High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens

The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.     

Determination of epimedin C in rat plasma by reversed-phase high-performance chromatography after oral administration of Herba Epimedii extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile -0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05-4.0 microg/mL. The lower limit of quantification was 0.05 microg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.     

Novel isoxazole carboxamides as growth hormone secretagogue receptor (GHS-R) antagonists

Novel isoxazole carboxamides have been identified as growth hormone secretagogue receptor (GHS-R) antagonists. Substituent modification off the 5-position of the isoxazole ring led to analogues with potent binding affinity and functional antagonism of GHS-R. A potent analogue (32) with high aqueous solubility and good GPCR selectivity was also identified as a potential pharmacological tool for in vivo studies.     

Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils

Dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Therefore, in the present study, we investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils. Male Mongolian gerbils were orally pretreated with either E-DHA (100, 150 mg/kg) or vehicle, once a day, for 4 weeks and were subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. The content of brain lipid AA at the termination of treatment, the survival ratio, change of regional cerebral blood flow (rCBF), brain free AA level, thromboxane B(2) (TXB(2)) production and cerebral edema formation following ischemia and reperfusion were evaluated. E-DHA (150 mg/kg) pretreatment significantly increased survival ratio, prevented post-ischemic hypoperfusion and attenuated cerebral edema after reperfusion compared with vehicle, which was well associated with the reduced levels of AA and TXB(2) in the E-DHA treated brain. These data suggest that the effects of E-DHA pretreatment on ischemic mortality and cerebral edema could be due to reduction of free AA liberation and accumulation, and its metabolite synthesis after ischemia and reperfusion by decreasing the content of membrane AA.     

西双版纳热带湿性季节雨林和次生林林下砂仁种植的比较研究

为保护热带雨林,改变现有砂仁栽培模式,探讨改雨林下种砂仁为次生林下种砂仁的可行性．比较研究了热带雨林和次生林下砂仁的生长状况和产量．砂仁种植对雨林生物量和生产力影响显著,对次生林的影响不显著．2种林下砂仁4类植株的密度和生物量的大小顺序均为壮株、衰老株、苗和笋．雨林和次生林下砂仁笋和苗的总数分别为衰老株数量的3．95和1．66倍,远多于衰老株数量,表明砂仁能够维持种群稳定．雨林下砂仁苗多为长势较弱的老苗,绝大多数最终不能生长成壮株．2种林下砂仁壮株密度和生物量差异不显著。但次生林下砂仁衰老株、苗和笋的密度和生物量显著高于雨林,表明次生林下砂仁更新比雨林下快．次生林下砂仁产量为78．068kg·hm^-2,略高于雨林．值得注意的是。砂仁产量很低,茎生物量比很高(0．6以上),果实生物量比极低(0．01),增产潜力较大．次生林的生物量低于雨林,透光率高于雨林。土壤水分含量与雨林接近,有利于砂仁的生长发育。可以在次生林下种植砂仁。     

Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.     

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.     

Proteomic characteristics of human sperm cryopreservation

Human sperm cryopreservation in assisted reproductive technology is the only proven method that enables infertile men to father their own children. However, freezing and thawing reduces spermatozoon motility, viability, and fertilizing ability. An association between dysfunctional spermatozoa due to cryoinjury and protein changes has not been established. We investigated through proteomic analysis the differential protein characteristics between freeze‐thawed and fresh sperm samples obtained from nine normozoospermic donors. Twenty‐seven proteins differed in abundance between the two groups, and results were verified for four proteins via Western blot and immunofluorescent staining. These proteins are putatively involved in sperm motility, viability, acrosomal integrity, ATP and isocitrate content, mitochondrial membrane potential, capacitation, acrosome reaction, and intracellular calcium concentration. These marked differences suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.     

A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen

Aims

A new liquid-phase piezoelectric immunosensor (LP-PEIS), which can detect Schistosoma japonicum (Sj) circulating antigens (SjCAg) quantificationally, was developed.

Methods

The IgG antibodies were purified from the sera of rabbits which had been infected or immunized by Sj and were immobilized on the surface of piezoelectric quartz crystal in LP-PEIS by staphylococcal protein A (SPA). It was used to detect SjCAg in sera of rabbits which had been infected by Sj in order to acquire some optimum conditions for detecting SjCAg. Finally, the LP-PEIS with optimum conditions was used to detect SjCAg in sera of patients who had been infected by Sj, and was compared with sandwich ELISA.

Results

A lot of optimum conditions of LP-PEIS for detecting SjCAg had been acquired. In the detection of patients' sera with acute Schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%). However, there were no significant difference between LP-PEIS and sandwich ELISA.

Conclusions

LP-PEIS can quantificationally detect SjCAg in patients' sera as well as sandwich ELISA.