morphology of the human dura mater motivated us to study dural lesions caused by the Whitacre 25 G and Quincke 26 G needles, using scanning electron microscopy with the aim of determining whether there is an anatomic basis for the different outcomes. METHOD: The dura mater from three fresh cadavers of individuals aged 65, 70 and 72 years were punctured 40 times at an angle of 90 degrees each time. The Whitacre 25 G needle was used for 20 punctures and the Quincke 26 G needle was used for the other 20. Half the punctures were performed with the bevel in the parallel alignment and the other half with the bevel perpendicular to the spinal column. Fifteen min after causing the punctures, specimens were fixed in solutions of glutaraldehyde phosphate buffer and dehydrated in acetone. After critical point removal of the acetone, after the specimens were treated with carbon and metallized with gold. The lesions were examined externally and internally and expressed as the ratio of area of lesion to diameter of the needle that had caused them. RESULTS: Whitacre needle: each lesion consisted in the superimposition of multiple damaged layers that started to close individually. After 15 min the outermost layers were 90% closed and the innermost ones had closed entirely. Layers in the arachnoid surface of the dura mater had closed from 86 to 88%, while deeper layers in the thick part had closed 97 to 98%. Quincke needle: lesions were V-shaped or half-moon shaped, much like the opening formed by a can opener, on both the external and internal surfaces. Alignment of the bevel of the needle parallel to the spinal column did not lead to a different shape of puncture. After 15 min the lesions had closed 94 to 95% on the epidural surface and 95 to 96% on the arachnoid side, a difference attributable to the retraction of the arachnoid layers over the spinal column. CONCLUSION: Non traumatic beveled dural needles, termed "pencil point needles", only partially separate dural fibers, and lesions caused by these needles develop in a more complex way. The Quincke 26G needle produced a puncture that is morphologically different from that caused by the Whitacre 25G needle, although lesions produced by both types close more than 94% after 15 min. We believe the size of the lesion caused by these needles does not explain the difference in post dural puncture headache due to loss of spinal fluid.