MALDI MSI Reveals the Spatial Distribution of Protein Markers in Tracheobronchial Lymph Nodes and Lung of Pigs after Respiratory Infection

Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, Actinobacillus pleuropneumoniae (APP) caused infections and still worries many pig breeders around the world. To better understand the influence of pathogenic effect of APP on a respiratory system—lungs and tracheobronchial lymph nodes (TBLN), we aimed to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI). In this study, six pigs were intranasally infected by APP and two were used as non-infected control, and 48 cryosections have been obtained. MALDI-TOF MSI and immunohistochemistry (IHC) were used to study spatial distribution of infectious markers, especially interleukins, in cryosections of porcine tissues of lungs (necrotic area, marginal zone) and tracheobronchial lymph nodes (TBLN) from pigs infected by APP. CD163, interleukin 1β (IL-1β) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL-1β were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL-1β and protegrin-4 precursor were all significantly (p < 0.001) more expressed in necrotic areas of lungs infected by APP than in marginal zone, TBLN and in control lungs.     

Succinic Acid Production from Cheese Whey using Actinobacillus succinogenes 130 Z

Actinobacillus succinogenes 130 Z was used to produce succinic acid from cheese whey in this study. At the presence of external CO₂ supply, the effects of initial cheese whey concentration, pH, and inoculum size on the succinic acid production were studied. The by-product formation during the fermentation process was also analyzed. The highest succinic acid yield of 0.57 was obtained at initial cheese whey concentration of 50 g/L, while the highest succinic acid productivity of 0.58 g h⁻¹ L⁻¹ was obtained at initial cheese whey concentration of 100 g/L. Increase in pH and inoculum size caused higher succinic acid yield and productivity. At the preferred fermentation condition of pH 6.8, inoculum size of 5% and initial cheese whey concentration of 50 g/L, succinic acid yield of 0.57, and productivity of 0.44 g h⁻¹ L⁻¹ were obtained. Acetic acid and formic acid were the main by-products throughout the fermentation run of 48 h. It is feasible to produce succinic acid using lactose from cheese whey as carbon resource by A. succinogenes 130 Z.     

反相HPLC同时测定产琥珀酸放线杆菌发酵液中的有机酸与葡萄糖

提出了一种利用高效液相色谱法同时分析产琥珀酸放线杆菌发酵液中葡萄糖和有机酸的方法。在EclipseXDB C8(150mm×Φ4.6mm,5μm)色谱柱上,以5mmol LH2SO4溶液(pH2.5)作流动相,流速为1mL min,采用示差折光检测器,一次进样可同时定性及定量分析待测试样中的有机酸及葡萄糖,每一样品的分析时间不超过9min。确定了测定各物质的工作曲线的回归方程、线性范围、相关系数和检出限。测定发酵液中葡萄糖、琥珀酸和副产物乳酸的相对标准偏差在0.41%～0.89%范围内,平均回收率在99.6%～100.8%之间。     

反相高效液相色谱法测定产琥珀酸放线杆菌发酵液中的有机酸

提出了一种利用高效液相色谱法分析产琥珀酸放线杆菌发酵液中有机酸的方法。在EclipseXDB C8(4 .6mmi.d .× 1 5 0mm ,5 μm)色谱柱上 ,以 0 .0 0 5mol L硫酸溶液 (pH 2 .5 )作流动相 ,流速为 1mL min ,紫外检测波长 2 1 0nm。 7min内可以把 6种混合酸标样完全分离定量。发酵液经离心后直接进样分离定量 ,其中的琥珀酸、乳酸的回收率大于 97%。经多次实验结果证明 :本方法是测定琥珀酸发酵液中各有机酸的快速、有效的定量测定方法。     

猪传染性胸膜肺炎快速诊断方法

针对猪胸膜肺炎放线杆菌的外膜脂蛋白(omlA)基因序列设计了1对特异性引物,研究了猪胸膜肺炎PCR诊断方法;探索了DNA的快速提取方法、PCR反应最适条件、特异性和灵敏度.结果显示:猪胸膜肺炎的6个分离株均能扩增出1个960 bp特异性条带,而对大肠杆菌、金黄色葡萄球菌的扩增结果为阴性;高浓度副猪嗜血杆菌6个分离株的菌液虽然有扩增产物,但是电泳谱带与猪胸膜肺炎明显不同.灵敏度测试表明,APP菌液最低检出浓度为7.7×105 个/mL.该方法有望成为应用于猪传染性炎的快速诊断和流行病学调查的新方法.     

Studies of Thermobifida fusca plant cell wall degrading enzymes

SYNOPSIS: I have been studying the Thermobifida fusca cellulose degrading proteins for the past 25 years. In this period, we have purified and characterized the six extracellular cellulases and an intracellular beta- glucosidase used by T. fusca for cellulose degradation, cloned and sequenced the structural genes encoding these enzymes, and helped to determine the 3-dimensional structures of two of the cellulase catalytic domains. This research determined the mechanism of a novel class of cellulase, family 9 processive endoglucanases, and helped to show that there were two types of exocellulases, ones that attacked the non-reducing ends of cellulose and ones that attacked the reducing ends. It also led to the sequencing of the T. fusca genome by the DOE Joint Genome Institute. We have studied the mechanisms that regulate T. fusca cellulases and have shown that cellobiose is the inducer and that cellulase synthesis is repressed by any good carbon source. A regulatory protein (CelR) that functions in the induction control has been purified, characterized, and its structural gene cloned and expressed in E. coli. I have also carried out research on two rumen bacteria, Prevotella ruminicola and Fibrobacter succinogenes, in collaboration with Professor James Russell, helping to arrange for the genomes of these two organisms to be sequenced by TIGR, funded by a USDA grant to the North American Consortium for Genomics of Fibrolytic Ruminal Biology.     

Production of succinate from glucose, cellobiose, and various cellulosic materials by the ruminai anaerobic bacteriaFibrobacter succinogenes andRuminococcus flavefaciens

The production of organic acids by two anaerobic ruminal bacteria,Fibrobacter succinogenes S85 andRuminococcus flavefaciens FD-1, was compared with glucose, cellobiose, microcrystalline cellulose, Walseth cellulose (acid swollen cellulose), pulped paper, and steam-exploded yellow poplar as substrates. The major end product produced byF. succinogenes from each of these substrates was succinate (69.5–83%), the principal secondary product was acetate (16–30.5%). Maximum succinate productivity ranged from 14.1 mg/L · h for steam-exploded yellow Poplar to 59.7 mg/L · h for pulped paper. ForR. flavefaciens, the major end product from cellobiose, microcrystalline cellulose, and acid-swollen Walseth cellulose was acetate (39–46%), pulped paper and steam-exploded yellow poplar yielded succinate (42–54%) as the major product. Maximum succinate productivity byR. flavefaciens ranged from 9.21 mg/L · h for cellobiose to 43.1 mg/L · h for pulped paper. In general, much less succinate was produced at a lower maximum productivity byR. flavefaciens than byF. succinogenes under similar fermentation conditions. The maximum succinate productivities by these two organisms are comparable to the previously reported value of 59 mg/L · h forAnderobiospirillum succiniciproducens grown on glucose and corn steep liquor.     

The antibacterial mechanism of berberine against Actinobacillus pleuropneumoniae

This study demonstrated berberine to be a potential natural compound against Actinobacillus pleuropneumoniae. Liquid doubling dilution, transmission electron microscopy (TEM), SDS-PAGE and 4′,6-diamidino-2-phenylindole (DAPI) staining were employed to elucidate the antibacterial activity and mechanism of berberine. The minimal inhibitory concentration of berberine was 0.3125 mg/mL, and time–kill curves showed concentration and time dependence. The TEM micrographs displayed damaged cell wall, concentrated cytoplasm, cytoplasmic content leakage and cell death. SDS-PAGE and DAPI assays revealed that berberine can restrain DNA and protein syntheses. Berberine inhibited the synthesis of proteins associated with the growth and cleavage of bacteria and then blocked the division and development of bacteria. The compound ultimately induced cytoplasm pyknosis and bacterial death.