Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.

Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.     

Development of sensitive esterase assays based on alpha-cyano-containing esters.

A novel approach is reported for the development of fluorogenic esterase reporters using alpha-cyano-containing esters as substrates. After ester hydrolysis, the released alcohol, a cyanohydrin, rapidly eliminates HCN to yield the corresponding aldehyde resulting in strong fluorescence. The pi conjugation of the resulting aldehyde also greatly enhances UV absorption and red shifts fluorescence emission relative to a corresponding alcohol or phenol. Two substrates, R/S-acetic acid cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound I) and trans/cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid R/S-cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound II), were synthesized and evaluated as substrates. Such alpha-cyano substrates possess very low background fluorescence and are more stable under enzyme assay conditions than phenolic substrates due to the aliphatic cyano group. The higher molar absorbtivity and quantum yield of the aldehyde, along with its larger Stokes' shift combined with the increased stability and lower background signal of the cyanohydrin substrate, increases the utility and sensitivity of the resulting assays over current methods. Moreover, compound II showed high selectivity to pyrethroid-cleaving esterases and may provide a direct tool to monitor pyrethroid resistance in insects.     

Nerve growth factor and neuronal cell death

The regulation of neuronal cell death by the neuronotrophic factor, nerve growth factor (NGF), has been described during neural development and following injury to the nervous system. Also, reduced NGF activity has been reported for the aged NGF-responsive neurons of the sympathetic nervous system and cholinergic regions of the central nervous system (CNS) in aged rodents and man. Although there is some knowledge of the molecular structure of the NGF and its receptor, less is known as to the mechanism of action of NGF. Here, a possible role for NGF in the regulation of oxidant--antioxidant balance is discussed as part of a molecular explanation for the known effects of NGF on neuronal survival during development, after injury, and in the aged CNS.     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

大鼠缰核与下丘脑外侧区在血压调节中的相互关系

电刺激乌拉坦麻醉的大鼠下丘脑外侧区(LH)可使缰核(Hb)内51.0％的单位兴奋,15.7％的单位抑制,其中发生兴奋反应的单位有15.4％可被逆行激活。双侧Hb内微量注射利多卡因,电刺激LH引起的升压反应可被阻断42.0±28.0％;反之,双侧LH内微量注射利多卡固,电刺激Hb引起的升压反应可被阻断62.0±26.4％。结果表明,LH与Hb在血压调节中相互依赖,具有协同作用。     

人酸性成纤维细胞生长因子神经营养作用的初步研究

本实验研究了人酸性成纤维细胞生长因子(haFGF)的体外神经营养作用。结果表明,haFGF在体外能明显促进鸡胚(E-8)脊髓组织神经突起的生长,并能明显改变新生大鼠脑星形胶质细胞的形态,使扁平、多角形紧密联接的细胞转化为具有纤维样突起的胶质细胞,同时对胶质细胞DNA合成也有一定促进作用。实验还证明,haFGF可增加体外培养新生大鼠海马神经元的存活,且大大增加神经元胞体体积及突起长度。     

大豆下胚轴线粒体产生超氧物自由基的效率

大豆下胚轴线粒体在呼吸基质存在下,显著地增加了肾上腺素氧化速率,这种氧化速率能为外源SOD抑制,表明线粒体呼吸时产生分子氧的单电子还原成O_2(?)。亚线粒体颗粒产生O_2(?)的效率略高于线粒体。大豆下胚轴线粒体吸链内O_2(?)的产生为NADH所支持并与交替途径无关。表明分子氧单电子还原的部位可能是NADH-黄素蛋白和UbQ-Cyt.B。