Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae.

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by ß-carotene hydroxylase (crtZ) and ß-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.

Efficacy and Safety of Teriflunomide in Chinese Patients with Relapsing Forms of Multiple Sclerosis: A Subgroup Analysis of the Phase 3 TOWER Study.

BACKGROUND: Disease-modifying therapy is the standard treatment for patients with multiple sclerosis (MS) in remission. The primary objective of the current analysis was to assess the efficacy and safety of two teriflunomide doses (7 mg and 14 mg) in the subgroup of Chinese patients with relapsing MS included in the TOWER study. METHODS: TOWER was a multicenter, multinational, randomized, double-blind, parallel-group (three groups), placebo-controlled study. This subgroup analysis includes 148 Chinese patients randomized to receive either teriflunomide 7 mg (n = 51), teriflunomide 14 mg (n = 43), or placebo (n = 54). RESULTS: Of the 148 patients in the intent-to-treat population, adjusted annualized relapse rates were 0.63 (95% confidence interval [CI]: 0.44, 0.92) in the placebo group, 0.48 (95% CI: 0.33, 0.70) in the teriflunomide 7 mg group, and 0.18 (95% CI: 0.09, 0.36) in the teriflunomide 14 mg group; this corresponded to a significant relative risk reduction in the teriflunomide 14 mg group versus placebo (-71.2%, P = 0.0012). Teriflunomide 14 mg also tended to reduce 12-week confirmed disability worsening by 68.1% compared with placebo (hazard ratio: 0.319, P = 0.1194). There were no differences across all treatment groups in the proportion of patients with treatment-emergent adverse events (TEAEs; 72.2% in the placebo group, 74.5% in the teriflunomide 7 mg group, and 69.8% in the teriflunomide 14 mg group); corresponding proportions for serious adverse events were 11.1%, 3.9%, and 11.6%, respectively. The most frequently reported TEAEs with teriflunomide versus placebo were neutropenia, increased alanine aminotransferase, and hair thinning. CONCLUSIONS: Teriflunomide was as effective and safe in the Chinese subpopulation as it was in the overall population of patients in the TOWER trial. Teriflunomide has the potential to meet unmet medical needs for MS patients in China. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00751881; https://clinicaltrials.gov/ct2/show/NCT00751881?term=NCT00751881&rank=1.

Neuroprotection and CD131/GDNF/AKT Pathway of Carbamylated Erythropoietin in Hypoxic Neurons.

Carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to neuroprotective effects without erythropoiesis. However, little is known about molecular mechanisms behind CEPO-mediated neuroprotection. In primary neurons with oxygen-glucose deprivation (OGD) and mice with hypoxia-reoxygenation, the neuroprotection and possible molecular mechanism of CEPO were performed by immunohistochemistry and immunocytochemistry, Western blot, RT-PCR, and ELISA. The comparisons were analyzed by ANOVA followed by unpaired two-tailed Student's t test. Both CEPO and EPO showed the neuroprotective effects in OGD model and hypoxic brain. CEPO did not trigger JAK-2 but activated AKT through glial cell line-derived neurotrophic factor (GDNF). It has been shown that CEPO acts upon a heteroreceptor complex comprising both the EPO receptor and the common ß receptor subunit (ßcR, also known as CD131). The blockage of CD131 reduced CEPO-mediated GDNF production, while GFR receptor blockage and GDNF neutralization inhibited CEPO-induced neurogenesis. Addition of GDNF to cultured neurons increased phosphorylation of AKT. CEPO protects neurons possible through the CD131/GDNF/AKT pathway.

The lack of CD131 and the inhibition of Neuro-2a growth by carbamylated erythropoietin.

Recombinant human erythropoietin (EPO), a glycohormone, is one of the leading biopharmaceutical products, while carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to its neuroprotective effects without erythropoiesis in several cells and animal models. However, exogenous EPO promotes an angiogenic response from tumor cells and is associated with tumor growth, but knowledge of CEPO on tumor growth is lacking. Here we show that CEPO, but not EPO, inhibited Neuro-2a growth and viability. As expected, CEPO--unlike EPO--did not activate JAK-2 either in primary neurons or in Neuro-2a cells. Interestingly, CEPO did not induce GDNF expression and subsequent AKT activation in Neuro-2a cells. Before CEPO/EPO treatment, glial cell line-derived neurotrophic factor (GDNF) neutralization and GFR receptor blocking decreased the viability of EPO-treated Neuro-2a cells but did not influence CEPO-treated Neuro-2a cells. As compared to primary neurons, the expression of CD131, as a receptor complex binding to CEPO, is almost lacking in Neuro-2a cells. In BABL/C-nu mice, CEPO did not promote the growth of Neuro-2a cells nor extended the survival time compared to mice treated with EPO. The results indicate that CEPO did not promote tumor growth because of lower expression of CD131 and subsequent dysfunction of CD131/GDNF/AKT pathway in Neuro-2a cells, revealing its therapeutic potential in future clinical application.

Novel mutations m.3959G>A and m.3995A>G in mitochondrial gene MT-ND1 associated with MELAS.

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are progressive neurodegenerative disorder associated with polygenetic, maternally inherited mutations in mitochondrial DNA. Approximately 80% of MELAS cases are caused by the mutation m.3243A>G of the mitochondrial tRNA(Leu (UUR)) gene (MT-TL1). We reported two probands with MELAS features. Muscle biopsy identified ragged-red fibers (RRF) in Gomori Trichrome staining. A respiratory chain function study showed decreased activity of mitochondrial respiratory chain complex I in both probands. Sequencing of the mitochondrial DNA revealed two novel MT-ND1 gene missense mutations, m.3959G>A and m.3995A>G, which are highly conserved among species. Protein secondary structure predictions demonstrated that these mutations may alter the peptide structure and may lead to decreased ND1 gene stability. Our findings suggest that these two novel mutations may contribute to the MELAS phenotypes of the patients in our study.

Histone decacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection against MPP+.

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disease, characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra. Recent investigations have shown that mitochondrial fragmentation, an early event during apoptosis, is implicated in the degeneration of DA neurons in PD, and more importantly, preventing mitochondrial fragmentation could rescue cell death in several PD models. Therefore, mitochondrial dynamics may be a therapeutic target for early intervention in PD. However, much remains unknown about the mechanism underlying mitochondrial fragmentation in PD. METHODS: The alterations in mitochondrial morphology, cell apoptosis, and mitochondrial shaping protein levels were detected after SH-SY5Y cells were treated with various doses of MPP+ or rotenone. RESULTS: Mitochondrial fragmentation is an early event during apoptosis caused by MPP+ but not rotenone, and Trichostatin A (TSA), a commonly used histone deacetylase (HDAC) inhibitor, selectively rescues mitochondrial fragmentation and cell death induced by lower doses of MPP+. Mitochondrial fragmentation triggered by lower doses of MPP+ may be a result of Mfn2 down-regulation, which could be completely reversed by TSA. Further investigation suggests that TSA prevents MPP+-induced Mfn2 down-regulation via inhibiting histone deacetylation over Mfn2 promoter and alleviating its transcriptional dysfunction. CONCLUSIONS: Histone deacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection in PD cell model induced by MPP+. Hence, HDAC inhibitors may be a potential early treatment for PD.

Carbamylated erythropoietin ameliorates hypoxia-induced cognitive and behavioral defects with the generation of choline acetyltransferase-positive neurons.

Carbamylated erythropoietin (CEPO) is attracting widespread interest because of its neuroprotective effects without influencing erythropoiesis. Here we show that CEPO, unlike EPO, does not stimulate erythropoiesis. Both CEPO and EPO inhibit the death/apoptosis of neurons in the hypoxic model of primary neurons and induce neuron proliferation and differentiation in hypoxic mice. Hypoxic mice show apparent memory deficits at 3 and 30 days after hypoxia. The administration of CEPO/EPO significantly improves cognitive and behavioral defects after hypoxic insults. Further investigation shows that CEPO/EPO induces neuron proliferation and differentiation and promotes the generation of choline acetyltransferase (ChAT)(+) neurons in hypoxic mice. Phosphorylated AKT was colabeled with ChAT(+) neurons and coexpressed in bromodeoxyuridine-positive cells, suggesting that the PI3K/AKT pathway may play a pivotal role in CEPO/EPO-cholinergic neuron generation. These results reveal that CEPO/EPO ameliorates hypoxia-induced cognitive and behavioral defects possibly through the generation of ChAT-positive neurons.

HLA-DQA1*03:02/DQB1*03:03:02 is strongly associated with susceptibility to childhood-onset ocular myasthenia gravis in Southern Han Chinese.

OBJECTIVE: Our aim was to investigate the correlation between onset age, clinical features and HLA-DQA1/DQB1 genetic variability in myasthenia gravis (MG) patients in Southern Han Chinese. METHODS: 205 MG patients and 100 controls were genotyped for HLA-DQA1 and -DQB1 using sequence-based typing (SBT) and analyzed for haplotype frequencies. Anti-acetylcholine receptor (AChR) autoantibodies were measured in all, and muscle-specific tyrosine kinase (MuSK) antibodies were tested in AChR antibody negative patients. RESULTS: HLA-DQA1/DQB1 haplotypes showed association only with childhood-onset MG. Haplotype DQA1*03:02/DQB1*03:03:02 (DQ9) was positively associated with the childhood-onset MG, while haplotype DQA1*02:01/DQB1*02:02 and DQA1*05:01:01/DQB1*02:01:01 (DQ2) were negatively associated with this group. Childhood-onset ocular MG patients had an extremely high phenotype frequency of DQ9 haplotype (90.1% of patients, 34.0% of controls, p≤0.0001, OR=17.8). CONCLUSIONS: The childhood-onset ocular MG in Southern Han Chinese may present a particular subgroup of distinct genetic background. Its correlation to the HLA haplotype DQA1*03:02/DQB1*03:03:02 might explain the phenotypic difference of MG between Han Chinese and Caucasians.

The polymorphism of the ATP-binding cassette transporter 1 gene modulates Alzheimer disease risk in Chinese Han ethnic population.

BACKGROUND: Recent studies highlight a potential role of cholesterol metabolic disturbance in the pathophysiology of Alzheimer disease (AD). The adenosine triphosphate (ATP)-binding cassette transporter 1 (ABCA1) gene resides within proximity of linkage peaks on chromosome 9q influence AD and plays a key role in cellular cholesterol efflux in the brain. METHODS: We studied the role of R219K and V825I polymorphisms of ABCA1 in modulating the risk of AD in 321 AD patients and 349 comparisons of Chinese Han. Genotyping of R219K and V825I were performed by PCR-restriction fragment length polymorphism analysis. RESULTS: The genotype distribution of R219K was different with more RK in total AD group (χ(2) = 8.705, df = 2, p = 0.013), late-onset AD (LOAD) group (χ(2) = 10.636, df = 2, p = 0.005), APOE non-ε4ε4 group (χ(2) = 9.900, df = 2, p = 0.007), and female AD group (χ(2) = 8.369, df = 2, p = 0.015). Logistic regression manifested the risk of AD increased in RK carriers in total AD group (Wald = 6.102, df = 1, p = 0.014, odds ratio [OR]: 1.546, 95% confidence interval [95% CI]: 1.094-2.185), LOAD group (Wald = 7.746, df = 1, p = 0.005, OR: 1.921, 95% CI: 1.213-3.041), and APOE non-ε4ε4 group (Wald = 6.399, df = 1, p = 0.011, OR: 1.586, 95% CI: 1.109-2.266). K allele (RK + KK) also increased the risk of AD compared with RR allele in LOAD group (Wald = 4.750, df = 1, p = 0.029, OR: 1.619, 95% CI: 1.050-2.497). However, no discrepancy was found in V825I. In R219K, age at onset (AAO) was significantly lower by 4.9 years on average in patients of KK genotype than those of RK in APOE ε4 carrying group and higher by 5.5 years in patients of KK genotype than those of RR in APOE ε4 noncarrying group. In V825I, AAO was diseased by 4.3 years in II genotype compared with VV genotype in APOE ε4 noncarrying group and 3.4 years in APOE ε4ε4 noncarrying group. CONCLUSION: The results indicated that the RK genotype or K allele (RK + KK) of R219K may relate to the development of AD in the east of China.

TSPO-specific ligand vinpocetine exerts a neuroprotective effect by suppressing microglial inflammation.

Vinpocetine has long been used for cerebrovascular disorders and cognitive impairment. Based on the evidence that the translocator protein (TSPO, 18 kDa) was expressed in activated microglia, while Vinpocetine was able to bind TSPO, we explored the role of Vinpocetine on microglia treated with lipopolysaccharide (LPS) and oxygen-glucose deprivation (OGD) in vitro. Our results show that both LPS and OGD induced the up-regulation of TSPO expression on BV-2 microglia by RT-PCR, western blot and immunocytochemistry. Vinpocetine inhibited the production of nitrite oxide and inflammatory factors such as interleukin-1ß (IL-1ß), IL-6 and tumour necrosis factor-α (TNF-α) in BV-2 microglia, in which cells were treated with LPS or exposed to OGD, regardless of the time Vinpocetine was added. Next, we measured cell death-related molecules Akt, Junk and p38 as well as inflammation-related molecules nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Vinpocetine did not change cell death-related molecules, but inhibited the expression of NF-κB and AP-1 in LPS-stimulated microglia, indicating that Vinpocetine has an anti-inflammatory effect by partly targeting NF-κB/AP-1. Next, conditioned medium from Vinpocetine-treated microglia protected from primary neurons. As compared with in vitro, the administration of Vinpocetine in hypoxic mice also inhibited inflammatory molecules, indicating that Vinpocetine as a unique anti-inflammatory agent may be beneficial for the treatment of neuroinflammatory diseases.

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice.

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1ß, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.

High frequency of Machado-Joseph disease identified in southeastern Chinese kindreds with spinocerebellar ataxia.

BACKGROUND: Machado-Joseph disease (MJD), caused by a CAG repeat expansion located in exon10 of the ATXN3 gene, is now regarded as one of the most common spinocerebellar ataxia (SCA) in the world. The relative frequency of MJD among SCA has previously been estimated at about 50% in the Chinese population and has been reported to be related to the frequency of large normal alleles in some populations. Taq polymerase has been used for PCR in nearly all studies reported previously. METHODS: Normal and expanded alleles of ATXN3 were detected via PCR using LA Taq DNA polymerase (better for GC-rich sequences) and denaturing polyacrylamide gel electrophoresis in 150 normal individuals and 138 unrelated probands from autosomal dominant SCA families. To compare reaction efficiency, 12 MJD patients' expanded alleles were amplified with La Taq and Taq polymerase respectively in the same amplifying systems and reaction conditions. RESULTS: Normal alleles ranged from 12 to 42 CAG repeats. The most common allele contained 14 repeats with a frequency of 23.3%, which corroborates previous reports. The frequency of large normal alleles (>27 repeats) was 0.28, which was very high relative to previous reports. The frequency of MJD in SCA patients was 72.5%, which was significantly higher than those in previous reports about the Chinese and other Asian populations. This frequency was one of the highest reported worldwide, with only Portuguese and Brazilian populations exhibiting higher proportions. All 12 expanded alleles were amplified in PCR with La Taq polymerase, whereas only 2 expanded alleles were amplified with Taq polymerase. CONCLUSION: We have first reported the highest relative frequency of MJD in Asia, and we attribute this high frequency to a more efficient PCR using LA Taq polymerase and hypothesized that large ANs may act as a reservoir for expanded alleles in the Southeastern Chinese population.

Predictors of vascular events after ischemic stroke: the China ischemic stroke registry study.

BACKGROUND/AIMS: To measure the risk of vascular event occurrence among postischemic stroke patients in mainland China. METHODS: In this multicenter prospective stroke registry study, we enrolled 1,951 patients diagnosed with acute ischemic stroke. Demographic data, prestroke risk factors, severity of neurological deficits and disability graded on the National Institutes of Health Stroke Scale (NIHSS), and modified Rankin Scale scores of each patient were measured and recorded. Patients were followed up regularly for 12 months after recruitment. Clinical endpoints were defined as occurrence of vascular events or death. RESULTS: We detected 103 cases with nonfatal vascular events and 27 cases that died of vascular events. Cumulative incidences of total vascular events, cerebrovascular events, and coronary artery diseases were 7.2, 5.0, and 1.8%, respectively, at 12 months after the initial ischemic stroke. Concomitant atherosclerotic-thrombotic diseases (HR, 1.68; 95% CI, 1.14-2.43) and baseline NIHSS (HR, 1.07; 95% CI, 1.03-1.11) were found to be the best predictors for further occurrence of a vascular event. Antiplatelet therapy (HR, 0.52; 95% CI, 0.35-0.77) is associated with a lower risk of further vascular events. CONCLUSION: Our study provided valuable data on prognosis of acute ischemic stroke in Chinese patients.

Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil, a Rho kinase inhibitor.

The migration of aberrant inflammatory cells into the central nervous system plays an important role in the pathogenesis of demyelinating diseases potentially through the Rho/Rho-kinase (Rock) pathway, but direct evidence from human and animal models remains inadequate. Here we further confirm that Fasudil, a selective Rock inhibitor, has therapeutic potential in a mouse model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). The results show that Fasudil decreased the development of EAE in C57BL/6 mice. Immunohistochemistry disclosed that expression of Rock-II in the perivascular spaces and vascular endothelial cells of spleens, spinal cords, and brains was elevated in EAE and was inhibited in the Fasudil-treated group. T-cell proliferation specific to MOG(35-55) was markedly reduced, together with a significant down-regulation of interleukin (IL)-17, IL-6, and MCP-1. In contrast, secretion of IL-4 was increased, and IL-10 was slightly elevated. There were no differences in the percentages of CD4(+)CD25(+), CD8(+)CD28(-), and CD8(+)CD122(+) in mononuclear cells. Histological staining disclosed a marked decrease of inflammatory cells in spinal cord and brain of Fasudil-treated mice. These results, together with previous studies showing the inhibitory effect of Fasudil on T-cell migration, might expand its clinical application as a new therapy for multiple sclerosis by decreasing cell migration and regulating immune balance.

Fasudil, a Rho kinase inhibitor, drives mobilization of adult neural stem cells after hypoxia/reoxygenation injury in mice.

Rho kinase (ROCK) is important in fundamental processes of cell proliferation and survival. Blockade of ROCK promotes stem cell survival in vitro and axonal regeneration in vivo, exhibiting therapeutic potential such as spinal cord injuries and stroke. Here, we used the model of hypoxia/reoxygenation (H/R) injury to explore the possibility whether Fasudil, a ROCK inhibitor in clinical application for subarachnoid hemorrhage and stroke, mobilizes adult neural stem cells in vivo. Most interestingly, Fasudil triggers neurogenesis especially in the subventricular zone after H/R. The increase of Brdu+ cholinergic neurons was observed in striatum and forebrain cortex of Fasudil-treated mice after 30 days. Further observation demonstrates that both levels of granulocyte colony-stimulating factor (G-CSF) and astrocytes expressing G-CSF were elevated in mice treated with Fasudil, as compared to mice injected with saline. In vitro H/R model of cultured astrocytes, Fasudil promoted astrocytes to produce G-CSF in a dose-dependent manner. In addition, antibody neutralization and receptor blocking of the G-CSF pathway clearly demonstrate that Fasudil-induced neurogenesis was mediated partially through astrocyte-derived G-CSF. Our results indicate that Fasudil might represent a promising therapeutic perspective by mobilizating endogenous adult neural stem cells in the CNS.

Association of antiplatelet therapy with lower risk of death and recurrent cerebrovascular events after ischemic stroke--results from the China Ischemic Stroke Registry Study.

BACKGROUND: Evidence of the beneficial effects of antiplatelet therapy after ischemic stroke is currently lacking in China. METHODS AND RESULTS: Demographic data, pre-stroke risk factors, severity of neurological deficit, and disability graded by the National Institutes of Health Stroke Scale (NIHSS) and Modified Rankin Scale (MRS) of 1,951 patients were measured and recorded at baseline. Regular follow-up by interview was performed for 12 months post-recruitment. The all-cause mortality was 1.88 per 100 person-years during the follow-up period. Recurrent fatal and nonfatal cerebrovascular events occurred in 90 patients, accounting for 4.24 per 100 person-years of cumulative incidence. After adjustment by other variables, antiplatelet therapy was identified as an independent protective predictor of all-cause death (hazard ratio (HR) 0.42; 95% confidence interval (CI) 0.21-0.86; P=0.017) and recurrent cerebrovascular events (HR 0.58; 95%CI 0.36-0.92; P=0.021). Among survivors, antiplatelet therapy was also an independent predictor for improvement in the NIHSS (HR 1.27; 95%CI 1.07-1.51; P=0.006) and the MRS (HR 1.25; 95%CI 1.02-1.52; P=0.031). CONCLUSIONS: The data from this multicenter, prospective study confirmed the association between antiplatelet therapy and decreased risk of all-cause mortality and recurrent cerebrovascular events after ischemic stroke in Chinese patients.

Granulocyte-colony stimulating factor is involved in low-dose LPS-induced neuroprotection.

Granulocyte-colony stimulating factor (G-CSF) has recently been noted for neuroprotective function. Evidence has been given to indicate that G-CSF is naturally expressed in neurons and directly activates anti-apoptosis pathways. Finding out the agents inducing G-CSF production is of value for understanding the neuroprotection network in central nervous system. It is known that lipopolysaccharide (LPS) can induce macrophages to produce G-CSF. Here we demonstrate that hippocampal neurons exhibited the expression of toll-like receptor-4, and prove that low-dose LPS treatment increased the expression and production of G-CSF mRNA and protein in cultured neurons. We further indicate that the neutralization of G-CSF with corresponding anti-G-CSF antibodies abolished the neuroprotective effect of LPS pretreatment in N-methyl-D-aspartic acid-induced neuronal injury by MTT/CCK-8 assays and LDH release. Thus our results reveal that G-CSF may be involved in LPS-mediated neuroprotection in vivo.

[Quantitative analysis of regional cerebral blood flow in elderly with white matter lesions].

OBJECTIVE: To observe whether the severity of white matter lesions (WML) is related to ischemia in elderly. METHODS: WML were divided into 2 categories (centrum semiovale and periventricular regions) and four grades (grade 0, grade 1, grade 2 and grade 3) according to the severity of WML showing on the FLAIR sequence of MRI using modified Fazekas scale. The values of regional cerebral blood flow (rCBF) within WML and other brain regions were measured using Xenon contrast CT. RESULTS: Mean rCBF (ml x 100 g(-1) x min(-1)) within lesions around periventricular areas, in right and left centrum semiovale were 20.8 +/- 2.8, 22.3 +/- 1.9 and 22.2 +/- 2.1 in grade 0; 20.3 +/- 2.5, 21.3 +/- 1.0 and 21.0 +/- 1.8 in grade1; 16.3 +/- 2.0, 15.6 +/- 1.7 and 15.9 +/- 0.9 in grade 2; 14.1 +/- 2.6, 14.5 +/- 2.2 and 14.2 +/- 1.9 in grade 3 respectively. The severity of WML is associated significantly with reduction of rCBF within lesions both in centrum semiovale and periventricular regions (all P < 0.05). There was no significant difference in rCBF values between grade 0 and 1, but significant differences existed between grade 0 and grades 2 and 3, between grade 1 and grades 2 and 3 (all P < 0.05). Statistical significance also existed between the severity of white matter lesions and rCBF in bilateral temporal lobes and lentiform nucleases (P < 0.05). CONCLUSIONS: The severity of WML both in centrum semiovale and periventricular regions is associated significantly with reduction of rCBF within lesions.

Rho kinase inhibitor Fasudil induces neuroprotection and neurogenesis partially through astrocyte-derived G-CSF.

Rho-kinases (ROCK) are serine/threonine kinases that play an important role in fundamental processes of cell migration, proliferation and survival. Blockade of ROCK promotes axonal regeneration and neuroprotection, thereby exhibiting therapeutic potentials for clinical application to spinal cord damage and stroke. Here we explored the mechanisms of Fasudil, a ROCK inhibitor, in neuroprotection and neurogenesis by using oxygen-glucose deprivation (OGD) as an in vitro ischemia model. Fasudil stimulates astrocytes to produce granulocyte colony-stimulating factor (G-CSF). Astrocyte-conditioned medium treated with Fasudil (ACM-F) contributes to the generation of neurospheres, and decreases neuron death. Neutralization of G-CSF in ACM-F and blocking of G-CSF receptor in neuronal cell cultures revealed that Fasudil-induced neuroprotection and/or neurogenesis are mediated partially through astrocyte-derived G-CSF. Our results indicate that ROCK inhibition by Fasudil, protecting neurons and mobilizating neural stem cells, might represent a useful therapeutic perspective for various neurological disorders characterized by neuron death.

Carbonic anhydrase III is insufficient in muscles of myasthenia gravis patients.

Myasthenia gravis (MG) is considered as an autoimmune disease mainly mediated by antibodies against acetylcholine receptor. In recent years, other targets related to MG have been the subject of interest. Our previous research found that protein P25 was lower in muscles of MG patients using two-dimensional electrophoresis. In present study, anti-serum to P25 was prepared, immunohistochemistry and ATPase staining revealed that P25 was a muscle specific cytosolic protein and was mainly distributed in type I muscle fibers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and precise molecular weight derived from mass spectrometer identified P25 as carbonic anhydrase III (CA III). Some members of CA family are related to autoimmune diseases and CA III is recently reported to be involved in rheumatoid arthritis. The results of immunoblot in this report showed that the level of CA III is specifically insufficient in the skeletal muscle of MG patients. The possible roles that CA III play in MG need further elucidation.