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Hiroshi Ueda - One of the best experts on this subject based on the ideXlab platform.
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involvement of snare protein interaction for non classical release of damps alarmins proteins Prothymosin Alpha and s100a13
2020Co-Authors: Hayato Matsunaga, Sebok Kumar Halder, Hiroshi UedaAbstract:Prothymosin Alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca2+-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca2+-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.
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Prothymosin Alpha and its mimetic hexapeptide improve delayed tissue plasminogen activator induced brain damage following cerebral ischemia
2020Co-Authors: Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:Tissue plasminogen activator (tPA) administration beyond 4.5 h of stroke symptoms is beneficial for patients but has an increased risk of cerebral hemorrhage. Thus, increasing the therapeutic window of tPA is important for stroke recovery. We previously showed that Prothymosin Alpha (ProTα) or its mimetic hexapeptide (P6Q) has anti-ischemic activity. Here, we examined the beneficial effects of ProTα or P6Q against delayed tPA-induced brain damage following middle cerebral artery occlusion (MCAO) or photochemically induced thrombosis in mice. Brain hemorrhage was observed by tPA administration during reperfusion at 4.5 and 6 h after MCAO. Co-administration of ProTα with tPA at 4.5 h inhibited hemorrhage and motor dysfunction 2-4 days, but not 7 days after MCAO. ProTα administration at 2 and 4.5 h after MCAO significantly inhibited tPA (4.5 h)-induced motor dysfunction and death more than 7 days. Administration of tPA caused the loss of tight junction proteins, zona occulden-1 and occludin, and up-regulation of matrix metalloproteinase-2/9, in a ProTα-reversible manner. P6Q administration abolished tPA (4.5 h)-induced hemorrhage and reversed tPA (6 h)-induced vascular damage and matrix metalloproteinase-2 and 9 up-regulation. Twice administrations of P6Q at 2 h alone and 6 h with tPA significantly improved motor dysfunction more than 7 days. In photochemically induced thrombosis ischemia, similar vascular leakage and neuronal damage (infarction and motor dysfunction) by late tPA (4.5 or 6 h) were also inhibited by P6Q. Thus, these studies suggest that co-administration with ProTα or P6Q would be beneficial to inhibit delayed tPA-induced hemorrhagic mechanisms in acute ischemic stroke.
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experimental evidence for the involvement of f0 f1 atpase and subsequent p2y12 receptor activation in Prothymosin Alpha induced protection of retinal ischemic damage
2020Co-Authors: Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:The inhibition of retinal ischemia-induced damage by post-ischemic Prothymosin Alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or β-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.
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gγ7 specific Prothymosin Alpha deletion causes stress and age dependent motor dysfunction and anxiety
2020Co-Authors: Sebok Kumar Halder, Keita Sasaki, Hiroshi UedaAbstract:Abstract We previously showed that Prothymosin Alpha (ProTα) improves cerebral ischemia-induced motor dysfunction. Our recent study also demonstrated that heterozygous ProTα deletion exhibited an enhanced anxiety-like behavior in mice. However, it remains elusive which brain regions or cells are related to these phenotypes. Here we generated conditional Gγ7-specific ProTα knockout mice using G protein γ7 subunit gene (Gng7)-cre promoter to see the brain robustness roles of ProTα in the striatum and hippocampus. The younger conditional ProTα (Gng7) knockout mice at the age of 10 weeks showed no significant phenotypes in motor dysfunction in the Rotarod test and locomotor activity in the open-field test, whereas significant motor dysfunction was obtained by 15 min transient middle cerebral artery occlusion (tMCAO)-induced cerebral ischemia. The aged conditional ProTα (Gng7) knockout mice at the age of 20 weeks showed hypolocomotor activity with less center time in the open-field test and impaired motor coordination in the Rotarod test without ischemia. Thus, this study suggests that ProTα has important roles in the maintenance of motor coordination and anxiety-like behavior.
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beneficial actions of Prothymosin Alpha mimetic hexapeptide on central post stroke pain reduced social activity learning deficit and depression following cerebral ischemia in mice
2020Co-Authors: Keita Sasaki, Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:Prothymosin Alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.
Sebok Kumar Halder - One of the best experts on this subject based on the ideXlab platform.
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involvement of snare protein interaction for non classical release of damps alarmins proteins Prothymosin Alpha and s100a13
2020Co-Authors: Hayato Matsunaga, Sebok Kumar Halder, Hiroshi UedaAbstract:Prothymosin Alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca2+-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca2+-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.
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experimental evidence for the involvement of f0 f1 atpase and subsequent p2y12 receptor activation in Prothymosin Alpha induced protection of retinal ischemic damage
2020Co-Authors: Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:The inhibition of retinal ischemia-induced damage by post-ischemic Prothymosin Alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or β-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.
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Prothymosin Alpha and its mimetic hexapeptide improve delayed tissue plasminogen activator induced brain damage following cerebral ischemia
2020Co-Authors: Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:Tissue plasminogen activator (tPA) administration beyond 4.5 h of stroke symptoms is beneficial for patients but has an increased risk of cerebral hemorrhage. Thus, increasing the therapeutic window of tPA is important for stroke recovery. We previously showed that Prothymosin Alpha (ProTα) or its mimetic hexapeptide (P6Q) has anti-ischemic activity. Here, we examined the beneficial effects of ProTα or P6Q against delayed tPA-induced brain damage following middle cerebral artery occlusion (MCAO) or photochemically induced thrombosis in mice. Brain hemorrhage was observed by tPA administration during reperfusion at 4.5 and 6 h after MCAO. Co-administration of ProTα with tPA at 4.5 h inhibited hemorrhage and motor dysfunction 2-4 days, but not 7 days after MCAO. ProTα administration at 2 and 4.5 h after MCAO significantly inhibited tPA (4.5 h)-induced motor dysfunction and death more than 7 days. Administration of tPA caused the loss of tight junction proteins, zona occulden-1 and occludin, and up-regulation of matrix metalloproteinase-2/9, in a ProTα-reversible manner. P6Q administration abolished tPA (4.5 h)-induced hemorrhage and reversed tPA (6 h)-induced vascular damage and matrix metalloproteinase-2 and 9 up-regulation. Twice administrations of P6Q at 2 h alone and 6 h with tPA significantly improved motor dysfunction more than 7 days. In photochemically induced thrombosis ischemia, similar vascular leakage and neuronal damage (infarction and motor dysfunction) by late tPA (4.5 or 6 h) were also inhibited by P6Q. Thus, these studies suggest that co-administration with ProTα or P6Q would be beneficial to inhibit delayed tPA-induced hemorrhagic mechanisms in acute ischemic stroke.
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gγ7 specific Prothymosin Alpha deletion causes stress and age dependent motor dysfunction and anxiety
2020Co-Authors: Sebok Kumar Halder, Keita Sasaki, Hiroshi UedaAbstract:Abstract We previously showed that Prothymosin Alpha (ProTα) improves cerebral ischemia-induced motor dysfunction. Our recent study also demonstrated that heterozygous ProTα deletion exhibited an enhanced anxiety-like behavior in mice. However, it remains elusive which brain regions or cells are related to these phenotypes. Here we generated conditional Gγ7-specific ProTα knockout mice using G protein γ7 subunit gene (Gng7)-cre promoter to see the brain robustness roles of ProTα in the striatum and hippocampus. The younger conditional ProTα (Gng7) knockout mice at the age of 10 weeks showed no significant phenotypes in motor dysfunction in the Rotarod test and locomotor activity in the open-field test, whereas significant motor dysfunction was obtained by 15 min transient middle cerebral artery occlusion (tMCAO)-induced cerebral ischemia. The aged conditional ProTα (Gng7) knockout mice at the age of 20 weeks showed hypolocomotor activity with less center time in the open-field test and impaired motor coordination in the Rotarod test without ischemia. Thus, this study suggests that ProTα has important roles in the maintenance of motor coordination and anxiety-like behavior.
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beneficial actions of Prothymosin Alpha mimetic hexapeptide on central post stroke pain reduced social activity learning deficit and depression following cerebral ischemia in mice
2020Co-Authors: Keita Sasaki, Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:Prothymosin Alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.
Hayato Matsunaga - One of the best experts on this subject based on the ideXlab platform.
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involvement of snare protein interaction for non classical release of damps alarmins proteins Prothymosin Alpha and s100a13
2020Co-Authors: Hayato Matsunaga, Sebok Kumar Halder, Hiroshi UedaAbstract:Prothymosin Alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca2+-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca2+-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.
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experimental evidence for the involvement of f0 f1 atpase and subsequent p2y12 receptor activation in Prothymosin Alpha induced protection of retinal ischemic damage
2020Co-Authors: Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:The inhibition of retinal ischemia-induced damage by post-ischemic Prothymosin Alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or β-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.
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Prothymosin Alpha and its mimetic hexapeptide improve delayed tissue plasminogen activator induced brain damage following cerebral ischemia
2020Co-Authors: Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:Tissue plasminogen activator (tPA) administration beyond 4.5 h of stroke symptoms is beneficial for patients but has an increased risk of cerebral hemorrhage. Thus, increasing the therapeutic window of tPA is important for stroke recovery. We previously showed that Prothymosin Alpha (ProTα) or its mimetic hexapeptide (P6Q) has anti-ischemic activity. Here, we examined the beneficial effects of ProTα or P6Q against delayed tPA-induced brain damage following middle cerebral artery occlusion (MCAO) or photochemically induced thrombosis in mice. Brain hemorrhage was observed by tPA administration during reperfusion at 4.5 and 6 h after MCAO. Co-administration of ProTα with tPA at 4.5 h inhibited hemorrhage and motor dysfunction 2-4 days, but not 7 days after MCAO. ProTα administration at 2 and 4.5 h after MCAO significantly inhibited tPA (4.5 h)-induced motor dysfunction and death more than 7 days. Administration of tPA caused the loss of tight junction proteins, zona occulden-1 and occludin, and up-regulation of matrix metalloproteinase-2/9, in a ProTα-reversible manner. P6Q administration abolished tPA (4.5 h)-induced hemorrhage and reversed tPA (6 h)-induced vascular damage and matrix metalloproteinase-2 and 9 up-regulation. Twice administrations of P6Q at 2 h alone and 6 h with tPA significantly improved motor dysfunction more than 7 days. In photochemically induced thrombosis ischemia, similar vascular leakage and neuronal damage (infarction and motor dysfunction) by late tPA (4.5 or 6 h) were also inhibited by P6Q. Thus, these studies suggest that co-administration with ProTα or P6Q would be beneficial to inhibit delayed tPA-induced hemorrhagic mechanisms in acute ischemic stroke.
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beneficial actions of Prothymosin Alpha mimetic hexapeptide on central post stroke pain reduced social activity learning deficit and depression following cerebral ischemia in mice
2020Co-Authors: Keita Sasaki, Sebok Kumar Halder, Hayato Matsunaga, Hiroshi UedaAbstract:Prothymosin Alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.
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neuroprotective impact of Prothymosin Alpha derived hexapeptide against retinal ischemia reperfusion
2016Co-Authors: Hiroshi Ueda, Sebok Kumar Halder, Hayato Matsunaga, Keita Sasaki, Shiori MaedaAbstract:Prothymosin Alpha (ProTα) has robustness roles against brain and retinal ischemia or serum-starvation stress. In the ProTα sequence, the active core 30-amino acid peptide/P30 (a.a.49-78) is necessary for the original neuroprotective actions against ischemia. Moreover, the 9-amino acid peptide sequence/P9 (a.a.52-60) in P30 still shows neuroprotective activity against brain and retinal ischemia, though P9 is less potent than P30. As the previous structure-activity relationship study for ProTα may not be enough, the possibility still exists that any sequence smaller than P9 retains potent neuroprotective activity. When different P9- and P30-related peptides were intravitreally injected 24h after retinal ischemia in mice, the 6-amino acid peptide/P6 (NEVDEE, a.a.51-56) showed potent protective effects against ischemia-induced retinal functional deficits, which are equipotent to the level of P30 peptide in electroretinography (ERG) and histological damage in Hematoxylin and Eosin (HE) staining. Further studies using ERG and HE staining suggested that intravitreal or intravenous (i.v.) injection with modified P6 peptide/P6Q (NEVDQE) potently inhibited retinal ischemia-induced functional and histological damage. In an immunohistochemical analysis, the ischemia-induced loss of retinal ganglion, bipolar, amacrine and photoreceptor cells were inhibited by a systemic administration with P6Q peptide 24h after the ischemic stress. In addition, systemic post-treatment with P6Q peptide significantly inhibited retinal ischemia-induced microglia and astrocyte activation in terms of increased ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) intensity, respectively, as well as their morphological changes, increased number and migration. Thus, this study demonstrates the therapeutic significance of modified P6 peptide P6Q (NEVDQE) derived from 6-amino acid peptide (P6) in ProTα against ischemic damage.
Shelby L Berger - One of the best experts on this subject based on the ideXlab platform.
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functional discontinuities in Prothymosin Alpha caused by caspase cleavage in apoptotic cells
2000Co-Authors: Steven A Enkemann, Mark W Trumbore, Ruihong Wang, Shelby L BergerAbstract:: Our study examines the effect of apoptosis on Prothymosin Alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, Prothymosin Alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of Prothymosin Alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact Prothymosin Alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified Prothymosin Alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated Prothymosin Alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of Prothymosin Alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.
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metabolic regulation of protein bound glutamyl phosphates insights into the function of Prothymosin Alpha
1999Co-Authors: Lian Tao, Steven A Enkemann, Mark W Trumbore, Ruihong Wang, Shelby L BergerAbstract:Prothymosin Alpha gene expression accompanies growth of all mammalian cells. The protein, which is abundant, exceedingly acidic, and localized to the nucleus, is further distinguished by the presence of clustered phosphorylated glutamic acid residues (Trumbore et al., 1997, J Biol Chem 272:26394-26404). These glutamyl phosphates are energy rich and unstable in vivo and in vitro (Wang et al., 1997, J Biol Chem 272:26405-26412). To understand the function of Prothymosin Alpha in greater detail, the turnover of its phosphates was examined in metabolically manipulated cells. Phosphate half-lives in growing, mock transfected, and vector-transfected COS cells were compared with the half-life in cells transfected with the Prothymosin Alpha gene to determine the fate of the predominantly ectopic phosphorylated protein. The values obtained--72-75 min in cells with normal levels of the protein, but 118 min in cells with surplus Prothymosin Alpha--led us to conclude that underutilized phosphates persist whereas functioning phosphates disperse. Cell-cycle-specific differences in the half-lives were observed in NIH3T3 cells: 72 min while cycling, 83 or 89 min during arrest in or progression through S phase, but 174 min during M-phase arrest. In the presence of actinomycin D, the value was about 145 min regardless of whether cells were quiescent or growing. In these experiments, reduced utilization of Prothymosin Alpha's glutamyl phosphates, signaled by an increase in their half-lives, accompanied the attenuation or abolition of transcription. Our data suggest that Prothymosin Alpha fuels an energy-requiring step in the production, processing, or export of RNA.
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Prothymosin Alpha is not found in yeast
1998Co-Authors: Mark W Trumbore, Richard E Manrow, Shelby L BergerAbstract:According to published accounts, Prothymosin Alpha exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. FEBS Lett. 257, 247-250, 1989). We report here our failure to find evidence for Prothymosin Alpha in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human Prothymosin Alpha coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human Prothymosin Alpha coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no Prothymosin Alpha-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a Prothymosin Alpha gene in animals other than mammals is highly unlikely.
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phosphorylation of human and bovine Prothymosin Alpha in vivo
1993Co-Authors: Adriana Sburlati, Richard E Manrow, Abel De La Rosa, David W Batey, Gloria L Kurys, Lewis K Pannell, Brian M Martin, Douglas M Sheeley, Shelby L BergerAbstract:Prothymosin Alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]Prothymosin Alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]Prothymosin Alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine Prothymosin Alpha and human [32P]Prothymosin Alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that Prothymosin Alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, Prothymosin Alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although Prothymosin Alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that Prothymosin Alpha does not directly govern mitosis.
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nuclear targeting of Prothymosin Alpha
1991Co-Authors: Richard E Manrow, A R Sburlati, J A Hanover, Shelby L BergerAbstract:Prothymosin Alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin Alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate Prothymosin Alpha as a nuclear protein: 1) in COS cells transfected with the human Prothymosin Alpha gene copious amounts of Prothymosin Alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human Prothymosin Alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of Prothymosin Alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin Alpha 1 sequence at the amino terminus failed to effect nuclear transport.
Fernando Dominguez - One of the best experts on this subject based on the ideXlab platform.
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Prothymosin Alpha mrna is expressed in competent and proliferating rat thyroid cells frtl 5 but is not sufficient to elicit cell progression through the cell cycle
1993Co-Authors: Clara V Alvarez, Esperanza Cancio, Juan Zalvide, B J Regueiro, Carlos Dieguez, F V Vega, Fernando DominguezAbstract:Using flow cytometry we observed the effects that different hormonal treatments had on the progression of rat thyroid (FRTL-5) cells through the cell cycle. The absence of hormones or the addition of TSH (6 mU/ml) did not induce DNA synthesis; however, the addition of IGF-I (30 ng/ml) promoted cell proliferation. The number of cells recruited by IGF-I was lower than when IGF-I and TSH were used. We therefore concluded that we had a model with three different types of cells: (1) quiescent cells, cells cultured in the absence of hormones, considered to be G0-arrested cells, (2) competent cells, TSH-treated cells that did not proliferate (being arrested in a cycle phase different from G0) and (3) actively proliferating cells, cells treated with TSH plus IGF-I. Prothymosin Alpha (PTA) mRNA levels were almost undetectable in cells cultured without hormones at all times studied, i.e. 8, 14 and 24 h. On the contrary, TSH and/or IGF-I greatly increased PTA mRNA. These data indicate that G0-arrested quiescent cells do not express PTA mRNA and that PTA mRNA is induced when FRTL-5 cells are committed to proliferate by the addition of TSH, in spite of being arrested by the lack of IGF-I. We therefore conclude that PTA mRNA expression may be an event that is necessary for cells to proliferate, but that it is not sufficient for the promotion of cell progression through the cell cycle.
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evolution of Prothymosin Alpha and proliferating cell nuclear antigen pcna immunoreactivity through the development of rat ovarian follicles
1993Co-Authors: E. Rosón, Fernando Dominguez, Maximo Fraga, Tomas Garciacaballero, R Gallego, A BeirasAbstract:The cellular distribution of Prothymosin Alpha (ProT) was studied in ovarian follicles of adult cycling rats. We found positive granulosa and theca cells throughout follicular maturation. When both ProT and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied, we observed that both proteins were expressed in the same granulosa and theca cells, although sometimes ProT immunoreactivity was weak or absent in the mitotic (M) phase. Moreover, both peptides share the nuclear distribution, but ProT immunoreactivity was never seen in nucleoli. Therefore, we conclude that in mitotic cells ProT is expressed only in actively proliferating cells, since all ProT-positive cells were also positive for PCNA. ProT and PCNA immunoreactivities during the meiotic division were studied in oocytes. The presence of PCNA was, unlike ProT, constant throughout follicle development (except atretic oocytes). Oocytes expressed ProT from primordial follicles to the eighth generation, but more developed oocytes and atretic oocytes were not immunoreactive. In hypophysectomized rats, all oocytes were immunoreactive. Interestingly, in hypophysectomized rats treated with follicle stimulating hormone (FSH) that promoted follicle development, the more developed oocytes did not show ProT immunoreactivity. Since hypophysectomized rats were not treated with luteinizing hormone we conclude that ProT expression is not required to complete meiotic division I.
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tissue concentrations of Prothymosin Alpha a novel proliferation index of primary breast cancer
1993Co-Authors: Fernando Dominguez, Carlos Magdalena, Esperanza Cancio, Elena Roson, Jesus P Paredes, Lourdes Loidi, Juan Zalvide, Maximo Fraga, Jeronimo Forteza, B J RegueiroAbstract:In 71 patents with classic invasive ductal carcinomas, levels of Prothymosin Alpha (PTα), as assayed by a radioimmunoassay that detects thymosin Alpha 1 (the NH 2 -terminal fragment of PTα), were significantly greater in tumour samples than in normal breast tissue. PTα levels were correlated with (a) the number of positive axillary lymph nodes ( r s = 0.5384, P r s = 0.5027, P
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immunohistochemical location of Prothymosin Alpha in regenerating human hepatocytes and hepatocellular carcinomas
1993Co-Authors: Maximo Fraga, Fernando Dominguez, Tomas Garciacaballero, Eugenio Perezbecerra, A Beiras, Jeronimo FortezaAbstract:In the present paper we analysed the presence of Prothymosin Alpha (ProT) in human liver. In normal liver, ProT immunostaining was found in the nuclei of bile duct cells, but not in the hepatocytes. In contrast an intense immunoreactivity was observed in regenerative hepatocytes of chronic hepatitis, cirrhosis and in hepatocellular carcinomas. In all cases the immunostaining was restricted to the nuclei, but the nucleoli were always negative. Similar results were obtained for proliferating cell nuclear antigen. These findings confirm that ProT is related to cell proliferation and provides a new immunohistochemical proliferation marker for routinely processed samples.
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Prothymosin Alpha mrna levels are invariant throughout the cell cycle
1992Co-Authors: Juan Zalvide, Esperanza Cancio, B J Regueiro, Clara V Alvarez, Fernando DominguezAbstract:Prothymosin Alpha (PT-Alpha) mRNA levels were evaluated at different stages during the cell cycle. NIH 3T3 cells were synchronized: (a) by serum deprivation, (b) by mitotic shake off after nocodazole arrest, and (c) by double thymidine block. Cell synchronism was estimated by flow cytometry. In cells grown in serum-free medium, PT-Alpha mRNA levels were almost undetectable. 14 h after serum restoration PT-Alpha mRNA was induced as had been described by others (Eschendfeldt, W. H., and Berger, S. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9403-9407). PT-Alpha mRNA induction seems to require the synthesis of proteic factor(s) since PT-Alpha mRNA response to serum restoration was abolished in the presence of cycloheximide. Interestingly, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake off after nocodazole arrest or double thymidine block did not show variations in the levels of PT-Alpha mRNA when progressed synchronously through the cycle. On the contrary, histone H4 mRNA was expressed only during the S phase. These data indicate that PT-Alpha mRNA was present in roughly the same amount through all phases of the cell cycle, arguing against the concept that PT-Alpha is a cell cycle-regulated gene.
