Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...
Mechanisms of aluminium neurotoxicity in oxidative stress-induced ... Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...
CHAPTER 1 followed by centrifugation at 13,000 rpm. The pellet was then discarded and the supernatant treated with trichloroacetic acid (1 M) followed consecutively by sonication and centrifugation in a microcentrifuge (model E, Beckman Instruments, Palo Alto, CA, USA) at 13,000 rpm for 5 min. The resulting pellet was reconstituted in NaOH (0.5 M) with vigorous vortexing for 3 min. Then, 10 mM 2,4-dinitrophenylhydrazine in 2 M chloric acid was added and the mixture incubated at room temperature for 1 h, in darkness, and with continuous agitation. After the addition of trichloroacetic acid (1 M), the resulting mixture was centrifuged at 13,000 rpm for 5 min. The resulting pellet was washed twice with ethyl acetate : ethanol (1:1, v/v). Then, the washed pellet was reconstituted with 6 M guanidine in a 20 mM KH2PO4 buffer (pH 2.3) and the absorbance of the solution measured at 370 nm. The PCC was calculated from the absorbance data using as absorption coefficient for dinitrophenylhydrazone ε = 22,000 M –1 cm –1 and expressing this parameter as nmol carbonyls/mg protein. Because of the numerous washing steps, protein content in the final pellet was estimated on an HCl blank pellet processed simultaneously using a BSA standard curve in 6 M guanidine, and reading the absorbance at 280 nm. Determination of protein thiol content (PTC) 90 The PTC of proteins was estimated spectrophotometrically using a modification introduced to a standard assay (Hermida-Ameijeiras et al. 2004). Briefly, an aliquot of the sample (200 μl) was treated with trichloroacetic acid (0.5 M) for protein precipitation. After vortexing and centrifugation at 13,000 rpm for 5 min, the resulting pellet was reconstituted in an 80 mM Na3PO4 and 2 mM EDTA buffer (pH 8.0) containing 70 mM sodium dodecylsulfate. Then, 100 μM 5,5‟-dithiobis-(2-nitrobenzoic acid) in a buffer 100 mM Na3PO4 (pH 8.0) were added and the mixture incubated at room temperature for 20 min with continuous mixing. After centrifugation at 13,000 rpm for 5 min, the absorbance of the resulting solution was measured at 412 nm and the PTC calculated from this data using as absorption coefficient for 2-nitro-5- mercaptobenzoic acid ε = 13,600 M –1 cm –1 and expressing the corresponding parameter as nmol thiols/mg protein.
Statistical analysis CHAPTER 1 Results were expressed as the mean ± SD from five animals. Statistical differences were tested using one-way ANOVA followed by LSD for multiple comparisons (p < 0.05). All statistical analyses were performed using Sigmastat 3.0 from Jandel Scientific (San Diego, CA, USA). 91
- Page 63 and 64: INTRODUCTION Oxidative damage happe
- Page 65 and 66: DA metabolism as a source of ROS IN
- Page 67 and 68: Contribution of oxidative and nitro
- Page 69 and 70: Excitotoxicity INTRODUCTION Glutama
- Page 71 and 72: INTRODUCTION al. 1996, Cicchetti et
- Page 73 and 74: Experimental models of PD INTRODUCT
- Page 75 and 76: INTRODUCTION induce selective DAerg
- Page 77 and 78: ALUMINIUM General features INTRODUC
- Page 79 and 80: INTRODUCTION Varying amounts of alu
- Page 81 and 82: INTRODUCTION and antiperspirants co
- Page 83 and 84: INTRODUCTION approximately 3% of al
- Page 85 and 86: Excretion INTRODUCTION As insoluble
- Page 87 and 88: INTRODUCTION 3.5 mg may be increase
- Page 89 and 90: INTRODUCTION Oteiza 1999), non-iron
- Page 91 and 92: Aluminium as a cell membrane menace
- Page 93 and 94: INTRODUCTION mobilization of Ca 2+
- Page 95: Aluminium impairs neurotransmission
- Page 99 and 100: AIMS OF THE PRESENT THESIS The main
- Page 101: OBJETIVOS
- Page 104 and 105: OBJETIVOS 3) Evaluar de forma preci
- Page 107: CHAPTER 1 Time-course of brain oxid
- Page 110 and 111: CHAPTER 1 INTRODUCTION 86 6-OHDA (2
- Page 112 and 113: CHAPTER 1 MATERIALS AND METHODS Che
- Page 116 and 117: CHAPTER 1 RESULTS Effects of 6-OHDA
- Page 118 and 119: CHAPTER 1 DISCUSSION 94 As shown by
- Page 120 and 121: CHAPTER 1 strategies or to study th
- Page 122 and 123: CHAPTER 1 Fig. 2 Changes in PCC in
- Page 125: CHAPTER 2
- Page 129 and 130: ABSTRACT CHAPTER 2 In the present w
- Page 131 and 132: MATERIALS AND METHODS Chemicals CHA
- Page 133 and 134: CHAPTER 2 atomization used were 1,5
- Page 135 and 136: DISCUSSION CHAPTER 2 Aluminium ente
- Page 137: Table 1. Graphite furnace programme
- Page 141: CHAPTER 3 Brain oxidative stress an
- Page 144 and 145: CHAPTER 3 INTRODUCTION 120 The huma
- Page 146 and 147: CHAPTER 3 MATERIALS AND METHODS Che
- Page 148 and 149: CHAPTER 3 1:15 for hippocampus and
- Page 150 and 151: CHAPTER 3 initially incorporated to
- Page 152 and 153: CHAPTER 3 RESULTS In vivo effects o
- Page 154 and 155: CHAPTER 3 Effects of aluminium admi
- Page 156 and 157: CHAPTER 3 DISCUSSION 132 Aluminium
- Page 158 and 159: CHAPTER 3 hippocampus, showed simil
- Page 160 and 161: CHAPTER 3 assume that the distinct
- Page 162 and 163: CHAPTER 3 Fig. 1 Levels of TBARS (A
Statistical analysis<br />
CHAPTER 1<br />
Results were expressed as the mean ± SD from five animals. Statistical<br />
differences were tested us<strong>in</strong>g one-way ANOVA followed by LSD for multiple<br />
comparisons (p < 0.05). All statistical analyses were performed us<strong>in</strong>g Sigmastat 3.0<br />
from Jandel Scientific (San Diego, CA, USA).<br />
91
Change language