PEGylation and antioxidant effects of a human glutathione peroxidase 1 mutant.

Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.

Astragalus protects PC12 cells from 6-hydroxydopamine-induced neuronal damage: A serum pharmacological study.

Accumulating evidence has already indicated that traditional Chinese medicine (TCM) possesses tremendous potential for treating neurodegenerative diseases. Astragalus, also named Huangqi, is a famous traditional medical herb that can be applied to treat cerebral ischemia and prevent neuronal degeneration. Nevertheless, the underlying mechanisms remain largely unexplored. In the present study, Astragalus-containing serum (ASMES) was prepared and added into the culture medium of PC12 cells to explore its neuroprotective effect on 6-hydroxydopamine (6-OHDA)-caused neuronal toxicity. Our data showed that ASMES significantly ameliorated the cellular viability of cultured PC12 cells against the neurotoxicity induced by 6-OHDA (P < 0.05). Moreover, ASMES significantly decreased the cell apoptosis triggered by 6-OHDA (P < 0.01). Furthermore, 2',7'-dichlorofluorescin diacetate assay was performed to detect the changes in oxidative stress, and we showed that 6-OHDA elevated the production of reactive oxygen species (ROS), whereas ASMES significantly reversed these changes (P < 0.01). Besides, mitochondrial membrane potential (MMP) assay showed that ASMES could restore 6-OHDA-damaged MMP in cultured PC12 cells (P < 0.05). In conclusion, Astragalus could protect PC12 cells from 6-OHDA-caused neuronal toxicity, and possibly, the ROS-mediated apoptotic pathway participated in this process. Collectively, our findings provided valuable insights into the potential in treatment of neurodegenerative diseases.

MRI Detectable Polymer Microspheres Embedded With Magnetic Ferrite Nanoclusters For Embolization: In Vitro And In Vivo Evaluation.

OBJECTIVE: The objective of this study was to develop magnetic embolic microspheres that could be visualized by clinical magnetic resonance imaging (MRI) scanners aiming to improve the efficiency and safety of embolotherapy. METHODS AND DISCUSSION: Magnetic ferrite nanoclusters (FNs) were synthesized with microwave-assisted solvothermal method, and their morphology, particle size, crystalline structure, magnetic properties as well as T2 relaxivity were characterized to confirm the feasibility of FNs as an MRI probe. Magnetic polymer microspheres (FNMs) were then produced by inverse suspension polymerization with FNs embedded inside. The physicochemical and mechanical properties (including morphology, particle size, infrared spectra, elasticity, etc.) of FNMs were investigated, and the magnetic properties and MRI detectable properties of FNMs were also assayed by vibrating sample magnetometer and MRI scanners. Favorable biocompatibility and long-term MRI detectability of FNMs were then studied in mice by subcutaneous injection. FNMs were further used to embolize rabbits' kidneys to evaluate the embolic property and detectability by MRI. CONCLUSION: FNMs could serve as a promising MRI-visualized embolic material for embolotherapy in the future.

A new multifunctional hydroxytyrosol-clofibrate with hypolipidemic, antioxidant, and hepatoprotective effects.

Oxidative stress has been regarded as the leading mechanism of the hepatotoxicity of clofibrate (CF). To achieve multifunctional novel hypolipidemic agents with hypolipidemia, antioxidant, and ameliorating liver injury, clofibric acid derivative hydroxytyrosol-clofibrate (CF-HT) was synthesized by molecular hybridization. CF-HT exhibited significant hypolipidemia, reducing serum triglyceride (TG), total cholesterol (TC), and malonaldehyde (MDA) by 30%, 33%, and 29% in hyperlipidemic mice induced by Triton WR 1339. CF-HT also shown hepatoprotective effect, a significant decrease in hepatic indices toxicity was observed, i.e. aspartate and lactate transaminases (AST and ALT) activities, alkalines phosphatases (ALP), and total bilirubin (TBIL) levels. The liver weight and liver coefficient were also ameliorated. Serum superoxide dismutase (SOD) was significantly elevated, and serum catalase (CAT) and malondialdehyde (MDA) content were remarkably restored. The hepatic glutathione (GSH) content was obviously increased and hepatic oxidized glutathione (GSSG) content was reduced dramatically by CF-HT, as compared to the CF treated mice (p < 0.05). Moreover, the histopathological damage that hepatocyte hyperplasia and hypertrophy was also significantly ameliorated by treatment with CF-HT. Therefore, the results indicated that CF-HT exerted more potent hypolipidemic activity and definite hepatoprotective effect which may mainly be associated with its antioxidative property in mice.

Hydroxytyrosol nicotinate, a new multifunctional hypolipidemic and hypoglycemic agent.

Hydroxytyrosol (HT) is a natural polyphenol antioxidant that exists in olive oil. In the study of multifunctional hypolipidemic of nicotinic derivatives, we found that hydroxytyrosol nicotinate (HT-N) incorporation of niacin with HT displayed ?-glucosidase inhibitory activities in vitro, such as yeast ?-glucosidase (IC50?=?117.72??M) and rat intestinal ?-glucosidases maltase (IC50?=?31.86??M) and sucrase (IC50?=?22.99??M), and had a good control of postprandial blood glucose (PBG). HT-N shown significantly hypoglycemic action by 16.9% and protection of pancreatic tissue in type 2 diabetic mellitus (T2DM) mouse model. HT-N also shown a potent antioxidant activity and property of anti-glycation in vitro, which were benefit for ameliorating diabetic complications. Moreover, HT-N exhibited much significant hypolipidemia, lowering plasma triglyceride (TG), total cholesterol (TC), and malonaldehyde (MDA) by 34.6%, 45.8% and 32.1% respectively, in hyperlipidemic mice induced by Triton WR 1339. The results indicated that HT-N has hypolipidemic, hypoglycemic and antioxidant actions. All these properties could be conducive to amelioration of oxidative stress, hyperlipidemia, and diabetes that HT-N may serve as a multifunctional potential therapeutic strategy in diabetic patients with hyperlipidemia.

Poly(acrylic acid) microspheres loaded with superparamagnetic iron oxide nanoparticles for transcatheter arterial embolization and MRI detectability: In vitro and in vivo evaluation.

To develop embolic microspheres with MRI detectability, superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized and mixed with monomer of acrylic acid to prepare SPIONs-loaded polymerized microspheres (SPMs) by inverse suspension polymerization method. The SPMs were evaluated for the ability of embolization by investigating the morphology, particle size, elasticity and renal arterial embolization to rabbits. Meanwhile, the loading of SPIONs was verified by optical microscope, transmission electron microscope, Fourier transform infrared spectrum, vibrating sample magnetometer, X-ray diffraction and X-ray photoelectron spectroscopy, and the content of SPIONs in SPMs was measured quantitatively. Furthermore, the MRI detectability of SPMs was testified in gel phantom, mice and rabbits respectively by a clinical 3.0T MRI scanner. The results revealed the SPMs were potential MRI detectable embolic microspheres for improving the effectiveness and safety of embolotherapy in the future.

Preparation and evaluation of MRI detectable poly (acrylic acid) microspheres loaded with superparamagnetic iron oxide nanoparticles for transcatheter arterial embolization.

To monitor the spatial distribution of embolic particles inside the target tissues during and after embolization, blank poly (acrylic acid) microspheres (PMs) were initially prepared by inverse suspension polymerization method and then loaded with superparamagnetic iron oxide (SPIO) nanoparticles by in situ precipitation method to obtain magnetic resonance imaging (MRI) detectable SPIO-loaded poly (acrylic acid) microspheres (SPMs). The loading of SPIO nanoparticles in SPMs was confirmed by vibrating sample magnetometer, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and infrared spectrum, respectively. The results showed that SPMs exhibited excellent superparamagnetism and the SPIO embedded in SPMs were proved to be inverse spinel magnetite. The content of SPIO loaded in wet SPMs of subgroups of 100-300, 300-500, 500-700 and 700-900µm was measured to be 11.84±0.07, 10.20±0.05, 9.98±0.00 and 8.79±0.01mg/ml, corresponding to the weight percentage in freeze-dried SPMs to be 18.07±0.28%, 18.54±0.13%, 18.66±0.01% and 18.50±0.07%, respectively. The SPMs were spherical in shape, had smooth surface, and were within the size range of clinical demands for embolization. The compression tests indicated that SPMs were more rigid than PMs and commercially used Embospheres (P<0.01). The MRI detectability of SPMs was evaluated with the SPMs embedded in gel phantom in vitro and injected subcutaneously into the back of mice in vivo. Both the results demonstrated that the SPMs could provide distinct negative contrast enhancement and be sensitively detected by T2-weighted MR imaging. All the results show that SPMs are potential MRI detectable embolic microspheres for the future embolotherapy.

Two New Armtype Polyoxometalates Grafted on Titanium Dioxide Films: Towards Enhanced Photoelectrochemical Performance.

Two new carboxyethyltin-functionalized polyoxometalates (POMs) were successfully obtained and confirmed with physicochemical and spectroscopic methods including X-ray crystallography. The lowest unoccupied molecular orbitals of both compounds are higher in energy than that of TiO2 , and the optical band gaps of these compounds are smaller than that of TiO2 . Grafting them onto a TiO2 film created two kinds of novel photoanode materials that showed significantly enhanced photovoltaic and photocurrent responses, as well as improved photoelectrooxidation activities for methanol relative to that shown by a single TiO2 film. Further, P2 W15 -Co-SnR produced the largest photocurrent by exploring the photoelectric activities of a series of carboxyethyltin POM derivatives. This work provides new insight into the photoelectrochemical functionalization of POM-based organic-inorganic hybrids.

First record of the genus Dialarnaca Gorochov, 2005 from China, with description of two new species (Orthoptera, Gryllacrididae, Gryllacridinae).

In the present paper, Dialarnaca Gorochov, 2005 is recorded from China for the first time, with two new species of the genus described, Dialarnaca longicerca Shi & Bian, sp. n. and Dialarnaca zhoui Shi & Bian, sp. n. A key and a distribution map of the genus Dialarnaca, are provided.

A new record of Larnaca Walker, 1869 (Orthoptera: Gryllacrididae: Gryllacridinae) from China with description of a new species.

In the paper, Larnaca is recorded from the Chinese fauna for the first time. A single new species, Larnaca (Larnaca) emarginata Bian, Guo & Shi, sp. nov., is described. This extends the distribution of the genus Larnaca from Lao Cai prov., Vietnam to Yunnan prov. in southwestern China.

[An exploration of induction methodology and experimental duration of Graves disease animal model].

OBJECTIVE: To compare the efficacy of Graves disease animal models induced by thyroid stimulating hormone receptor (TSHR) plasmid DNA (pcDNA3.1-TSHR) and by TSHR A subunit recombinant adenovirus (Ad-TSHR289), and to investigate the influence of duration for preparing animal model induced by Ad-TSHR289 on Graves hyperthyroidism and its related indices. METHODS: The plasmid group and the adenovirus group were set up respectively. The plasmid group: 21 female BALB/c mice were randomly divided into model group (n = 12) and control group (n = 9). The model group were injected intradermally with pcDNA3.1-TSHR 50 µg, once every 3 weeks, totally 3 times. Then 4 weeks after the last immunization, the mice were euthanized to obtain blood for testing TSHR antibody (TRAb), total T(4), and thyroid tissue for histological examination. The controls were injected with the same dose of pcDNA3.1 in the same way. The adenovirus group: 52 female BALB/c mice were divided into 10-week model group (n = 8), 14-week model group (n = 10) and 18-week model group (n = 8), and the respective controls (n = 8, n = 10, n = 8) were set up. All model groups were injected intramuscularly with Ad-TSHR289, three times at three weekly intervals. Then the mice were euthanized at 4, 8 and 12 weeks to test TRAb, total T(4) level and to observe the change of thyroid histology. The controls were treated with the same dose of Ad-lacz in the same way. Another 8 mice were scheduled to test the dynamic variation of TRAb before and after the 3 times immunization. RESULTS: In the plasmid model group, only two of 12 mice developed weak antibody responses against TSHR, and no elevated total T(4) level and no hyperplasia changes of thyroid were observed. In the 10-week model group, all mice had high level TRAb [(807.65 ± 136.33) U/L], Six-eighths mice had hyperthyroidism exhibited hyperplasia changes. In the 14-week model group, the TRAb level [(650.12 ± 192.88) U/L] and the incidence of hyperthyroidism (3/10) were lower than those in 10-week group. Histologically, the degree of thyroid hyperplasia lightened to a small extent, but its positive rate did not decline. In the 18-week model group, only 2 of 8 mice displayed slightly elevated TRAb level, and no mice showed increased total T(4) level. Additionally, thyroid tissues of 2 mice were mildly abnormal. Compared with the model groups at different time, the change of antibody levels of the mice for TRAb dynamic observation exhibited the similar trend. CONCLUSIONS: Being good at repeatability and high incidence of hyperthyroidism, the animal model of Graves disease induced by Ad-TSHR289 is still an ideal research tool presently. The duration of model can be maintained 18 weeks, and 10 weeks is the best period to sustain characteristic of Graves disease.

[Effect of overdose fluoride on the expression of enamelin in rat mandibular incisor].

OBJECTIVE: To observe the effect of overdose fluoride on the expression of enamelin in rat mandibular incisor. METHODS: Twenty Wistar rats were divided randomly into two groups. Animals were maintained in standard environment with free access to food and distilled water (control group) or water added with 100 mg/L F-(experimental group). The rats were killed in the eighth week. HE staining was used to observe the morphology of ameloblasts. Immunohistochemical staining was adopted to study the expressions of enamelin in rat incisor. RESULTS: The ameloblasts of the treated rat were arranged in multi-layer. The ameloblasts in group II were thinner than those in group I. The structure of enamel matrix was in disorder. The expressions of enamelin in ameloblasts and odontoblasts were obviously inhibited in group II (P < 0.01). CONCLUSION: The overdose fluoride inhibits the secretion of enamelin and leads to the abnormal development of enamel matrix.

[Effects of overdosed fluoride on rat's incisor expression of matrixmetalloproteinase-20 and tissue inhibitors of metalloproteinase-2].

OBJECTIVE: To investigate the effects of overdosed fluoride on the expression of MMP-20 and TIMP-2 in rat incisor. METHODS: 20 Wistar rats were randomly divided into experiment group and control group. The distilled water was given in control group. 100 mg/L fluoride- was given in experiment group. After 8 weeks treatment, the rats were killed. Immunohistochemical staining was used to observe the expression of MMP-20 and TIMP-2 in rat incisors. RESULTS: Immunohistochemical results demonstrated the presence of MMP-20 and TIMP-2 protein in ameloblasts, odontoblasts, stratum intermedium and the stellate reticulum of rat incisor. The imagination analysis results showed that the expression of MMP-20 was reduesed in experiment group (P<0.01), and the expression of TIMP-2 had no significant difference (P>0.05). CONCLUSION: The overdosed fluoride inhibits the secretion of MMP-20 and leads to the disturbed balance between MMP-20, TIMP-2 in rat incisor, which leads to the delay of the amelogenin removal and the enamel demineralization.

[Effect of overdose fluoride on expression of bone sialoprotein in developing dental tissues of rats].

PURPOSE: To investigate the changes of bone sialoprotein (BSP) in developing dental tissues of rats exposed to fluoride. METHODS: Twenty rats were randomly divided into two groups, one was with distilled water (control group), the other was with distilled water treated by fluoride (experimental group). When the fluorosis model was established, the changes of the expression of BSP were investigated and compared between the two groups. HE staining was used to observe the morphology of the cell, and immunohistochemisty assay was used to determine the expression of BSP in rat incisor. Student's t test was used for statistical analysis. RESULTS: The ameloblasts had normal morphology and arranged orderly. Immunoreactivitis of BSP was present in matured ameloblasts, dentinoblasts, cementoblasts, and the matrix in the control group. But in the experimental group the ameloblasts arranged in multiple layers, the enamel matrix was confused and the expression of BSP was significantly lower than that of the control group. Statistical analysis showed significant differences between the two groups (P<0.01). CONCLUSION: Fluoride can inhibit the expression of BSP in developing dental tissues of rats, and then inhibit differentiation of the tooth epithelial cells and secretion of matrix. This is a probable intracellular mechanism of dental fluorosis.

Expression of Nerve Growth Factor from Agkistrodon halys Pallas in Silkworm Larvae.

The cDNA encoding nerve growth factor (NGF) precursor from Agkistrodon halys Pallas (a Chinese snake species) was cloned into pBacPAK8 a baculovirus shuttle vector. The recombinant shuttle vector pBacPAK-NGF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. The SDS-PAGE and the NGF activity by bioassay of PC12 cells have shown a high expression level of NGF of good biological activity in the larvae.

Isolation and Characterization of a Neurotrophic Factor-like Substance from Venom of Agkistrodon acutus.

A neurotrophic factor-like substance from Agkistrodon acutus was isolated by ion exchange and gel filtration chromatography and was found to be homogenous by electrophoresis. Itsmolecular weight was estimated to be approximately 26 kD by gel filtration. A characteristic of the substance was that the protein consisted of two subunits which were bound one to another noncovalently. The analytical isoelectric focusing revealed its isoelectric point of 7.8. The biological activity of the substance was comparable to that of mouse 2.5 S NGF. It rescued PC12 cells from apoptosis at the concentration interval of 1 100 &mgr;g/L and could induce PC12 cells differentiate to sympathetic-like neurons.