Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Effect of leukotrienes B4, C4, and D4 on segmental pulmonary vascular pressures

Leukotrienes (LTs) C4 and D4 are vasoconstrictors and are thought to increase both systemic and pulmonary vascular permeability. However, we and others have observed that LTC4 and LTD4 cause pulmonary vasoconstriction but do not increase the fluid filtration coefficient of excised guinea pig lungs perfused with a cell-depleted perfusate. To determine what vascular segments were exposed to an LT-induced increase in intravascular hydrostatic pressure we measured pulmonary arterial (Ppa), pulmonary arterial occlusion (Po,a), venous (Po,v) and double occlusion (Pdo) pressures in isolated guinea pig lungs perfused with a cell-depleted buffered salt solution before and after injecting 4 micrograms of LTB4, LTC4, or LTD4 into the pulmonary artery. All three LTs increased airway pressures and also increased Ppa, Po,a, and Pdo. Histamine (15 micrograms) as well as serotonin (20 or 200 micrograms) had the same effect. In excised rabbit lungs, histamine and serotonin increased only Ppa, and Po,a. LTC4 had no vasoactivity. There are marked species variations with regard to the activity and site of action of histamine, serotonin, and LTC4 on the pulmonary circulation.     

Expression of the p80 region of bovine viral diarrhea virus and identification of specific antibodies to this recombinant protein in bovine sera.

A 917-base pair segment of the bovine viral diarrhea virus (BVDV) genome encoding part of the p80 region was cloned into plasmid Gex-2T expression vector for expression as a fusion protein with glutathione-S-transferase (GST). When the p80 and GST sequences were in the same reading frames, the resulting GST-p80 fusion protein had a molecular mass of 58 kilodaltons (kDa) in SDS-PAGE. Extracts of control E. coli carrying only the vector plasmid (Gex-2T) did not contain this new 58-kDa protein band. Mouse monoclonal antibody specific to BVDV-p80 recognized this recombinant protein. Seventy cattle sera that had an SN titer (to TGAC isolate of cytopathic BVDV) greater than 1:8 reacted with this recombinant protein in Western blots. Of 28 cattle sera that had SN titers less than or equal to 1:8, only one serum tested positive on Western blots.     

Propagation of Geotrichum candidum in Acid Brine

Geotrichum candidum completely neutralized the acid brine and reduced its biochemical oxygen demand (BOD) by 88%. Yield of dry mycelium was 62 g per 100 g of BOD utilized.     

MacELISA、RPHI和IFAT用于流行性出血热早期诊断的比较

比较了IgM捕获ELISA(MacELISA)、反向间接血凝抑制试验(RPHI)和间接免疫荧光抗体试验(IFAT)检测流行性出血热(EHF)病人血清特异性抗体的结果。MacELISA对急性期血清IgM抗体的阳性检出率与RPHI对总抗体的阳性检出率相近,两法都具有较高的敏感性。而IFAT检测IgG抗体的阳性率则较低。总抗体滴度(RPHI)与IgG抗体滴度(IFAT)相关(r=0.542,P<0.01),而与IgM抗体滴度(MacELISA)无明显相关(r<0.1)。但进一步研究发现,3日内血清IgM抗体滴度与总抗体滴度(RPHI)存在相关关系(r=0.701,P<0.01),表明IgM抗体可能也与发病初期RPHI的较高的阳性检出率有关。本工作表明,MacELISA作为一种早期诊断方法具有高度的特异性和敏感性,而RPHI操作简便、快速、敏感性高,但存在一定的非特异性。研究还发现,流行区临床诊断为EHF的病人,IFAT(IgG)和RPHI检测均阳性,而MacELISA(IgM)阴性,提示用RPHI进行血清学诊断时,检查双份血清是必要的。     

流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

Grape pomace: A novel substrate for microbial production of citric acid

Summary Grape pomace was used as substrate for microbial production of citric acid. Of the five cultures examined,Aspergillus niger NRRL 567 was found to produce the greatest amount of citric acid from grape pomace in the presence of methanol at a concentration of 3% (vol/wt). The yield was 60% based on the amount of fermentable sugar consumed.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.