Sensitization of mice with wild-type and cold-adapted influenza virus variants: immune response to two H1N1 and H3N2 viruses

Two A strain influenza viruses, A/Hong Kong/123/77 (A/HK/123/77) (H1N1) and A/Queensland/6/72 (A/Qld/6/72) (H3N2), and the two cold-adapted reassortants which possess the surface antigens of these strains (CR35 and CR6, respectively) were tested for their ability both to induce primary cytotoxic T-cell (Tc cell) responses in mice and to sensitize mice for a second Tc cell response when challenged with a distantly related A strain virus, A/Shearwater/72 (H6N5). After intranasal inoculation, A/Qld/6/72 replicated to higher titers in the lung (1 to 2 log10 50% egg infective doses) than did A/HK/123/77 or either of the reassortants. A/Qld/6/72 induced higher Tc cell responses in the lung than did CR6, and both were more effective than either A/HK/123/77 or CR35 in this respect. When similar doses (10 or 10(3) hemagglutinin units) of each virus were injected intravenously into mice and the spleens were tested for Tc cell activity 6 days later, both A/Qld/6/72 and CR6 were ca. 100-fold better at inducing a primary Tc cell response than A/HK/123/77 or CR35. In contrast, the H1N1 and H3N2 viruses gave rather similar anti-hemagglutinin antibody titers (after intravenous injection) and delayed-type hypersensitivity reactions (after subcutaneous injection). If mice were primed with a low dose of these viruses (10(4) 50% egg infective doses intranasally), A/Qld/6/72 and CR6 were more effective than A/HK/123/77 or CR35 at sensitizing for a secondary Tc cell response when challenged with A/Shearwater/72, but if larger doses were given either intranasally (10(6) 50% egg infective doses) or intravenously (10 to 10(3) hemagglutinin units), all viruses sensitized the mice equally well, despite the fact the A/Shearwater/72 gives a poor primary Tc cell response in mice. Thus, the viral glycoprotein antigens can be important in determining the immunogenicity of the virus and, particularly, the class I antigen-restricted Tc cell response of the host.     

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.     

Partial purification and characterization of locust allatotropin I

Methanol extracts of locust brains, corpora cardiaca (CC), and suboesophageal ganglia (SOG) were separated by gradient and/or isocratic reverse-phase high-performance liquid chromatography (HPLC) and allatotropic activity monitored in the eluted fractions. A major peak of activity, separated by isocratic separation with 12% 2-propanol, designated allatotropin I, exhibited identical retention times in the three tissue extracts. Doseresponse curves of allatotropin I indicate similar content in brain and CC-equivalents, whereas optic lobes, similarly separated by isocratic HPLC, contain only one-tenth of this amount of allatotropin. Allatotropin I is resistant to boiling and is susceptible to tryptic and chymotryptic digestion. Methanol extracts of thoracic muscle, Malpighian tubules, fat body or ovaries, similarly prepared and boiled, did not exhibit allatotropic activity at high doses of tissue equivalents.     

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。     

Influenza virus-specific antibody-secreting cells in the murine lung during primary influenza virus infection.

An enzyme-linked immunosorbent plaque assay is described which can reliably enumerate influenza virus-specific antibody-secreting cells and exhibits specificity similar to that of the indirect enzyme-linked immunosorbent assay. The assay was used to characterize the development of specific antibody-secreting cells, principally within lung tissue, during primary murine influenza virus infection after intranasal inoculation. Cells secreting influenza virus-specific immunoglobulin M (IgM), IgG, and IgA were detected in greatest numbers in lung tissue, and the data presented indicated that the cells may have originated from specific B-cell precursors in lung tissue which are demonstratable in vitro. At 11 months after infection, cells secreting IgG and IgA were still present in lung tissue. Influenza virus-specific antibody-secreting cells were also detected in spleen tissue and blood. Antibody-secreting cells appeared earlier in spleen than in lung tissue and declined more rapidly in spleen tissue.     

Studies on Analogues of Succinic Semialdehyde as Substrates for Succinate Semialdehyde Dehydrogenase from Rat Brain

The methyl ester of succinic semialdehyde (SSA) was examined as a substrate for succinate semialdehyde dehydrogenase (SSADH) from rat brain. It was found that the ester can be oxidized by the enzyme. Values of Km for SSA-Me were higher than for those for SSA, and for this substrate the enzyme showed a substrate-dependent inhibition. This finding suggests that the carboxylate group of SSA is not essential in the process of inhibition of SSADH by the substrate. Cyclopropyl analogues of SSA, cis- and trans-1-formyl-cyclopropan-2-carboxylic acids, were also individually tested as substrates of SSADH. Only the trans isomer was found to be oxidized to the corresponding dicarboxylic acid; it inhibited the enzyme in the same range of concentrations as SSA. The above data suggest that, as for gamma-aminobutyric acid, SSA is present in an unfolded, transoid conformation at the active site of SSADH.