Palmitic acid, but not other long-chain saturated fatty acids, increases S100B protein and TNF-α secretion by astrocytes.

Obesity is a health problem that involves fat accumulation in adipose and other tissues and causes cell dysfunction. Long-chain saturated fatty acids can induce and propagate inflammation, which may also contribute to the brain alterations found in individuals with obesity. Fatty acids accumulate in astrocytes in situations of blood‒brain barrier disruption, such as inflammatory conditions. Furthermore, the increase in tumor necrosis factor-alpha (TNF-α) and S100 calcium-binding protein B (S100B) secretion is considered an essential component of the inflammatory response. We hypothesize that through their action on astrocytes, long-chain saturated fatty acids mediate some of the brain alterations observed in individuals with obesity. Here, we investigate the direct effect of long-chain fatty acids on astrocytes. Primary astrocyte cultures were incubated for 24 hours with myristic, palmitic, stearic, linoleic, or α-linolenic acids (25-100 µM). All saturated fatty acids tested led to an increase in TNF-α secretion, but only palmitic acid, one of the most common fatty acids, increased S100B secretion, indicating that S100B secretion is probably not caused in response to TNF-α release. Palmitic acid also caused nuclear migration of nuclear factor kappa B. Long-chain saturated fatty acids did not alter cell viability or redox status. In conclusion, long-chain saturated fatty acids can alter astrocytic homeostasis and may contribute to brain disorders associated with obesity, such as neuroinflammation.

Glutamine supplementation accelerates functional recovery of EDL muscles after injury by modulating the expression of S100 calcium-binding proteins.

The aim of the current study was to investigate the effect of glutamine supplementation on the expression of HSP70 and the calcium-binding proteins from the S100 superfamily in the recovering extensor digitorum longus (EDL) muscle after injury. Two-month-old Wistar rats were subjected to cryolesion of the EDL muscle and then randomly divided into two groups (with or without glutamine supplementation). Starting immediately after the injury, the supplemented group received daily doses of glutamine (1 g/kg/day, via gavage) for 3 and 10 days orally. Then, muscles were subjected to histological, molecular, and functional analysis. Glutamine supplementation induced an increase in myofiber size of regenerating EDL muscles and prevented the decline in maximum tetanic strength of these muscles evaluated 10 days after injury. An accelerated upregulation of myogenin mRNA levels was detected in glutamine-supplemented injured muscles on day 3 post-cryolesion. The HSP70 expression increased only in the injured group supplemented with glutamine for 3 days. The increase in mRNA levels of NF-κB, the pro-inflammatory cytokines IL-1ß and TNF-α, and the calcium-binding proteins S100A8 and S100A9 on day 3 post-cryolesion in EDL muscles was attenuated by glutamine supplementation. In contrast, the decrease in S100A1 mRNA levels in the 3-day-injured EDL muscles was minimized by glutamine supplementation. Overall, our results suggest that glutamine supplementation accelerates the recovery of myofiber size and contractile function after injury by modulating the expression of myogenin, HSP70, NF-κB, pro-inflammatory cytokines, and S100 calcium-binding proteins.

S100 proteins and their implication in the thalamus: an integrative review / Las proteínas S100 y su implicación en el tálamo: una revisión integradora

SUMMARY: S100 proteins belong group of calcium-binding proteins and are present in physiological intracellular and extracellular regulatory activities, such as cell differentiation, and act in inflammatory and neoplastic pathological processes. Recently, its expressions in the nervous system have been extensively studied, seeking to elucidate its action at the level of the thalamus: A structure of the central nervous system that is part of important circuits, such as somatosensory, behavioral, memory and cognitive, as well as being responsible for the transmission and regulation of information to the cerebral cortex. This article is an integrative review of scientific literature, which analyzed 12 studies present in Pubmed. The analysis showed that the relationship of S100 proteins and the thalamus has been described in neoplastic processes, mental disorders, hypoxia, trauma, stress, infection, Parkinson's disease and epilepsy. In summary, it is possible to conclude that this protein family is relevant as a marker in processes of thalamic injury, requiring further studies to better understand its clinical, preclinical meanings and its prognostic value.

Las proteínas S100 pertenecen al grupo de proteínas fijadoras de calcio y están presentes en actividades reguladoras fisiológicas intracelulares y extracelulares, como la diferenciación celular, y actúan en procesos patológicos inflamatorios y neoplásicos. Recientemente, sus expresiones en el sistema nervioso han sido ampliamente estudiadas, buscando dilucidar su acción a nivel del tálamo: una estructura del sistema nervioso central que forma parte de importantes circuitos, como el somatosensorial, conductual, de memoria y cognitivo, así como además de ser responsable de la transmisión y regulación de la información a la corteza cerebral. Este artículo es una revisión integradora de la literatura científica, que analizó 12 estudios presentes en Pubmed. El análisis mostró que la relación de las proteínas S100 y el tálamo ha sido descrita en procesos neoplásicos, trastornos mentales, hipoxia, trauma, estrés, infección, enfermedad de Parkinson y epilepsia. En resumen, es posible concluir que esta familia de proteínas es relevante como marcador en procesos de lesión talámica, requiriendo más estudios para comprender mejor su significado clínico, preclínico y su valor pronóstico.

Why antidiabetic drugs are potentially neuroprotective during the Sars-CoV-2 pandemic: The focus on astroglial UPR and calcium-binding proteins.

We are living in a terrifying pandemic caused by Sars-CoV-2, in which patients with diabetes mellitus have, from the beginning, been identified as having a high risk of hospitalization and mortality. This viral disease is not limited to the respiratory system, but also affects, among other organs, the central nervous system. Furthermore, we already know that individuals with diabetes mellitus exhibit signs of astrocyte dysfunction and are more likely to develop cognitive deficits and even dementia. It is now being realized that COVID-19 incurs long-term effects and that those infected can develop several neurological and psychiatric manifestations. As this virus seriously compromises cell metabolism by triggering several mechanisms leading to the unfolded protein response (UPR), which involves endoplasmic reticulum Ca2+ depletion, we review here the basis involved in this response that are intimately associated with the development of neurodegenerative diseases. The discussion aims to highlight two aspects-the role of calcium-binding proteins and the role of astrocytes, glial cells that integrate energy metabolism with neurotransmission and with neuroinflammation. Among the proteins discussed are calpain, calcineurin, and sorcin. These proteins are emphasized as markers of the UPR and are potential therapeutic targets. Finally, we discuss the role of drugs widely prescribed to patients with diabetes mellitus, such as statins, metformin, and calcium channel blockers. The review assesses potential neuroprotection mechanisms, focusing on the UPR and the restoration of reticular Ca2+ homeostasis, based on both clinical and experimental data.

RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths.

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix's properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.

Calcium, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD Localization in Testis of Leptodactylus chaquensis (Anura: Leptodactylidae).

The intracellular localization of Ca2+, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD have been studied in testes of the toad Leptodactylus chaquensis, using ultracytochemical and immunohistochemical techniques. The Ca2+ presences in the nucleus and into the mitochondria of the germ cells, together with the activity of Ca2+-ATPase detected in the nuclear envelope and mitochondrial crests, suggest the participation of this transporter in the storage of Ca2+. In Sertoli cells, Ca2+ deposits were also found in vesicles and lamellar bodies. Calmodulin and Calbindin D-28KD were revealed in the cytoplasm of both cell types. At the spermatozoon level, the cation deposits were located in the subacrosomal space and in the acrosomal vesicle. Ca2+-ATPase activity was observed in the acrosomal and plasma membranes of the gamete that suggests the existence of a transport system responsible for maintaining low cytoplasmic Ca2+ levels. The activity of Ca2+-ATPase and the location of Ca2+ deposits in gamete tail would be related to flagellar movement. The colocalization of Ca2+ deposits and their binding proteins in efferent duct cells would probably be associated with secretory activity. Considering that intracellular Ca2+ is present in different gonadal cells, this work would provide a better understanding of the cation importance in the testicular functions of this species.

Engineering a calcium-dependent conformational change in Calbindin D9k by secondary elements replacement.

EF-hand is a common motif in Ca2+-binding proteins, some of which present a conformational change upon Ca2+-binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D9k, a modulator protein with a high affinity for Ca2+ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D9k. Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca2+ affinity and restricting motif conformation.

Simulations on Simple Models of Connexin Hemichannels Indicate That Ca2+ Blocking Is Not a Pure Electrostatic Effect.

Connexin hemichannels allow the unspecific but regulated interchange of molecules from ions to second messenger and ATP, between the eukariotic cell and its extracellular space. The transport of ions and water through hemichannels is important for physiological functions and also in the progression of several pathological conditions. Extracellular Ca2+ concentration is one of the regulators that drives the channel to a closed state. However the relation between their functional and structural states is far for being totally understood. In this work, we modelled connexin hemichannels using simple systems based on a fixed array of carbon atoms and assess the Ca2+ regulation using molecular dynamics simulations. The two proposed mechanism described so far for calcium action were studied combined, e.g., an electrostatic effect and a pore stretching. Our results show that the addition of positive charge density inside the channel cannot stop the flow of potassium, chloride nor water. Only a pore stretching at the center of the pore can explain the channel blocking.

Aging Alters Daily and Regional Calretinin Neuronal Expression in the Rat Non-image Forming Visual Thalamus.

Aging affects the overall physiology, including the image-forming and non-image forming visual systems. Among the components of the latter, the thalamic retinorecipient inter-geniculate leaflet (IGL) and ventral lateral geniculate (vLGN) nucleus conveys light information to subcortical regions, adjusting visuomotor, and circadian functions. It is noteworthy that several visual related cells, such as neuronal subpopulations in the IGL and vLGN are neurochemically characterized by the presence of calcium binding proteins. Calretinin (CR), a representative of such proteins, denotes region-specificity in a temporal manner by variable day-night expression. In parallel, age-related brain dysfunction and neurodegeneration are associated with abnormal intracellular concentrations of calcium. Here, we investigated whether daily changes in the number of CR neurons are a feature of the aged IGL and vLGN in rats. To this end, we perfused rats, ranging from 3 to 24 months of age, within distinct phases of the day, namely zeitgeber times (ZTs). Then, we evaluated CR immunolabeling through design-based stereological cell estimation. We observed distinct daily rhythms of CR expression in the IGL and in both the retinorecipient (vLGNe) and non-retinorecipient (vLGNi) portions of the vLGN. In the ZT 6, the middle of the light phase, the CR cells are reduced with aging in the IGL and vLGNe. In the ZT 12, the transition between light to dark, an age-related CR loss was found in all nuclei. While CR expression predominates in specific spatial domains of vLGN, age-related changes appear not to be restricted at particular portions. No alterations were found in the dark/light transition or in the middle of the dark phase, ZTs 0, and 18, respectively. These results are relevant in the understanding of how aging shifts the phenotype of visual related cells at topographically organized channels of visuomotor and circadian processing.

Immunolocalización de s100a4 y α-sma en tejidos gingivales de pacientes con hipertrofia gingival por tratamiento ortodóntico: estudio preliminar / Immunolocalization of s100a4 and α-sma in gingival tissues of patients with gingival hypertrophy due to orthodontic treatment: preliminary study

Objective: to determine the presence and distribution of markers of the epithelialmesenchymal transition (EMT) (S-100A4 and alpha-smooth muscle actin-α-SMA) in gingival tissues of patients affected by Gingival hypertrophy (GH) due to orthodontics.GH is an exaggerated increase in gingival tissue whose pathogenesis is unknown. However, it has been reported that the epithelial-mesenchymal transition as a process involved in other types of GH. Materials and methods: descriptive study that included the analysis of gingival tissues of healthy individuals (n = 6) and patients with GH by orthodontic treatment (n = 6). Before gingival surgery, the patients underwent a periodontal hygiene phase. The gingival tissue samples obtained were processed and embedded in paraffin. The cuts were made with a microtome and deposited on polysine adhesion slides. Histological hematoxylin-eosin staining was performed.The identification and location of S-100A4 and α-SMA markers was determined by immunohistochemistry with monoclonal antibodies. The reading of the findings was carried out by oral pathologists. Results: in healthy individuals, an S100A4 label was observed in Langerhans cells, while α-SMA was identified in the vascular endothelium of all samples analysed. However, in patients with GH due to orthodontics, they registered an intense staining of S100A4 in gingival fibroblasts, Langerhans cells, vascular endothelium, and areas adjacent to the rupture of blood vessel. α-SMA expression in GO was detected in the vascular endothelium and gingival fibroblasts. Conclusion: the differential immunostaining of EMT markers in gingival tissues of patients with orthodontic GH suggests an eventual role of EMT in the pathogenesis of this pathology..Au

Objective: to determine the presence and distribution of markers of the epithelialmesenchymal transition (EMT) (S-100A4 and alpha-smooth muscle actin-α-SMA) in gingival tissues of patients affected by Gingival hypertrophy (GH) due to orthodontics. GH is an exaggerated increase in gingival tissue whose pathogenesis is unknown. However, it has been reported that the epithelial-mesenchymal transition as a process involved in other types of GH. Materials and methods: descriptive study that included the analysis of gingival tissues of healthy individuals (n = 6) and patients with GH by orthodontic treatment (n = 6). Before gingival surgery, the patients underwent a periodontal hygiene phase. The gingival tissue samples obtained were processed and embedded in paraffin. The cuts were made with a microtome and deposited on polysine adhesion slides. Histological hematoxylin-eosin staining was performed. The identification and location of S-100A4 and α-SMA markers was determined by immunohistochemistry with monoclonal antibodies. The reading of the findings was carried out by oral pathologists. Results: in healthy individuals, an S100A4 label was observed in Langerhans cells, while α-SMA was identified in the vascular endothelium of all samples analysed. However, in patients with GH due to orthodontics, they registered an intense staining of S100A4 in gingival fibroblasts, Langerhans cells, vascular endothelium, and areas adjacent to the rupture of blood vessel. α-SMA expression in GO was detected in the vascular endothelium and gingival fibroblasts. Conclusion: the differential immunostaining of EMT markers in gingival tissues of patients with orthodontic GH suggests an eventual role of EMT in the pathogenesis of this pathology..Au

Calcium Binding Protein Ncs1 Is Calcineurin Regulated in Cryptococcus neoformans and Essential for Cell Division and Virulence.

Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+ binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca2+ sensor in C. neoformans, working with calcineurin to regulate Ca2+ homeostasis and, consequently, promote fungal growth and virulence.IMPORTANCECryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca2+ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.

A Proline Derivative-Enriched Fraction from Sideroxylon obtusifolium Protects the Hippocampus from Intracerebroventricular Pilocarpine-Induced Injury Associated with Status Epilepticus in Mice.

The N-methyl-(2S,4R)-trans-4-hydroxy-l-proline-enriched fraction (NMP) from Sideroxylon obtusifolium was evaluated as a neuroprotective agent in the intracerebroventricular (icv) pilocarpine (Pilo) model. To this aim, male mice were subdivided into sham (SO, vehicle), Pilo (300 µg/1 µL icv, followed by the vehicle per os, po) and NMP-treated groups (Pilo 300 µg/1 µL icv, followed by 100 or 200 mg/kg po). The treatments started one day after the Pilo injection and continued for 15 days. The effects of NMP were assessed by characterizing the preservation of cognitive function in both the Y-maze and object recognition tests. The hippocampal cell viability was evaluated by Nissl staining. Additional markers of damage were studied-the glial fibrillary acidic protein (GFAP) and the ionized calcium-binding adaptor molecule 1 (Iba-1) expression using, respectively, immunofluorescence and western blot analyses. We also performed molecular docking experiments revealing that NMP binds to the γ-aminobutyric acid (GABA) transporter 1 (GAT1). GAT1 expression in the hippocampus was also characterized. Pilo induced cognitive deficits, cell damage, increased GFAP, Iba-1, and GAT1 expression in the hippocampus. These alterations were prevented, especially by the higher NMP dose. These data highlight NMP as a promising candidate for the protection of the hippocampus, as shown by the icv Pilo model.

The Leptospira interrogans LIC10774 is a multifunctional surface protein that binds calcium and interacts with host components.

Leptospirosis is a global re-emerging zoonosis, caused by pathogenic bacteria of the genus Leptospira. Humans are infected mainly through contact with contaminated water or soil. The understanding of the molecular mechanisms of leptospirosis through the characterization of unknown outer membrane proteins may contribute to the development of new treatments, diagnostic methods and vaccines. We have identified using bioinformatics analysis a protein that is encoded by the gene LIC10774, predicted to be localized at the leptospiral outer membrane and exhibit beta-roll folding. Surface exposure was confirmed by flow cytometry, ELISA and immunofluorescence-based confocal microscopy. Through circular dichroism spectroscopy and hydrophobic dye binding we have shown that rLIC10774 binds calcium ions, which imposes changes to secondary and tertiary structures. The recombinant protein was capable of binding to several host extracellular matrix and serum components. Therefore, we describe LIC10774 as a calcium-binding protein exposed in the outer surface of pathogenic leptospires with possible multifunctional roles in adhesion to host tissues, evasion of the immune system and participation in dissemination processes during leptospirosis. In addition, we hypothesize that the calcium binding is important for temperature-dependent functional roles during leptospirosis.

The Leptospira interrogans LIC10774 is a multifunctional surface protein that binds calcium and interacts with host components

Leptospirosis is a global re-emerging zoonosis, caused by pathogenic bacteria of the genus Leptospira. Humans are infected mainly through contact with contaminated water or soil. The understanding of the molecular mechanisms of leptospirosis through the characterization of unknown outer membrane proteins may contribute to the development of new treatments, diagnostic methods and vaccines. We have identified using bioinformatics analysis a protein that is encoded by the gene LIC10774, predicted to be localized at the leptospiral outer membrane and exhibit beta-roll folding. Surface exposure was confirmed by flow cytometry, ELISA and immunofluorescence-based confocal microscopy. Through circular dichroism spectroscopy and hydrophobic dye binding we have shown that rLIC10774 binds calcium ions, which imposes changes to secondary and tertiary structures. The recombinant protein was capable of binding to several host extracellular matrix and serum components. Therefore, we describe LIC10774 as a calcium-binding protein exposed in the outer surface of pathogenic leptospires with possible multifunctional roles in adhesion to host tissues, evasion of the immune system and participation in dissemination processes during leptospirosis. In addition, we hypothesize that the calcium binding is important for temperature-dependent functional roles during leptospirosis

A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica.

Oxygen or nitrogen oxidative species and chemical stress induce the programmed cell death (PCD) of Entamoeba histolytica trophozoites. PCD caused by the aminoglycoside G418 is reduced by incubation with the cysteine protease inhibitor E-64; however, no typical caspases or metacaspases have been detected in this parasite. Calpain, a cysteine protease activated by calcium, has been suggested to be part of a specific PCD pathway in this parasite because the specific calpain inhibitor Z-Leu-Leu-Leu-al diminishes the PCD of trophozoites. Here, we predicted the hypothetical 3D structure of a calpain-like protein of E. histolytica and produced specific antibodies against it. We detected the protein in the cytoplasm and near the nucleus. Its expression gradually increased during incubation with G418, with the highest level after 9 h of treatment. In addition, a specific calpain-like siRNA sequence reduced the cell death rate by 65%. All these results support the hypothesis that the calpain-like protein is one of the proteases involved in the execution phase of PCD in E. histolytica. The hypothetical interactome of the calpain-like protein suggests that it may activate or regulate other proteins that probably participate in PCD, including those with EF-hand domains or other calcium-binding sites.

Glial fibrillary acidic protein levels are associated with global histone H4 acetylation after spinal cord injury in rats.

Emerging evidence has suggested global histone H4 acetylation status plays an important role in neural plasticity. For instance, the imbalance of this epigenetic marker has been hypothesized as a key factor for the development and progression of several neurological diseases. Likewise, astrocytic reactivity - a well-known process that markedly influences the tissue remodeling after a central nervous system injury - is crucial for tissue remodeling after spinal cord injury (SCI). However, the linkage between the above-mentioned mechanisms after SCI remains poorly understood. We sought to investigate the relation between both glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100B) (astrocytic reactivity classical markers) and global histone H4 acetylation levels. Sixty-one male Wistar rats (aged ~3 months) were divided into the following groups: sham; 6 hours post-SCI; 24 hours post-SCI; 48 hours post-SCI; 72 hours post-SCI; and 7 days post-SCI. The results suggested that GFAP, but not S100B was associated with global histone H4 acetylation levels. Moreover, global histone H4 acetylation levels exhibited a complex pattern after SCI, encompassing at least three clearly defined phases (first phase: no changes in the 6, 24 and 48 hours post-SCI groups; second phase: increased levels in the 72 hours post-SCI group; and a third phase: return to levels similar to control in the 7 days post-SCI group). Overall, these findings suggest global H4 acetylation levels exhibit distinct patterns of expression during the first week post-SCI, which may be associated with GFAP levels in the perilesional tissue. Current data encourage studies using H4 acetylation as a possible biomarker for tissue remodeling after spinal cord injury.

In silico analysis of the EF-hand proteins in the genome of Giardia intestinalis assembly A.

Giardia intestinalis is a parasite that inhabits the small intestine of humans and other mammals, causing a disease that can manifest itself with acute diarrhea. This parasite is an early divergent eukaryote with a compact genome and a life cycle composed of two distinct cell types: the trophozoite, the replicative form, and the cyst, the infectious form. Signal transduction pathways implicated in differentiation processes of G. intestinalis are largely unknown. Calcium, considered an essential messenger in cell signaling, has been shown to regulate a myriad of key cell processes including metabolism, motility, and exocytosis, among other important functions, through calcium-binding proteins (CaBPs). The most important and largest family of CaBPs is the EF-hand protein family. To investigate the nature of calcium signaling pathways present in this protozoan, an in silico analysis of the genome to identify genes encoding EF-hand proteins was undertaken. Twenty-eight sequences containing EF-hand domains were found; most of which have only a pair of domains, and half of the sequences were divergent or unique to Giardia. In addition, the transcription pattern for eight genes encoding EF-hand proteins was assessed during encystation. It was found that all the genes were differentially transcribed suggesting a different function in this process. The in silico results suggest that in G. intestinalis, calcium is involved in the regulation of protein phosphorylation through kinases and phosphatases.

Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity.

Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.

Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca2+ Ion.

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca2+ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a µs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca2+, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca2+-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca2+ affinity in site II, thus opening up new insights into the HCM phenotype.

La inmunorreactividad a calbindina en células de purkinje del cerebelo de ratones no es afectada por la infección con virus de la rabia / Immunoreactivity to calbindin in purkinje cells in the cerebellum of mice is not affected by rabies virus infection

Introducción: La calbindina (CB) es una proteína reguladora del calcio intracelular y la célula de Purkinje del cerebelo es la neurona con más alta concentración de CB. Se ha demostrado pérdida de inmunorreactividad a CB en diferentes áreas del sistema nervioso en ratones inoculados con virus de la rabia, pero faltaba estudiar este fenómeno en el cerebelo. Objetivo: Determinar el efecto de la inoculación con virus de la rabia sobre la expresión de CB en células de Purkinje del cerebelo de ratones. Metodología: Se inocularon ratones con el virus por vía intramuscular. Se sacrificaron los animales cuando alcanzaron la fase avanzada de la enfermedad y se fijaron mediante perfusión intracardiaca con paraformaldehído al 4%. Se les extrajo el cerebelo y se hicieron cortes sagitales de 50 micrómetros de espesor en un vibrátomo. Estos se procesaron mediante inmunohistoquímica para revelar la presencia de CB o de antígenos del virus de la rabia. El mismo procedimiento se realizó con animales no infectados (controles). Resultados: Las células de Purkinje fueron masivamente infectadas con el virus de la rabia. En las imágenes panorámicas observadas en el microscopio se comprobó que sólo estas células fueron inmunorreactivas a CB. No se hallaron diferencias significativas en la inmunorreactividad a CB, evaluada por densitometría óptica, entre los animales infectados y los controles. Conclusión: La expresión de CB en las células de Purkinje del cerebelo parece no afectarse por la infección con rabia, a diferencia de lo que se ha demostrado en otras áreas del sistema nervioso del ratón.

Introduction: Calbindin (CB) is a regulatory protein of intracellular calcium, and the cerebellar Purkinje cell is the neuron with the highest concentration of CB. Loss of CB immunoreactivity has been demonstrated in different areas of the nervous system in rabies virus-infected mice, but the study of this phenomena in the cerebellum lacked. Objective: To determine the effect of inoculation with rabies virus on the expression of CB in Purkinje cells of the cerebellum of mice. Methodology: Mice were intramuscularly inoculated with rabies virus. Animals were sacrificed when they reached an advanced stage of the disease and then they were fixed by intracardiac perfusion with 4% paraformaldehyde. Cerebellums were extracted and sagittal sections 50 microns thick were obtained in a vibratome. These were processed by immunohistochemistry to reveal the presence of CB protein or rabies virus antigens. The same procedure was performed with uninfected animals (controls). Results: Purkinje cells were massively infected with rabies virus. In the microscopic panoramic images observed was found that only these cells are immunoreactive to CB. No significant difference in CB immunoreactivity evaluated by optical densitometry was found between infected animals and controls. Conclusion: The expression of CB in Purkinje cells of the cerebellum appears not to be affected by infection with rabies unlike what has been shown in other areas of the mouse nervous system.