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تحميل الدليل التدريبي

أسئلة شائعة


 

Name: Dr. Amany A. Aly

PhD degree from UK, University of Leeds, School of Biochemistry and Molecular Biology, April 2001.

Title: Biophysical Studies on Phospholamban

 

Abstract of Thesis:

 

Phospholamban is an inhibitor of the sarcoplasmic reticulum Ca2+-pump of cardiac muscle. This inhibition is achieved by physical interaction between the two proteins, which suppresses the rate of Ca2+ transport into the SR to bring about relaxation of the muscle. Inhibition is relieved upon phosphorylation of PLB on one of a number of residues, including Ser-16. In addition to its interaction with the Ca2+-pump, PLB forms apparently stable interactions with itself, adopting an oligomeric structure in the membrane, which has been modelled to be a pentamer. The oligomeric structure of PLB is reminiscent of an ion channel, and some evidence has been presented in support of PLB possessing cation-channel properties. This property of a Ca2+-channel is difficult to reconcile with its established role in the regulation of Ca2+-sequestration by the SR.

This project aimed to investigate whether PLB possessed ion channel properties and to determine how these were regulated.

In these studies, PLB has been expressed in Escherichia coli as a fusion protein with an N-terminal affinity purification tag (His6-thrombin cleavage site) immediately preceding the PLB protein. This was used to purify (tagged) PLB in significant amounts (3mg/L bacterial culture) in a single chromatographic step from a sample of SDS-solubilised inclusion body material. For ion channel studies the PLB protein was purified to homogeneity by reverse phase HPLC chromatography on C4 columns, which allowed recovery of 40% of protein loaded onto the column. The tagged PLB protein was reconstituted into vesicles and then incorporated into a black lipid bilayer, but this protein (taggedPLB) did not exhibit ion channel properties.

To investigate whether the affinity tag affected PLB function, the fusion protein was digested with thrombin and the PLB product was purified to homogeneity by reverse phase HPLC. This was then incorporated into black lipid membranes after reconstitution of PLB into vesicles, as before. In this format PLB displayed ion channel activity. Channel opening was rather erratic in character, producing current amplitudes of short duration but variable amplitude.  Standard electrophysiological analysis of these current traces was not possible, and so we developed a simple, logical methodology for the analysis of these data. The closed state of the PLB channel was readily identified in all experiments, and thus the absolute values (of current) that represent the closed state were deducted from all other current values in the data set (at that holding potential). An average conductance per unit time was then calculated for each data set, and used to construct average current/voltage relationships. In the presence of a ten-fold gradient of KCl, PLB displayed voltage dependent channel activity with mild anion selectivity (PCl/PK = 3.33 ± 1.5 (n=7)).

To investigate the regulation of this ion channel we examined the effect of oligomeric status on channel function, and the effect of two important physiological regulators of PLB, namely Ser-16 phosphorylation and Ca2+ ions. A mutant form of PLB, C41A/C46A (called m5 throughout this study), was expressed in E. coli and purified to homogenity. This protein was monomeric in SDS-PAGE experiments, however it was able to form ion channels indistinguishable from wild type PLB when incorporated in black lipid membranes. From this we suspect that the oligomeric status of m5PLB in SDS does not reflect the oligomeric state of the protein in a lipid bilayer.

Wild type PLB was phosphorylated (on Ser-16) to stoichiometry using purified protein kinase A. Phosphorylated PLB exhibited voltage dependent ion channel activity with mild anion selectivity similar to the dephosphorylated channel. However the channel differed from dephosphorylated PLB in two respects: firstly, the channel activity was more orthodox, with channel openings generating a current amplitude of more uniform size within each data sweep. Secondly, the average conductance of the phosphorylated channel was significantly greater than that of the dephosphorylated PLB.

Finally, we investigated the regulatory effects of Ca2+-ions. PLB and phosphorylated PLB channels were exposed to CaCl2 in the presence of the ten-fold gradient of KCl. In all cases it appeared that Ca2+, K+ and Cl- were permeant species, however in the case of the dephosphorylated PLB Ca2+ also altered the ion selectivity of the channel, causing it to become more permeable to cations.

 

The publications:

 

1- Dielectric relaxation and light scattering studies for lipid/cholesterol liposomes, W. A. Khalil, A. Soltan Monem, M. Abutaleb, A. Abdalla and M. Ali Fadel

Egyptian J. of Biophysics, Special Issue. “Molecular and Molecular Biophysics” (1994) 128

 

 

2- Studies of the induced effects of exposures to 0.1 mT 50 Hz magnetic fields on the biophysical properties of RBCs and ECG of rats (in vivo studies), Reem El-Gebaly and Amany A. Aly, Egyptian J. of Biophysics, December2003

Abstract

In this work the effect of exposure to sine wave 0.1 mT homogeneous magnetic field for a period of one month on the osmotic fragility, solubilization of red blood cells (RBCs) membrane and on the molecular structure of haemoglobin from albino rats  is studied. The rats were exposed to the magnetic field five days/week for 8 hs/day, similar to occupational exposures. The changes in the molecular structure of haemoglobin were measured through measurements of the dielectric relaxation in the frequency range 0.1-10 MHz. The electrocardiograms (ECGs) for the exposed and unexposed rat groups were also studied. The results indicated that exposure to magnetic fields induce changes in the RBCs membrane fragility and solubilization with non-ionic detergents, which were confirmed by histological studies. Heart injuries were also recorded for the exposed animals. 

 

 

 

3- Control of Ehrlich tumour growth by electromagnetic waves at resonance frequency (In vivo studies)

Fadel M. A., Reem H.El-Gebaly, Amany A. Aly and Fakhry F. Ibrahim*

 

Department of Biophysics , Faculty of Science ,Cairo University Egypt

 *  Department of Clinical pathology,Cairo University hospitals Egypt

Electromagnetic Biology And Medicine, volume24, number1, 2005

3- Control of Ehrlich tumour growth by electromagnetic waves at resonance frequency (In vivo studies)

Fadel M. A., Reem H.El-Gebaly, Amany A. Aly and Fakhry F. Ibrahim*, Electromagnetic Biology And Medicine, volume24, number1, 2005

Abstract: In this work Ehrlich tumour growth in mice was controlled by applying extremely low frequency electromagnetic field (ELF EF) at a resonance frequency of 4.5 Hz. The electromagnetic waves at this frequency were conducted deeply in the tumour tissue using  amplitude modulated waves (AMW).Sixty female Balb/c mice carrying Ehrlich tumour in the thigh were divided into three equal groups namely A, B and C.  Group A was the  control while Group B and C both were exposed to 4.5Hz square amplitude modulated waves (QAMW) for a period of  ten hours (hs) starting day10 and day16 post tumour implantation respectively. Tumour size, telomerase enzyme activity, histopathological examination and dielectric relaxation of the tumour tissue were used to investigate the tumour activity of the treated and untreated groups of animals. The results indicated that irradiating the tumour tissue by 4.5 Hz QAMW for a period of 10hs inhibited tumour growth. Early treatment of the tumour by ELFEF gave better results than delayed treatments.

 

 

4-Effect of microwave radiation on some biophysical and cardiovascular properties of exposed rats. The 3rd meeting of sauid physical scientific society, 2-18 December 2006, in King saud university.  

 

 

 

 
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