Thermal stability and aggregation of sulfolobus solfataricus b-glycosidase are dependent upon the N-e-methylation of specific lysyl residues: critical role of in vivo post-translational modifications.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricu

Metabolic control and sex: A focus on inflammatory-linked mediators

Men and women have many differing biological and physiological characteristics. Thus, it is no surprise that the control of metabolic processes and the mechanisms underlying metabolic-related diseases have sex-specific components. There is a clear metabolic sexual dimorphism in that up until midlife, men have a far greater likelihood of acquiring cardio-metabolic disease than women. Following menopause, however, this difference is reduced, suggestive of a protective role of the female sex hormones. Inflammatory processes have been implicated in the pathogenesis of cardio-metabolic disease with human studies correlating metabolic disease acquisition or risk with levels of various inflammatory markers. Rodent studies employing genetic modifications or novel pharmacological approaches have provided mechanistic insight into the role of these inflammatory mediators. Sex differences impact inflammatory processes and the subsequent biological response. As a consequence, this may affect how inflammation alters metabolic processes between the sexes. Recently, some of our work in the field of inflammatory genes and metabolic control identified a sexual dimorphism in a preclinical model and caused us to question the frequency and scale of such findings in the literature. This review concentrates on inflammatory-related signalling in relation to obesity, insulin resistance, and type 2 diabetes and highlights the differences observed between males and females. Differences in the activation and signalling of various inflammatory genes and proteins present another reason why studying both male and female patients or animals is important in the context of understanding and finding therapeutics for metabolic-related disease

Reduced glycogen availability is associated with increased AMPKα2 activity, nuclear AMPKα2 protein abundance, and GLUT4 mRNA expression in contracting human skeletal muscle

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK [alpha]2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK [alpha]2 activity reduced the association of AMPK with glycogen and increased AMPK [alpha]2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at ~70% [VO.sub.2] peak on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK [alpha]2 activity and acetyl-CoA carboxylase [beta] phosphorylation, increased translocation of AMPK [alpha]2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK [alpha]2 under conditions of low muscle glycogen enhances AMPK [alpha]2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle. <br /

Bone marrow chimeric mice reveal a dual role for CD36 in Plasmodium berghei ANKA infection

BACKGROUND: Adhesion of Plasmodium-infected red blood cells (iRBC) to different host cells, ranging from endothelial to red blood cells, is associated to malaria pathology. In vitro studies have shown the relevance of CD36 for adhesion phenotypes of Plasmodium falciparum iRBC such as sequestration, platelet mediated clumping and non-opsonic uptake of iRBC. Different adhesion phenotypes involve different host cells and are associated with different pathological outcomes of disease. Studies with different human populations with CD36 polymorphisms failed to attribute a clear role to CD36 expression in human malaria. Up to the present, no in vivo model has been available to study the relevance of different CD36 adhesion phenotypes to the pathological course of Plasmodium infection. METHODS: Using CD36-deficient mice and their control littermates, CD36 bone marrow chimeric mice, expressing CD36 exclusively in haematopoietic cells or in non-haematopoietic cells, were generated. Irradiated CD36(-/- )and wild type mice were also reconstituted with syngeneic cells to control for the effects of irradiation. The reconstituted mice were infected with Plasmodium berghei ANKA and analysed for the development of blood parasitaemia and neurological symptoms. RESULTS: All mice reconstituted with syngeneic bone marrow cells as well as chimeric mice expressing CD36 exclusively in non-haematopoietic cells died from experimental cerebral malaria between day 6 and 12 after infection. A significant proportion of chimeric mice expressing CD36 only in haematopoietic cells did not die from cerebral malaria. CONCLUSION: The analysis of bone marrow chimeric mice reveals a dual role of CD36 in P. berghei ANKA infection. Expression of CD36 in haematopoietic cells, most likely macrophages and dendritic cells, has a beneficial effect that is masked in normal mice by adverse effects of CD36 expression in non-haematopoietic cells, most likely endothelial cells

Infection by and protective immune responses against Plasmodium berghei ANKA are not affected in macrophage scavenger receptors A deficient mice

BACKGROUND: Scavenger receptors (SRs) recognize endogenous molecules modified by pathological processes as well as components of diverse microorganisms. Mice deficient for both SR-AI and II are more susceptible to infections by a variety of bacterial and viral pathogens. RESULTS: Here we show that SR-A deficient mice and wild type mice are equally susceptible to malaria infection both during liver and blood stages. Moreover, like wild type mice, SR-A deficient mice are able to mount a protective immune response against radiation attenuated sporozoites. CONCLUSION: Our results do not reveal a function of SR-A I and II receptors in the Plasmodium berghei ANKA infection, both in the development of CM and parasitemia control. Moreover, these receptors appear not to be required for the establishment of a protective immune response against the malaria liver stages

Multiple Metabolic Hits Converge on CD36 as Novel Mediator of Tubular Epithelial Apoptosis in Diabetic Nephropathy

BACKGROUND: Diabetic nephropathy (DNP) is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED) and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood. METHODS AND FINDINGS: We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC) apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA) and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis. CONCLUSION: Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP

CD11b/CD18 mediates production of reactive oxygen species by mouse and human macrophages adherent to matrixes containing oxidized LDL

Production of reactive oxygen species (ROS) and other proinflammatory substances by macrophages adherent to matrix proteins that contain or have been modified by oxidized LDL (oxLDL) may play an important role in atherogenesis. In vitro, human macrophages adhere to matrixes containing oxLDL via scavenger receptors and are signaled to produce ROS partly by interactions of the class B scavenger receptor (SR-B) CD36 with ligands on the matrix. In this report, we show that macrophages from mice genetically deficient in SR-A or CD36 adhered equally as well and produced equal amounts of ROS on interaction with matrix-associated oxLDL. In contrast, macrophages from mice genetically deficient in the CD18 chain of β2-integrins produced insignificant amounts of ROS on interaction with oxLDL-containing matrixes, even though they adhered to these matrixes as efficiently as did macrophages from wild-type mice. Antibodies against CD18, CD11b, or EDTA, the last of which chelates divalent cations required for integrin function, had no effect on adhesion of normal mouse or human macrophages to matrixes containing oxLDL but almost completely inhibited ROS production by macrophages adherent to this matrix. Thus, CD11b/CD18 plays an important role in regulating production of ROS by mouse and human macrophages adherent to matrixes containing oxLDL. It may play a hitherto-unsuspected role in regulating macrophage signaling pathways involved in inflammation and atherogenesis

Relevance of arginine residues in Cu(II)-induced DNA breakage and Proteinase K resistance of H1 histones

This work analyzes the involvement of arginines in copper/H2O2-induced DNA breakage. Copper is a highly redox active metal which has been demonstrated to form compounds with arginines. For this aim we used mixtures of pGEM3 DNA plasmid and two types of H1 histones which differ only in their arginine content. The sperm H1 histone from the annelid worm Chaetopterus variopedatus (arginine content 12.6 mol% K/R ratio 2) and the somatic H1 histone from calf thymus (arginine content 1.8 mol% and K/R ratio 15). Copper/H2O2-induced DNA breakage was observed only in presence of sperm H1 histones, but it was more relevant for the native molecule than for the deguanidinated derivative (K/R ratio 14), in which 80% of arginine residues were converted to ornithine. Further, copper induced proteinase K resistance and increase of DNA binding affinity on native sperm H1 histones. These observations are consistent with a copper induced reorganization of the side-chains of arginine residues. Copper, instead, did not affect DNA binding affinity of somatic and deguanidinated H1 histones, which show similar K/R ratio and DNA binding mode. These results indicate that arginine residues could affect these H1 histones properties and provide new insights into copper toxicity mechanisms

Point-of-care diagnostics of covid-19: From current work to future perspectives

Coronaviruses have received global concern since 2003, when an outbreak caused by SARS‐CoV emerged in China. Later on, in 2012, the Middle‐East respiratory syndrome spread in Saudi Arabia, caused by MERS‐CoV. Currently, the global crisis is caused by the pandemic SARS‐ CoV‐2, which belongs to the same lineage of SARS‐CoV. In response to the urgent need of diagnostic tools, several lab‐based and biosensing techniques have been proposed so far. Five main areas have been individuated and discussed in terms of their strengths and weaknesses. The cell‐culture detection and the microneutralization tests are still considered highly reliable methods. The genetic screening, featuring the well‐established Real‐time polymerase chain reaction (RT‐PCR), represents the gold standard for virus detection in nasopharyngeal swabs. On the other side, immunoassays were developed, either by screening/antigen recognition of IgM/IgG or by detecting the whole virus, in blood and sera. Next, proteomic mass‐spectrometry (MS)‐based methodologies have also been proposed for the analysis of swab samples. Finally, virus-biosensing devices were efficiently designed. Both electrochemical immunosensors and eye‐based technologies have been described, showing detection times lower than 10 min after swab introduction. Alternative to swab‐based techniques, lateral flow point‐of‐care immunoassays are already commercially available for the analysis of blood samples. Such biosensing devices hold the advantage of being portable for on‐site testing in hospitals, airports, and hotspots, virtually without any sample treatment or complicated lab precautions