苦参素对肾间质纤维化大鼠单核巨噬细胞浸润及MCP-1表达的影响

目的 观察苦参素对肾间质纤维化大鼠单核巨噬细胞（MC/MP）浸润,MCP-1及Ⅰ型胶原表达的影响。方法 大鼠行单侧输尿管结扎（UUO）建立肾小管间质纤维化模型。实验分为3组：假手术组,UUO组,苦参素治疗组。治疗组在UUO的基础上每天以苦参素100mg/Kg腹腔注射。各组于术后第14d分别处死5只大鼠。用PAS及Masson染色法观察肾脏病理改变。用免疫组织化学法观察肾间质ED-1阳性的MC/MP细胞浸润,单核细胞趋化蛋白-1（MCP-1）及Ⅰ型胶原（ColⅠ）的表达。结果 苦参素治疗组肾间质MC/MP细胞浸润数及MCP-1,ColⅠ的表达显著低于UUO组,肾小管变性和肾间质纤维化的程度也明显减轻。结论 苦参素可下调UUO大鼠肾间质MCP-1的表达,减少MC/MP细胞浸润,减轻肾间质纤维化。     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.     

Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase

The OspF family of phosphothreonine lyase, including SpvC from Salmonella, irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination. We determined crystal structures of SpvC and its complex with a phosphopeptide substrate. SpvC adopts a unique fold of alpha/beta type. The disordered N terminus harbors a canonical D motif for MAPK substrate docking. The enzyme-substrate complex structure indicates that recognition of the phosphotyrosine followed by insertion of the threonine phosphate into an arginine pocket places the phosphothreonine into the enzyme active site. This requires the conformational flexibility of pT-X-pY, which suggests that p38 (pT-G-pY) is likely the preferred physiological substrate. Structure-based biochemical and enzymatic analysis allows us to propose a general acid/base mechanism for beta elimination reaction catalyzed by the phosphothreonine lyase. The mechanism described here provides a structural understanding of MAPK inactivation by a family of pathogenic effectors conserved in plant and animal systems and may also open a new route for biological catalysis.     

Effect of temperature on tether extraction, surface protrusion, and cortical tension of human neutrophils

Neutrophil rolling on endothelial cells, the initial stage of its migrational journey to a site of inflammation, is facilitated by tether extraction and surface protrusion. Both phenomena have been studied extensively at room temperature, which is considerably lower than human body temperature. It is known that temperature greatly affects cellular mechanical properties such as viscosity. Therefore, we carried out tether extraction, surface protrusion, and cortical tension experiments at 37 degrees C with the micropipette aspiration technique. The experimental temperature was elevated using a custom-designed microscope chamber for the micropipette aspiration technique. To evaluate the constant temperature assumption in our experiments, the temperature distribution in the whole chamber was computed with finite element simulation. Our simulation results showed that temperature variation around the location where our experiments were performed was less than 0.2 degrees C. For tether extraction at 37 degrees C, the threshold force required to pull a tether (40 pN) was not statistically different from the value at room temperature (51 pN), whereas the effective viscosity (0.75 pN.s/microm) decreased significantly from the value at room temperature (1.5 pN.s/microm). Surface protrusion, which was modeled as a linear deformation, had a slightly smaller spring constant at 37 degrees C (40 pN/microm) than it did at room temperature (56 pN/microm). However, the cortical tension at 37 degrees C (5.7+/-2.2 pN/microm) was substantially smaller than that at room temperature (23+/-8 pN/microm). These data clearly suggest that neutrophils roll differently at body temperature than they do at room temperature by having distinct mechanical responses to shear stress of blood flow.     

Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50 °C with Thermoanaerobacterium saccharolyticum ALK2

Studies were undertaken to understand phenomena operative during simultaneous saccharification and fermentation (SSF) of a model cellulosic substrate (Avicel) at 50°C with enzymatic hydrolysis mediated by a commercial cellulase preparation (Spezyme CP) and fermentation by a thermophilic bacterium engineered to produce ethanol at high yield, Thermoanaerobacterium saccharolyticum ALK2. Thermal inactivation at 50 °C, as shown by the loss of 50% of enzyme activity over 4 days in the absence of ethanol, was more severe than at 37 °C, where only 25% of enzyme activity was lost. In addition, at 50 °C ethanol more strongly influenced enzyme stability. Enzyme activity was moderately stabilized between ethanol concentrations of 0 and 40 g/L, but ethanol concentrations above 40 g/L accelerated enzyme inactivation, leading to 75% loss of enzymatic activity in 80 g/L ethanol after 4 days. At 37 °C, ethanol did not show a strong effect on the rate of enzyme inactivation. Inhibition of cellulase activity by ethanol, measured at both temperatures, was relatively similar, with the relative rate of hydrolysis inhibited 50% at ethanol concentrations of 56.4 and 58.7 g/L at 50 and 37 °C, respectively. A mathematical model was developed to test whether the measured phenomena were sufficient to quantitatively describe system behavior and was found to have good predictive capability at initial Avicel concentrations of 20 and 50 g/L.     

Ethylene removal efficiency and bacterial community diversity of a natural zeolite biofilter

To establish an economical and environmentally friendly technology for ethylene removal from horticultural facilities and industrial point sources, a bench-scale natural zeolite biofiltration system was developed in this study. The system was evaluated for its performance in removing ethylene from an artificially contaminated air stream and characterized for its bacterial diversity under varied ethylene concentrations, and in different spatial stages of the filter. The biofilter enabled to approximately 100% remove ethylene at loading rates of 0.26-3.76 g m⁻³ h⁻¹ when operated with inoculum containing enriched ethylene-degrading bacteria. The bacterial diversity and abundance varied with the height of the biofilter. Moreover, the occurrence and predominance of specific bacterial species varied with the concentrations of ethylene introduced into the biofilter, as observed by PCR-DGGE methods. Phylogenetic analysis indicated that the biofilter system supported a diverse community of ethylene-degrading bacteria, with high similarity to species in the classes Betaproteobacteria, Gammaproteobacteria, Bacilli, and Actinobacteria.     

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1^−/− mice. Four-month-old male Srd5a1 ^−/− mice had reduced trabecular bone mineral density (−36%, p<0.05) and cortical bone mineral content (−15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1 ^−/− mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1 ^−/− mice. Male Srd5a1 ^−/− mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1 ^−/− mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1 ^−/− mice, is an indirect effect mediated by elevated circulating androgen levels.     

Habenula--a new target for treatment of intractable depression

Despite substantial advancement in psychopharmacological and electro-magnetic treatments over the last decades on the depression patients, there are non-responders remain with a chronic disease and high suicidal risk yet. Deep brain stimulation (DBS) is now being experimentally to treat the intractable depression and yielded an impressive therapeutic benefit, and especially few adverse effect occurred. The beneficial action of DBS is closely related to the stimulation sit. And the efficacy of high frequency stimulation of lateral habenula is one of the best choice. In depression, the concentration of 5-HT released by the raphe nuclei is decreased. It's due to mainly the overactivation of the lateral habenula. High frequency stimulation of lateral habenula impairs the activation of lateral habenula, and the inhibitory effect of lateral habenula on raphe nuclei is decreased. Then, the 5-HT concentration released by the raphe nuclei is increased, the pathological changes of depression is eliminated. The lateral habenula could be a promising novel target for BDS in the cases of intractable depression.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.