Inhibiting von Hippel‒Lindau protein-mediated Dishevelled ubiquitination protects against experimental parkinsonism.

Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von Hippel‒Lindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.

Enhanced nuclear gene delivery via integrating and streamlining intracellular pathway.

The essential challenge of gene therapy is to develop safe and efficient vectors that escort genes to target sites. However, due to the cumbersome workflow of gene transfection into cells, successive gene loss occurs. This leads to considerable reductions in nuclear gene uptake, eventually causing low gene expression. Herein, we designed a gene vector named CA3S2 (C: N,N'-cystamine-bis-acrylamide [CBA], A: agmatine dihydrochloride [Agm], S: 4-(2-aminoethyl) benzenesulfonamide [ABS]) with excellent gene transfection ability. This vector can promote gene delivery to the nucleus via enhanced endoplasmic reticulum (ER) targeting through integrating and streamlining of the complex intracellular pathway. Briefly, ABS endowed CA3S2/DNA nanoparticles with not only a natural ER-targeting tendency attributed to the caveolae-mediated pathway but also direct receptor-binding capacity on the ER surface. Agm enabled CA3S2 to enhance lysosomal escape and nuclear uptake ability. The gene delivery efficiency of CA3S2 was significantly better than that of polyethyleneimine 25K (PEI 25K). Therefore, CA3S2 is a promising gene carrier, and the ER-targeting strategy involving intracellular pathway integration and streamlining has potential for gene therapy.

Monocyte-derived multipotent cell delivered programmed therapeutics to reverse idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal disease. However, IPF treatment has been limited by the low drug delivery efficiency to lungs and dysfunctional "injured" type II alveolar epithelial cell (AEC II). Here, we present surface-engineered nanoparticles (PER NPs) loading astaxanthin (AST) and trametinib (TRA) adhered to monocyte-derived multipotent cell (MOMC) forming programmed therapeutics (MOMC/PER). Specifically, the cell surface is designed to backpack plenty of PER NPs that reach directly to the lungs due to the homing characteristic of the MOMC and released PER NPs retarget injured AEC II after responding to the matrix metalloproteinase-2 (MMP-2) in IPF tissues. Then, released AST can enhance synergetic effect of TRA for inhibiting myofibroblast activation, and MOMC can also repair injured AEC II to promote damaged lung regeneration. Our findings provide proof of concept for developing a strategy for cell-mediated lung-targeted delivery platform carrying dual combined therapies to reverse IPF.

Fibroblast growth factor 21 ameliorates neurodegeneration in rat and cellular models of Alzheimer's disease.

Our understanding of the mechanisms underlying process in Alzheimer's disease (AD) is far from completion and new therapeutic targets are urgently needed. Recently, the link between dementia and diabetes mellitus (DM) prompted us to search for new therapeutic strategies from glucose metabolism regulators for neurodegeneration. Previous studies have indicated that fibroblast growth factor 21 (FGF21), an attractive and potential therapeutic treatment for DM, may exert diverse effects in the central nervous system. However, the specific biological function and mechanisms of FGF21 on AD is still largely unknown. We report here a study in vivo and in vitro of the neuroprotective effects of FGF21 on cell apoptosis, tau hyperphosphorylation and oxidative stress induced by amyloid ß-peptide 25-35. In the present study, the results also further provided evidence for molecular mechanisms by which FGF21 exerted its beneficial effects in neuron and suggested that the regulation of protein phosphatase 2A / mitogen-activated protein kinases / hypoxia-inducible factor-1α pathway may play a key role in mediating the neuroprotective effects of FGF21 against AD-like pathologies.

Evidence of metabolic memory-induced neurodegeneration and the therapeutic effects of glucagon-like peptide-1 receptor agonists via Forkhead box class O.

Metabolic memory, which refers to diabetic stresses that persist after glucose normalization, is considered a major factor in addition to hyperglycaemia for diabetes complications, including dementia. We previously reported that glucagon-like peptide-1 receptor agonist (GLP-1RA) alleviated neuronal injury in diabetes-related dementia models. However, our understanding of the effects and mechanisms of GLP-1RA on metabolic memory-induced neurodegeneration are limited. The present study mainly focuses on the mechanisms of action of GLP-1RA on metabolic memory-induced neurotoxicity in vivo and in vitro. Thus, in this study, aiming at mimicking metabolic memory phenomena, in vivo and in vitro models were exposed to high glucose first and then normal glucose. We also used advanced glycation end products, which are key metabolic memory-related factors, to induce neuronal injury in vitro. Based on the models, here, we report that GLP-1RA alleviated neurodegeneration in db/db mice with normalized blood glucose levels controlled with metformin and neuronal damage induced by high glucose treatment followed by withdrawal. GLP-1RA ameliorated metabolic memory-induced amyloid-ß and tau pathologies in vivo and in vitro. Furthermore, the data suggested that GLP-1RA can protect neurons against metabolic memory via Forkhead box class O (FoxO) pathways, and silent information regulator 2 homolog 1-dependent deacetylation and protein kinase B-dependent phosphorylation of FoxO1 were involved in the mechanisms underlying protective effects. This study provides evidence of the beneficial effects of GLP-1RA on neuronal cell metabolic memory, as well as GLP-1 analogues and metformin combination therapy efficiency on cognitive impairment.

The novel α-glucan YCP improves the survival rates and symptoms in septic mice by regulating myeloid-derived suppressor cells.

Sepsis is a life-threatening health condition that is initially characterized by uncontrolled inflammation, followed by the development of persistent immunosuppression. YCP is a novel α-glucan purified from the mycelium of the marine fungus Phoma herbarum YS4108, which has displayed strong antitumor activity via enhancing host immune responses. In this study, we investigated whether YCP could influence the development of sepsis in a mouse model. Caecal ligation and puncture (CLP)-induced sepsis was established in mice that were treated with YCP (20 mg/kg, ip or iv) 2 h before, 4 and 24 h after the CLP procedure, and then every other day. YCP administration greatly improved the survival rate (from 39% to 72% on d 10 post-CLP) and ameliorated disease symptoms in the septic mice. Furthermore, YCP administration significantly decreased the percentage of myeloid-derived suppressor cells (MDSCs) in the lungs and livers, which were dramatically elevated during sepsis. In cultured BM-derived cells, addition of YCP (30, 100 µg/mL) significantly decreased the expansion of MDSCs; YCP dose-dependently decreased the phosphorylation of STAT3 and increased the expression of interferon regulatory factor-8 (IRF-8). When BM-derived MDSCs were co-cultured with T cells, YCP dose-dependently increased the production of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS), and activated the NF-κB pathway. In addition, the effects of YCP on MDSCs appeared to be dependent on toll-like receptor (TLR) 4. These results reveal that YCP inhibits the expansion of MDSCs via STAT3 while enhancing their immunosuppressive function, partially through NF-κB. Our findings suggest that YCP protects mice against sepsis by regulating MDSCs. Thus, YCP may be a potential therapeutic agent for sepsis.

Glycoengineering of pertuzumab and its impact on the pharmacokinetic/pharmacodynamic properties.

Pertuzumab is an antihuman HER2 antibody developed for HER2 positive breast cancer. Glycosylation profiles are always the important issue for antibody based therapy. Previous findings have suggested the impact of glycosylation profiles on the function of antibodies, like pharmacodynamics, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, the roles of fucose and sialic acid in the function of therapeutic antibodies still need further investigation, especially the role of sialic acid in nonfucosylated antibodies. This study focused on the pharmacokinetic and pharmacodynamic properties of pertuzumab after glycoengineering. Herein, nonfucosylated pertuzumab was produced in CHOFUT8-/- cells, and desialylated pertuzumab was generated by enzymatic hydrolysis. Present data indicated that fucose was critical for ADCC activity by influencing the interaction between pertuzumab and FcγRIIIa, nevertheless removal of sialic acid increased the ADCC and CDC activity of pertuzumab. Meanwhile, regarding to sialic acid, sialidase hydrolysis directly resulted in asialoglycoprotein receptors (ASGPRs) dependent clearance in hepatic cells in vitro. The pharmacokinetic assay revealed that co-injection of asialofetuin can protect desialylated pertuzumab against ASGPRs-mediated clearance. Taken together, the present study elucidated the importance of fucose and sialic acid for pertuzumab, and also provided further understanding of the relationship of glycosylation/pharmacokinetics/pharmacodynamics of therapeutic antibody.

Establishment of a fluorescence-based method to evaluate endocytosis of desialylated glycoproteins in vitro.

Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins.

Inhibiting receptor for advanced glycation end product (AGE) and oxidative stress involved in the protective effect mediated by glucagon-like peptide-1 receptor on AGE induced neuronal apoptosis.

Our previous study has demonstrated that glucagon-like peptide-1 (GLP-1) receptor agonist could protect neurons from advanced glycation end products (AGEs) toxicity in vitro. However, further studies are still needed to clarify the molecular mechanism of this GLP-1 receptor -dependent action. The present study mainly focused on the effect of GLP-1 receptor agonists against the receptor for advanced glycation end products (RAGE) signal pathway and the mechanism underlying this effect of GLP-1. Firstly the data based on the SH-GLP-1R(+) and SH-SY5Y cells confirmed our previous finding that GLP-1 receptor could mediate the protective effect against AGEs. The assays of the protein activity and of the mRNA level revealed that apoptosis-related proteins such as caspase-3, caspase-9, Bax and Bcl-2 were involved. Additionally, we found that both GLP-1 and exendin-4 could reduce AGEs-induced reactive oxygen species (ROS) accumulation by suppressing the activity of nicotinamide adenine dinucleotide phosphate-oxidase. Interestingly, we also found that GLP-1 receptor activation could attenuate the abnormal expression of the RAGE in vitro and in vivo. Furthermore, based on the analysis of the protein expression and translocation level of transcription factor nuclear factor-κB (NF-κB), and the use of GLP-1 receptor antagonist exendin(9-39) and NF-κB inhibitor pyrrolidine dithiocarbamate, we found that the effect mediated by GLP-1 receptor could alleviate the over expression of RAGE induced by ligand via the suppression of NF-κB. In summary, the results indicated that inhibiting RAGE/oxidative stress was involved in the protective effect of GLP-1 on neuron cells against AGEs induced apoptosis.

Immunomodulatory effects of polysaccharide from marine fungus Phoma herbarum YS4108 on T cells and dendritic cells.

YCP, as a kind of natural polysaccharides from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune response, but little is known about the molecular mechanisms. In the present study, we mainly focused on the effects and mechanisms of YCP on the specific immunity mediated by dendritic cells (DCs) and T cells. T cell /DC activation-related factors including interferon- (IFN-) γ, interleukin-12 (IL-12), and IL-4 were examined with ELISA. Receptor knock-out mice and fluorescence-activated cell sorting are used to analyze the YCP-binding receptor of T cells and DCs. RT-PCR is utilized to measure MAGE-A3 for analyzing the tumor-specific killing effect. In our study, we demonstrated YCP can provide the second signal for T cell activation, proliferation, and IFN-γ production through binding to toll-like receptor- (TLR-) 2 and TLR-4. YCP could effectively promote IL-12 secretion and expression of markers (CD80, CD86, and MHC II) via TLR-4 on DCs. Antigen-specific immunity against mouse melanoma cells was strengthened through the activation of T cells and the enhancement of capacity of DCs by YCP. The data supported that YCP can exhibit specific immunomodulatory capacity mediated by T cells and DCs.

Preparation, optimization and application of affinity absorbent with a polysaccharide YCP as the ligand.

YCP, an α-glucan from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9 (3)(4) was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate-protein interaction.

Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage migration in vitro via the NF-κB pathway.

AIM: To investigate the effects of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 on oxidized low-density lipoprotein (ox-LDL)-induced inhibition of macrophage migration and the mechanisms underlying the effects of exendin-4. METHODS: Primary peritoneal macrophages were extracted from the peritoneal cavity of mice treated with 3% thioglycollate (2 mL, ip). Migration of the macrophages was examined using a cell migration assay. Macrophage migration-related factors including leptin-like ox-LDL receptor (LOX-1), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin-1 (IL-1)ß, matrix metalloproteinase-2 (MMP-2), intercellular adhesion molecule (ICAM)-1 and macrophage migration inhibitory factor (MIF) were measured using semi-quantitative RT-PCR. Expression of MIF and ICAM-1 proteins was examined with ELISA. Gelatin zymography was used to evaluate the activity of MMP-9. Activation of the NF-κB pathway was determined by confocal laser scanning microscopy. RESULTS: Treatment of the macrophages with ox-LDL (50 µg/mL) markedly suppressed the macrophage migration. Furthermore, ox-LDL treatment substantially increased the expression of the macrophage migration-related factors, the activity of MMP-9 and the translocation of the NF-κB p65 subunit. These effects of ox-LDL were significantly ameliorated by pretreatment with the specific NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (100 µmol/L). These effects of ox-LDL were also significantly ameliorated by pretreatment with exendin-4 (25 and 50 nmol/L). CONCLUSION: Exendin-4 ameliorates the inhibition of ox-LDL on macrophage migration in vitro, via suppressing ox-LDL-induced expression of ICAM-1 and MIF, which is probably mediated by the NF-κB pathway.

Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry.

DNA methylation is one of the major epigenetic modifications and has been involved in a number of biological processes in mammalian cells. Yeast is widely used as a model organism for studying cell metabolism, cell cycle regulation, and signal transduction. However, it remains controversial whether methylated cytosine (5-methylcytosine, 5mC) exists in the yeast genome. In the current study, we developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of 5mC in 19 yeast strains, which represent 16 yeast species. Our results showed that DNA methylation is widespread in yeast and the genome-wide DNA methylation of the studied yeast strains ranged from 0.014 to 0.364%, which were 1 to 2 orders of magnitude lower than that in mammalian cells (i.e., 3-8%). Furthermore, we found that the 5mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor 5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells. The demonstration of the universal presence of DNA cytosine methylation in yeast constituted the first and essential step toward understanding the functions of this methylation in yeast.

[Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS].

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.

[Prognostic value of circulating catestatin levels for in-hospital heart failure in patients with acute myocardial infarction].

OBJECTIVE: To determine whether circulating level of catestatin (CST) could provide prognostic information independently of conventional risk markers for the development of in-hospital heart failure in patients with ST-segment elevation myocardial infarction (STEMI). METHODS: The data of 120 STEMI patients (mean age: 61 years, 73% male) were collected from the Second Hospital of Shanxi Medical University and Taiyuan Central Hospital between November 2010 and September 2011.The patients were categorized into 4 groups according to CST (ng/L) quartile: ≤ 74.72, 74.73-79.67, 79.68 - 84.21 and ≥ 84.22 ng/L. Clinical features, therapeutic approaches were compared among groups. The patients were also grouped according to Killip class: Killip level I (n = 68), Killip level II (n = 23), Killip level III (n = 18), Killip level IV (n = 11). CST, NE and NT-proBNP were compared among groups. The Spearma rank correlation and multivariate logistic regression analysis were applied to determine the association between risk factors and in-hospital heart failure. Receiver-operator characteristic (ROC) curve was performed to evaluate the power of CST and NT-proBNP on predicting in-hospital heart failure. RESULTS: Gender, hospital days, past history of smoking, hypertension, myocardial infarction, CK-MB peak level, TnI peak level, heart rate, blood pressure, blood glucose, blood lipid levels on admission and early reperfusion therapy were similar among groups. Patients with higher CST values were more likely to be older, to have lower body mass index, to have higher white blood cell count, CysC, hs-CRP, NE, NT-proBNP, past history of angina, diabetes mellitus, being diuretic users, and to have a lower ejection fraction (all P < 0.05). Higher CST levels were also associated with increased risk of heart failure (P < 0.05). In proportion with the deterioration of the cardiac function, CST, NE, NT-proBNP concentration gradually increased (all P < 0.05). Spearman rank correlation analysis showed that the CST was negatively correlated with LVEF (r(s) = -0.923, P < 0.001) and positively correlated with NT-proBNP (r(s) = 0.884, P < 0.001). After multivariate adjustment, CST remained to be an independent risk factor for the development of in-hospital heart failure (OR = 1.125, 95%CI: 1.056 - 1.198;P < 0.001). The area under the ROC curve of CST and NT-proBNP was 0.777 and 0.874. Using CST = 77.29 ng/L as a cut-off value, the sensitivity was 92.8% and specificity was 70.6% for predicting the development of in-hospital heart failure. CONCLUSION: The plasma CST level is an independent predictor for the development of in-hospital heart failure in patients with STEMI.

Amelioration of neurodegenerative changes in cellular and rat models of diabetes-related Alzheimer's disease by exendin-4.

Growing evidence suggests that type 2 diabetes mellitus (DM) is associated with age-dependent Alzheimer's disease (AD), the latter of which has even been considered as type 3 diabetes. Several physiopathological features including hyperglycemia, oxidative stress, and dysfunctional insulin signaling relate DM to AD. In this study, high glucose-, oxidative stress-induced neuronal injury and intracerebroventricular-streptozotocin (ICV-STZ) animals as the possible models for diabetes-related AD were employed to investigate the effects of exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, on diabetes-associated Alzheimer-like changes as well as the molecular mechanisms involved. Our study demonstrated that GLP-1/Ex-4 could exert a protective effect against reduced viability of PC12 cells caused by high glucose and that this protective effect was mediated via the PI3-kinase pathway. In addition, GLP-1/Ex-4 ameliorated oxidative stress-induced injury in PC12 cells. In rat models, bilateral ICV-STZ administration was used to produce impaired insulin signaling in the brain. Fourteen days following ICV-STZ injection, rats treated with twice-daily Ex-4 had better learning and memory performance in the Morris water maze test compared with rats treated with saline. Additionally, histopathological evaluation confirmed the protective effects of Ex-4 treatment on hippocampal neurons against degeneration. Furthermore, we demonstrated that Ex-4 reversed ICV-STZ-induced tau hyperphosphorylation through downregulation of GSK-3ß activity, a key kinase in both DM and AD. Our findings suggests that Ex-4 can protect neurons from diabetes-associated glucose metabolic dysregulation insults in vitro and from ICV-STZ insult in vivo, and that Ex-4 may prove of therapeutic value in the treatment of AD especially DM-related AD.

[Quality of life among patients receiving chemotherapy in the outpatient departments of a general hospital and its correlation with pain].

OBJECTIVE: To analyze the relationship of quality of life and pain in patients receiving chemotherapy in the outpatient departments in a general hospital. METHOD: A questionnaire-based survey was conducted to collect data on pain status, sleep disorder,vomiting,constipation as well as the social function status. RESULTS: Among the 428 patient-times of survey, 12.2% of the patients had moderate to severe pain,of which 78.8% was consistent pain. The occurrence of pain was closely related to the metastasis of cancer (P = 0.000). The intensity and location of pain were independently correlated to the origin of cancer (P = 0.035, P = 0.000). Nearly a quarter of patients (24.5%) agreed that talking with his/her family was helpful to relieve pain, while 17.3% of patients had ambulatory restriction or changes in social function. The changes in social function were closely related with sleeping disorder, constipation, fatigue, and intensity of pain (all ,0.05). Furthermore, sleeping disorder was related to pain intensity (P=0.000), while constipation had no relationship with analgesic medicines (P = 0.798). CONCLUSIONS: A significantly large proportion of patients who are receiving chemotherapies in the outpatient departments of general hospital are suffering from moderate to severe pain. Their decreased social functions correlate with the cancer-related somatic symptoms.

[Cerebral lymphatic blockage aggravates apoptosis of cortical neurons after subarachnoid hemorrhage in rats in vivo].

This present study was performed to investigate the influence of cerebral lymphatic blockage (CLB) on apoptosis of cortical neurons after subarachnoid hemorrhage (SAH) in rats in vivo. Healthy adult Wistar rats were randomly divided into normal control group, SAH group and SAH+CLB group. SAH model was made by double injection of autologous blood into the cisterna magna. On the third day after the second cisternal injection, morphological changes of cortical cells were observed by hematoxylin-eosin (HE) combined with propidium iodide (PI) staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was applied to determine in situ apoptosis in the cerebral cortex. Immunohistochemistry was conducted to detect the expression of Caspase-3 and Bcl-2 in cortical neurons. HE and PI staining showed that cortical neurons of SAH rats were partly shrinkable; the nuclei showed wavy, folded or wrinkled appearance, and some nuclei had the shape of crescent. The cortical neurons in SAH+CLB group distributed sparsely and the nuclear fragmentation, apoptotic bodies were found, surrounded by the formation of vacuoles. The numbers of TUNEL-positive cells in SAH group and SAH+CLB group were higher than that in the normal control group, while the number in SAH+CLB group was significantly higher than that in the SAH group. Caspase-3 expressions in SAH group and SAH+CLB group were higher than that in the normal control group, while the expression in SAH+CLB group was significantly higher than that in the SAH group. Bcl-2 expressions in SAH group and SAH+CLB group were higher than that in the normal control group, while the expression in the SAH+CLB group was significantly lower than that in SAH group. The results obtained suggest that CLB exacerbates the apoptosis of cortical neurons in rats after SAH by up-regulating Caspase-3 expression and down-regulating Bcl-2 expression.

Quantitative determination of pegylated consensus interferon in rhesus monkey serum using a competitive enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay (ELISA) with generated mouse anti-consensus interferon (CIFN) antibody was developed for quantitative determination of pegylated consensus interferon (PEG-CIFN) in rhesus monkey serum. The operational concentrations of the original assay were determined using the chessboard method. ELISA working range was 10-5000 ng/ml, corresponding to a limit of quantification of 8.4 ng/ml in rhesus monkey serum. In a precision test, intra-assay CV ranged 1.8-9.6% and inter-assay CV ranged 3.5-12.7%. Relative recovery rate of this ELISA assay ranged from 102.65-115.77%, with RSD values ranging from 2.26-5.44%. Three groups of rhesus monkeys received 1250 microg/kg, 300 microg/kg, 150 microg/kg PEG-CIFN by subcutaneous administration, and blood samples were drawn via the femoral vein at the specified time points. PEG-CIFN in rhesus monkey serum was determined using the competitive ELISA, and the results were compared with antiviral activity assay. In conclusion, the competitive ELISA assay we developed has sufficient sensitivity, precision, and accuracy for the analysis of a rhesus monkey serum sample.

Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108.

AIM: YCP, a novel (1,4)-alpha-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation. METHODS: Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production. RESULTS: YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation. CONCLUSION: We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.Acta Pharmacologica Sinica (2009) 30: 1008-1014; doi: 10.1038/aps.2009.93.