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Purification of alpha-crystallin protein from calf eye lens
S258
Abstracts
E. poster — [A-10-565-2] Novel cilostamide analogs, PDE3 inhibitors, produce positive inotropic but differential lusitropic and chronotropic effects on isolated rat atria Azar Hosseinia, Reza Shafiee-Nicka, Hamid Sadeghianb, Heydar Parsaeea a Pharmacological Research Center of Medicinal Plants, Dept. of Pharmacology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b Paramedic School, Mashhad University of Medical Sciences, Iran E-mail addresses: [email protected] (A. Hosseini), [email protected] (R. Shafiee-Nick), [email protected] (H. Sadeghian), [email protected] (H. Parsaee) Introduction: In previous work, we synthesized several analogs of cilostamide (4-(1,2-dihydro-2-oxoquinolin-6-hydroxy)-N-cyclohexyl-Nmethylbutanamide), a selective PDE3 inhibitor, via modification of the amide and carbostiryl moiety and examined their inotropic and chronotropic effects. Among the synthetic compounds, some piperidine and piperazine derivatives showed high inotropic beside their low chronotropic activity in comparison with IBMX, a non-selective PDE inhibitor. Therefore, we made a detailed comparative study on the potential cardiotonic activity of the mentioned compounds by using the spontaneously beating atria model. Materials and methods: In each experiment, the atrium of reserpinetreated rat was isolated and the possible inotropic, lusitropic and chronotropic effects of each test-compound, alone or in combination with different concentrations of isoprenaline, were assessed. Results: Most of the test compounds significantly increased atria contraction force and produced deferential effects on heart rate. Cilostamide, MCPIP, mc1, mc2 and mc5 synergistically increased the effect of isoprenaline on ACF. No such effect was observed for mc6. In enzyme assay, mc6 inhibited PDE3 activity with high potency. Higher hydrophilic nature of mc3 and mc4 was assumed as the major factor for describing their low potency. It was interesting that; mc2 with the highest inotropic activity did not affect the basal contraction rate. Furthermore, it reduced the isoprenaline chronotropic effect. Conclusion: By considering the synergistic inotropic effect of mc2–isoprenaline combination, attenuation of isoprenaline chronotropic effect cannot be mediated via reducing B-adrenergic receptors activity. The differential effects of the test compounds are not correlated with their PDE3 inhibitory effects and are subjects for further investigations. Keywords: PDE inhibitor, Inotropic activity, Rat atria, Isoprenaline, Cilostamide doi:10.1016/j.clinbiochem.2011.08.645
Poster – [A-10-572-1] Purification of alpha-crystallin protein from calf eye lens Fereshte Bahmania, Zahra Bathaeib, Arezou Ghahghaeic a Tehran, Iran b Tehran, Jalal-e-Ale Ahmad Highway, Iran c Zahedan, University of Sistan and Baluchestan, Iran E-mail addresses: [email protected] (F. Bahmani), [email protected] (Z. Bathaei), [email protected] (A. Ghahghaei) Introduction: Alpha-crystallin, a member of the small heat shock protein family, is a major structural protein of the vertebrate eye lens. It is a large heteropolymer composed of two subunits αA and αB, each of 20 kDa. In addition to its structural role, it can act as molecular
chaperone. The chaperoning activity of alpha-crystallin is believed to be essential for the maintenance of transparency of the lens, thus preventing the formation of cataract. In this study we have attempted to isolate alpha-crystallin from calf eye lens and investigated the effects of glycation on its structure and chaperoning function. Materials and methods: Calf eyeballs were obtained from a local abattoir, and lenses were dissected out. Lenses were homogenized in 50 mM Tris buffer, pH 7.2, 5 mM EDTA, 1 mM dithiothreitol (DTT), 0.04% (W/V) NaN3, 0.1 mM PMSF and centrifuged at 12,000 rpm for 30 min at 5 °C. The supernatant was fractionated by size-exclusion chromatography on a Sephacryl S300HR column. Protein was eluted as the column buffer (50 mM Tris, 0.04% (W/V) NaN3, pH 7.5) was passed through at a flow rate 20 ml/h. Fractions containing alpha-crystallin were freeze-dried and stored. The purity of alpha-crystallin was checked by SDS-PAGE and its chaperoning activity was assessed by DTT-induced aggregation of insulin. Results: Our results indicate the purity of isolated alpha-crystallin and it can suppress DTT-induced aggregation of insulin. The study about glycation effects on the structure and chaperoning activity of this protein is continuing. Keywords: Alpha-crystallin, Glycation, Chaperone activity, Cataract doi:10.1016/j.clinbiochem.2011.08.646
Oral – [A-10-654-1] Study of non-enzymatic glycosylation effects on the structure of human serum albumin via Differential Scanning Calorimetry Ahmad Mohamadinejada, Ali.A Moosavi-Movahedib a Mashhad, Iran b Tehran, Iran E-mail addresses: [email protected] (A. Mohamadinejad), [email protected] (A.A. Moosavi-Movahedi) Introduction: Human serum albumin is the major carrier in blood with almost the same three domains of energy as were showed by X-ray crystallography, so that each of them has two fairly similar subdomains. Completely separate the two parts, one part hard and compact makes head of albumin whiles the other part somewhat more stretched and loose makes its tail. And each contains three almost identical subdomains. Materials and methods: Extracted albumin after purification, was dialyzed in phosphates buffer 2.5 mM, pH7.4 at 37 °C, and incubated with glucose in concentrations 1.5, 3 and 5 mg/ml (the different conditions of diabetes) for fifteen days at 37 °C. Binding of glucose with albumin as a non-enzymatic manner, glycated albumin (GHSA) is formed. After this stage each of glycosylated and non-glycosylated albumins were studied, by “Differential Scanning Calorimetry (DSC)”. Results: non-glycosylated albumin denaturation occurs in two stages separately, the first phase related to tail of albumin and the second to its head. Whiles the glycosylated albumin denaturations show three distinct stages. Increased concentration of glucose causes: firstly formation of new energetic domain and secondly sustainable structure that will become more compact the head and subdomains in tail of albumin. Conclusion: Therefore, glucose squeezed out the head of albumin and its subdomains located in the tail namely the fundamental changes in the structure and hence alter its function fundamentally. Keywords: Human serum albumin, DSC, Structure, Function, Glycosylated albumin doi:10.1016/j.clinbiochem.2011.08.647
Abstracts
E. poster — [A-10-565-2] Novel cilostamide analogs, PDE3 inhibitors, produce positive inotropic but differential lusitropic and chronotropic effects on isolated rat atria Azar Hosseinia, Reza Shafiee-Nicka, Hamid Sadeghianb, Heydar Parsaeea a Pharmacological Research Center of Medicinal Plants, Dept. of Pharmacology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b Paramedic School, Mashhad University of Medical Sciences, Iran E-mail addresses: [email protected] (A. Hosseini), [email protected] (R. Shafiee-Nick), [email protected] (H. Sadeghian), [email protected] (H. Parsaee) Introduction: In previous work, we synthesized several analogs of cilostamide (4-(1,2-dihydro-2-oxoquinolin-6-hydroxy)-N-cyclohexyl-Nmethylbutanamide), a selective PDE3 inhibitor, via modification of the amide and carbostiryl moiety and examined their inotropic and chronotropic effects. Among the synthetic compounds, some piperidine and piperazine derivatives showed high inotropic beside their low chronotropic activity in comparison with IBMX, a non-selective PDE inhibitor. Therefore, we made a detailed comparative study on the potential cardiotonic activity of the mentioned compounds by using the spontaneously beating atria model. Materials and methods: In each experiment, the atrium of reserpinetreated rat was isolated and the possible inotropic, lusitropic and chronotropic effects of each test-compound, alone or in combination with different concentrations of isoprenaline, were assessed. Results: Most of the test compounds significantly increased atria contraction force and produced deferential effects on heart rate. Cilostamide, MCPIP, mc1, mc2 and mc5 synergistically increased the effect of isoprenaline on ACF. No such effect was observed for mc6. In enzyme assay, mc6 inhibited PDE3 activity with high potency. Higher hydrophilic nature of mc3 and mc4 was assumed as the major factor for describing their low potency. It was interesting that; mc2 with the highest inotropic activity did not affect the basal contraction rate. Furthermore, it reduced the isoprenaline chronotropic effect. Conclusion: By considering the synergistic inotropic effect of mc2–isoprenaline combination, attenuation of isoprenaline chronotropic effect cannot be mediated via reducing B-adrenergic receptors activity. The differential effects of the test compounds are not correlated with their PDE3 inhibitory effects and are subjects for further investigations. Keywords: PDE inhibitor, Inotropic activity, Rat atria, Isoprenaline, Cilostamide doi:10.1016/j.clinbiochem.2011.08.645
Poster – [A-10-572-1] Purification of alpha-crystallin protein from calf eye lens Fereshte Bahmania, Zahra Bathaeib, Arezou Ghahghaeic a Tehran, Iran b Tehran, Jalal-e-Ale Ahmad Highway, Iran c Zahedan, University of Sistan and Baluchestan, Iran E-mail addresses: [email protected] (F. Bahmani), [email protected] (Z. Bathaei), [email protected] (A. Ghahghaei) Introduction: Alpha-crystallin, a member of the small heat shock protein family, is a major structural protein of the vertebrate eye lens. It is a large heteropolymer composed of two subunits αA and αB, each of 20 kDa. In addition to its structural role, it can act as molecular
chaperone. The chaperoning activity of alpha-crystallin is believed to be essential for the maintenance of transparency of the lens, thus preventing the formation of cataract. In this study we have attempted to isolate alpha-crystallin from calf eye lens and investigated the effects of glycation on its structure and chaperoning function. Materials and methods: Calf eyeballs were obtained from a local abattoir, and lenses were dissected out. Lenses were homogenized in 50 mM Tris buffer, pH 7.2, 5 mM EDTA, 1 mM dithiothreitol (DTT), 0.04% (W/V) NaN3, 0.1 mM PMSF and centrifuged at 12,000 rpm for 30 min at 5 °C. The supernatant was fractionated by size-exclusion chromatography on a Sephacryl S300HR column. Protein was eluted as the column buffer (50 mM Tris, 0.04% (W/V) NaN3, pH 7.5) was passed through at a flow rate 20 ml/h. Fractions containing alpha-crystallin were freeze-dried and stored. The purity of alpha-crystallin was checked by SDS-PAGE and its chaperoning activity was assessed by DTT-induced aggregation of insulin. Results: Our results indicate the purity of isolated alpha-crystallin and it can suppress DTT-induced aggregation of insulin. The study about glycation effects on the structure and chaperoning activity of this protein is continuing. Keywords: Alpha-crystallin, Glycation, Chaperone activity, Cataract doi:10.1016/j.clinbiochem.2011.08.646
Oral – [A-10-654-1] Study of non-enzymatic glycosylation effects on the structure of human serum albumin via Differential Scanning Calorimetry Ahmad Mohamadinejada, Ali.A Moosavi-Movahedib a Mashhad, Iran b Tehran, Iran E-mail addresses: [email protected] (A. Mohamadinejad), [email protected] (A.A. Moosavi-Movahedi) Introduction: Human serum albumin is the major carrier in blood with almost the same three domains of energy as were showed by X-ray crystallography, so that each of them has two fairly similar subdomains. Completely separate the two parts, one part hard and compact makes head of albumin whiles the other part somewhat more stretched and loose makes its tail. And each contains three almost identical subdomains. Materials and methods: Extracted albumin after purification, was dialyzed in phosphates buffer 2.5 mM, pH7.4 at 37 °C, and incubated with glucose in concentrations 1.5, 3 and 5 mg/ml (the different conditions of diabetes) for fifteen days at 37 °C. Binding of glucose with albumin as a non-enzymatic manner, glycated albumin (GHSA) is formed. After this stage each of glycosylated and non-glycosylated albumins were studied, by “Differential Scanning Calorimetry (DSC)”. Results: non-glycosylated albumin denaturation occurs in two stages separately, the first phase related to tail of albumin and the second to its head. Whiles the glycosylated albumin denaturations show three distinct stages. Increased concentration of glucose causes: firstly formation of new energetic domain and secondly sustainable structure that will become more compact the head and subdomains in tail of albumin. Conclusion: Therefore, glucose squeezed out the head of albumin and its subdomains located in the tail namely the fundamental changes in the structure and hence alter its function fundamentally. Keywords: Human serum albumin, DSC, Structure, Function, Glycosylated albumin doi:10.1016/j.clinbiochem.2011.08.647