ABSTRACT
Lactose is commonly crystallized in the presence of whey proteins, forming co-crystals of lactose and proteins. This work hypothesized that flavonoids such as rutin or epigallocatechin-3-gallate (EGCG) could be incorporated into the lactose and protein co-crystal structure since flavonoids may interact with both lactose and proteins. The interactions between whey proteins and flavonoids were first studied. Then, lactose-protein solutions were crystallized with and without flavonoids, measuring the kinetic parameters of crystallization and characterizing the resulting crystals. The incorporation of flavonoids in lactose-protein co-crystals depended on the hydrophilic nature of flavonoids. The hydrophilic EGCG was scarcely enclosed in the crystal lattice of lactose and avoided the inclusion of whey proteins in the crystals. In contrast, the less water-soluble rutin interacted with whey proteins and lactose, leading to the formation of co-crystals containing lactose, protein, and a large concentration of rutin (3.468 ± 0.392 mg per 100 mg of crystals).
Subject(s)
Flavonoids , Lactose , Crystallization/methods , Kinetics , Lactose/chemistry , Whey Proteins/chemistryABSTRACT
This phase 2 study evaluated the activity and safety of ibrutinib, a Bruton's tyrosine kinase inhibitor, plus rituximab in adults with previously untreated follicular lymphoma. Patients received once-daily ibrutinib 560 mg continuously plus once-weekly rituximab 375 mg/m2 for 4 weeks beginning Week 1 (Arm 1, n = 60) or Week 9 (following an 8-week ibrutinib lead-in) to explore biomarkers (Arm 2, n = 20). The primary endpoint was the best overall response rate (ORR). The median age was 58 years; most had an Eastern Cooperative Oncology Group Performance Status of 0 (74%) and Stage III/IV disease (84%). At a median study follow-up of 34 months in Arm 1 and 29 months in Arm 2, ORRs were 85% [95% confidence interval (CI) 73-93] and 75% (95% CI 51-91), respectively, with complete responses in 40% and 50%. The median duration of response was not reached in either arm; 30-month progression-free and overall survival rates were 67% and 97% (Arm 1) and 65% and 100% (Arm 2). The most common adverse events were fatigue, diarrhoea and nausea. Higher grade (Grade 3/4) haematological, haemorrhagic and cardiac events occurred infrequently. Ibrutinib plus rituximab was active and tolerable in first-line follicular lymphoma.
Subject(s)
Adenine/analogs & derivatives , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lymphoma, Follicular/drug therapy , Piperidines/therapeutic use , Rituximab/therapeutic use , Adenine/pharmacology , Adenine/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Female , Humans , Lymphoma, Follicular/pathology , Male , Middle Aged , Piperidines/pharmacology , Rituximab/pharmacologyABSTRACT
Lactose is recovered by crystallization from cheese whey that is a by-product of cheesemaking. The whey used for the recovery of lactose usually has a residual content of protein that alters the crystallization of lactose. In addition, the pH of whey may fluctuate depending on the cheese variety. However, there is little information on how the pH modifies the effect that whey proteins have on lactose crystallization. Accordingly, this work aimed to evaluate the individual and combined effect of whey proteins and pH on the kinetics of crystallization, the crystal size distribution and the crystallinity of lactose. The addition of whey proteins in lactose solutions (25% v/v) modified the process of lactose crystallization. However, the effect that whey proteins had on lactose crystallization heavily depended on the pH. The number of crystals per milliliter as well as the growth and size distribution of crystals was the most affected with the changes in pH (pHs of 7, 5.5 and 4) and the addition of whey proteins (0 and 0.63%). All the treatment produced mostly α-lactose monohydrated but some treatments also generated crystals of ß-lactose (pHâ¯5.5, 0% of proteins). Amorphous lactose was observed mainly in lactose solutions adjusted at pHâ¯7 and added with whey proteins. This particular treatment also incorporated the highest amount of protein into the lattice of lactose crystals. The results of this work highlight the importance of controlling the pH of lactose crystallization, especially if there is a presence of whey proteins.
Subject(s)
Lactose/chemistry , Whey Proteins/chemistry , Blood Proteins , Crystallization/methods , Hydrogen-Ion Concentration , Kinetics , Particle SizeABSTRACT
PURPOSE: The aurora A kinase inhibitor alisertib demonstrated single-agent clinical activity and preclinical synergy with vincristine/rituximab in B-cell non-Hodgkin lymphoma (B-NHL). This phase I study aimed to determine the safety and recommended phase II dose (RP2D) of alisertib in combination with rituximab ± vincristine in patients with relapsed/refractory aggressive B-NHL. PATIENTS AND METHODS: Patients with relapsed/refractory, diffuse, large, or other aggressive B-NHL received oral alisertib 50 mg b.i.d. days 1 to 7, plus i.v. rituximab 375 mg/m2 on day 1, for up to eight 21-day cycles (MR). Patients in subsequent cohorts (3 + 3 design) received increasing doses of alisertib (30 mg starting dose; 10 mg increments) b.i.d. days 1 to 7 plus rituximab and vincristine [1.4 mg/m2 (maximum 2 mg) days 1, 8] for 8 cycles (MRV). Patients benefiting could continue single-agent alisertib beyond 8 cycles. Cell-of-origin and MYC/BCL2 IHC was performed on available archival tissue. RESULTS: Forty-five patients participated. The alisertib RP2D for MR was 50 mg b.i.d. For MRV (n = 32), the RP2D was determined as 40 mg b.i.d. [1 dose-limiting toxicity (DLT) at 40 mg; 2 DLTs at 50 mg]. Drug-related adverse events were reported in 89% of patients, the most common was neutropenia (47%). Seven patients had complete responses (CR), 7 had partial responses (PRs); 9 of 20 (45%) patients at the MRV RP2D responded (4 CRs, 5 PRs), all with non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL). CONCLUSIONS: The combination of alisertib 50 mg b.i.d. plus rituximab or alisertib 40 mg b.i.d. plus rituximab and vincristine was well tolerated and demonstrated activity in non-GCB DLBCL.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aurora Kinase A/antagonists & inhibitors , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/pathology , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Azepines/administration & dosage , Azepines/pharmacokinetics , Disease Progression , Drug Monitoring , Drug Resistance, Neoplasm , Female , Humans , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/mortality , Male , Maximum Tolerated Dose , Middle Aged , Molecular Targeted Therapy , Neoplasm Metastasis , Neoplasm Staging , Pyrimidines/administration & dosage , Pyrimidines/pharmacokinetics , Recurrence , Retreatment , Rituximab/administration & dosage , Rituximab/pharmacokinetics , Treatment Outcome , Vincristine/administration & dosage , Vincristine/pharmacokineticsABSTRACT
Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article "Identification of proteins that are differentially expressed in brains with Alzheimer's disease using iTRAQ labeling and tandem mass spectrometry" (Minjarez et al., 2016) [1].
ABSTRACT
Alzheimer's disease (AD) is a degenerative and irreversible disorder whose progressiveness is dependent on age. It is histopathologically characterized by the massive accumulation of insoluble forms of tau and amyloid-ß (Aß) asneurofibrillary tangles and neuritic plaques, respectively. Many studies have documented that these two polypeptides suffer several posttranslational modifications employing postmortem tissue sections from brains of patients with AD. In order to elucidate the molecular mechanisms underlying the posttranslational modifications of key players in this disease, including Aß and tau, several transgenic mouse models have been developed. One of these models is the 3×Tg-AD transgenic mouse, carrying three transgenes encoding APPSWE, S1M146V, and TauP301L proteins. To further characterize this transgenicmouse, we determined the accumulation of fibrillar Aß as a function of age in relation to the hyperphosphorylation patterns of TauP301L at both its N- and C-terminus in the hippocampal formation by immunofluorescence and confocal microscopy. Moreover, we searched for the expression of activated protein kinases and mediators of inflammation by western blot of wholeprotein extracts from hippocampal tissue sections since 3 to 28 months as well. Our results indicate that the presence of fibrillar Aß deposits correlates with a significant activation of astrocytes and microglia in subiculum and CA1 regions of hippocampus. Accordingly, we also observed a significant increase in the expression of TNF-α associated to neuritic plaques and glial cells. Importantly, there is an overexpression of the stress activated protein kinases SAPK/JNK and Cdk-5 in pyramidal neurons, which might phosphorylate several residues at the C-terminus of TauP301L. Therefore, the accumulation of Aß oligomers results in an inflammatory environment that upregulates kinases involved in hyperphosphorylation of TauP301L polypeptide.
Subject(s)
Aging/immunology , Alzheimer Disease/immunology , Amyloid beta-Peptides/metabolism , Hippocampus/immunology , tau Proteins/metabolism , Aged , Aged, 80 and over , Aging/pathology , Alzheimer Disease/pathology , Amyloid beta-Peptides/genetics , Animals , Disease Models, Animal , Female , Hippocampus/pathology , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Neuroglia/immunology , Neuroglia/pathology , Phosphorylation/immunology , Plaque, Amyloid/immunology , Plaque, Amyloid/pathology , Presenilin-1/genetics , Presenilin-1/metabolism , Pyramidal Cells/immunology , Pyramidal Cells/pathology , tau Proteins/geneticsABSTRACT
Alzheimer's disease is one of the leading causes of dementia in the elderly. It is considered the result of complex events involving both genetic and environmental factors. To gain further insights into this complexity, we quantitatively analyzed the proteome of cortex region of brains from patients diagnosed with Alzheimer's disease, using a bottom-up proteomics approach. We identified 721 isobaric-tagged polypeptides. From this universe, 61 were found overexpressed and 69 subexpressed in three brains with Alzheimer's disease in comparison to a normal brain. We determined that the most affected processes involving the overexpressed polypeptides corresponded to ROS and stress responses. For the subexpressed polypeptides, the main processes affected were oxidative phosphorylation, organellar acidification and cytoskeleton. We used Drosophila to validate some of the hits, particularly those non-previously described as connected with the disease, such as Sideroflexin and Phosphoglucomutase-1. We manipulated their homolog genes in Drosophila models of Aß- and Tau-induced pathology. We found proteins that can either modify Aß toxicity, Tau toxicity or both, suggesting specific interactions with different pathways. This approach illustrates the potential of Drosophila to validate hits after MS studies and suggest that model organisms should be included in the pipeline to identify relevant targets for Alzheimer's disease. BIOLOGICAL SIGNIFICANCE: We report a set of differentially expressed proteins in three Alzheimer's disease brains in comparison to a normal brain. Our analyses allowed us to identify that the main affected pathways were ROS and stress responses, oxidative phosphorylation, organellar acidification and cytoskeleton. We validated some identified proteins using genetic models of Amyloid-ß and Tau-induced pathology in Drosophila melanogaster. With this approach, Sideroflexin and Phosphoglucomutase-1 were identified as novel proteins connected with Alzheimer's disease.
Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Gene Expression Regulation , Mass Spectrometry/methods , Nerve Tissue Proteins/biosynthesis , Aged , Aged, 80 and over , Animals , Disease Models, Animal , Drosophila Proteins/metabolism , Drosophila melanogaster , Female , HumansABSTRACT
Abundant evidence indicates that iron accumulation, oxidative damage and mitochondrial dysfunction are common features of Huntington's disease, Parkinson's disease, Friedreich's ataxia and a group of disorders known as Neurodegeneration with Brain Iron Accumulation. In this study, we evaluated the effectiveness of two novel 8-OH-quinoline-based iron chelators, Q1 and Q4, to decrease mitochondrial iron accumulation and oxidative damage in cellular and animal models of PD. We found that at sub-micromolar concentrations, Q1 selectively decreased the mitochondrial iron pool and was extremely effective in protecting against rotenone-induced oxidative damage and death. Q4, in turn, preferentially chelated the cytoplasmic iron pool and presented a decreased capacity to protect against rotenone-induced oxidative damage and death. Oral administration of Q1 to mice protected substantia nigra pars compacta neurons against oxidative damage and MPTP-induced death. Taken together, our results support the concept that oral administration of Q1 is a promising therapeutic strategy for the treatment of NBIA.
Subject(s)
Cell Death/drug effects , Hydroxyquinolines/pharmacology , Iron Chelating Agents/pharmacology , Mitochondria/drug effects , Neurons/drug effects , Oxidative Stress/drug effects , Animals , Cell Line, Tumor , Humans , Iron/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Neurons/cytology , Rotenone/pharmacologyABSTRACT
We previously demonstrated that, in the early stages of tau processing in Alzheimer's disease, the N-terminal part of the molecule undergoes a characteristic cascade of phosphorylation and progressive misfolding of the proteins resulting in a structural conformation detected by Alz-50. In this immunohistochemical study of AD brain tissue, we have found that C-terminal truncation of tau at Asp-421 was an early event in tau aggregation and analyzed the relationship between phospho-dependent tau epitopes located at the C-terminus with truncation at Glu-391. The aim of this study was to determine whether C-terminal truncation may trigger events leading to the assembly of insoluble PHFs from soluble tau aggregates present in pre-tangle cells. Our findings suggest that there is a complex interaction between phosphorylated and truncated tau species. A model is presented here in which truncated tau protein represents an early neurotoxic species while phosphorylated tau species may provide a neuroprotective role in Alzheimer's disease.
ABSTRACT
Objetivo. Describir la distribución normalizada de los valores del índice de masa corporal (IMC) en la población adulta mayor mexicana. Material y método. Se utilizó la fórmula original de Quetelet para determinar el IMC de los participantes en SABE. Este es un estudio de campo, descriptivo, transversal, simultaneo, de corte comparativo. Fue llevado a cabo en 12 entidades federativas mexicanas en personas adultas mayores de 60 y más años de edad, residentes habituales de su domicilio actual al momento de realizarse la encuesta. La muestra abarcó a 12 411 individuos con representatividad estadística. El estudio se basó en estadística descriptiva. En el manejo estadístico de la información se utilizó como prueba no parametrica la χ² con un grado de libertad, y un intervalo de confianza > 95%. Se consideraron como valores estadísticamente significativos aquellos con p≤0,005. Resultados. Se encontró que los conceptos de IMC "normal", "sobrepeso" y "obesidad I" en SABE fueron mayores que los aportados por las actuales tablas estandarizadas de la OMS, lo que coincide en lo relativo a la obesidad grados II y III.
Objective. To describe the standard distribution of the values of the of body mass index in the Mexican older population. Material and methods. BMI was determined by the original Quetelet's formula in the participants of SABE study. This is a descriptive, cross-sectional, simultaneous and comparative field study. It was carried out a survey in 12 Mexican States in 60 year-old and elder usual adult residents. The representative sample comprised 12 411 individuals. The study was based on descriptive statistics. For analysis, it was used the non-parametric χ² test with one degree of freedom and an interval confidence<95%. It was considered as statistically significant those values with p≤0,005. Results. We found that the concepts of "normal" BMI, "overweight" and "obesity I" in SABE study were greater than the provided by the current standardized tables, and there coincidence with obesity grades II and III.
Subject(s)
Humans , Aged, 80 and over , Aged , Anthropometry , Weight by Height , Body Mass Index , Epidemiology, Descriptive , Cross-Sectional Studies , MexicoABSTRACT
The main amyloid-ß peptide (Aß) variants detected in the human brain are Aß1-40 and Aß1-42; however, a significant proportion of Aß in Alzheimer's disease (AD) brain also consists of N-terminal truncated/modified species. AßN3(pE), Aß peptide bearing amino-terminal pyroglutamate at position 3, has been demonstrated to be a major N-truncated/modified constituent of intracellular, extracellular, and vascular Aß deposits in AD and Down syndrome brain tissue. It has been previously demonstrated that rabbits fed a diet enriched in cholesterol and given water containing trace copper levels developed AD-like pathology including intraneuronal and extracellular Aß accumulation, tau hyperphosphorylation, vascular inflammation, astrocytosis, microgliosis, reduced levels of acetylcholine, as well as learning deficits and thus, may be used as a non-transgenic animal model of sporadic AD. In the present study, we have demonstrated for the first time the presence of AßN3(pE) in blood vessels in cholesterol-enriched diet-fed rabbit brain. In addition, we detected AßN3(pE) immunoreactivity in all postmortem AD brain samples studied. We believe that our results are potentially important for evaluation of novel therapeutic molecules/strategies targeting Aß peptides in a suitable non-transgenic animal model.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/blood supply , Brain/metabolism , Cholesterol, Dietary/administration & dosage , Aged , Aged, 80 and over , Animals , Antibodies/metabolism , Astrocytes/metabolism , Cerebral Cortex/blood supply , Cerebral Cortex/metabolism , Extracellular Space/metabolism , Hippocampus/blood supply , Hippocampus/metabolism , Humans , Intracellular Space/metabolism , Male , Mice , Mice, Transgenic , Neurons/metabolism , Peptide Fragments/metabolism , RabbitsABSTRACT
Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.
Subject(s)
Alzheimer Disease/pathology , Brain/metabolism , Caspase 3/pharmacology , tau Proteins/drug effects , tau Proteins/metabolism , Aged, 80 and over , Amino Acid Chloromethyl Ketones/pharmacology , Aspartic Acid/metabolism , Brain/pathology , Brain/ultrastructure , Caspase 3/metabolism , Female , Humans , Male , Microscopy, Electron , Molecular Weight , Neurofibrillary Tangles/metabolism , Neurofibrillary Tangles/pathology , Neurofibrillary Tangles/ultrastructure , Neurofibrils/metabolism , Neurofibrils/pathology , Neurofibrils/ultrastructure , Neuroprotective Agents/pharmacology , Oligopeptides/pharmacology , tau Proteins/ultrastructureABSTRACT
Abnormal intracellular aggregation of tau protein is a pathological condition leading to neuronal death in Alzheimer's disease. Fibrillar and nonfibrillar aggregates of tau protein alter the normal functioning of neurons by disturbing important cellular processes and distinct membranous organelles. However, tau-caused alterations in the nuclear compartment are not totally established so far. In our study we evaluated whether tau protein and its Asp421-truncated variant produce alterations in the normal architecture of the nucleus when expressed in cultured neuroblastoma cells. After 48 hours of transfection, significant deformity of the nuclear compartment with extensive lobulations along the nuclear envelope was observed in SH-SY5Y cells expressing either full-length tau or Asp421-truncated tau. This aberrant formation did not involve either nuclear fragmentation or cell death. The lobulated nuclei were devoid of tau protein, which mostly remained in the cytoplasm in a nonfibrillar state. Degradation of nuclear Lamins was not observed in tau-expressing SH-SY5Y cells, and a cell-cycle analysis did not show aberrant chromosome accumulation. Thus multiple division defects leading to multinucleation were discarded. The lobulated nuclei in tau-expressing SH-SY5Y cells seem to more resemble the multilobular phenotype of the nuclear envelope seen in Lamin-mutated cells from those pathological conditions leading to premature aging. Nevertheless, in our tau-expressing cells, the abnormal formation of cortical and perinuclear rings of tubulin generated by tau binding may be a more feasible mechanism of a nuclear-cytoskeleton generating force that causes the nuclear deformation.
Subject(s)
Cell Nucleus/metabolism , Cytoskeleton/metabolism , Neuroblastoma/metabolism , tau Proteins/metabolism , Cell Line, Tumor , Cell Nucleus/genetics , Cytoskeleton/genetics , Humans , Microtubules/genetics , Microtubules/metabolism , Neuroblastoma/genetics , Neurons/metabolism , Phosphorylation , Tubulin/metabolism , tau Proteins/geneticsABSTRACT
Thorn-shaped astrocytes (TsA) are mainly localized in the periventricular white matter of the temporal lobe in a subgroup of aged individuals usually in the context of Alzheimer's disease (AD). Immunohistochemistry of TsA shows 4Rtau deposition, tau phosphorylation at different sites recognized with phosphospecific anti-tau antibodies Thr181, Ser202, Ser214, Thr231, Ser396, Ser422, and clones AT8 and PHF-1, and conformational changes revealed with Alz50 and MC-1 antibodies; TsA are also immunostained with antibodies to active tau kinases MAPK/ERK-P, SAPK/JNK-P, p38-P and GSK-3ß. These findings are common to neurofibrillary tangles in AD. However, TsA are not stained with 3Rtau antibodies, and they are seldom stained or not at all with phosphospecific tauSer262 and with Tau-C3 antibody, which recognizes the latter tau truncation at aspartic acid 421. Previous studies have shown that tau phosphorylation at Ser262 reduces tau binding to microtubules and increases caspase-3 activity, whereas tau truncation at aspartic acid 421 is associated with tau ubiquitination, and toxic effects of tau. In this line, ubiquitin is not accumulated in TsA, and in situ end-labeling of nuclear DNA fragmentation shows absence of degeneration in TsA. These observations support the concept that tau lesions in neurons differ from those seen in TsA in AD.
Subject(s)
Alzheimer Disease/pathology , Astrocytes/pathology , Cell Shape , Nerve Fibers, Myelinated/pathology , Temporal Lobe/pathology , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Astrocytes/metabolism , Axons/metabolism , Axons/pathology , Caspase 3/metabolism , Female , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Male , Mitogen-Activated Protein Kinase 8/metabolism , Nerve Fibers, Myelinated/metabolism , Neurofibrillary Tangles/metabolism , Neurofibrillary Tangles/pathology , Neurons/metabolism , Neurons/pathology , Phosphorylation , Temporal Lobe/metabolism , tau Proteins/metabolismABSTRACT
Alzheimer's disease (AD) is the most common cause of dementia in the elderly. AD brains are characterized by the presence of neurofibrillary tangles (NFTs) and neuritic plaques. NFTs are constituted of paired helical filaments, which are structurally composed by assembled hyperphosphorylated and truncated tau polypeptides. To date, the integral constituents of NFTs remain unknown mainly due to the high insolubility of NFTs. The aim of this study was to identify by tandem mass spectrometry, the polypeptides contained in both isolated NFTs by laser capture microdissection and total homogenates, using tissue sections from paraformaldehyde-fixed AD brains. In the first case, we isolated 2,000 NFTs from tissue samples of hippocampus from each of the three Mexican AD brains used in our study. These were previously stained with anti-hyperphosphorylated tau AT-100 antibodies. After the removal of paraformaldehyde and delipidation with organic solvents, we tested three solubilization methods. We identified 102 polypeptides from total homogenates and 41 from isolated NFTs. We selected UCH-L1, transferrin, and GAPDH polypeptides to be studied by immunofluorescence and confocal microscopy. Only UCH-L1 and GAPDH colocalized with hyperphosphorylated tau in NFTs.
Subject(s)
Alzheimer Disease/pathology , Brain/metabolism , Neurofibrillary Tangles/metabolism , Peptides/metabolism , Tandem Mass Spectrometry , Aged , Aged, 80 and over , Brain/ultrastructure , Chromatography, High Pressure Liquid , Computational Biology , Female , Humans , Laser Capture Microdissection , Male , Microscopy, Electron, Transmission , Neurofibrillary Tangles/ultrastructureABSTRACT
Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aß 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aß 1-42 in ELISA as well as to Aß aggregates present in AD brain. Aß 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aß 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aß 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.
Subject(s)
Amyloid beta-Peptides/metabolism , Electron Transport Complex IV/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Animals , Cattle , Electron Transport Complex IV/chemistry , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Peptide Fragments/chemistry , Protein Binding , Protein Multimerization , Protein Structure, SecondaryABSTRACT
RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.
Subject(s)
Breast Neoplasms/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Neoplasm Recurrence, Local/genetics , Paraffin Embedding , Sequence Analysis, RNA , Tissue Fixation , Base Sequence , Biomarkers, Tumor/genetics , DNA, Intergenic/genetics , Female , Formaldehyde , Humans , Introns/genetics , Proportional Hazards Models , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm , Risk FactorsABSTRACT
Neonatal ventral hippocampal lesion (nVHL) in rats has been widely used as a neurodevelopmental model to mimic schizophrenia-like behaviors. Recently, we reported that nVHLs result in dendritic retraction and spine loss in prefrontal cortex (PFC) pyramidal neurons and medium spiny neurons of the nucleus accumbens (NAcc). Cerebrolysin (Cbl), a neurotrophic peptide mixture, has been reported to ameliorate the synaptic and dendritic pathology in models of aging and neurodevelopmental disorder such as Rett syndrome. This study sought to determine whether Cbl was capable of reducing behavioral and neuronal alterations in nVHL rats. The behavioral analysis included locomotor activity induced by novel environment and amphetamine, social interaction, and sensoriomotor gating. The morphological evaluation included dendritic analysis by using the Golgi-Cox procedure and stereology to quantify the total cell number in PFC and NAcc. Behavioral data show a reduction in the hyperresponsiveness to novel environment- and amphetamine-induced locomotion, with an increase in the total time spent in social interactions and in prepulse inhibition in Cbl-treated nVHL rats. In addition, neuropathological analysis of the limbic regions also showed amelioration of dendritic retraction and spine loss in Cbl-treated nVHL rats. Cbl treatment also ameliorated dendritic pathology and neuronal loss in the PFC and NAcc in nVHL rats. This study demonstrates that Cbl promotes behavioral improvements and recovery of dendritic neuronal damage in postpubertal nVHL rats and suggests that Cbl may have neurotrophic effects in this neurodevelopmental model of schizophrenia. These findings support the possibility that Cbl has beneficial effects in the management of schizophrenia symptoms.
Subject(s)
Amino Acids/administration & dosage , Behavior, Animal/drug effects , Hippocampus/injuries , Neurons , Neuroprotective Agents/administration & dosage , Schizophrenia , Acoustic Stimulation , Analysis of Variance , Animals , Animals, Newborn , Dendritic Spines/drug effects , Dendritic Spines/pathology , Dendritic Spines/ultrastructure , Disease Models, Animal , Female , Inhibition, Psychological , Interpersonal Relations , Motor Activity/drug effects , Neurons/drug effects , Neurons/pathology , Neurons/ultrastructure , Pregnancy , Rats , Rats, Sprague-Dawley , Reflex, Startle , Schizophrenia/drug therapy , Schizophrenia/etiology , Schizophrenia/pathology , Silver StainingABSTRACT
La población de personas adultas mayores crece de forma paulatina y sostenida en el mundo y por ende en nuestro país. Sin embargo, la información que se tiene sobre la forma de vida, salud y bienestar de este grupo poblacional es limitada debido a que la mayor parte de las investigaciones en adultos mayores es derivada del estudio de grupos pequeños con patología bien circunscrita y características especiales. Por otro lado, la realización de estudios, en la población abierta, que permitan establecer la demografía y epidemiología de las personas mayores de 60 años requieren de una gran infraestructura y recursos tanto humanos como económicos. El Proyecto-Encuesta Salud Bienestar y Envejecimiento (SABE), mediante la realización de un estudio tipo encuesta, tiene la finalidad de obtener dicha información contando con la participación del Sector Salud, instituciones de educación superior así como organizaciones no gubernamentales lo que constituye un logro interinstitucional. Para la realización del proyecto SABE se ha seguido con rigurosidad científica el protocolo y para su puesta en marcha hubo la necesidad de la integración intersectorial de las instituciones que brindan atención a los adultos mayores mexicanos, ya sea dentro del contexto de la seguridad o asistencia social. Los alcances de SABE permitirán la creación y difusión del conocimiento derivado del mismo. En este documento, se expone y analiza de forma breve y concreta la metodología operativa de SABE México, lo que significa dar el primer paso para que otros investigadores interesados en el tema, realicen este tipo de estudios. Dada la experiencia que se ha ido generando en la realización de SABE México, este tipo de documentos sirven como base para proponer e iniciar las acciones al respecto.
The elderly population grows so gradual and sustained in the world and therefore in our country. However, information that is on the way of life, health and well-being of this population group is limited since most of the research in older adults is derived from the study of small groups with well circumscribed pathology and special features. On the other hand, open population studies to establish the demography and epidemiology of people older than 60 years require a large infrastructure and resources both human and financial. Project- Survey Health, Wellbeing and Ageing (SABE, in Spanish)), through a study type survey, has the purpose of obtaining such information with the participation of the Health Sector, institutions of higher education as well as organizations NGO constituting an interinstitutional achievement. For the implementation of the project SABE the Protocol has been followed with scientific rigor and for it simplementation underway there was the need to cross-sectoral integration of institutions providing care to Mexican older adults, either within the context of the social security or welfare. The reaches of SABE will enable the creation and dissemination of the knowledge derived from it. In this document, it exposes and analyzes operational methodology of a brief and concrete way of SABE Mexico, which means the first step so that other researchers interested in the topic, take place this type of study. Given the experience that has been generated in the implementation of SABE Mexico, this type of documents serves as the basis to propose and initiate actions in this regard.