Cristina Osorio

Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca(2+)-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit β (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity.

Analysis of proteins using DIGE and MALDI mass spectrometry.

Difference gel electrophoresis (DIGE) is a common technique for characterizing differential protein expression in quantitative proteomics. Usually a combination of enzymatic digestion and peptide analysis by mass spectrometry is used to identify differentially expressed proteins following separation and statistical analysis by DIGE. In this chapter, methods for gel spot picking, enzymatic digestion, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for protein identification of DIGE-analyzed proteins are discussed. Two examples are given: first, a specific protein is used to test the sensitivity of the 2D DIGE/MALDI MS combination for protein quantification and identification, and second, several proteins with and without the labels typically used in DIGE are identified to demonstrate that these labels do not alter MS-based protein identification. Technical variations of protein gel spot preparation, in-gel digestion, and mass spectral protein identification are discussed.

Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics

Difference gel electrophoresis (DIGE) is most often used to assess relative changes in the expression levels of individual proteins in multiple complex samples, and this information is valuable in making inferences about relative protein activity. However, a proteins activity is not solely dependent upon its expression level. A change in activity may also be influenced by myriad posttranslational modifications (PTMs), including palmitoylation, ubiquitination, oxidation, and phosphorylation. In this chapter, we describe the use of DIGE to determine specific PTMs by introducing specific labels or changes in pI and/or molecular weight.

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress

BACKGROUND S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. METHODS In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. RESULTS We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. CONCLUSIONS Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. GENERAL SIGNIFICANCE Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Diabetes mellitus in patients withAlzheimer´s disease: clinical descriptionand correlation with the APOEgenotype in a sample population from the province of Antioquia, Colombia

INTRODUCTION The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. OBJECTIVE Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. MATERIALS AND METHODS The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. RESULTS On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). CONCLUSION This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.INTRODUCTION Alzheimers disease is a multifactorial disease affecting approximately twenty million people worldwide. Numerous variables are associated with increased risk of developing this severe neurological disorder. Among the risk factors, diabetes mellitus, and the ε4 isoform of the APOE gene have been amply demonstrated as increasing the risk of developing this disease. OBJECTIVE To determine if a correlation exists between APOE genotype, diabetes mellitus and Alzheimers disease. MATERIALS AND METHODS Clinical studies were carried out by surveying the clinical histories in a group of patients in the province of Antioquia, Colombia. Forty-three Alzheimers patients were compared with 43 control subjects, paired by age and gender. Commercially available methods were used to determine whether the patients had diabetes, and restriction enzyme-based genotyping was used to determine the APOE genotypes. RESULTS The most common non-neurological comorbidities were: arterial hypertension, acute myocardial infarction, chronic obstructive pulmonary disease and hypothyroidism. From the many variables investigated, two were conclusive: (1) the presence of Alzheimers disease was higher in patients with diabetes mellitus, and (2) no correlation between late-onset sporadic Alzheimers disease and APOE was found in the target population. CONCLUSIONS To detect any association with the APOE genotype, a study involving much a larger population samples must be undertaken.

Differentially charged isoforms of apolipoprotein E from human blood are potential biomarkers of Alzheimer’s disease

IntroductionAlzheimer’s disease (AD) is the major cause of dementia among the elderly. Finding blood-based biomarkers for disease diagnosis and prognosis is urgently needed.MethodsWe studied protein distributions in brain tissues, cerebrospinal fluid (CSF), and blood of AD patients by using proteomics and a new proteomic method that we call “2D multiplexed Western blot” (2D mxWd). This method allows us to determine in multiple samples the electrophoretic patterns of protein isoforms with different isoelectric points.ResultsApolipoprotein E (ApoE) displays a unique distribution of electrophoretic isoforms in the presence of AD and also a unique pattern specific to the APOE genotype.ConclusionsThe isoelectric distribution of differentially charged ApoE isoforms was used to determine the presence of AD in a small group of samples. Further studies are needed to validate their use as predictors of disease onset and progression, and as biomarkers for determining the efficacy of therapeutic treatments.

Proteomic analysis of mice expressing human ApoE demonstrates no differences in global protein solubility between APOE 3 and APOE 4 young mice.

Apolipoprotein E (ApoE) is a major lipid carrier protein. In humans, ApoE is expressed in three polymorphic isoforms, which are encoded by three different alleles APOE2, APOE3, and APOE4. In the brains of Alzheimers disease (AD) patients, each one of these three allelic isoforms is found in several “isoelectric” protein isoforms (qPI), i.e. protein isoforms resulting from PTMs altering the net charge (q) of the polypeptide. AD is a complex disease in which multiple causes and several risk factors affect the onset and disease outcome. A major risk factor for AD is ApoE4; therefore, it is important to characterize the different ApoE qPIs. We have implemented a detergent‐based method for isolation and quantitation of protein isoforms, and we found differences in the solubility of protein isoforms depending on the type of solvent used. In this manuscript, we describe these methods and applied them to young human‐ApoE targeted replacement mice. Our results indicate that there are no significant differences in the hippocampus proteome of these mice as a function of the APOE genotype.

Protein Engineering of Bacillus thuringiensis δ-Endotoxins

Protein engineering of insecticidal Bt δ-endotoxins is a powerful tool for designing novel Cry toxins with altered properties, including changing the toxin’s specificity. By following some elementary rules governing the structure/function relationship, it has been possible to create new toxins with modified properties including increased toxicity and binding affinity, enhanced ion-transport activity, and changes in insect specificity. These methods have also produced valuable information and have led to an improved understanding of the mode of action of these important biopesticides. The results discussed in this chapter derive from rational molecular design where protein structure is modified by incorporating single or multiple amino acid substitutions aimed at modifying specific protein functions. In this review, we analyze several protein modifications that have been successfully used for creating stable, functional proteins with minimal structural alterations. The understanding and proper use of protein engineering approaches may help in implementing appropriate pest management strategies by improving the efficacy of these toxins against insect pests.

Diabetes mellitus en pacientes con enfermedad de Alzheimer: descripción clínica y correlación con el genotipo APOE en una muestra de población del departamento de Antioquia, Colombia

Introduccion. La enfermedad de Alzheimer es compleja y afecta, aproximadamente, a 20 millones de personas en todo el mundo. Muchas variables parecen aumentar el riesgo de desarrollar esta alteracion neurologica. Entre los factores de riesgo, se ha demostrado ampliamente que la diabetes mellitus y la isoforma e4 del gen APOE tienen incidencia positiva en el desarrollo de la enfermedad. Se reporta un estudio en el cual se investigo la posible correlacion entre APOE, diabetes mellitus yla enfermedad de Alzheimer, en un grupo especifico de pacientes del departamento de Antioquia, Colombia. Objetivo. Determinar si existe una correlacion entre APOE, diabetes mellitus y la enfermedad de Alzheimer, en un grupo de pacientes de Antioquia, Colombia. Materiales y metodos. Se buscaron y analizaron las historias clinicas de los pacientes con diagnostico de enfermedad de Alzheimer. Se seleccionaron aquellos que cumplian los criterios de inclusion. Se utilizaron metodos comercialmente disponibles para confirmar la presencia de diabetes mellitus. La genotipificacion de APOE se hizo con un metodo basado en la PCR y la digestion con enzimas de restriccion, en muestras de todos los participantes en el estudio. Resultados. En este estudio se analizan 43 casos de enfermedad de Alzheimer y 43 individuos sanos controles, pareados por edad y sexo. Las enfermedades concomitantes no neurologicas mas comunes fueron: hipertension arterial, infarto agudo del miocardio, enfermedad pulmonar obstructiva cronica e hipotiroidismo.Conclusiones. De las diferentes variables investigadas, dos arrojaron resultados concluyentes: i) la presencia de la enfermedad de Alzheimer es mas frecuente en pacientes con diabetes mellitus, y 2) no se encontro correlacion entre la enfermedad de Alzheimer de inicio tardio esporadico y el genotipo de APOE. Es importante indicar que debe llevarse a cabo un estudio con un tamano de poblacion mayor, para determinar cualquier posible correlacion o inferencia con el genotipo de APOE. doi: http://dx.doi.org/10.7705/biomedica.v32i2.579