PARTICIPACIÓN DE RESIDUOS ÁCIDOS EN EL FUNCIONAMIENTO DE LA PROTEÍNA ChrA DE Pseudomonas aeruginosa

ChrA es una proteína de membrana que confiere resistencia al oxianión cromato en Pseudomonas aeruginosa, a través de la expulsión del cromato al exterior celular. La topología de ChrA en la membrana consiste de 13 segmentos transmembranales (STMs), unidos por seis asas citoplásmicas (C1-C6) y seis asas periplásmicas (P1-P6). ChrA pertenece a la familia de transportadores LCHR, a la cual también pertenece la proteína ChrA de Cupriavidus metallidurans y otras 75 proteínas homólogas no caracterizadas. Empleando la técnica de mutagénesis sitio dirigida se identificaron algunos residuos neutros y básicos esenciales para el funcionamiento de ChrA, a los cuales se les ha asignado un papel estructural o funcional. El objetivo de este trabajo fue identificar residuos ácidos (Aspartato y Glutamato) esenciales para el funcionamiento de la proteína ChrA. La selección de residuos ácidos como blanco de mutagénesis dirigida se hizo tomando en cuenta aquellos residuos que presentaran una conservación de su carga negativa igual o mayor al 50% en homólogos de ChrA. En total se mutaron ocho residuos ácidos por Alanina. Las mutantes obtenidas fueron sometidas a pruebas de susceptibilidad a cromato, determinándose a partir de este ensayo la esencialidad para ChrA de los residuos mutados. Los residuos Glu60, Glu230 y Asp281 (ubicados en asas hidrofílicas) mostraron no ser importantes para el funcionamiento de ChrA. El residuo Asp59 (ubicado en el asa P1) resultó ser importante para el funcionamiento de la proteína. El residuo Glu56 (ubicado en el asa periplásmica P1), así como los residuos Glu83, Glu86 y Asp162 (ubicados en STMs), resultaron ser esenciales para el funcionamiento de la proteína ChrA. Los resultados indican que el asa P1 de ChrA participa de manera importante en el proceso de expulsión de cromato. Los datos también sugieren que los residuos ácidos presentes en STMs están involucrados en generar una estructura correcta de ChrA en la membrana, relacionada con su función como transportador de cromato

Neutrophil Functions in Periodontal Homeostasis

Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed

GSK3α: An Important Paralog in Neurodegenerative Disorders and Cancer

The biological activity of the enzyme glycogen synthase kinase-3 (GSK3) is fulfilled by two paralogs named GSK3α and GSK3β, which possess both redundancy and specific functions. The upregulated activity of these proteins is linked to the development of disorders such as neurodegenerative disorders (ND) and cancer. Although various chemical inhibitors of these enzymes restore the brain functions in models of ND such as Alzheimer’s disease (AD), and reduce the proliferation and survival of cancer cells, the particular contribution of each paralog to these effects remains unclear as these molecules downregulate the activity of both paralogs with a similar efficacy. Moreover, given that GSK3 paralogs phosphorylate more than 100 substrates, the simultaneous inhibition of both enzymes has detrimental effects during long-term inhibition. Although the GSK3β kinase function has usually been taken as the global GSK3 activity, in the last few years, a growing interest in the study of GSK3α has emerged because several studies have recognized it as the main GSK3 paralog involved in a variety of diseases. This review summarizes the current biological evidence on the role of GSK3α in AD and various types of cancer. We also provide a discussion on some strategies that may lead to the design of the paralog-specific inhibition of GSK3α

Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens

Abstract Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.</p

Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens

Background: Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.Non UBCScience, Faculty ofMicrobiology and Immunology, Department ofReviewedFacult

Behavior of Eosinophil Counts in Recovered and Deceased COVID-19 Patients over the Course of the Disease

Knowledge about the immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, particularly regarding the function of eosinophils, has been steadily emerging recently. There exists controversy regarding the implications of eosinophils in the coronavirus disease 2019 (COVID-19)’s pathology. We report a retrospective cohort study including the comparison of leukocyte counts in COVID-19 patients, considering the outcomes of recovery (n = 59) and death (n = 60). Among the different types of leukocytes, the eosinophil counts were those that showed the greatest difference between recovered and deceased patients. Eosinopenia (eosinophil count &lt; 0.01 × 109/L) was more frequently observed in deceased than recovered patients (p = 0.0012). The eosinophil counts more rapidly increased and showed a greater proportion over the course of the disease in the recovered than deceased patients. Furthermore, the estimated survival rate was greater in patients without eosinopenia than in patients with eosinopenia (p = 0.0070) during hospitalization. Importantly, recovered but not deceased patients showed high negative correlations of the eosinophils with the neutrophil-to-lymphocyte ratio (NLR) and neutrophil counts at Day 9 of the onset of clinical symptoms (p ≤ 0.0220). Our analysis suggests that eosinopenia may be associated with unfavorable disease outcomes and that the eosinophils have a beneficial function in COVID-19 patients, probably contributing by controlling the exacerbated inflammation induced by neutrophils

The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus

PGN induces IL-12p40 expression through TLR2/Akt activation and GSK3 inhibition in BEC.

<p>A) BEC were left untreated and unstimulated (0) or stimulated with 10 μg/mL of PGN-prep for 2, 4 or 8 h. B) BEC were stimulated with 10 μg/mL of PGN for 4 h, treated with 5 μg/mL of neutralizing anti-TLR2 for 60 min, or treated with 5 μg/mL of anti-TLR2 for 60 min and then stimulated with 10 μg/mL of PGN for 4 h. C) BEC were stimulated with 10 μg/mL of PGN for 9 h or treated with 5 μg/mL of anti-TLR2 for 60 min and then stimulated with 10 μg/mL of PGN for 9 h. D) BEC were stimulated with 10 μg/mL of PGN for 9 h or pretreated with 1 μM of Akt inhibitor IV (Akt-i IV) for 30 min and then stimulated with 10 μg/mL for 9 h. E) BEC were treated with 10 mM NaCl for 60 min, stimulated with 10 μg/mL of PGN for 9 h, treated with 10 mM of LiCl for 60 min or treated with 10 mM of LiCl for 60 min and then stimulated with 10 μg/mL of PGN for 9 h. F) BEC were treated with 10 μM of DMSO, stimulated with 10 μg/mL of PGN for 9 h, treated with 10 μM of SB 216763 (SB) for 30 min or treated with 10 μM of SB 216763 (SB) for 30 min and then stimulated with 10 μg/mL of PGN for 9 h. As controls, in B-F BEC were left untreated and stimulated (-). Total RNA was extracted and relative transcript level of IL-12p40 was quantitated by qRT-PCR using the delta-delta Ct method, and amplification of β-actin as a reference gene (A-B). Cell-free supernatants were analyzed by ELISA for production of IL-12p40 (C-F). Results are expressed as the mean ± S.E.M. (<i>n</i> = 3). *<i>p</i> <0.05; **<i>p</i> <0.01. All data were compared with the untreated and unstimulated controls.</p

PGN induces PI3K/Akt-dependent phosphorylation of GSK3α and GSK3β in BEC.

<p>A-B) BEC were untreated and unstimulated (U1 and U2), stimulated with 10 μg/mL of PGN for 30 min (P1 and P2) or treated with either 10 μM of LY294002 (LY), 100 nM of Wortmannin (Wort) or 10 μM of SH-5 for 30 min, and then stimulated with 10 μg/mL of PGN for 30 min. Protein extracts were analyzed by western blot and probed with monoclonal antibodies against the phosphorylated forms of GSK3α (pGSK3α Ser21) or GSK3β (pGSK3β Ser9. To verify for equal amount of proteins, blots were stripped and reprobed with an antibody that recognizes the nonphosphorylated form of GSK3β. Blots are representative of three independent experiments. Graphs indicate the band intensity obtained by densitometric analysis. In each graph, the densitometric control values plotted were the average of U1 + U2 while the values plotted for the PGN-stimulated cells were the average of P1 + P2. Results are expressed as the mean ± S.E.M. (<i>n</i> = 3). *<i>p</i> < 0.05; **<i>p</i> < 0.01, compared with the unstimulated control.</p