Isolation and purification of BVⅠ-2H from bee venom and analysis of its biological action

The medical use of bee venom for rheumatoid arthritis (RA) has a very long tradition. In this study, isolation and purification of polypeptides from bee venom were carried out on sephadex chromatography, heparin sepharose CL-6B chromatography and HPLC. Several fractions were extracted, and their effects on activation of splenocyte and THP-1 cell were studied. The inhibitory fraction was selected for further studies. Finally, BVⅠ-2H that the HPLC elution profiles was a single peak was isolated by C8 column. ESI- MS detection results showed that BVⅠ-2H was a fraction of bee venom, and the molecular weight of the major component was 644.8. BVⅠ-2H could inhibit ConA-induced splenocyte proliferation, IL-1 production and interfere with splenocyte cycle in mice. Moreover, BVⅠ-2H could inhibit PMA-induced TNFa production in THP-1 cells, which was due to its inhibitory effects on TNFa mRNA expression and protein phosphorylation of IkBa. Our studies indicated that BVⅠ-2H was one of the anti-inflammatory components of bee venom.     

Positive role of nitrite as electron acceptor on anoxic denitrifying phosphorus removal process

Literatures revealed that the electron acceptor-nitrite could be inhibitory or toxic in the denitrifying phosphorus removal process. Batch test experiments were used to investigate the inhibitory effect during the anoxic condition. The inoculated activated sludge was taken from a continuous double-sludge denitrifying phosphorus and nitrogen removal system. Nitrite was added at the anoxic stage. One time injection and sequencing batch injection were carried on in the denitrifying dephosphorus procedure. The results indicated that the nitrite concentration higher than 30 mg/L would inhibit the anoxic phosphate uptake severely,and the threshold inhibitory concentration was dependent on the characteristics of the activated sludge and the operating conditions; instead,lower than the inhibitory concentration would not be detrimental to anoxic phosphorus uptake,and it could act as good electron acceptor for the anoxic phosphate accumulated. Positive effects performed during the denitrifying biological dephosphorus all the time. The utility of nitrite as good electron acceptor would provide a new feasible way in the denitrifying phosphorus process.     

Nerve growth factor activity of several immunal related serine proteases

Rabbit was immuned by the previously purified protein with high nerve growth factor (NGF) bioactivity (NGF_like protease) from \%Agkistrodon halys Pallas\% and the antisera were collected. The polyclonal antibodies were tentatively purified and then used as ligands of an affinity column. The \%A.h.Pallas\% crude venom was fractionated by this affinity column and then by Mono Q on fast protein liquid chromatography (FPLC). As a result, fraction Ⅱ and fraction Ⅲ were purified respectively, whose N_terminal amino acid sequences show high homology with the serine proteases in snake venoms, as well as the previous NGF_like protease. However, they possessed different levels of NGF bioactivity. The NGF activity of the previous NGF_like protease is equivalent to that of NGF, while the activity of fraction Ⅱ seems relatively low in contrast to fraction Ⅲ which had no NGF activity.     

Effects of various pretreatments on biohydrogen production from sewage sludge

The sewage sludge of wastewater treatment plant is a kind of biomass which contains many organics, mainly carbohydrates and proteins. Four pretreatments, acid pretreatment, alkaline pretreatment, thermal pretreatment and ultrasonic pretreatment, were used to enhance biohydrogen production from sewage sludge. The experimental results showed that the four pretreatments could all increase the soluble chemical oxygen demand （SCOD） of sludge and decrease the dry solid （DS） and volatile solid （VS） because the pretreatments could disrupt the floc structure and even the microbial cells of sludge. The results of batch anaerobic fermentation experiments demonstrated that all of the four pretreat- ments could select hydrogen-producing microorganisms from the microflora of sludge and enhance the hydrogen production. The hydrogen yield of the alkaline pretreated sludge at initial pH of 11.5 was the maximal （11.68 mL H2/g VS） and that of the thermal pretreated sludge was the next （8.62 mL H2/g VS）. The result showed that the hydrogen yield of pretreated sludge was correlative with its SCOD. The hydrogen yields of acid pretreated sludge and alkaline pretreated sludge were also influenced by their initial pH. No methane could be detected in the anaerobic fermentation of alkaline pretreated sludge and thermal pretreated sludge, which suggested that these pretreatments could fully inhibit the activity of methanogens. The volatile fatty acids （VFA） production in anaerobic fermentation of alkaline pretreated sludge was the maximum and the next is that of thermal pretreated sludge.     

Neuronal growth inhibitory factor inhibits pheochromocytoma PC12 in vitro

Neuronal growth inhibitory factor (GIF), named Metallothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF’s neuronal growth inhibitory activity.     

Isolation, chemical characteristics and immunity activity of an extracellular polysaccharide EPSⅠ isolated from Antarctic bacterium Pseudoalteromonas sp. S-15-13

A new extracelluar polysaccharide (EPS) was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions (EPSⅠand EPSⅡ) were obtained. EPSⅠwas composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPSⅡwas composed of mannose only with a molecular weight of 62kDa.The effect of the polysaccharide EPSⅠ on the cellular immune response of mice was investigated. Results demonstrated that EPSⅠ could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.     

Isolation, chemical characteristics and immunity activity of an extracellular polysaccharide EPSⅠ isolated from Antarctic bacterium Pseudoalteromonas sp. S-15-13

A new extracelluar polysaccharide (EPS) was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions (EPSⅠ and EPSⅡ ) were obtained. EPSⅠ was composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPSⅡ was composed of mannose only with a molecular weight of 62kDa. The effect of the polysaccharide EPSⅠ on the cellular immune response of mice was investigated. Results demonstrated that EPSⅠ could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Mechanism of the interaction between Au nanoparticles and polymerase in nanoparticle PCR

Nanoparticle PCR is a novel method to optimize DNA amplification. It performs well in improving specificity, enhancing sensitivity and speed. Several mechanisms were proposed in previous studies: one was based on the interaction between gold nanoparticles (AuNPs) and DNA while the other was attributed to the heat transfer property of AuNPs. In this paper, we propose that the interaction between AuNPs and DNA polymerase can significantly influence PCR. First, the addition of DNA polymerase can eliminate the inhibitory effects of excess AuNPs. Second, the addition of AuNPs will increase yield of the desired PCR product and make the optimum concentration of DNA polymerase move to higher value. Third, while excess polymerase might inhibit amplification efficiency, AuNPs can reverse this process and the yield of PCR amplification. Based on these results we propose a possible mechanism that AuNPs might modulate the activity of polymerase and improve PCR amplification.     

Molecular mechanism of the inhibitory effect of methylation outside the recognition sequence of restriction endonuclease Pvu Π on its cleavage activity

In 1991, we found that methylation outside the PvuⅡ recognition sequence could partially inhibit its cleavage activity. To clarify the molecular mechanism, three plasmids with different methylation states were constructed. Then, together with the original one, four plasmids were digested with different amounts of PvuⅡ. Results show that methylation on both sites results in 90% inhibition; moving the methylated site one base further away decreases the inhibitory effect to about 30%; with the adjacent dam methylation site eliminated, the inhibitory effect disappears. The data suggest that the inhibition of cleavage activity caused by outside methylation is not "all or none", and the degree of inhibition is dependent on the position and the number of methylated bases.     

Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

Some monoclonal antibodies （mAbs） could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env^＋ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion （syncytium formation） by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.     

Activated Sludge Ozonation to Reduce Sludge Production in MBR

The total experimental period was divided into two stages. At the first stage, a series of batch studies were carried out to get an understanding of the effect of ozouation on sludge properties. At the following stages, three MBRs with different amounts of activated sludge to be ozonated were run in parallel for a long period to evaluate the influence of sludge ozonation on sludge yield and permeate quality. Through batch study, it was found that ozone could disrupt the cell walls and caused the release of plasm from the cells, then the amounts of soluble organics in the solution increased with ozouation time. With the rise of soluble organics, the amount of soluble organics to be mineralized increased as well, which would reduce the soluble organics content. For the counteraction between these two aspects, a pseudo-balanee could be achieved, and soluble organics would vary in a limited range. Sludge ozonation also increased the contents of nitrogen and phosphorus in the solution. In addition, ozouation was effective in improving sludge settling property. On the basis of batch study, a suitable ozone dosage of 0.16 kgO3/kgMLSS was determined. Three systems were run in parallel for a total period of 39 days, it was demonstrated that a part of activated sludge ozonation could reduce sludge production significantly, and biological perfonnanee of mineralization and nitrification would not be inhibited due to sludge ozouation. Experimental results proved that the combination of ozonation unit with MBR unit could achieve an excellent quality of permeate as well as a small quantity of sludge production, and economic analysis indicated that an additional ozonation operating cost for treatment of both wastewater and sludge was only 0.096Yuan （US$ 0.011,5 ）/m^3 wastewater.     

Equilibrium,Kinetics and Thermodynamics of the Adsorption of Methylene Blue onto a Metal-Organic Frameworks Material,Copper Coordination Polymer with Dithiooxamide

The equilibrium, kinetics and thermodynamics of the adsorption of methylene blue （MB） from aqueous solution onto copper coordination polymer with dithiooxamide （H2dtoaCu） , one of the metal-organic frameworks （MOFs）, were investigated in a batch adsorption system as a function of initial pH, adsorbent concentration, contact time, initial dye concentration, and temperature. The Langmnir, Freundlich, and Dubinin- Radushkevich （D-R） isotherm models were used for modeling the adsorption equilibrium. It was found that Langmuir model yielded a much better fit than the Freundlich model under different temperatures. The maximum monolayer adsorption capacities of MB were 192.98, 229.86, and 297.38 mg/g at 298, 308, and 318 K, respectively. The calculated mean adsorption energy （8.26- 11.04 kJ/mol） using D-R model indicated that the adsorption process might take place by chemical adsorption mechanism. Otherwise, the kinetic studies revealed that the adsorption process could be well explained by pseudo-second-order rate kinetics and intraparticle diffusion was not the rate-limiting step. Thermodynamic studies indicated that this system was feasible, spontaneous, and endothermic process. Based on these studies, H2dtoaCu can be considered as a potential adsorbent for the removal of MB from aqueous solution.     

The study of antioxidant activities of fucoidan from Laminaria japonica

Two large molecular weight fucoidan fractions F-A and F-B were obtained by water extraction and anion-exchange chromatography and then L-A and L-B with low molecular weight and uronic acid content were prepared by radical process degradation of F-A and F-B. The antioxidant activities of the fucoidan fractions on two oxygen free radicals and Cu2 -induced low-density lipoprotein (LDL) oxidation were studied. The results show that all the fucoidan fractions had excellent scavenging capacities on superoxide radical and hypochlorous acid, except the highly sulfated fraction L-B. In LDL oxidation system, low molecular weight fractions L-A and L-B exhibited great inhibitory effects on LDL oxidation induced by Cu2 , however F-A and F-B had little inhibitory effects in this system due to their large molecular weights. The present results suggest that fucoidan from Laminaria japonica is an excellent natural antioxidant and has great potential for preventing the free radical-mediated diseases.     

Mechanism of Mg2+ dissolution from olivine and serpentine: Implication for bioleaching of high-magnesium nickel sulfide ore at elevated pH

To inhibit the dissolution of Mg2+ during the bioleaching process of high-magnesium nickel sulfide ore, the effect of major bioleaching factors on the dissolution of Mg2+ from olivine and serpentine was investigated and kinetics studies were carried out. The results indicated that the dissolution rate-controlling steps are chemical reaction for olivine and internal diffusion for serpentine. The most influential factor on the dissolution of Mg2+ from olivine and serpentine was temperature, followed by pH and particle size. A novel method of bioleaching at elevated pH was used in the bioleaching of Jinchuan ore. The results showed that elevated pH could significantly reduce the dissolution of Mg2+ and acid consumption along with slightly influencing the leaching efficiencies of nickel and cobalt. A model was used to explain the leaching behaviors of high-magnesium nickel sulfide ore in different bioleaching systems. The model suggested that olivine will be depleted eventually, whereas serpentine will remain because of the difference in the rate-controlling steps. Bioleaching at elevated pH is a suitable method for treating high-magnesium nickel sulfide ores.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cos14 and cos56, which represents the HSV-1 genome was used for generation of this rHSV. Rep and cap genes of AAV-2 were inserted into Xba Ⅰ site of UL2 gene on cos6, generating cos6-rcΔUL2. After being digested with Pac Ⅰ , cos6-rcΔUL2 and the other 4 cosmids were cotrans-fected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carrying rep and cap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence of rep and cap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

Climate instability during the last glaciation recorded in the Yuanbu loess section

Abrupt events within the Linxia Yuanbu loess section in the western Loess Plateau in China were investigated. The climatic proxy index of the content of mean grain-size and >4 μm grain-size fraction of the section was used as a climatic indicator, and a moving average method was applied in the statistical analysis to extract the abrupt events recorded in the section. It was found that the loess of the last glaciation recorded not only the abrupt events teleconnected with the high-latitude North Atlantic and Greenland regions but also as many as 15 abrupt events in addition to the Heinrich (H) and Younger Dryas (YD) events. This indicates that abrupt climate changes were typical of the climate changes occurring in the Loess Plateau during the last glaciation. The widely recognized mechanisms of the abrupt climate changes, including the heat transfer theory of the North Atlantic Ocean, the atmospheric circulation theory, and the theory of the interaction between low-latitude ocean and the atmosphere remains to be discussed. Perhaps this problem could be resolved by revealing the relation between the evolution of the features of the East Asian monsoon climate and the climate in the surrounding areas. Through a detailed study of the features of the Heinrich and YD events, criteria on which the abrupt events recorded in the loess could be distinguished were tentatively defined. The events were associated with a large grain size, high carbonate content, high lightness, low susceptibility, low redness and low yellowness. In addition, the variation amplitude of the adjacent peak and valley on the curve of the content of the mean grain-size and >40 μm grain-size fraction was larger than 3.4 μm and the content of >40 μm grain-size fraction was larger than 2.0 μm. Lastly, the length of the variation time was less than 500 years. If all these criteria were met, the event was viewed as an abrupt event.     

Effects of nitrogen sources and glucose on the consumption of ethylene and methane by temperate volcanic forest surface soils

There is limited knowledge with regard to the consumption of ethylene (C2H4) and methane (CH4) in volcanic forest soils containing low microbial carbon-to-organic carbon ratio, and to the responses of both consumptions to nitrogen and carbon additions. Temperate volcanic forest surface soils under three forest stands (e.g. Pinus sylvestris L., Cryptomeria japonica and Quercus serrata) were used to compare CH4 and C2H4 consumption by forest soils, and to study the effects of nitrogen sources and glucose on both consumptions. There was a good parallel between CH4 and C2H4 consumption by for- est soils, but mineralization reduced CH4 consumption rather than C2H4 consumption in forest soils, particularly in a Pinus forest soil. The stimulatory effect of glucose addition on both CH4 and C2H4 consumption by forest soils was increased by increasing the pre-incubation period after glucose addi- tion, and a largest stimulation occurred in the Pinus forest soil. The addition of KNO3-N at the rate of 100 μg·g1 significantly reduced the consumptions of both C2H4 and CH4 by forest soils (P≤0.05). In the presence of urea plus dicyandiamide, the consumption rates of C2H4 and CH4 by forest soils were higher than those in the KNO3-N and urea-N treated soils at the same N rate (P≤0.05), but were similar to those of the control. Hence, under experimental conditions, there was a strong inhibitory effect of NO3 rather than NH4 addition on the CH4 and C2H4 consumption in these forest soils. When amount of the added NO3-N increased up to more than 2―3 times the soil initial NO3-N concentrations, both C2H4 and CH4 consumption rates were reduced to 10%―20% of the rates in soils without nitrate addition. By comparing the three forest stands, it was shown that there was a smallest effective concentration of the added nitrate that could inhibit C2H4 and CH4 consumption in the Pinus forest soil, which indicated that C2H4 and CH4 consumption of the soil was more sensitive to NO3-N addition.     

Inducible expression pattern of rice Bowman-Birk inhibitor gene<Emphasis Type="Italic">OsWIP1-2</Emphasis> and its protease inhibitory activity

The WIP1-2 gene was cloned from rice. It be-longs to the Bowman-Birk inhibitor gene family. Northernblot showed that expression of this gene was induced bywounding and jasmonic acid (JA). It indicates that the OsWIPI gene plays an important role in the rice defense sys-tem. The OsWIP1-2 was cloned into pET28a and expressed inE. coli. Its expressed product was purified in the form offusion protein and tested for the inhibitory activities againsttrypsin and chymotrypsin. It was found that the fusion pro-tein could inhibit chymotrypsin, but not trypsin. It was alsofound that the His tag at its C-terminal affected its inhibitoryactivity significantly. The fusion protein with a naturalC-terminal had the inhibitory activity, while no inhibitoryactivity was detected in the fusion protein with a (His)6-tag atits C-terminal. This implies that extra amino acid residues atthe C-terminal of OsWIP1-2 may interfere with its correctfolding. The inhibitory assay indicated that the members ofrice Bowman-Birk inhibitor gene family probably differenti-ated both in their structure and function.