Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Epr Studies on the Effects of Complexation of Heme by Hemopexin upon its Reactions with Organic Peroxides

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.     

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Time-course of Biosynthesis of Phenolics and Lignin in Roots of Wheat Genotypes Differing in Manganese Efficiency and Resistance to Take-all Fungus

Inhibition of lignin biosynthesis in Triticum aestivum L. rootsby Mn deficiency has been suggested as the mechanism of reducedresistance of Mn-deficient wheat roots to infection by the take-allfungus (Gaeumannomyces graminis var. tritici). This study evaluatedphenolics and lignin accumulation in roots of wheat genotypesdiffering in Mn efficiency (measured as growth and yield inMn-deficient soils) and take-all resistance. Seedlings of theMn-inefficient, take-all sensitive genotype Bayonet and theMn-efficient, more take-all resistant genotype C8MM were grownin nutrient solution without added Mn for 18 d and then transferredto a Mn-deficient sandy soil fertilized with Mn at 0 or 30 mgkg^-1. Both genotypes had Mn-deficient roots and shoots at thetime of transfer to the soil. Roots of both genotypes were inoculatedwith the take-all fungus 0, 1, 3 and 7 d after transfer. Twenty-fourhours after inoculation, take-all fungus penetrated the rootstele of take-all sensitive Bayonet but not of more resistantC8MM wheat. Rates of phenolics and lignin accumulation in rootsdeclined steadily during growth in soil for up to 8 d, werehigher in mature, fully differentiated parts of the root systemcompared to distal, younger root tissue, and were higher inBayonet than in C8MM. Manganese fertilization did not significantlyinfluence rates of phenolics and lignin accumulation but reduceddepth of radial penetration by hyphae in both genotypes. Therate of phenolics accumulation was positively (r = 0·91to 0·96) correlated with the rate of lignin accumulation.Mn-efficient C8MM had a higher rate of lignin accumulation perunit of phenolics than Mn-inefficient Bayonet over a wide rangeof phenolics synthesis rates. From this we suggest that C8MMhas a more efficient mechanism for conversion of phenolics tolignin, the trait which appears related to higher take-all resistanceof this genotype.Copyright 1994, 1999 Academic Press Gaeumannomyces graminis var. tritici, lignin, manganese, phenolics, resistance, roots, Triticum aestivum     

The Preparation of Subcellular Organelles from Mouse Liver in Self-Generated Gradients of Iodixanol

This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.     

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.