Systematic studies of the interaction between amyloid {β-protein [beta-protein] and lipids [Elektronische Ressource] / vorgelegt von Haipeng Song

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Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2005
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Systematic Studies of the interaction between
Amyloid ß-Protein and Lipids



Dissertation zur Erlangung des Grades
“Doktor der Naturwissenschafen”





am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz





vorgelegt von
Haipeng Song
aus Changchun, Jilin, V. R. China



Mainz, June, 2005 - ii -


thTag der mündlichen Prüfung: 28 , June 2005




















Die vorliegende Arbeit wurde unter Betreuung von Herrn Prof. Dr. H. Paulsen und Herrn
Prof. Dr. W. Knoll im Zeitraum zwischen August 2001 bis Februar 2005 am Max-Planck-
Institute für Polymerforschung, Mainz, Deutschland angefertigt.

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CONTENTS
CONTENTS........................................................................................................................I
1 INTRODUCTION..................................................................................................... 1
1.1 THE AMYLOID HYPOTHESIS OF ALZHEIMER’S DISEASE..................................................1
1.2 FACTORS AFFECTING Aß AGGREGATION..........................................................................3
1.3 AIM OF THE STUDY.........................................................................................................5
2 METHODS AND THEORIES................................................................................. 8
2.1 LANGMUIR FILMS............................................................................................................8
2.1.1 Surface pressure-area isotherms ...................................................................................................... 9
2.1.2 Forces at the subphase surface........................................................... 10
2.1.3 Surface pressure measurement 13
2.1.4 Langmuir-Blodgett (LB) films............................................................................................................ 15
2.2 CONTACT ANGLE MEASUREMENT .................................................................................18
2.3 PREPARATION OF LIPOSOMES (VESICLES) .....................................................................22
2.3.1 Mechanism of vesicle formation....................................................................................................... 22
2.3.2 Procedures of liposome preparation ..................................................... 23
2.3.2.1 Preparation of lipid for hydration ............................................................ 23
2.3.2.2 Hydration of lipid film/cake ................................................ 23
2.3.2.3 Sizing of lipid suspension.............................................................................................. 25
2.4 FLUORESCENCE MICROSCOPY ......................................................................................26
2.4.1 Experimental apparatus for florescence microscopy .................................................................... 26
2.4.2 Image acquisition and data analysis ..................................................... 28
2.5 SURFACE PLASMON RESONANCE SPECTROSCOPY (SPR) & SURFACE PLASMON FIELD-
ENHANCED FLUORESCENCE SPECTROSCOPY (SPFS) .................................................................29
2.5.1 SPR................................................................................................................................... 29
2.5.2 SPFS........................................................................................................ 33
2.5.2.1 Field Enhancement........................................ 34
2.5.2.2 Fluorescence at the Metal/Dielectric Interface ............................................................. 35
2.5.2.3 Home-Made SPFS Setup..................................................................................................................... 38
2.6 INTERFACIAL KINETICS BASED ON LANGMUIR ADSORPTION MODEL ...........................39
3 CONSTRUCTION OF PEPTIDE-TETHERED LIPID MEMBRANES.......... 41
3.1 INTRODUCTION/BACKGROUND.....................................................................................41
3.2 MATERIALS ...................................................................................................................44
3.3 THE CONSTRUCTION OF PEPTIDE-TETHERED LIPID BILAYERS (T-LBS) .........................45
3.4 RESULTS AND DISCUSSIONS..........................................................................................48
3.5 CONCLUSION ................................................................................................................51
4 INVESTIGATION ON THE INTERACTION BETWEEN AMYLOID ß-
PROTEIN (Aß) AND LIPIDS BY SURFACE PLASMON FIELD-ENHANCED
FLUORESCENCE SPECTROSCOPY (SPFS) ........................................................... 53
4.1 INTRODUCTION/BACKGROUND.....................................................................................53
4.2 MATERIALS ...................................................................................................................55
4.3 FLUORESCENCE LABELLING OF THE MONOCLONAL ANTIBODY....................................56
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4.4 INTERACTION OF ANTI-Aß-MAB AND T-LBS.................................................................58
4.5 BINDING SPECIFICITY OF Aß TO THE MODEL MEMBRANES............................................64
4.6 CONCLUSIONS...............................................................................................................69
5 THE INTERACTIONS OF THE STEROLS (CHOLESTEROL AND 25-
HYDROXYCHOLESTEROL) WITH SPHINGOMYELIN...................................... 70
5.1 INTRODUCTION/BACKGROUND .....................................................................................70
5.2 MAT E R I A L S ...................................................................................................................71
5.3 FLUORESCENCE IMAGING .............................................................................................71
5.4 DETERGENT SOLUBILIZATION OF EXPERIMENTS...........................................................80
5.5 CONCLUSIONS...............................................................................................................81
6 CONFORMATION OF AMYLOID ß-PROTEIN ADSORBED ON THE
TETHERED ARTIFICIAL MEMBRANE PROBED BY SURFACE PLASMON
FIELD-ENHANCED FLUORESCENCE SPECTROSCOPY................................... 83
6.1 INTRODUCTION .............................................................................................................83
6.4 PAIR-WISE BINDING OF THE FLUORESCENT ANTIBODIES TO Aß ....................................87
6.5 THE PREPARATION OF MELATONIN SAMPLE ..................................................................89
6.6 THE ORIENTATION OF Aß ADSORBED ON THE MODEL MEMBRANES ..............................89
6.7 EFFECTS OF MELATONIN ON THE BINDING OF THE MABS TO Aß ...................................96
6.8 CONCLUSIONS.............................................................................................................101
7 SUMMARY ........................................................................................................... 102
8 SUPPLEMENT ..................................................................................................... 104
8.1 LIST OF FIGURES.........................................................................................................104
8.2 LIST OF TABLES...........................................................................................................108
8.3 BIBLIOGRAPHY ...........................................................................................................109

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1 INTRODUCTION
1.1 The Amyloid hypothesis of Alzheimer’s disease
Alzheimer’s disease (AD) is the most frequent form of intellectual deterioration in elderly
individuals and the fourth leading cause of death in developed nations. Although the
cause of AD remains unknown, this disorder is a progressive degenerative disorder of
insidious onset and is characterized by memory loss, confusion and a variety of cognitive
disabilities. Clearly, this disease is a major social and health-care problem for which there
is no cure or effective treatment. AD can be divided into a familial form, which usually
2has an early onset (before 60 years), and a sporadic late-onset form.
The molecular mechanisms and hypothesis of Alzheimer’s disease can be incredibly
complex. So far, the prevalent hypothesis is that amyloid ß-peptides (Aß), aggregating
3outside nerve cells in the brain, are the primary influence driving AD pathogenesis. Two
variants of Aß have been found in these plaques: Aß1-40 and Aß1-42. The evidence that
Aß aggregates play an important role in the early pathogenesis of AD comes mainly from
studies of individuals affected by the familial form of the disease or by the so-called
4Down syndrome. Almost all patients with Down syndrome develop AD neuropathology
5at an early age. In a survey of brains from Down syndrome patients, diffuse amyloid
deposits