Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

Experimental acute pancreatitis is enhanced in mice with tissue nonspecific alkaline phoshatase haplodeficiency due to modulation of neutrophils and acinar cells

Tissue nonspecific alkaline phosphatase (TNAP) has a well established role in bone homeostasis and in hepatic/biliary conditions. In addition, TNAP is expressed in the inflamed intestine and is relevant to T and B lymphocyte function. TNAP KO mice are only viable for a few days, but TNAP^+/? haplodeficient mice are viable. Acute pancreatitis was induced by repeated caerulein injection in WT and TNAP^+/? mice. TNAP^+/? mice presented an increased expression of Cxcl2, Ccl2, Selplg (P-selectin ligand), Il6 and Il1b in the pancreas. Freshly isolated acinar cells showed a dramatic upregulation of Cxcl1, Cxcl2, Ccl2, Il6, Selpg or Bax in both pancreatitis groups. TNAP^+/? cells displayed a 2-fold higher expression of Cxcl2, and a smaller increase in Il6. These findings could be partly replicated by in vitro treatment of primary acinar cells with caerulein. Furthermore, the proinflammatory effect on acinar cells could be partially reproduced in wild type cells treated with the TNAP inhibitor levamisole. TNAP mRNA levels were also markedly upregulated by pancreatitis in acinar cells. Neutrophil infiltration (MRP8+ cells) and activation (IL-6 and TNF production in LPS treated primary neutrophils) were increased in TNAP^+/? vs WT mice. Neutrophil depletion greatly attenuated inflammation, indicating that this cell type is mainly responsible for the higher inflammatory status of TNAP^+/? mice. In conclusion, our results show that altered TNAP expression results in heightened pancreatic inflammation, which may be explained by an augmented response of neutrophils and by a higher sensitivity of acinar cells to caerulein injury.     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Root Pressure of Tropical Lianas and Their Relationships with Phylogeny and Environments

Lianas usually possess large vessels, which are vulnerable to cavitation. Root pressure may play an important role in embolism repair of vessels. However, little is known about the generality of root pressure in tropical lianas. To characterize root pressure of lianas in tropical rainforests, we used pressure transducers to measure root pressure in the rainy and dry seasons for a total of 32 lianas from 14 families common in Xishuangbanna. We further analyzed the associations of maximum root pressure with phylogeny and of transient root pressure with environmental factors. We found that all lianas we selected had root pressure, with maximum root pressure ranging from 2-138kPa. In the dry season, about 72% (23 species) of the lianas had relatively low root pressure (<15kPa) and maintained positive throughout the day. This may be important for water balance for roots and basal stems of lianas. There were three types of diurnal changes in liana root pressure. In Type I, root pressure had obvious diurnal variation in the dry and rainy seasons. In Type II, root pressure did not show obvious diurnal variation in the dry and rainy seasons. In Type III, either in the dry or in the rainy season, root pressure showed obvious diurnal variation. Root pressure varied substantially among lianas, with lianas from two families, Fabaceae and Vitaceae, usually having relatively higher root pressure, suggesting that phylogeny may influence root pressure. Transient root pressure closely responded to photosynthetically active radiation. In most cases, however, it was not related to rainfall and vapour pressure deficit. These results suggest that the associations of liana root pressure with environments need further investigation.     

NGF 与Neuritin 在脑外伤伴四肢骨折病人血清中的表达及意义

目的:通过检测脑外伤患者、脑外伤合并骨折患者、骨折患者及正常人外周血中NGF,neuritin不同时间段的表达,根据其含量的变化,判断与骨折愈合速度的相关性,寻找骨折愈合的关键因子。方法:收集单纯脑外伤、单纯骨折患者各80例、脑外伤合并骨折患者60例、健康体检人群20例。选取外伤后3天、10天、2周抽取所有实验对象的静脉血,应用ELISA技术测定标本中NGF与Neuritin的含量。结果:损伤后每个时间段里,患者血清中NGF、neuritin含量有不同程度升高,均高于正常对照组,其中又以脑外伤合并骨折组最高。血清NGF在骨折合并脑外伤组伤后第3天含量明显升高,为(0.86±0.21),伤后10天为(1.47±0.29),14天为(2.0 7±0.21),脑外伤合并四肢骨折组neuritin血清含量在伤后第3天略有升高为(83.47±18.85),10天(108.50±31.65),2周(91.86±21.12).脑外伤合并骨折病人血清NGF、neuritin表达明显高于其他对照组,差别均有统计学意义(P<0.05)。结论:脑外伤合并骨折病人血清NGF、neuritin表达明显高于其他对照组,说明与骨折愈合有着密切的相关性,两种因子可能在骨折愈合修复过程中共同起作用,从而,推测可能是脑外伤后骨折愈合加速的重要因素。     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.