A new sesquiterpenoid from the aconitum-derived fungus Aspergillus fumigatus M1.

A new bergamotane sesquiterpenoid, fumigatanol (1), along with nine known compounds (2-10) were isolated from the Aconitum-derived fungus Aspergillus fumigatus M1. Their structures were established on the basis of extensive spectroscopic analyses, ECD experiment and NMR computational method. Antibacterial and cytotoxic activities of compound 1 were evaluated and no obvious antibacterial and cytotoxic activities were observed at concentrations of 256 µg/mL and 40.00 µM, respectively.

Osteoblastic Swedish mutant APP expedites brain deficits by inducing endoplasmic reticulum stress-driven senescence.

Patients with Alzheimer's disease (AD) often have osteoporosis or osteopenia. However, their direct link and relationship remain largely unclear. Previous studies have detected osteoporotic deficits in young adult Tg2576 and TgAPPsweOCN mice, which express APPswe (Swedish mutant) ubiquitously and selectively in osteoblast (OB)-lineage cells. This raises the question, whether osteoblastic APPswe contributes to AD development. Here, we provide evidence that TgAPPsweOCN mice also exhibit AD-relevant brain pathologies and behavior phenotypes. Some brain pathologies include age-dependent and regional-selective increases in glial activation and pro-inflammatory cytokines, which are accompanied by behavioral phenotypes such as anxiety, depression, and altered learning and memory. Further cellular studies suggest that APPswe, but not APPwt or APPlon (London mutant), in OB-lineage cells induces endoplasmic reticulum-stress driven senescence, driving systemic and cortex inflammation as well as behavioral changes in 6-month-old TgAPPsweOCN mice. These results therefore reveal an unrecognized function of osteoblastic APPswe to brain axis in AD development.

TFEB insufficiency promotes cardiac hypertrophy by blocking autophagic degradation of GATA4.

Autophagosome-lysosome pathway (ALP) insufficiency has been suggested to play a critical role in the pathogenesis of cardiac hypertrophy. However, the mechanisms underlying ALP insufficiency remain largely unknown, and strategies to specifically manipulate ALP insufficiency for treating cardiac hypertrophy are lacking. Transcription factor EB (TFEB), as a master regulator of ALP, regulates the generation and function of autophagosomes and lysosomes. We found that TFEB was significantly decreased, whereas autophagosome markers were increased in phenylephrine (PE)-induced and transverse aortic constriction-induced cardiomyocyte hypertrophy and failing hearts from patients with dilated cardiomyopathy. Knocking down TFEB induced ALP insufficiency, as indicated by increased autophagosome markers, decreased light chain 3II flux, and cardiomyocyte hypertrophy manifested through increased levels of atrial natriuretic peptide and ß-myosin heavy chain and enlarged cell size. The effects of TFEB knockdown were abolished by promoting autophagy. TFEB overexpression improved autophagic flux and attenuated PE-stimulated cardiomyocyte hypertrophy and transverse aortic constriction-induced hypertrophic remodeling, fibrosis, and cardiac dysfunction. Curcumin analog compound C1, a specific TFEB activator, similarly attenuated PE-induced ALP insufficiency and cardiomyocyte hypertrophy. TFEB knockdown increased the accumulation of GATA4, a transcription factor for several genes causing cardiac hypertrophy by blocking autophagic degradation of GATA4, whereas knocking down GATA4 attenuated TFEB downregulation-induced cardiomyocyte hypertrophy. Both TFEB overexpression and C1 promoted GATA4 autophagic degradation and alleviated PE-induced cardiomyocyte hypertrophy. In conclusion, TFEB downregulation plays a vital role in the development of pressure overload-induced cardiac hypertrophy by causing ALP insufficiency and blocking autophagic degradation. Activation of TFEB represents a potential therapeutic strategy for treating cardiac hypertrophy.

Hippocampal astrocytic neogenin regulating glutamate uptake, a critical pathway for preventing epileptic response.

Epilepsy, a common neurological disorder, is featured with recurrent seizures. Its underlying pathological mechanisms remain elusive. Here, we provide evidence for loss of neogenin (NEO1), a coreceptor for multiple ligands, including netrins and bone morphological proteins, in the development of epilepsy. NEO1 is reduced in hippocampi from patients with epilepsy based on transcriptome and proteomic analyses. Neo1 knocking out (KO) in mouse brains displays elevated epileptiform spikes and seizure susceptibility. These phenotypes were undetectable in mice, with selectively depleted NEO1 in excitatory (NeuroD6-Cre+) or inhibitory (parvalbumin+) neurons, but present in mice with specific hippocampal astrocytic Neo1 KO. Additionally, neurons in hippocampal dentate gyrus, a vulnerable region in epilepsy, in mice with astrocyte-specific Neo1 KO show reductions in inhibitory synaptic vesicles and the frequency of miniature inhibitory postsynaptic current(mIPSC), but increase of the duration of miniature excitatory postsynaptic current and tonic NMDA receptor currents, suggesting impairments in both GABAergic transmission and extracellular glutamate clearance. Further proteomic and cell biological analyses of cell-surface proteins identified GLAST, a glutamate-aspartate transporter that is marked reduced in Neo1 KO astrocytes and the hippocampus. NEO1 interacts with GLAST and promotes GLAST surface distribution in astrocytes. Expressing NEO1 or GLAST in Neo1 KO astrocytes in the hippocampus abolishes the epileptic phenotype. Taken together, these results uncover an unrecognized pathway of NEO1-GLAST in hippocampal GFAP+ astrocytes, which is critical for GLAST surface distribution and function, and GABAergic transmission, unveiling NEO1 as a valuable therapeutic target to protect the brain from epilepsy.

Linking cortical astrocytic neogenin deficiency to the development of Moyamoya disease-like vasculopathy.

Moyamoya-like vasculopathy, the "puff of smoke"-like small vessels in the brain, is initially identified in patients with Moyamoya disease (MMD), a rare cerebrovascular disease, and later found in patients with various types of neurological conditions, including Down syndrome, Stroke, and vascular dementia. It is thus of interest to understand how this vasculopathy is developed. Here, we provided evidence for cortical astrocytic neogenin (NEO1) deficiency to be a risk factor for its development. NEO1, a member of deleted in colorectal cancer (DCC) family netrin receptors, was reduced in brain samples of patients with MMD. Astrocytic Neo1-loss resulted in an increase of small blood vessels (BVs) selectively in the cortex. These BVs were dysfunctional, with leaky blood-brain barrier (BBB), thin arteries, and accelerated hyperplasia in veins and capillaries, resembled to the features of moyamoya-like vasculopathy. Additionally, we found that both MMD patient and Neo1 mutant mice exhibited altered gene expression in their cortex in proteins critical for not only angiogenesis [e.g., an increase in vascular endothelial growth factor (VEGFa)], but also axon guidance (e.g., netrin family proteins) and inflammation. In aggregates, these results suggest a critical role of astrocytic NEO1-loss in the development of Moyamoya-like vasculopathy, providing a mouse model for investigating mechanisms of Moyamoya-like vasculopathy.

Critical Roles of Embryonic Born Dorsal Dentate Granule Neurons for Activity-Dependent Increases in BDNF, Adult Hippocampal Neurogenesis, and Antianxiety-like Behaviors.

BACKGROUND: Dentate gyrus (DG), a "gate" that controls information flow into the hippocampus, plays important roles in regulating both cognitive (e.g., spatial learning and memory) and mood behaviors. Deficits in DG neurons contribute to the pathogenesis of not only neurological, but also psychiatric, disorders, such as anxiety disorder. Whereas DG's function in spatial learning and memory has been extensively investigated, its role in regulating anxiety remains elusive. METHODS: Using c-Fos to mark DG neuron activation, we identified a group of embryonic born dorsal DG (dDG) neurons, which were activated by anxiogenic stimuli and specifically express osteocalcin (Ocn)-Cre. We further investigated their functions in regulating anxiety and the underlying mechanisms by using a combination of chemogenetic, electrophysiological, and RNA-sequencing methods. RESULTS: The Ocn-Cre+ dDG neurons were highly active in response to anxiogenic environment but had lower excitability and fewer presynaptic inputs than those of Ocn-Cre- or adult born dDG neurons. Activating Ocn-Cre+ dDG neurons suppressed anxiety-like behaviors and increased adult DG neurogenesis, whereas ablating or chronically inhibiting Ocn-Cre+ dDG neurons exacerbated anxiety-like behaviors, impaired adult DG neurogenesis, and abolished activity (e.g., voluntary wheel running)-induced anxiolytic effect and adult DG neurogenesis. RNA-sequencing screening for factors induced by activation of Ocn-Cre+ dDG neurons identified BDNF, which was required for Ocn-Cre+ dDG neurons mediated antianxiety-like behaviors and adult DG neurogenesis. CONCLUSIONS: These results demonstrate critical functions of Ocn-Cre+ dDG neurons in suppressing anxiety-like behaviors but promoting adult DG neurogenesis, and both functions are likely through activation of BDNF.

Astrocytic neogenin/netrin-1 pathway promotes blood vessel homeostasis and function in mouse cortex.

Astrocytes have multiple functions in the brain, including affecting blood vessel (BV) homeostasis and function. However, the underlying mechanisms remain elusive. Here, we provide evidence that astrocytic neogenin (NEO1), a member of deleted in colorectal cancer (DCC) family netrin receptors, is involved in blood vessel homeostasis and function. Mice with Neo1 depletion in astrocytes exhibited clustered astrocyte distribution and increased BVs in their cortices. These BVs were leaky, with reduced blood flow, disrupted vascular basement membranes (vBMs), decreased pericytes, impaired endothelial cell (EC) barrier, and elevated tip EC proliferation. Increased proliferation was also detected in cultured ECs exposed to the conditioned medium (CM) of NEO1-depleted astrocytes. Further screening for angiogenetic factors in the CM identified netrin-1 (NTN1), whose expression was decreased in NEO1-depleted cortical astrocytes. Adding NTN1 into the CM of NEO1-depleted astrocytes attenuated EC proliferation. Expressing NTN1 in NEO1 mutant cortical astrocytes ameliorated phenotypes in blood-brain barrier (BBB), EC, and astrocyte distribution. NTN1 depletion in astrocytes resulted in BV/BBB deficits in the cortex similar to those in Neo1 mutant mice. In aggregate, these results uncovered an unrecognized pathway, astrocytic NEO1 to NTN1, not only regulating astrocyte distribution, but also promoting cortical BV homeostasis and function.

Microglial VPS35 deficiency regulates microglial polarization and decreases ischemic stroke-induced damage in the cortex.

BACKGROUND: Vacuolar sorting protein 35 (VPS35), a critical component of retromer, is essential for selective endosome-to-Golgi retrieval of membrane proteins. It is highly expressed in microglial cells, in addition to neurons. We have previously demonstrated microglial VPS35's functions in preventing hippocampal, but not cortical, microglial activation, and in promoting adult hippocampal neurogenesis. However, microglial VPS35's role in the cortex in response to ischemic stroke remains largely unclear. METHODS: We used mice with VPS35 cKO (conditional knockout) in microglial cells and examined and compared their responses to ischemic stroke with control mice. The brain damage, cell death, changes in glial cells and gene expression, and sensorimotor deficits were assessed by a combination of immunohistochemical and immunofluorescence staining, RT-PCR, Western blot, and neurological functional behavior tests. RESULTS: We found that microglial VPS35 loss results in an increase of anti-inflammatory microglia in mouse cortex after ischemic stroke. The ischemic stroke-induced brain injury phenotypes, including brain damage, neuronal death, and sensorimotor deficits, were all attenuated by microglial VPS35-deficiency. Further analysis of protein expression changes revealed a reduction in CX3CR1 (CX3C chemokine receptor 1) in microglial VPS35-deficient cortex after ischemic stroke, implicating CX3CR1 as a potential cargo of VPS35 in this event. CONCLUSION: Together, these results reveal an unrecognized function of microglial VPS35 in enhancing ischemic brain injury-induced inflammatory microglia, but suppressing the injury-induced anti-inflammatory microglia. Consequently, microglial VPS35 cKO mice exhibit attenuation of ischemic brain injury response.

NMDA receptors inhibit axonal outgrowth by inactivating Akt and activating GSK-3ß via calcineurin in cultured immature hippocampal neurons.

Neurons are highly polarized cells with an axon and dendritic arbors. It is still not well studied that how formation and elaboration of axon and dendrites is controlled by diffusible signaling factors such as glutamate via specific receptors. We found that N-methyl-D-aspartate (NMDA) receptors were enriched (stage 2-3) but decreased expression (stage 4-5) at tip of axon of cultured hippocampal neurons during distinct development stages. Inhibition of NMDA receptor activity by competitive antagonist DL-2-amino-5-phosphonovalerate (APV) or channel blocker MK801 promoted axonal outgrowth at the early stages, whereas inhibited dendritic development in later stages. Meanwhile, knockdown of NMDA receptors also promoted axonal outgrowth and branch in immature neurons. Furthermore, GluN2B but not GluN2A subunit inhibited axonal outgrowth in immature hippocampal neurons. Finally, we found that NMDA receptors inhibited axonal outgrowth by inactivating Akt and activating GSK-3ß signaling in a calcineurin-dependent manner. Taken together, our results demonstrate that stabilization GSK-3ß activation in the axon growth cone by Ca2+ influx through NMDA receptors may be involved in regulation of axon formation in immature neurons at early stages.

Factors affecting cerebrospinal fluid opening pressure in patients with spontaneous intracranial hypotension.

OBJECTIVE: Spontaneous intracranial hypotension (SIH) is recognized far more commonly than ever before. Though usually characterized by low cerebrospinal fluid (CSF) pressure, some patients with SIH are observed to have normal pressure values. In this study, we aimed to confirm the proportion of patients with normal CSF opening pressure (CSF OP) and explore the factors affecting CSF OP in SIH patients. METHODS: We retrospectively reviewed 206 consecutive SIH patients and analyzed their clinical and imaging variables (including demographic data, body mass index (BMI), duration of symptoms, and brain imaging findings). Univariate and multivariate analyses were performed to identify the potential factors affecting CSF OP. RESULTS: In a total of 114 (55.3%) cases the CSF OP was ≤60 mmH2O (1 mmH2O=9.806 65 Pa), in 90 (43.7%) cases it was between 60 and 200 mmH2O, and in 2 (1.0%) cases it was >200 mmH2O. Univariate analysis showed that the duration of symptoms (P<0.001), BMI (P<0.001), and age (P=0.024) were positively correlated with CSF OP. However, multivariate analysis suggested that only the duration of symptoms (P<0.001) and BMI (P<0.001) were strongly correlated with CSF OP. A relatively high R2 of 0.681 was obtained for the multivariate model. CONCLUSIONS: Our study indicated that in patients without a low CSF OP, a diagnosis of SIH should not be excluded. BMI and the duration of symptoms can influence CSF OP in SIH patients, and other potential factors need further investigation.

S100B promotes injury-induced vascular remodeling through modulating smooth muscle phenotype.

S100B is a biomarker of nervous system injury, but it is unknown if it is also involved in vascular injury. In the present study, we investigated S100B function in vascular remodeling following injury. Balloon injury in rat carotid artery progressively induced neointima formation while increasing S100B expression in both neointimal vascular smooth muscle (VSMC) and serum along with an induction of proliferating cell nuclear antigen (PCNA). Knockdown of S100B by its shRNA delivered by adenoviral transduction attenuated the PCNA expression and neointimal hyperplasia in vivo and suppressed PDGF-BB-induced VSMC proliferation and migration in vitro. Conversely, overexpression of S100B promoted VSMC proliferation and migration. Mechanistically, S100B altered VSMC phenotype by decreasing the contractile protein expression, which appeared to be mediated by NF-κB activity. S100B induced NF-κB-p65 gene transcription, protein expression and nuclear translocation. Blockade of NF-κB activity by its inhibitor reversed S100B-mediated downregulation of VSMC contractile protein and increase in VSMC proliferation and migration. It appeared that S100B regulated NF-κB expression through, at least partially, the Receptor for Advanced Glycation End products (RAGE) because RAGE inhibitor attenuated S100B-mediated NF-κB promoter activity as well as VSMC proliferation. Most importantly, S100B secreted from VSMC impaired endothelial tube formation in vitro, and knockdown of S100B promoted re-endothelialization of injury-denuded arteries in vivo. These data indicated that S100B is a novel regulator for vascular remodeling following injury and may serve as a potential biomarker for vascular damage or drug target for treating proliferative vascular diseases.

Phenylephrine protects cardiomyocytes from starvation-induced apoptosis by increasing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be a "housekeeping" protein; studies in non-cardiomyocytic cells have shown that GAPDH plays pro-apoptotic role by translocating from cytoplasm to the nucleus or to the mitochondria. However, the cardiovascular roles of GAPDH are unknown. We observed that phenylephrine (PE) (100 µM) protected against serum and glucose starvation -induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and mitochondrial membrane potential depolarization. GAPDH glycolysis activity was positively correlated with the antiapoptotic action of PE. GAPDH activity inhibition blunted PE-induced protection of the mitochondrial membrane potential and cardiomyocytes. PE-induced Bcl-2 protein increase, Bax mitochondrial decrease and inhibition of cytochrome C release and Caspase 3 activation, as well as ROS production were blunted by GAPDH activity inhibition. Moreover, GAPDH overexpression provided protection against starvation-induced cardiomyocyte apoptosis in vitro and ischemia-induced cardiac infarction in vivo. Inhibition of Akt prevented PE-induced GAPDH activity increase and cardiomyocytes protection. In conclusion, the present study provides the first direct evidence of an antiapoptotic role of GAPDH in PE-induced cardiomyocytes protection; GAPDH activity elevation mainly affects the mitochondria-induced apoptosis.

Expression and ultrastructural localization of Mint2 in the spinal cord of rats.

Mint protein family, as adaptor molecules, contains three members, Mint1, Mint2 and Mint3. Although Mint3 is ubiquitously expressed, Mint1 and Mint2 have been reported to express specifically in neuron. Here we demonstrated Mint1 and Mint2 expression pattern in rat spinal cord. The protein level of Mint2 was found to be higher than that of Mint1 in rat spinal by western blot. In an attempt to know Mint2 distribution in the spinal cord of rat, in situ hybridization was carried out, Mint2 mRNA was showed to be ubiquitously distributed in cervical, thoracic and lumbar sections of rat spinal cord, and high intensive signal was detected in motor neurons. These were further confirmed by fluorescent immunohistochemistry, Mint2 was also found to exist throughout gray matter especially motor neurons where Mint2 was mainly located in perikaryon, however, Mint1 was showed to be relatively lower. By electron microscope, Mint2 was found to be mainly located in vesicles in perikaryon in motor neuron of lumbar section, and at the same time Mint2 was located in axons in myelin and presynaptic terminals. These data suggest that Mint2 may play more important role in spinal cord than the other two family members.

Hydrogen sulfide protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by preventing GSK-3beta-dependent opening of mPTP.

Hydrogen sulfide (H(2)S) is an endogenously generated gaseous transmitter, which has recently been suggested to regulate cardiovascular functions. The present study aims to clarify the mechanisms underlying the cardioprotective effects of H(2)S. Signaling elements were examined in cardiomyocytes cultured under hypoxia/reoxygenation conditions and in a rat model of ischemia-reperfusion. In cultured cardiomyocytes, sodium hydrosulfide (NaHS; 10, 30, and 50 mumol/l) showed concentration-dependent inhibitory effects on cardiomyocyte apoptosis induced by hypoxia/reoxygenation. These effects were associated with an increase in phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) (Ser9) and a decrease in Bax translocation, caspase-3 activation, and mitochondrial permeability transition pore (mPTP) opening. Transfection of a phosphorylation-resistant mutant of GSK-3beta at Ser9 attenuated the effects of NaHS in reducing cardiomyocyte apoptosis, Bax translocation, caspase-3 activation, and mPTP opening. In a rat model of ischemia-reperfusion, NaHS administration reduced myocardial infarct size and increased the phosphorylation of GSK-3beta (Ser9) at a dose of 30 mumol/kg. In conclusion, the H(2)S donor prevents cardiomyocyte apoptosis by inducing phosphorylation of GSK-3beta (Ser9) and subsequent inhibition of mPTP opening.

Survivin is involved in the anti-apoptotic effect of edaravone in PC12 cells.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a newly developed hydroxy radical scavaging agent which has been widely used for protection against ischemia-reperfusion injury is highly effective in preventing cell apoptosis. However, the exact intracellular mechanism(s) underlying the protective action of edaravone is not clear. We observed that in PC12 cells cultured under serum deprivation (DEPV) condition, the levels of survivin were positively correlated with the anti-apoptotic action of edaravone. Survivin RNA interference (RNAi) increased DEPV-induced PC12 cell apoptosis, whereas the anti-apoptotic effect of edaravone was blunted by survivin RNAi. Moreover, survivin overexpression provided protection against DEPV-induced PC12 cell apoptosis. Inhibition of ERK and PI(3)-K/AKT prevented edaravone's ability to decrease apoptosis and increase survivin. In conclusion, the present study provides the first direct evidence that survivin involves in the anti-apoptotic effects of edaravone via a pathway involving ERK and PI(3)-K/AKT.

Bone morphogenetic protein-2 acts upstream of myocyte-specific enhancer factor 2a to control embryonic cardiac contractility.

OBJECTIVE: Cardiac contractility is regulated tightly as an extrinsic and intrinsic homeostatic mechanism to the heart. The molecular basis of the intrinsic system is largely unknown. Here, we test the hypothesis that bone morphogenetic protein-2 (BMP-2) mediates embryonic cardiac contractility upstream of myocyte-specific enhancer factor 2A (MEF2A). METHODS: The BMP-2 and MEF2A expression pattern was analyzed by RT-PCR, Western blotting, whole-mount in situ hybridization, and an in vivo transgenic approach. The cardiac phenotype of BMP-2 and MEF2A knock-down zebrafish embryos was analysed. Cardiac contractions were recorded with a video camera. Myofibrillar organization was observed with transmission electron microscopy. Gene expression profiles were performed by quantitative real-time PCR analysis. RESULTS: We demonstrate that BMP-2 and MEF2A are co-expressed in embryonic and neonatal cardiac myocytes. Furthermore, we provide evidence that BMP-2 is required for cardiac contractility in vitro and in vivo and that MEF2A expression can be activated by BMP-2 signaling in neonatal cardiomyocytes. BMP-2 is involved in the assembly of the cardiac contractile apparatus. Finally, we find that exogenous MEF2A is sufficient to rescue ventricular contractility defects in the absence of BMP-2 function. CONCLUSIONS: In all, these observations indicate that BMP-2 and MEF2A are key components of a pathway that controls the cardiac ventricular contractility and suggest that the BMP2-MEF2A pathway can offer new opportunities for the treatment of heart failure.

Survivin mediates the anti-apoptotic effect of delta-opioid receptor stimulation in cardiomyocytes.

Survivin is known to be essential for cell division and to inhibit apoptosis during embryonic development and in adult cancerous tissues. However, the cardiovascular role of survivin is unknown. We observed that in cardiomyocytes cultured under conditions of serum and glucose deprivation (DEPV), the levels of survivin, Bcl-2 and extracellular signal-regulated kinase (ERK) were positively correlated with the anti-apoptotic action of a delta-opioid receptor agonist, [D-Ala2, D-Leu5]-enkephalin acetate (DADLE). By contrast, Bax translocation, mitochondrial membrane damage and reactive oxygen species (ROS) production were inversely correlated with the changes of survivin and Bcl-2. The use of RNA interference (RNAi) targeting survivin increased DEPV-induced cardiomyocyte apoptosis, whereas the anti-apoptotic effect of DADLE was blunted by survivin RNAi. Moreover, survivin transfection and overexpression provided protection against DEPV-induced cardiomyocyte apoptosis. Inhibition of ERK prevented the DADLE-induced decrease in apoptosis and Bax translocation, and increase in survivin and Bcl-2. DADLE-induced increase in survivin was also blunted by phosphoinositol 3-kinase (PI 3-kinase) inhibition. In conclusion, the present study provides the first direct evidence of an anti-apoptotic role of survivin mediating the anti-apoptotic effect of delta-opioid receptor activation in cardiomyocytes. ERK and PI 3-kinase were found to be upstream regulators of survivin. Mitochondrial membranes as well as ROS, Bcl-2 and Bax were also involved in this anti-apoptotic action.

[Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein].

OBJECTIVE: To construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-1-CAT. METHODS: Using pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2(+)-CAT plasmid, and the PCR product was added with "A" using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3' end was ligated with pGEM-T Easy vector and transformed into DH5alpha. The correct recombinant was identified by PCR and Sal I/Bgl II digestion and named as pGEM-T-CAT. Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho I-digested pET15b. The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and Sal I/Bgl II, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E. coli BL21(DE3) which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni(2+)-NTA-resin column. The fusion protein PEP-1-CAT was produced and confirmed by specific enzyme activity in vitro. RESULTS: Sequence analysis showed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase cDNA sequence in GenBank (accession No. AY028632), respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-1-CAT fusion protein with specific activity of 77.15 U/g. CONCLUSION: The prokaryotic expression plasmid pET15b-PEP-1-CAT has been constructed successfully, and the successful expression and purification of PEP-1-CAT provides a basis for prevention and therapy of various disorders related to oxidative stress.

[The protective effect of PEP-1-CAT fusion protein on hydrogen peroxide-induced oxidative stress injury in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell (HUVECs) and the effects on hydrogen-peroxide (H2O2)-induced oxidative stress injury in these cells. METHODS: With the use of TA-cloning program and isocaudamer technique, the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) and the protein expression was induced by IPTG. The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni2+-NTA-resin column. The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro. The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro. The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity. The cells were treated with H2O2 (0.5 mmol/L) alone and in combination with PEP-1-CAT fusion protein for 4 h. Then, the cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) contents were measured. RESULTS: The PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose- and time-dependent manner and be stable for at least 48 h. After H2O2 administration, cell viability was significantly reduced compared with control group (37.23%+/-5.68% vs. 100%, P<0.05), while LDH leakage (849.3 U/L+/-95.1 U/L) and MDA (8.23 nmol/L+/-1.58 nmol/L) content were significantly higher than that in control group (540.6 U/L+/-65.7 U/L and 2.46 nmol/L+/-1.42 nmol/L, respectively, all P<0.05). Preincubation with PEP-1-CAT proteins at various concentrations (0.25-2 micromol/L) significantly attenuated H2O2-induced cell injury. CONCLUSION: The PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.