Neuroglobin(Ngb)is a 17 kDa monomeric hexa-coordinated heme protein belonging to the globin family.Ngb is mainly expressed in neurons of the central and peripheral nervous system,although moderate levels of Ngb have b...Neuroglobin(Ngb)is a 17 kDa monomeric hexa-coordinated heme protein belonging to the globin family.Ngb is mainly expressed in neurons of the central and peripheral nervous system,although moderate levels of Ngb have been detected in non-nervous tissues.In the past decade,Ngb has been studied for its neuroprotective role in a large number of neurological disorders such as Alzheimer’s disease,Huntington’s disease,brain ischemia and hypoxia.This review discusses and summarizes the natural compounds and the small synthetic molecules capable of modulating Ngb expression that exhibits a protective role against various neurodegenerative diseases.展开更多
In this study, we used a rat model of severe closed traumatic brain injury to explore the relationship between neuroglobin, brain injury and neuronal apoptosis. Real-time PCR showed that neuroglobin mRNA expression ra...In this study, we used a rat model of severe closed traumatic brain injury to explore the relationship between neuroglobin, brain injury and neuronal apoptosis. Real-time PCR showed that neuroglobin mRNA expression rapidly increased in the rat cerebral cortex, and peaked at 30 minutes and 48 hours following traumatic brain injury. Immunohistochemical staining demonstrated that neuroglobin expression increased and remained high 2 hours to 5 days following injury. The rate of increase in the apoptosis-related Bax/Bcl-2 ratio greatly decreased between 30 minutes and 1 hour as well as between 48 and 72 hours post injury. Expression of neuroglobin and the anti-apoptotic factor Bcl-2 greatly increased, while that of the proapoptotic factor decreased, in the cerebral cortex post severe closed traumatic brain injury. It suggests that neuroglobin might protect neurons from apoptosis after traumatic injury by regulating Bax/Bcl-2 pathway.展开更多
BACKGROUND： Adenovirus has been used to develop neuroglobin （Ngb） vectors. Although transfection efficiency is high, induced gene mutation, cytotoxicity, inflammation, and low exogenous gene content have limited it...BACKGROUND： Adenovirus has been used to develop neuroglobin （Ngb） vectors. Although transfection efficiency is high, induced gene mutation, cytotoxicity, inflammation, and low exogenous gene content have limited its application. OBJECTIVE： To observe the effects of recombinant Ngb plasmid in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Genetically engineered, randomized, controlled, animal experiment was performed at the Laboratory of Chongqing Medical University from May 2006 and January 2007. MATERIALS： 2, 3, 5-triphenyltetrazolium chloride was purchased from Shanghai Sangon Biological Engineering Technology and Services. Rabbit anti-rat Bcl-2 polyclonal antibody, rabbit anti-rat β-actin monoclonal antibody, and FITC-labeled goat anti-rabbit IgG were purchased from Sigma, USA. TUNEL apoptosis kit was purchased from Roche, Germany. METHODS： A total of 54 male, adult, Wistar rats were randomly assigned to 3 groups （n=18）： normal saline, plasmid control, and recombinant Ngb （pCDNA3.1 （＋）/Ngb）. Normal saline, plasmid pCDNA3.1 （＋）, and recombinant plasmid pCDNA3.1 （＋）/Ngb were separately injected into two sites in the rat cerebral cortex, and models of focal ischemia were established by occlusion of the right middle cerebral artery after 24 hours. MAIN OUTCOME MEASURES： Local ischemic damage was detected by 2, 3, 5- triphenyltetrazolium chloride staining, apoptosis in the penumbra was confirmed using the TUNEL method, and Bcl-2 protein expression in the penumbra was determined by indirect immunofluorescent staining and Western blot analysis. RESULTS： Compared with the normal saline and plasmid control groups, cerebral infarction size and the number of apoptotic cells in the pCDNA3.1 （＋）/Ngb group were significantly reduced （P 〈 0.01）. The percentage of Bcl-2-positive cells in the penumbra of the pCDNA3.1 （＋）/Ngb group was significantly increased （P 〈 0.01）. The relative expression level of Bcl-2 protein was increased by 40%-50%. CONCLUSION： Recombinant plasmid pCDNA3.1/Ngb provides neuroprotection by upregulating Bcl-2 expression and inhibiting cell apoptosis in the penumbra.展开更多
In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound in...In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound intensity of 0.8 W/cm2, a 60-second exposure duration, 50% duty cycle, and 20% microbubble concentration, pAcGFP1-C1-Ngb-transfected cells exhibited the highest cell viability and transfection efficiency. The efficiency of plasmid delivery was significantly higher with UTMD than transfection with plasmid alone, transfection with plasmid using microbubbles, or transfection of plasmid by ultrasound. In addition, during cobalt chloride-induced hypoxia, caspase-3 activity in pAcGFP1-C1-Ngb-transfected cells was significantly lower than in untransfected cells. Ngb protein and mRNA expression were significantly higher in cells transfected by UTMD than in cells transfected with the other methods. These results demonstrate that UTMD can very efficiently mediate exogenous gene delivery, and that Ngb overexpression protects cells against cobalt chloride-induced hypoxia.展开更多
AIM: To investigate the Expression of neuroglobin (Ngb) in the retina of rats with ocular hypertension induced acute retinal hypoxic-ischemic injury. METHODS: Seventy Wistar rats were divided into 7 groups randomly. T...AIM: To investigate the Expression of neuroglobin (Ngb) in the retina of rats with ocular hypertension induced acute retinal hypoxic-ischemic injury. METHODS: Seventy Wistar rats were divided into 7 groups randomly. The Experimental model was induced by elevation of intraocular pressure via anterior chamber canula insertion in the left eyes and the fellow eyes were preserved as normal controls. The retinal tissues were taken at 1, 5, 10, 15, 20, 30 and 60 minutes after hypoxic-ischemia injury. Protein was extracted, and then analyzed by Western-blot method. SPSS was used for statistical analysis. RESULTS: The time-depended expressions of Ngb were observed. The level of Ngb increased rapidly at 1 minute after ischemia and reached to the peak at 5 minutes, which had significant difference from that of control group (P<0.05). It kept in high level during 5-15 minutes (P <0.05), then decreased after 20 minutes till 60 minutes. There were no significant differences between experimental and control group in the latter period (P> 0.05). CONCLUSION: The expression of Ngb in retinal tissue increased rapidly after hypoxic-ischemic injury in rats, suggesting that Ngb may play an important role in the process of acute retinal hypoxic-ischemic injury.展开更多
This study sought to examine neuroglobin （NGB） in the serum of acute cerebral infarction patients with double-antibody sandwich enzyme-linked immunosorbent assay to identify all risk factors, calculate infarct size,...This study sought to examine neuroglobin （NGB） in the serum of acute cerebral infarction patients with double-antibody sandwich enzyme-linked immunosorbent assay to identify all risk factors, calculate infarct size, assess neurological impairment, and analyze the relation between NGB and each of these factors. The double-antibody sandwich assay indicated that levels of NGB in serum were unaltered within 6 hours following acute cerebral infarction compared with normal levels. NGB levels then underwent a distinct change, peaking at 24 hours then returning to normal levels in 72 hours. The results suggest that the level of NGB might be related to infarct size and low-density lipoprotein at 24 hours after acute cerebral infarction. There were no significant differences in neurological impairment scores and infarct size at different periods following infarction. The findings indicated that the level of NGB in serum of acute cerebral infarction patients was correlated with infarct time.展开更多
BACKGROUND： Despite a large amount of resuscitation research, the survival rate after cardiac arrest remains low, and brain injury is the key issue. Neuroglobin （NGB） is an oxygen-binding heme protein found in the...BACKGROUND： Despite a large amount of resuscitation research, the survival rate after cardiac arrest remains low, and brain injury is the key issue. Neuroglobin （NGB） is an oxygen-binding heme protein found in the brain with a protection role against ischemic-hypoxic brain injury. Hemin is an effective activator of neuroglobin. This study was undertaken to assess the effect of hemin on expression of neuroglobin （NGB） in the cerebral cortex, neuro-defi cit score （NDS） and pathological changes after cardiopulmonary resuscitation （CPR） in rats.METHODS： A total of 120 male Sprague-Dawley （SD） rats were randomly divided into a control group （A）, a CPR group （B） and a Hemin group （C）. The animal model of cardiac arrest （CA） induced by asphyxia and CPR was established. NGB expression in the cerebral cortex with immunohistochemistry, NDS and pathological changes in the cerebral cortex were examined at 3, 6, 12, 24 hours after recovery of spontaneous circulation （ROSC） in each group. Experimental data were treated as one-factor analysis of variance and the Tukey test.RESULTS： In comparison with group A, NGB expression was increased signifi cantly at 12 and 24 hours after ROSC （P〈0.05 or P〈0.01）, NDS was decreased signifi cantly at each time point after ROSC （P〈0.01）, and pathological changes were severe at each time point after ROSC in group B. In comparison with group A, NGB expression was increased signifi cantly at 6, 12, 24 hours after ROSC （P〈0.05 or P〈0.01）, NDS was decreased signifi cantly at 3, 6, 12 hours after ROSC （P〈0.01） in group C. In comparison with group B, NGB expression was increased signifi cantly at 12 and 24 hours after ROSC, NDS was increased signifi cantly at 12 and 24 hours after ROSC, and pathological changes were milder in group C.CONCLUSION： There were increased NGB expression in the cerebral cortex, decreased NDS, and severe pathological changes after CPR in rats. Hemin treatment up-regulated expression of NGB, improved NDS, mitigated pathological changes, and alleviated cerebral injury after CPR.展开更多
^1H NMR has been used to determine the 2-, 4-vinyl orientation of heme active site from oxidized mouse neuroglobin （mNgb）. The NOEs between 3-methyl and Hα, Hβ of 2-vinyl, together with the NOEs between 5-methyl a...^1H NMR has been used to determine the 2-, 4-vinyl orientation of heme active site from oxidized mouse neuroglobin （mNgb）. The NOEs between 3-methyl and Hα, Hβ of 2-vinyl, together with the NOEs between 5-methyl and Hα, Hβ of 4-vinyl, allowed the unambiguous determination of trans and cis orientations for the 2- and 4-vinyl groups in the mNgb, respectively.展开更多
Introduction: The mechanisms of overexpression of neuroglobin in patients with severe glaucoma (CG+) remain hypothetical. Objective: To study the anti-apoptotic, anti-hypoxic and anti-oxidant properties of neuroglobin...Introduction: The mechanisms of overexpression of neuroglobin in patients with severe glaucoma (CG+) remain hypothetical. Objective: To study the anti-apoptotic, anti-hypoxic and anti-oxidant properties of neuroglobin in CG+. Population and Methods: The visual field, as well as plasma dosage of neuroglobin (CmNgb, ng/ml), hypoxia inductible factor-1alpha (CmHIF-1α, pg/ml), glutathione peroxidase (CmGpx, pg/ml), and cytochrome C oxidase (CmCyt C, pg/ml) were carried out in 45 CG+ and 45 controls (CG-). The chi-2 test compared the proportions, and Spearman’s test studied the correlations between quantitative variables (p Results: CmNgb was 4.1 in CG+, versus 2.3 in CG- (p = 1.52 × 10-5). CmGpx was 1144.7 in CG+, versus 752.8 in GC- (p = 0.0199). CmHIF-1α was 4.1 in CG+, versus 3.5 in CG- (p = 0.4530). CmCyt C was 2303.26 in CG+, versus 1750.44 in CG- (p = 0.0450). In CG+, there was a correlation between CmNgb and CmGpx (r = 0.417;p = 0.004), CmNgb and CmHIF-1α (r = 0.644;p = 1.8 × 10-6), and between CmHIF-1α and CmGpx (r = 0.447;p = 0.002), CmHIF-1α and CmCyt C (r = 0.371;p = 0.012). None correlation was found between CmNgb and CmCyt C (r = 0.126;p = 0.370), as well as CmGpx and CmCyt C (r = 0.102;p = 0.505). Conclusion: The variations of apoptosis, hypoxic, and oxidative stress biomarkers were found between CG+ and CG-, as well as their correlations, suggesting that neuroglobin overexpression is related to its anti-apoptotic, anti-oxidative, and anti-hypoxic properties.展开更多
Introduction: Neuroglobin (Ngb) owes its name to its preferred location in the nervous system. Its plasma concentration increases during cerebral ischemia. However, the interest of its dosage in the diagnosis and the ...Introduction: Neuroglobin (Ngb) owes its name to its preferred location in the nervous system. Its plasma concentration increases during cerebral ischemia. However, the interest of its dosage in the diagnosis and the prognosis of the strokes in the adult is not defined. Objectives: To determine if plasmatic Ngb can be used as a diagnostic biomarker and prognostic for stroke in adults at the acute phase. Population and Methods: This was a prospective study in 69 people, including 39 suspected stroke (Cerebral ischemia or CI, Intracerebral hemorrhage or ICH) and 30 healthy volunteers (controls). The plasma concentration of Ngb (CmNgb in ng/ml) of the patients was determined at admission day (d1), at the third day (d3) and seventh day (d7). CmNgbtaken at d1 was compared between patients and controls. Its evolution over time, as well as its relation with the clinical parameters, including the Glasgow coma scale and the short-term mortality in stroke subjects was analyzed by the Mann and Whitney tests and the Wilcoxon test (p Results: At d1, the CmNgb of all types of stroke was 3.140 ± 2.700 ng/ml, and did not differ significantly from controls (0.303 ± 0.114 ng/ml, p = 0.070). On the other hand, it was higher in CI victims (5.800 ± 0.720 ng/ml) than in ICH (1.750 ± 0,090 ng/ml) (p = 0.030). It then decreased on d3 in CI victims (2.600 ± 0.112 ng/ml) and ICH (0.420 ± 0.211 ng/ml), returning to normal on d7 (0.420 ± 0.200 ng/ml for CI’s, p = 0.001, and 0.360 ± 0.300 ng/ml for ICH, p = 0.002). There was a relationship between CmNgb, delay of occurrence of the first symptoms of the stroke (3.140 ± 2.700 ng/ml before the 6th hour, and 0.643 ± 0.244 ng/ml after the 6th hour (p = 0.003) and the volume of the hematoma (p = 0.0027). None relationship existed between CmNgb, Glasgow coma scale (p = 0.427) and short-term mortality (CmNgb = 3.95 ng/ml in survivors versus 2.65 ng/ml in deceased p = 0.060). Conclusion: This study shows that the plasma concentration of Neuroglobin is high during stroke in humans in the acute phase. This elevation follows triphasic?kinetics and appears to be more important during infarction than hemorrhage. These results suggest that CmNgb can be used as a diagnostic marker for stroke in adult at the acute phase, by differentiating ischemia from hemorrhage. However, this work needs to be confirmed on a larger sample of patients.展开更多
Neuroglobin(Ngb) is a respiratory protein that is preferentially expressed in brain of mouse and man.In this article,Tibetan antelope,living at altitude of 3 000～5 000 m for millions of years,was selected as the mode...Neuroglobin(Ngb) is a respiratory protein that is preferentially expressed in brain of mouse and man.In this article,Tibetan antelope,living at altitude of 3 000～5 000 m for millions of years,was selected as the model of hypoxia-tolerant adaptation species.Using reverse transcription polymerase chain reaction(RT-PCR) and Western blot techniques,expression of Ngb gene was amplified and analyzed in antelope brain tissue.Our results showed that Ngb homology protein in Tibetan antelope was identified with more sequence similarity with cattle(96%),sheep(95%),and human(95%).We detected that there were some mutations occurred in the Open Reading Frame of Ngb in Tibetan antelope compared with sheep.Phylogenetic analysis of Ngb chain showed that it was closer to cattle than the others.This study suggests possible roles of central nervous system enriched Ngb in adaptation of Tibetan antelope to extremely high altitude.展开更多
The discovery of neuroglobin(Ngb),a brain-or neuron-specific member of the hemoglobin family,has revolutionized our understanding of brain oxygen metabolism.Currently,how Ngb plays such a role remains far from clear.H...The discovery of neuroglobin(Ngb),a brain-or neuron-specific member of the hemoglobin family,has revolutionized our understanding of brain oxygen metabolism.Currently,how Ngb plays such a role remains far from clear.Here,we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia.We found that Ngb was present in,co-localized to,and co-migrated with mitochondria in the cell body and neurites of neurons.Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane(CM)or cell surface in living neurons,and this was accompanied by the mitochondria.In vivo,hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio.Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase(SDH)and ATPase activity in neuronal N2a cells.Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia.Mutation of Ngb at its oxygen-binding site(His64)significantly increased SDH activity and reduced ATPase activity in N2a cells.Taken together,Ngb was physically and functionally linked to mitochondria.In response to an insufficient oxygen supply,Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation.This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer’s disease and diseases that cause hypoxia in the brain such as anemia.展开更多
Neuroglobin (NGB) is a newly discov- ered member of the hemoglobin superfamily that is primarily expressed in the brain of humans and other vertebrates. The effects of protein concentration, solvent, pH and temperatur...Neuroglobin (NGB) is a newly discov- ered member of the hemoglobin superfamily that is primarily expressed in the brain of humans and other vertebrates. The effects of protein concentration, solvent, pH and temperature on the secondary structure of NGB were investigated by employing far UV circular dichroism (CD) spectroscopy. The results show that NGB exists mainly in α-helix form when its concentration is less than 10 μmol/L. However, its α-helix content decreases with the increase of con- centration in the range of 10―40 μmol/L and remains unchanged when the concentration is higher than 40 μmol/L, which suggest that NGBs form intermolecular disulfide bond and aggregate in higher concentration. The α-helix content of NGB in methanol and ethanol is a little higher than that in water, indicating a higher stability of NGB in these solvents. NGB loses its α-helical secondary structure in either acidic or alka- line solution to some extent. Although increased temperature destabilizes the α-helices of NGB, over 16% of α-helices can be kept at 110°C. Therefore, NGB is a protein with hyperthermal stability.展开更多
The expression, purification and spectra char-acterization of recombinant human neuroglobin (NGB) are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 (DE3) plys cells and expressed in ...The expression, purification and spectra char-acterization of recombinant human neuroglobin (NGB) are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 (DE3) plys cells and expressed in TB culture medium. The results indicated that the expression amount of NGB is about 10 percent of the total protein in cells. The NGB protein was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and a Hiprep 16/10 Q FF anion exchange column, and a red solu-ble protein was obtained which showed a single band in elec-trophoresis. Electrospray ionization mass spectrometry (ESI-MS) showed that its molecular weight is 16930.0 Da. UV-spectra indicated that the reduced NGB has a strong absorption peak at 425 nm, and two weak peaks at 531 and 559 nm, which can be assigned to γ, β and α bands of por-phyrin, respectively, and the oxidized NGB has a strong ab-sorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maxi-mal excitation wavelength is at 281 nm and its maximal emission wavelength is at 338 nm. CD spectra indicated that its secondary structure is a typical α helix, and has a positive peak at 410 nm induced by heme. The NGB protein is stable when the pH is higher than 4.展开更多
基金This work was supported by the Italian Ministero dell’Istruzione,dell’Universitáe della Ricerca PRIN 2017SNRXH3(to EO and SN)PRA_2018_20 University of Pisa(to EO).
文摘Neuroglobin(Ngb)is a 17 kDa monomeric hexa-coordinated heme protein belonging to the globin family.Ngb is mainly expressed in neurons of the central and peripheral nervous system,although moderate levels of Ngb have been detected in non-nervous tissues.In the past decade,Ngb has been studied for its neuroprotective role in a large number of neurological disorders such as Alzheimer’s disease,Huntington’s disease,brain ischemia and hypoxia.This review discusses and summarizes the natural compounds and the small synthetic molecules capable of modulating Ngb expression that exhibits a protective role against various neurodegenerative diseases.
基金supported by General Program of National Natural Science Foundation of China,No. 30400465,30571903Open Project from Medical Neurobiology of State Key Laboratory (09-08) of Fudan University
文摘In this study, we used a rat model of severe closed traumatic brain injury to explore the relationship between neuroglobin, brain injury and neuronal apoptosis. Real-time PCR showed that neuroglobin mRNA expression rapidly increased in the rat cerebral cortex, and peaked at 30 minutes and 48 hours following traumatic brain injury. Immunohistochemical staining demonstrated that neuroglobin expression increased and remained high 2 hours to 5 days following injury. The rate of increase in the apoptosis-related Bax/Bcl-2 ratio greatly decreased between 30 minutes and 1 hour as well as between 48 and 72 hours post injury. Expression of neuroglobin and the anti-apoptotic factor Bcl-2 greatly increased, while that of the proapoptotic factor decreased, in the cerebral cortex post severe closed traumatic brain injury. It suggests that neuroglobin might protect neurons from apoptosis after traumatic injury by regulating Bax/Bcl-2 pathway.
基金a Grant from Chongqing Health Bureau, No. 06-2-177
文摘BACKGROUND： Adenovirus has been used to develop neuroglobin （Ngb） vectors. Although transfection efficiency is high, induced gene mutation, cytotoxicity, inflammation, and low exogenous gene content have limited its application. OBJECTIVE： To observe the effects of recombinant Ngb plasmid in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Genetically engineered, randomized, controlled, animal experiment was performed at the Laboratory of Chongqing Medical University from May 2006 and January 2007. MATERIALS： 2, 3, 5-triphenyltetrazolium chloride was purchased from Shanghai Sangon Biological Engineering Technology and Services. Rabbit anti-rat Bcl-2 polyclonal antibody, rabbit anti-rat β-actin monoclonal antibody, and FITC-labeled goat anti-rabbit IgG were purchased from Sigma, USA. TUNEL apoptosis kit was purchased from Roche, Germany. METHODS： A total of 54 male, adult, Wistar rats were randomly assigned to 3 groups （n=18）： normal saline, plasmid control, and recombinant Ngb （pCDNA3.1 （＋）/Ngb）. Normal saline, plasmid pCDNA3.1 （＋）, and recombinant plasmid pCDNA3.1 （＋）/Ngb were separately injected into two sites in the rat cerebral cortex, and models of focal ischemia were established by occlusion of the right middle cerebral artery after 24 hours. MAIN OUTCOME MEASURES： Local ischemic damage was detected by 2, 3, 5- triphenyltetrazolium chloride staining, apoptosis in the penumbra was confirmed using the TUNEL method, and Bcl-2 protein expression in the penumbra was determined by indirect immunofluorescent staining and Western blot analysis. RESULTS： Compared with the normal saline and plasmid control groups, cerebral infarction size and the number of apoptotic cells in the pCDNA3.1 （＋）/Ngb group were significantly reduced （P 〈 0.01）. The percentage of Bcl-2-positive cells in the penumbra of the pCDNA3.1 （＋）/Ngb group was significantly increased （P 〈 0.01）. The relative expression level of Bcl-2 protein was increased by 40%-50%. CONCLUSION： Recombinant plasmid pCDNA3.1/Ngb provides neuroprotection by upregulating Bcl-2 expression and inhibiting cell apoptosis in the penumbra.
文摘In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound intensity of 0.8 W/cm2, a 60-second exposure duration, 50% duty cycle, and 20% microbubble concentration, pAcGFP1-C1-Ngb-transfected cells exhibited the highest cell viability and transfection efficiency. The efficiency of plasmid delivery was significantly higher with UTMD than transfection with plasmid alone, transfection with plasmid using microbubbles, or transfection of plasmid by ultrasound. In addition, during cobalt chloride-induced hypoxia, caspase-3 activity in pAcGFP1-C1-Ngb-transfected cells was significantly lower than in untransfected cells. Ngb protein and mRNA expression were significantly higher in cells transfected by UTMD than in cells transfected with the other methods. These results demonstrate that UTMD can very efficiently mediate exogenous gene delivery, and that Ngb overexpression protects cells against cobalt chloride-induced hypoxia.
文摘AIM: To investigate the Expression of neuroglobin (Ngb) in the retina of rats with ocular hypertension induced acute retinal hypoxic-ischemic injury. METHODS: Seventy Wistar rats were divided into 7 groups randomly. The Experimental model was induced by elevation of intraocular pressure via anterior chamber canula insertion in the left eyes and the fellow eyes were preserved as normal controls. The retinal tissues were taken at 1, 5, 10, 15, 20, 30 and 60 minutes after hypoxic-ischemia injury. Protein was extracted, and then analyzed by Western-blot method. SPSS was used for statistical analysis. RESULTS: The time-depended expressions of Ngb were observed. The level of Ngb increased rapidly at 1 minute after ischemia and reached to the peak at 5 minutes, which had significant difference from that of control group (P<0.05). It kept in high level during 5-15 minutes (P <0.05), then decreased after 20 minutes till 60 minutes. There were no significant differences between experimental and control group in the latter period (P> 0.05). CONCLUSION: The expression of Ngb in retinal tissue increased rapidly after hypoxic-ischemic injury in rats, suggesting that Ngb may play an important role in the process of acute retinal hypoxic-ischemic injury.
基金the Science and Technology Plan of Anhui Province, No.08020304111
文摘This study sought to examine neuroglobin （NGB） in the serum of acute cerebral infarction patients with double-antibody sandwich enzyme-linked immunosorbent assay to identify all risk factors, calculate infarct size, assess neurological impairment, and analyze the relation between NGB and each of these factors. The double-antibody sandwich assay indicated that levels of NGB in serum were unaltered within 6 hours following acute cerebral infarction compared with normal levels. NGB levels then underwent a distinct change, peaking at 24 hours then returning to normal levels in 72 hours. The results suggest that the level of NGB might be related to infarct size and low-density lipoprotein at 24 hours after acute cerebral infarction. There were no significant differences in neurological impairment scores and infarct size at different periods following infarction. The findings indicated that the level of NGB in serum of acute cerebral infarction patients was correlated with infarct time.
文摘BACKGROUND： Despite a large amount of resuscitation research, the survival rate after cardiac arrest remains low, and brain injury is the key issue. Neuroglobin （NGB） is an oxygen-binding heme protein found in the brain with a protection role against ischemic-hypoxic brain injury. Hemin is an effective activator of neuroglobin. This study was undertaken to assess the effect of hemin on expression of neuroglobin （NGB） in the cerebral cortex, neuro-defi cit score （NDS） and pathological changes after cardiopulmonary resuscitation （CPR） in rats.METHODS： A total of 120 male Sprague-Dawley （SD） rats were randomly divided into a control group （A）, a CPR group （B） and a Hemin group （C）. The animal model of cardiac arrest （CA） induced by asphyxia and CPR was established. NGB expression in the cerebral cortex with immunohistochemistry, NDS and pathological changes in the cerebral cortex were examined at 3, 6, 12, 24 hours after recovery of spontaneous circulation （ROSC） in each group. Experimental data were treated as one-factor analysis of variance and the Tukey test.RESULTS： In comparison with group A, NGB expression was increased signifi cantly at 12 and 24 hours after ROSC （P〈0.05 or P〈0.01）, NDS was decreased signifi cantly at each time point after ROSC （P〈0.01）, and pathological changes were severe at each time point after ROSC in group B. In comparison with group A, NGB expression was increased signifi cantly at 6, 12, 24 hours after ROSC （P〈0.05 or P〈0.01）, NDS was decreased signifi cantly at 3, 6, 12 hours after ROSC （P〈0.01） in group C. In comparison with group B, NGB expression was increased signifi cantly at 12 and 24 hours after ROSC, NDS was increased signifi cantly at 12 and 24 hours after ROSC, and pathological changes were milder in group C.CONCLUSION： There were increased NGB expression in the cerebral cortex, decreased NDS, and severe pathological changes after CPR in rats. Hemin treatment up-regulated expression of NGB, improved NDS, mitigated pathological changes, and alleviated cerebral injury after CPR.
基金This research was supported by the grants from the National Natural Science Foundation of China(No.10572097)Shanghai Natural Science Foundation(No.05ZR14111)Shanghai leading Academic Discipline Project(No.T0901).
文摘^1H NMR has been used to determine the 2-, 4-vinyl orientation of heme active site from oxidized mouse neuroglobin （mNgb）. The NOEs between 3-methyl and Hα, Hβ of 2-vinyl, together with the NOEs between 5-methyl and Hα, Hβ of 4-vinyl, allowed the unambiguous determination of trans and cis orientations for the 2- and 4-vinyl groups in the mNgb, respectively.
文摘Introduction: The mechanisms of overexpression of neuroglobin in patients with severe glaucoma (CG+) remain hypothetical. Objective: To study the anti-apoptotic, anti-hypoxic and anti-oxidant properties of neuroglobin in CG+. Population and Methods: The visual field, as well as plasma dosage of neuroglobin (CmNgb, ng/ml), hypoxia inductible factor-1alpha (CmHIF-1α, pg/ml), glutathione peroxidase (CmGpx, pg/ml), and cytochrome C oxidase (CmCyt C, pg/ml) were carried out in 45 CG+ and 45 controls (CG-). The chi-2 test compared the proportions, and Spearman’s test studied the correlations between quantitative variables (p Results: CmNgb was 4.1 in CG+, versus 2.3 in CG- (p = 1.52 × 10-5). CmGpx was 1144.7 in CG+, versus 752.8 in GC- (p = 0.0199). CmHIF-1α was 4.1 in CG+, versus 3.5 in CG- (p = 0.4530). CmCyt C was 2303.26 in CG+, versus 1750.44 in CG- (p = 0.0450). In CG+, there was a correlation between CmNgb and CmGpx (r = 0.417;p = 0.004), CmNgb and CmHIF-1α (r = 0.644;p = 1.8 × 10-6), and between CmHIF-1α and CmGpx (r = 0.447;p = 0.002), CmHIF-1α and CmCyt C (r = 0.371;p = 0.012). None correlation was found between CmNgb and CmCyt C (r = 0.126;p = 0.370), as well as CmGpx and CmCyt C (r = 0.102;p = 0.505). Conclusion: The variations of apoptosis, hypoxic, and oxidative stress biomarkers were found between CG+ and CG-, as well as their correlations, suggesting that neuroglobin overexpression is related to its anti-apoptotic, anti-oxidative, and anti-hypoxic properties.
文摘Introduction: Neuroglobin (Ngb) owes its name to its preferred location in the nervous system. Its plasma concentration increases during cerebral ischemia. However, the interest of its dosage in the diagnosis and the prognosis of the strokes in the adult is not defined. Objectives: To determine if plasmatic Ngb can be used as a diagnostic biomarker and prognostic for stroke in adults at the acute phase. Population and Methods: This was a prospective study in 69 people, including 39 suspected stroke (Cerebral ischemia or CI, Intracerebral hemorrhage or ICH) and 30 healthy volunteers (controls). The plasma concentration of Ngb (CmNgb in ng/ml) of the patients was determined at admission day (d1), at the third day (d3) and seventh day (d7). CmNgbtaken at d1 was compared between patients and controls. Its evolution over time, as well as its relation with the clinical parameters, including the Glasgow coma scale and the short-term mortality in stroke subjects was analyzed by the Mann and Whitney tests and the Wilcoxon test (p Results: At d1, the CmNgb of all types of stroke was 3.140 ± 2.700 ng/ml, and did not differ significantly from controls (0.303 ± 0.114 ng/ml, p = 0.070). On the other hand, it was higher in CI victims (5.800 ± 0.720 ng/ml) than in ICH (1.750 ± 0,090 ng/ml) (p = 0.030). It then decreased on d3 in CI victims (2.600 ± 0.112 ng/ml) and ICH (0.420 ± 0.211 ng/ml), returning to normal on d7 (0.420 ± 0.200 ng/ml for CI’s, p = 0.001, and 0.360 ± 0.300 ng/ml for ICH, p = 0.002). There was a relationship between CmNgb, delay of occurrence of the first symptoms of the stroke (3.140 ± 2.700 ng/ml before the 6th hour, and 0.643 ± 0.244 ng/ml after the 6th hour (p = 0.003) and the volume of the hematoma (p = 0.0027). None relationship existed between CmNgb, Glasgow coma scale (p = 0.427) and short-term mortality (CmNgb = 3.95 ng/ml in survivors versus 2.65 ng/ml in deceased p = 0.060). Conclusion: This study shows that the plasma concentration of Neuroglobin is high during stroke in humans in the acute phase. This elevation follows triphasic?kinetics and appears to be more important during infarction than hemorrhage. These results suggest that CmNgb can be used as a diagnostic marker for stroke in adult at the acute phase, by differentiating ischemia from hemorrhage. However, this work needs to be confirmed on a larger sample of patients.
基金supported by the"973"National Basic Research Program(2012CB51820500)National Natural Science Foundation of China(NSFC,31160219).
文摘Neuroglobin(Ngb) is a respiratory protein that is preferentially expressed in brain of mouse and man.In this article,Tibetan antelope,living at altitude of 3 000～5 000 m for millions of years,was selected as the model of hypoxia-tolerant adaptation species.Using reverse transcription polymerase chain reaction(RT-PCR) and Western blot techniques,expression of Ngb gene was amplified and analyzed in antelope brain tissue.Our results showed that Ngb homology protein in Tibetan antelope was identified with more sequence similarity with cattle(96%),sheep(95%),and human(95%).We detected that there were some mutations occurred in the Open Reading Frame of Ngb in Tibetan antelope compared with sheep.Phylogenetic analysis of Ngb chain showed that it was closer to cattle than the others.This study suggests possible roles of central nervous system enriched Ngb in adaptation of Tibetan antelope to extremely high altitude.
基金This work was supported by the National Natural Science Foundation of China(81972362,82173197,and 81672504).
文摘The discovery of neuroglobin(Ngb),a brain-or neuron-specific member of the hemoglobin family,has revolutionized our understanding of brain oxygen metabolism.Currently,how Ngb plays such a role remains far from clear.Here,we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia.We found that Ngb was present in,co-localized to,and co-migrated with mitochondria in the cell body and neurites of neurons.Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane(CM)or cell surface in living neurons,and this was accompanied by the mitochondria.In vivo,hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio.Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase(SDH)and ATPase activity in neuronal N2a cells.Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia.Mutation of Ngb at its oxygen-binding site(His64)significantly increased SDH activity and reduced ATPase activity in N2a cells.Taken together,Ngb was physically and functionally linked to mitochondria.In response to an insufficient oxygen supply,Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation.This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer’s disease and diseases that cause hypoxia in the brain such as anemia.
基金This work was supported by the National Natural Science Foundation of China(Grant No.20471025).
文摘Neuroglobin (NGB) is a newly discov- ered member of the hemoglobin superfamily that is primarily expressed in the brain of humans and other vertebrates. The effects of protein concentration, solvent, pH and temperature on the secondary structure of NGB were investigated by employing far UV circular dichroism (CD) spectroscopy. The results show that NGB exists mainly in α-helix form when its concentration is less than 10 μmol/L. However, its α-helix content decreases with the increase of con- centration in the range of 10―40 μmol/L and remains unchanged when the concentration is higher than 40 μmol/L, which suggest that NGBs form intermolecular disulfide bond and aggregate in higher concentration. The α-helix content of NGB in methanol and ethanol is a little higher than that in water, indicating a higher stability of NGB in these solvents. NGB loses its α-helical secondary structure in either acidic or alka- line solution to some extent. Although increased temperature destabilizes the α-helices of NGB, over 16% of α-helices can be kept at 110°C. Therefore, NGB is a protein with hyperthermal stability.
文摘The expression, purification and spectra char-acterization of recombinant human neuroglobin (NGB) are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 (DE3) plys cells and expressed in TB culture medium. The results indicated that the expression amount of NGB is about 10 percent of the total protein in cells. The NGB protein was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and a Hiprep 16/10 Q FF anion exchange column, and a red solu-ble protein was obtained which showed a single band in elec-trophoresis. Electrospray ionization mass spectrometry (ESI-MS) showed that its molecular weight is 16930.0 Da. UV-spectra indicated that the reduced NGB has a strong absorption peak at 425 nm, and two weak peaks at 531 and 559 nm, which can be assigned to γ, β and α bands of por-phyrin, respectively, and the oxidized NGB has a strong ab-sorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maxi-mal excitation wavelength is at 281 nm and its maximal emission wavelength is at 338 nm. CD spectra indicated that its secondary structure is a typical α helix, and has a positive peak at 410 nm induced by heme. The NGB protein is stable when the pH is higher than 4.
