1 |
Molecular Mechanism of Connector-RNA Interaction of Bacteriophage Phi29 DNA Packaging Motor and Applications of Motor Components in NanotechnologyXiao, Feng January 2009 (has links)
No description available.
|
2 |
The effect of PEG-insulin and insulin hexamer assembly on stability in solution and dry powders : hexamer assembly of PEGylated-insulin and insulin studied by multi-angle light scattering to rationally choose the pH and zinc content for analytical methods and formulations of dry powdersBueche, Blaine January 2010 (has links)
The objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin's systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin's charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID's in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.
|
3 |
The Effect of PEG-Insulin and Insulin Hexamer Assembly on Stability in Solution and Dry Powders. Hexamer Assembly of PEGylated-Insulin and Insulin Studied by Multi-Angle Light Scattering to Rationally Choose the pH and Zinc Content for Analytical Methods and Formulations of Dry Powders.Bueche, Blaine January 2010 (has links)
The objective of this research is to further define the relationship between the charge state
of insulin, and the self assembly properties of insulin and PEGylated insulin in solution.
Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate
their impact on insulin¿s systemic duration of action after pulmonary dosing. This thesis
will focus on the assembly properties of the PEG-insulin and insulin, and also
demonstrate how the charge state, which was modified by the covalent attachment of
PEG, relates to different modes of behavior by anion and cation exchange
chromatography. In addition, explain how modifying the assembly state extends to
improving formulation properties of spray-dried insulin powders.
This thesis is an investigation into the relationship of insulin¿s charge state controlled by
pH and how the charge state affects the self assembly of insulin, especially when the zinc
ion is removed. Ionic interaction is one of the major forces affecting insulin assembly.
The theory that a change in the charge state of insulin could modulate the ionic
interaction and reduce hexamer formation at alkaline conditions was investigated.
Experiments were designed to measure the level of hexamer with light scattering, and the
amount of hexamer was then correlated with the pH and zinc content of the solutions. The
importance of the charge state of the monomer and its behavior extends to
chromatography and purification modes as well. Specifically, the purification of various
species of PEGylated insulin presents a challenge. By varying mobile phase pH which
induces the charge to insulin, an ion exchange method demonstrated very high resolution
and controllable interaction between the ion exchange media and the insulin derivatives.
A highly accurate method for determining molecular weight and thus the average
associated state of insulin in solution has been developed using the MALS (Multi-Angle
Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in
pharmaceutically relevant ranges. The MALS method was developed to evaluate how the
parameters above affect the self-assembly properties of insulin, and use the assembly
properties to improve formulations of insulin or PEGylated insulin. To use the light
scattering technique the dn/dc (change in refractive index with change in concentration) is
required. During the method development, the dn/dc of insulin was measured at 690 nm,
and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based
on maintaining the dissociation of hexamers in solution during the spray drying process
was developed. The mode presented here is to remove the zinc ions from solution,
increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin
approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer
population in solution, but by combining all three factors, hexamers are driven to very
low levels of equilibrium. The increased stability of the powders is predominately related
to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced
hexamer population in the solution with lower levels of CID¿s in the dry powder
compared to controls. The CID formation rate was reduced by 40% compared to a
control.
|
4 |
Počítačové modelování interakcí iont ů s proteiny: Allosterický efekt iont ů a fenolických ligand ů na strukturu insulinového hexameru / Computer modeling of ion protein interactions: Allo steric effects of phenolic ligands and ions on insulin hexamer struct urePalivec, Vladimír January 2016 (has links)
Title: Computer modeling of ion protein interactions: Allosteric effects of phenolic ligands and ions on insulin hexamer structure Author: Vladimír Palivec Department: Department of Physical and Macromolecular Chemistry Faculty of Science UK Advisor: prof. RNDr. Pavel Jungwirth, DSc., IOCB AS CR, v.v.i. Advisor's email address: pavel.jungwirth@uochb.cas.cz Abstract: Insulin hexamer is an allosteric protein capable of undergoing conformational changes between three states: T6, T3R3, and R6. Transitions between them, as well as the formation of insulin hexamers, are mediated through binding of phenolic ligands or ions. This thesis presents a molecular dynamics study of allosteric behavior of insulin using empirical force fields. Two effects are closely inspected - cation (Zn2+ , Ca2+ , K+ , and Na+ ) binding to the insulin hexamers and a possible binding of two neurotransmitters - dopamine and serotonin to the phenolic pocket. The results show that high charge density cations (Zn2+ and Ca2+ ) are mostly localized in the B13 glutamate cavity, slow- down diffusion, while preventing other cations from entering. In contrast, low charge density cations (Na+ and K+ ) do not have this effect. Concerning neurotransmitters, dopamine does not bind to the phenolic pocket whereas serotonin binds in a similar way like...
|
5 |
Caractérisation des structures de septines hautement organisées chez la drosophile et leur interaction avec le cytosquelette d’actineDragieva, Zlatina 06 1900 (has links)
Les septines sont des GTPases conservées dérégulées dans le cancer et les maladies neurodégénératives. Elles servent de protéines d’échafaudage et forment une barrière de diffusion à la membrane plasmique et au corps central lors de la cytokinèse. Elles interagissent avec l’actine et s’organisent en complexes qui polymérisent et forment des structures hautement organisées (anneaux et filaments). Leur dynamique d’assemblage et leur rôle dans la cellule restent à être élucidés. La Drosophile est un modèle simple pour l’étude des septines puisqu’on n’y retrouve que 5 gènes (sep1, sep2, sep4, sep5, peanut) comparativement aux 13 gènes chez l’humain.
À l’aide d’un anticorps contre Pnut, nous avons identifié des structures tubulaires dans 30% des cellules S2 de Drosophile. Mon projet a comme but de caractériser ces tubes en élucidant leurs constituants, leur comportement et leurs propriétés pour mieux clarifier le mécanisme par lequel les septines forment des structures hautement organisées et interagissent avec le cytosquelette d’actine.
Par immunofluorescence, j’ai pu démontrer que ces tubes sont cytoplasmiques, en mitose ou interphase, ce qui suggère qu’ils ne sont pas régulés par le cycle cellulaire. Pour investiguer la composition et les propriétés dynamiques de ces tubes, j’ai généré une lignée cellulaire exprimant Sep2-GFP qui se localise aux tubes et des ARNi contre les cinq septines. Trois septines sont importantes pour la formation de ces tubes et anneaux notamment Sep1, Sep2 et Pnut. La déplétion de Sep1 cause la dispersion du signal GFP en flocons, tandis que la déplétion de Sep2 ou de Pnut mène à la dispersion du signal GFP uniformément dans la cellule. Des expériences de FRAP sur la lignée Sep2-GFP révèlent un signal de retour très lent, ce qui indique que ces structures sont très stables.
J’ai aussi démontré une relation entre l’actine et les septines. Le traitement avec la Latrunculin A (un inhibiteur de la polymérisation de l’actine) ou la Jasplakinolide (un stabilisateur des filaments d’actine) mène à la dépolymérisation rapide (< 30 min) des tubes en anneaux flottants dans le cytoplasme, même si ces tubes ne sont pas reconnus suite à un marquage de la F-actine. L’Actin05C-mCherry se localise aux tubes, tandis que le mutant déficient de la polymérisation, Actin05C-R62D-mCherry perd cette localisation. On observe aussi que la déplétion de la Cofiline et de l’AIP1 (ce qui déstabilise l’actine) mène au même phénotype que le traitement avec la Latrunculine A ou la Jasplakinolide. Alors on peut conclure qu’un cytosquelette d’actine dynamique est nécessaire pour la formation et le maintien des tubes de septines.
Les futures études auront comme but de mieux comprendre l’organisation des septines en structures hautement organisées et leur relation avec l’actine. Ceci sera utile pour l’élaboration du réseau d’interactions des septines qui pourra servir à expliquer leur dérégulation dans le cancer et les maladies neurodégénératives. / Septins are highly conserved GTP-binding proteins deregulated in diseases such as cancer and neurodegenerative diseases. Septins scaffold other proteins and act as diffusion barriers at the plasma membrane and midbody during cytokinesis. They interact with the actin cytoskeleton and have been observed to form ordered complexes that can polymerize into higher-order structures such as filaments and rings. The principles of assembly and disassembly of such filaments and rings and their cellular roles are yet to be elucidated. Drosophila offers a simple system, as there are only 5 septin genes: peanut, sep1, sep2, sep4, and sep5 in contrast to 13 found in humans.
We have previously found that Drosophila S2 cells contain unusual tubular structures that label with Peanut antibody. The goal of my Master’s project has been to characterise these structures, by defining their constituents, behaviours and properties, in the hope that this will shed light on the mechanisms by which septins can form higher-ordered structures and how they interact with other cytoskeletal elements such as actin.
Using fluorescence microscopy, I show that these tubes are cytoplasmic and present in 30% of cells, both during mitosis and interphase, suggesting they are not cell cycle regulated. To investigate their composition and dynamic properties, I generated S2 cell lines stably expressing Sep2-GFP, which localizes to septin tubes and double stranded RNAs against all septins. The products of three septin genes were found to be essential for the assembly of septin tubes: Sep1, Sep2, and Pnut. The depletion of Sep1 led to the dispersal of the GFP signal into cytoplasmic clumps, whereas the depletion of Sep2 and Pnut led to its uniform distribution through the cell. FRAP analysis of Sep2-GFP revealed only slow recovery after many hours, indicating that the structures are very stable.
I also discovered an unusual relationship between septin tubes and the actin cytoskeleton. Although the tubes did not label with conventional F-actin probes (Phalloidin, LifeAct), treatment with inhibitors of F-actin assembly (Latrunculin A) or disassembly (Jasplakinolide) led to their rapid (<30 min) dispersal into scattered rings. Furthermore, overexpressed Actin05C-mCherry localised to the septin tubes, while a polymerization-deficient mutant Actin05C-R62D-mCherry did not. Depletion of the actin severing protein Cofilin and the actin capping protein AIP1 also disrupted septin tubes dispersing them into cytoplasmic rings. A dynamic actin cytoskeleton is thus required for the formation and/or maintenance of higher ordered structures such as rings and tubes.
Conclusion and Relevance: Ongoing studies aim to further elucidate how septins organize into such ordered structures and how actin regulates the process. This will clarify the septin network of interactions and facilitate the comprehension of their implication in cancer and neurodegenerative diseases.
Key words: actin, septin, tube, ring, higher-order, hexamer, cofilin, AIP1
|
6 |
Functional Characterization of Hereditary Spastic Paraplegia Proteins Spastin and ZFYVE27Pantakani, Dasaradha Venkata Krishna 02 July 2009 (has links)
No description available.
|
Page generated in 0.0425 seconds