Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

使用MUG培养基定量检测植物药中大肠杆菌时荧光猝灭现象的发生与克服方法

实验中观察到,用ＭＵＧ培养基对植物药中的大肠杆菌定量时多发生荧光猝灭现象,影响检测结果。本文对此现象产生的原因与克服方法进行了系统的考察,发现以一种简便的转接方法可排除植物药介质对菌检的干扰。该方法由两组检验系列构成,当怀疑正常稀释系列（第一系列）４０ｈ培养液的荧光结果可能因猝灭现象呈假阴性时,立即分别将该系列的１—３号管培养液以０．５ｍｌ的接种量转接入新鲜的ＭＵＧ培养基（第二系列）,重新培养２４ｈ,荧光猝灭现象即可克服。综合两系列的荧光、产气和吲哚三项生化特征得出检品中大肠杆菌含量。实际应用表明,此法能显著提高使用该培养基时菌检结果的可靠性。     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.     

A survey of porcine kidneys and chicken liver for ochratoxin A in Spain

Contamination studies by ochratoxin A on pork kidney and chicken liver has been carried out in Catalonia (Spain). 73% of the pork kidney samples analyzed did not contain an amount of ochratoxin A over our detection limit (0.5 ng/g) whereas only 7% had contamination higher than 1 ng/g. None of the chicken samples analyzed were contaminated by this toxin above the detection limit. All contamination levels found are below the maximum levels accepted by several countries for this kind of material. A confirmative test is necessary before discarding false positive samples.     

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.     

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.     

Predictions for oxygen supply control to enhance population stability of engineered production strains

The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. (c) 1994 John Wiley & Sons, Inc.     

Efficient use of lactose for the lac promotercontrolled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions

Abstract: Derivatives of the lac promoter (tac, pac, rac) belong to the strongest bacterial promoters which are frequently used for the induced overexpression of foreign genes in Escherichia coli . However, their use in fermentation processes is strongly restricted because of the high cost of the inducer iso-propyl-β-D-thiogalactopyranoside (IPTG). The aim of this work was to investigate the possibility of using lac-derived promoters in high cell density processes resulting in a high yield of the induced recombinant protein if glucose is the main carbon and energy source. Lactose is tested as inducer of the main antigenic coat protein (VP1) of the foot and mouth disease (FMD) virus in a T7-RNA polymerase expression system. It was shown that lactose is able to induce the expression of the recombinant gene to an amount of the VP1 protein corresponding to 20% of the total cell protein.