CTL-Derived Granzyme B Participates in Hippocampal Neuronal Apoptosis Induced by Cardiac Arrest and Resuscitation in Rats.

Hippocampal neuronal apoptosis is a devastating consequence of cardiac arrest (CA) and subsequent cardiopulmonary resuscitation (CPR). In this study, we assessed the contribution of cytotoxic T lymphocyte (CTL)-derived toxic mediator granzyme B (Gra-b) to the hippocampal neuronal apoptosis following CA/CPR in rats. Rats that experienced CA/CPA presented with cytosomal shrinkage, dense cytoplasm, and intensive eosinophilic staining in the CA1 region of dorsal hippocampus. CA/CPR rats also exhibited inability in spatial navigation and a local infiltration of peripheral CD8+ T cells into the hippocampus. The protein levels of Gra-b, cleaved Caspase-3, and cleaved PARP1 were significantly elevated in rats undergoing CA/CPR. Pretreatment with Gra-b inhibitor suppressed Gra-b release, attenuated hippocampal neuronal apoptosis, as well as improved cognitive impairment. Together, this study indicates that CTL-derived Gra-b is involved in the CA/CPR-induced neuronal apoptosis, and pharmacological manipulation of Gra-b may represent a novel avenue for the treatment of brain injury following CA/CPR.

Diversity and Dynamics of Microbial Community Structure in Different Mangrove, Marine and Freshwater Sediments During Anaerobic Debromination of PBDEs.

Little is known about the diversity and succession of indigenous microbial community during debromination of polybrominated diphenyl ethers (PBDEs). This study examined the diversity and dynamics of microbial community structure in eight saline (mangrove and marine) and freshwater sediment microcosms exhibiting different debrominating capabilities for hexa-BDE 153, a common congener in sediments, using terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses. The results showed that microbial community structure greatly differed between the saline and freshwater microcosms, likely leading to distinct variations in their debrominating capabilities and pathways. Higher relative abundances of Chloroflexi and Deltaproteobacteria succeed by Alphaproteobacteria and Betaproteobacteria were detected in the two mangrove microcosms with the fastest debrominating capabilities mainly via para pathway, respectively; the dominance of Alphaproteobacteria resulted in less accumulation of tetra-BDEs and more complete debromination of lower brominated congeners (from di- to tetra-BDEs). Meanwhile, the shifts in both microbial community structure and PBDE profiles were relatively small in the less efficient freshwater microcosms, with relatively more ortho and meta brominated products of BDE-153 resulted. Coincidently, one of the freshwater microcosms showed sudden increases of Chloroflexi and Deltaproteobacteria by the end of incubation, which synchronized with the increase in the removal rate of BDE-153. The significant relationship between microbial community structure and PBDEs was confirmed by redundancy analysis (18.7% of total variance explained, P = 0.002). However, the relative abundance of the well-known dechlorinator Dehalococcoides showed no clear correlation with the debrominating capability across different microcosms. These findings shed light in the significance of microbial community network in different saline environments on enhancement of PBDE intrinsic debromination.

The uptake of mixed PAHs and PBDEs in wastewater by mangrove plants under different tidal flushing regimes.

Wastewater often contains mixed toxic pollutants, and the contribution of plant uptake in constructed wetland treatment systems is affected by environmental conditions, particularly tidal flushing. In this study, the uptake of wastewater-borne pollutants, including a mixture of polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenyl ethers (PBDEs) congeners, by two mangrove plant species, namely Excoecaria agallocha L. and Kandelia obovata Sheue, Liu & Yong, under different tidal flushing regimes was investigated. Results showed that Fe plaque formed on root surfaces could immobilize wastewater-borne PAHs and PBDEs. At the end of 8-month wastewater treatment, most of the pollutants removed by plants ended up in Fe plaque, with 0.12-20.83% of total PAHs and 0.78-24.76% of total PBDEs added to the microcosm retained in Fe plaque. On the contrary, the percentages of PAHs and PBDEs taken up by plant tissues were relatively small, ranging from not detected to 0.09% and from 0.01 to 2.00%, respectively. More uptake of Fe plaque-immobilized PAHs and PBDEs was found in K. obovata than in E. agallocha, leading to more plant damages in the former species due to its weaker root outer layers. While E. agallocha with stronger root protective outer layer was able to uptake more PAHs and PBDEs from wastewater but immobilize in Fe plaque than that of K. obovata. In both plant species, tidal flushing regimes significantly affected the immobilization of PAHs and PBDEs in Fe plaque, and more frequent tidal flushing led to higher percentages of immobilization. This is the first study demonstrating that E. agallocha was a more suitable mangrove plant species to remove wastewater-borne PAHs and PBDEs than K. obovata, and the significance of tidal flushing on performance of constructed mangrove wetlands.

Bioactivity, pharmacokinetics, and immunogenicity assays in preclinical and clinical trials for recombinant human endostatin.

AIM: To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients. METHODS: The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People's Republic of China (2005 edition, part III). Its in vitro and in vivo bioactivities were assayed via proliferation-inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method. RESULTS: The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4 x 10(7), 6.7 x 10(7), and 3.8 x 10(8) U/mg, and the in vivo antitumoral potency was 4.04 x 10(7) U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase I clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44+/-91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients. CONCLUSION: RhEndostatin exhibited a definite proliferation- inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEndostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.

Adsorption and cosorption of tetracycline and copper(II) on montmorillonite as affected by solution pH.

Land application of wastes generated from concentrated animal feeding operations may result in accumulation of tetracyclines (TCs) and metals in agricultural soils. Adsorption of TCs and metals on soil minerals strongly affects their mobility. This study was conducted to evaluate the interaction between tetracycline (TC) and Cu(ll) with regard to their adsorption and cosorption on montmorillonite as affected by solution pH. When solution pH was below 6.5, the presence of TC increased Cu(ll) adsorption on montmorillonite, which could be due to increasing Cu(II) adsorption via the TC bridge, or due to the stronger affinity of TC-Cu(II) complex to the mineral than Cu2+ ion itself. Zeta potential of the montmorillonite significantly decreased after the adsorption of TC, suggesting a strong interaction between TC and montmorillonite. Addition of Cu(ll) ions increased TC adsorption on the mineral in a wide range of pH. The experimental data were well fit with the weighted sum model. The complexes of TC and Cu(II) (CuH2L(2+), CuHL+, and CuL) had higher sorption coefficients (K(d)) than that of the corresponding TC species (H3L+, H2L, and HL-). Increasing adsorption of TC and Cu(II) on montmorillonite as they coexist in the normal pH environment may thus reduce their mobility.

Zinc adsorption on goethite as affected by glyphosate.

Cosorption of metals with herbicides on minerals affects their mobility and their environmental effect. Batch experiments were conducted to evaluate the interaction between Zn and glyphosate [N-(phosphonomethyl)glycine (GPS; H3L)] with regard to the effect of GPS on Zn adsorption on goethite. The herbicide GPS markedly affected Zn adsorption on goethite when they coexisted in a goethite suspension. When solution pH was not intentionally adjusted, addition of GPS decreased Zn adsorption on goethite, since the equilibrium solution pH was significantly decreased in the presence of GPS and correspondingly the negative surface charges of goethite decreased. Zinc adsorption on goethite in the presence and absence of GPS at different pH of the equilibrium solution was studied in order to know if pH was the only variable for Zn adsorption with coexisting GPS. At lower pH (pH<5), the presence of GPS increased the adsorption of Zn, because Zn adsorbed on the sites of goethite via GPS bridge. However, at higher pH (pH>5), the presence of GPS decreased the adsorption of Zn on goethite, because GPS reacted with solution Zn to form water-soluble complexes that had lower affinity to the goethite surface in comparison with Zn itself. Zeta potential of goethite significantly decreased after adsorption of GPS, suggesting a chemical bond occurred between GPS and the mineral. FTIRs also show that GPS adsorbs on goethite by coordinating through caboxylate group.

A quantitative method for measuring the antitumor potency of recombinant human endostatin in vivo.

Recombinant human endostatin (rhEndostatin) has been shown to inhibit tumor growth, but the variable antitumor activity of different rhEndostatin preparations has necessitated the development of an accurate, reproducible in vivo bioassay for evaluating the rhEndostatin activity. To assess the in vivo antitumor efficacy of rhEndostatin, H22 tumor-bearing mice received three doses of rhEndostatin and the potency of rhEndostatin preparations in inhibiting tumor growth was determined by ED(50)-potency assay and validated by dose-response parallel-line assay. There was a consistent and highly reproducible linear regression relationship between rhEndostatin dosage and tumor growth inhibition rate. The ED(50) values were determined from dose-response regression lines for seven rhEndostatin preparations with high reproducibility. On the basis of the current study, the potency of rhEndostatin preparations was assigned a value of 6.09 x 10(5) U/ampoule and a 95% confidence limit of 5.96 x 10(5)-6.22 x 10(5). We consider that this procedure can be served as a potential candidate pharmacopoeial method for potency measurement of different rhEndostatin preparations.

The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin.

In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.