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One of the hallmarks of Alzheimer's disease (AD) is the accumulation of beta-amyloid peptide (Aß) leading to formation of soluble neurotoxic Aß oligomers and insoluble amyloid plaques in various parts of the brain. Aß undergoes post-translational modifications that alter its pathogenic properties. Aß is produced not only in brain, but also in the peripheral tissues. Such Aß, including its post-translationally modified forms, can enter the brain from circulation by binding to RAGE and contribute to the pathology of AD. However, the transport of modified forms of Aß across the blood-brain barrier (BBB) has not been investigated. Here, we used a transwell BBB model as a controlled environment for permeability studies. We found that Aß42 containing isomerized Asp7 residue (iso-Aß42) and Aß42 containing phosphorylated Ser8 residue (pS8-Aß42) crossed the BBB better than unmodified Aß42, which correlated with different contribution of endocytosis mechanisms to the transport of these isoforms. Using microscale thermophoresis, we observed that RAGE binds to iso-Aß42 an order of magnitude weaker than to Aß42. Thus, post-translational modifications of Aß increase the rate of its transport across the BBB and modify the mechanisms of the transport, which may be important for AD pathology and treatment.
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In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.
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Doença de Alzheimer , Neoplasias , Camundongos , Humanos , Animais , Cobre , Doença de Alzheimer/diagnóstico , Íons , Técnicas EletroquímicasRESUMO
Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis (ΔgmhA) prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress. We found that the derepression of purine biosynthesis (∆purR) normalizes the metabolic equilibrium in PPP in ΔgmhA mutants, suppressing the negative effects of gmhA mutation likely via the over-expression of the glycine-serine pathway that is under the negative control of PurR and might be responsible for the enhanced synthesis of NADPH and glutathione. Consistently, the activity of the soxRS system, as well as the level of glutathionylation and oxidation of proteins, indicative of oxidative stress, were reduced in the double ΔgmhAΔpurR mutant compared to the ΔgmhA mutant.
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Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , NADP/metabolismo , Purinas/farmacologia , Purinas/metabolismo , Heptoses/química , Heptoses/metabolismo , Glutationa/metabolismo , Via de Pentose FosfatoRESUMO
ß-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of ß-amyloid (Aß) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss, etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules and consequently alterations of cell mechanical properties. The effect of amyloid oligomers on cells' Young's modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on the local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young's modulus mapping, and confocal imaging of Aß aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aß leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aß aggregates on living cell surfaces.
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Peptídeos beta-Amiloides , Microscopia , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/química , Citoesqueleto/metabolismo , Membrana Celular/metabolismo , Amiloide/química , Fragmentos de Peptídeos/químicaRESUMO
Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein's thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells.
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Antioxidantes , Oxiemoglobinas , Humanos , Triptofano , Hemoglobinas , Glutationa , Heme , OxigênioRESUMO
Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.
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Doença de Alzheimer , Humanos , Animais , Camundongos , Doença de Alzheimer/metabolismo , Zinco/metabolismo , Taiwan , Placa Amiloide , Peptídeos beta-Amiloides/metabolismo , Isoformas de Proteínas/genética , Mutação , ÍonsRESUMO
The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme-a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of C-methylated analogs of Spd. The biochemical properties of these compounds and the possibility for the regulation of their activity by moving a methyl group along the polyamine backbone and by changing the stereochemistry of the chiral center(s) are discussed.
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Poliaminas Biogênicas , Espermidina , Poliaminas/metabolismo , Espermina/metabolismo , HomeostaseRESUMO
The deposition of beta-amyloid (Aß) aggregates in the brain, accompanied by impaired cognitive function, is a characteristic feature of Alzheimer's disease (AD). An important role in this process is played by vascular disorders, in particular, a disturbance of the blood-brain barrier (BBB). The BBB controls the entry of Aß from plasma to the brain via the receptor for advanced glycation end products (RAGE) and the removal of brain-derived Aß via the low-density lipoprotein receptor-related protein (LRP1). The balance between the input of Aß to the brain from the periphery and its output is disturbed during AD. Aß changes the redox-status of BBB cells, which in turn changes the functioning of mitochondria and disrupts the barrier function of endothelial cells by affecting tight junction proteins. Aß oligomers have the greatest toxic effect on BBB cells, and oligomers are most rapidly transferred by transcytosis from the brain side of the BBB to the blood side. Both the cytotoxic effect of Aß and the impairment of barrier function are partly due to the interaction of Aß monomers and oligomers with membrane-bound RAGE. AD therapies based on the disruption of this interaction or the creation of decoys for Aß are being developed. The question of the transfer of various Aß isoforms through the BBB is important, since it can influence the development of AD. It is shown that the rate of input of Aß40 and Aß42 from the blood into the brain is different. The actual question of the transfer of pathogenic Aß isoforms with post-translational modifications or mutations through the BBB still remains open.
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Bacterial cystathionine γ-lyase (bCSE) is the main producer of H2S in pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, etc. The suppression of bCSE activity considerably enhances the sensitivity of bacteria to antibiotics. Convenient methods for the efficient synthesis of gram quantities of two selective indole-based bCSE inhibitors, namely (2-(6-bromo-1H-indol-1-yl)acetyl)glycine (NL1), 5-((6-bromo-1H-indol-1-yl)methyl)- 2-methylfuran-3-carboxylic acid (NL2), as well as a synthetic method for preparation 3-((6-(7-chlorobenzo[b]thiophen-2-yl)-1H-indol-1-yl)methyl)- 1H-pyrazole-5-carboxylic acid (NL3), have been developed. The syntheses are based on the use of 6-bromoindole as the main building block for all three inhibitors (NL1, NL2, and NL3), and the designed residues are assembled at the nitrogen atom of the 6-bromoindole core or by the substitution of the bromine atom in the case of NL3 using Pd-catalyzed cross-coupling. The developed and refined synthetic methods would be significant for the further biological screening of NL-series bCSE inhibitors and their derivatives.
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Antibacterianos , Cistationina gama-Liase , Antibacterianos/química , Indóis/química , BactériasRESUMO
The pathogenesis of Alzheimer's disease (AD) is associated with the formation of cerebral amyloid plaques, the main components of which are the modified Aß molecules as well as the metal ions. Aß isomerized at Asp7 residue (isoD7-Aß) is the most abundant isoform in amyloid plaques. We hypothesized that the pathogenic effect of isoD7-Aß is due to the formation of zinc-dependent oligomers, and that this interaction can be disrupted by the rationally designed tetrapeptide (HAEE). Here, we utilized surface plasmon resonance, nuclear magnetic resonance, and molecular dynamics simulation to demonstrate Zn2+-dependent oligomerization of isoD7-Aß and the formation of a stable isoD7-Aß:Zn2+:HAEE complex incapable of forming oligomers. To demonstrate the physiological importance of zinc-dependent isoD7-Aß oligomerization and the ability of HAEE to interfere with this process at the organismal level, we employed transgenic nematodes overexpressing human Aß. We show that the presence of isoD7-Aß in the medium triggers extensive amyloidosis that occurs in a Zn2+-dependent manner, enhances paralysis, and shortens the animals' lifespan. Exogenous HAEE completely reverses these pathological effects of isoD7-Aß. We conclude that the synergistic action of isoD7-Aß and Zn2+ promotes Aß aggregation and that the selected small molecules capable of interrupting this process, such as HAEE, can potentially serve as anti-amyloid therapeutics.
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ß-amyloid (Aß) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aß aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aß aggregation have intensively being investigated using synthetic Aß peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aß aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aß (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aß oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aß, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aß are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aß aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Oxirredução , Aminoácidos , Fragmentos de Peptídeos/químicaRESUMO
Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.
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Glaucoma de Ângulo Aberto , Glaucoma , Humanos , Glaucoma de Ângulo Aberto/tratamento farmacológico , Proteômica/métodos , Inteligência Artificial , Glaucoma/diagnóstico , Glaucoma/complicações , Olho/metabolismo , Pressão Intraocular , Biomarcadores/metabolismoRESUMO
Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms on L. monocytogenes uptake and intracellular proliferation in human macrophages. Three isoforms of the receptor binding domain (idInlB) were derived from phylogenetically distinct L. monocytogenes strains belonging to the highly virulent CC1 (idInlBCC1), medium-virulence CC7 (idInlBCC7), and low-virulence CC9 (idInlBCC9) clonal complexes. The constant dissociation increased in the order idInlBCC1 << idInlBCC7 < idInlBCC9 for interactions with c-Met, and idInlBCC1 ≈ idInlBCC7 < idInlBCC9 for interactions with gC1q-R. The comparison of uptake and intracellular proliferation of isogenic recombinant strains which expressed full-length InlBs revealed that the strain expressing idInlBCC1 proliferated in macrophages twice as efficiently as other strains. Macrophage pretreatment with idInlBCC1 followed by recombinant L. monocytogenes infection disturbed macrophage functions decreasing pathogen uptake and improving its intracellular multiplication. Similar pretreatment with idInlBCC7 decreased bacterial uptake but also impaired intracellular multiplication. The obtained results demonstrated that InlB impaired macrophage functions in an idInlB isoform-dependent manner. These data suggest a novel InlB function in L. monocytogenes virulence.
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Listeria monocytogenes , Listeria , Listeriose , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Fatores de Virulência/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.
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Phosphorylation of beta-amyloid peptide (Aß) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic properties. Phosphorylated Aß (pS8-Aß) was detected in vivo in AD model mice and in the brains of patients with AD. However, the pS8-Aß production and the regulation of its levels have not been previously studied in detail. In this paper, immunochemical methods together with radioactive labelling were used to study the Aß phosphorylation by intracellular and surface protein kinases of HEK293 cells and brain endothelial cells (bEnd.3). It was found that HEK293 robustly phosphorylated Aß, likely with contribution from casein kinase 2 (CK2), whereas in bEnd.3, the activity of Aß phosphorylation was relatively low. Further, the study showed that both HEK293 and bEnd.3 could dephosphorylate pS8-Aß, mainly due to the activity of protein phosphatases PP1 and PP2A. The Aß dephosphorylation efficiency in bEnd.3 was three times higher than in HEK293, which correlated with the reduced abundance of pS8-Aß in vascular amyloid deposits of patients with AD compared to senile plaques. These data suggest an important role of CK2, PP1, and PP2A as regulators of Aß phosphorylation, and point to the involvement of the blood-brain barrier in the control of Aß modification levels.
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A hallmark of Alzheimer's disease (AD) are the proteinaceous aggregates formed by the amyloid-beta peptide (Aß) that is deposited inside the brain as amyloid plaques. The accumulation of aggregated Aß may initiate or enhance pathologic processes in AD. According to the amyloid hypothesis, any agent that has the capability to inhibit Aß aggregation and/or destroy amyloid plaques represents a potential disease-modifying drug. In 2023, a humanized IgG1 monoclonal antibody (lecanemab) against the Aß-soluble protofibrils was approved by the US FDA for AD therapy, thus providing compelling support to the amyloid hypothesis. To acquire a deeper insight on the in vivo Aß aggregation, various animal models, including aged herbivores and carnivores, non-human primates, transgenic rodents, fish and worms were widely exploited. This review is based on the recent data obtained using transgenic animal AD models and presents experimental verification of the critical role in Aß aggregation seeding of the interactions between zinc ions, Aß with the isomerized Asp7 (isoD7-Aß) and the α4ß2 nicotinic acetylcholine receptor.
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Doença de Alzheimer , Animais , Doença de Alzheimer/genética , Animais Geneticamente Modificados , Placa Amiloide , Peptídeos beta-Amiloides , Proteínas AmiloidogênicasRESUMO
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc amidase that catalyzes the second step of the biosynthesis of lipid A, which is an outer membrane essential structural component of Gram-negative bacteria. Inhibitors of this enzyme can be attributed to two main categories, non-hydroxamate and hydroxamate inhibitors, with the latter being the most effective given the chelation of Zn2+ in the active site. Compounds containing diacetylene or acetylene tails and the sulfonic head, as well as oxazoline derivatives of hydroxamic acids, are among the LpxC inhibitors with the most profound antibacterial activity. The present article describes the synthesis of novel functional derivatives of hydroxamic acids-bioisosteric to oxazoline inhibitors-containing 1,2,4- and 1,3,4-oxadiazole cores and studies of their cytotoxicity, antibacterial activity, and antibiotic potentiation. Some of the hydroxamic acids we obtained (9c, 9d, 23a, 23c, 30b, 36) showed significant potentiation in nalidixic acid, rifampicin, and kanamycin against the growth of laboratory-strain Escherichia coli MG1655. Two lead compounds (9c, 9d) significantly reduced Pseudomonas aeruginosa ATCC 27853 growth in the presence of nalidixic acid and rifampicin.
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Antibacterianos , Ácidos Hidroxâmicos , Oxidiazóis , Antibacterianos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Ácido Nalidíxico , Rifampina , Escherichia coliRESUMO
Alzheimer's disease (AD) is the most common cause of dementia in the elderly, characterised by the accumulation of senile plaques and tau tangles, neurodegeneration, and neuroinflammation in the brain. The development of AD is a pathological cascade starting according to the amyloid hypothesis with the accumulation and aggregation of the ß-amyloid peptide (Aß), which induces hyperphosphorylation of tau and promotes the pro-inflammatory activation of microglia leading to synaptic loss and, ultimately, neuronal death. Modelling AD-related processes is important for both studying the molecular basis of the disease and the development of novel therapeutics. The replication of these processes is often achieved with the use of a purified Aß peptide. However, Aß preparations obtained from different sources can have strikingly different properties. This review aims to compare the structure and biological effects of Aß oligomers and aggregates of a higher order: synthetic, recombinant, purified from cell culture, or extracted from brain tissue. The authors summarise the applicability of Aß preparations for modelling Aß aggregation, neurotoxicity, cytoskeleton damage, receptor toxicity in vitro and cerebral amyloidosis, synaptic plasticity disruption, and cognitive impairment in vivo and ex vivo. Further, the paper discusses the causes of the reported differences in the effect of Aß obtained from the sources mentioned above. This review points to the importance of the source of Aß for AD modelling and could help researchers to choose the optimal way to model the Aß-induced abnormalities.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Idoso , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Placa Amiloide/patologia , Encéfalo/metabolismo , Desenvolvimento de MedicamentosRESUMO
Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin-myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.
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Cardiomiopatias , Tropomiosina , Troponina , Humanos , Actinas/metabolismo , Cálcio/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Mutação , Tropomiosina/genética , Troponina/genética , Troponina T/metabolismoRESUMO
Heptose phosphates-unique linkers between endotoxic lipid A and O-antigen in the bacterial membrane-are pathogen-associated molecular patterns recognized by the receptors of the innate immune system. Understanding the mechanisms of immune system activation is important for the development of therapeutic agents to combat infectious diseases and overcome antibiotic resistance. However, in practice, it is difficult to obtain a substantial amount of heptose phosphates for biological studies due to the narrow scope of the reported synthetic procedures. We have optimized and developed an inexpensive and convenient synthesis for the first performed gram-scale production of 1-O-methyl d-glycero-α-d-gluco-heptoside 7-phosphate from readily available d-glucose. Scaling up to such amounts of the product, we have increased the efficiency of the synthesis and reduced the number of steps of the classical route through the direct phosphorylation of the O6,O7-unprotected heptose. The refined method could be of practical value for further biological screening of heptose phosphate derivatives.