用重组痘苗病毒表达pGHcDNA的研究

构建了含有ｐＧＨｃＤＮＡ的重组痘苗病毒,用ＥＬＩＳＡ证明该重组病毒在被感染的ｈ１４３细胞中,可表达出猪生长激素并将之分泌到培养基中,表达量约为１．０５μｇ／１０￣６细胞（２４ｈ）。用定位免疫化学法进一步证明该病毒可感染小鼠并在小鼠体内表达ｐＧＨｃＤＮＡ。同时还构建了含双拷贝ｐＧＨｃＤＮＡ的重组痘苗病毒,并证明其ｐＧＨ表达量比单拷贝重组病毒有明显提高,约为１．５０μｇ／１０￣６细胞（２４ｈ）。     

重组水蛭素HV2的稳定性

重组水蛭素ＨＶ２是凝血酶的特异性抑制剂,是一种非常稳定的蛋白质。温度的升高（１００℃水浴）和ｐＨ（１─１３）的改变不影响其活力,在某些变性剂（８ｍｏｌ／Ｌ尿素、１％ＳＤＳ和６ｍｏｌ／Ｌ盐酸胍）存在的条件下也非常稳定,０．１ｍｏｌ／Ｌ的ＤＴＴ在７０℃时使其部分失活,只有ｐＨ和温度同时升高其活力才开始下降,ｐＨ１３、８０℃处理１５ｍｉｎ即完全失活,氨基酸组成和活性分析发现失活样品的Ｃｙｓ和Ｌｙｓ被破坏。重组水蛭素ＨＶ２含有一个结构紧密的Ｎ端核心区和一个无序的Ｃ端尾部。其Ｎ端的３个Ｌｙｓ－Ｘａａ键均不被胰蛋白酶水解;胃蛋白酶及糜蛋白酶消化后,分离所得片段,氨基酸组成分析发现Ｎ端核心区依然保持很高的抗凝血酶活性,继续消化２４ｈ,核心区不被进一步降解。     

钙离子和蛋白激酶C对大鼠缺血海马脑片谷氨酸堆积的影响

采用大鼠海马脑片体外缺血模型观察钙离子和蛋白激酶Ｃ（ＰＫＣ）对神经元胞外谷氨酸（ＧＬＵ）堆积的影响,结果显示：海马脑片在体外“缺血”１０ｍｉｎ,ＧＬＵ在胞外的浓度增加４倍（从３２±４升高到１１３±１０ｐｍｏｌ／（ｍｉｎ．ｍｇＰｒ）．ｎ＝６）．Ｎ型钙通道拮抗剂蝙蝠葛苏林碱（ＤＳＬ）或无钙培养液均能有效抑制这种浓度的升高（Ｐ＜０．０１）．提示缺血１０ｍｉｎ引发的ＧＬＵ浓度升高是受Ｃａ２＋内流调控的．当脑片在缺血状况下孵育３０ｍｉｎ,ＤＳＬ只部分抑制这种ＧＬＵ堆积,而无钙培养液则无影响,但这额外的ＧＬＵ堆积可被ＰＫＣ抑制剂Ｈ－７完全阻断,而被ＰＫＣ激动剂ＰＤＢ所加强;且不受钙调蛋白抑制剂Ｃａｌｍｄａｚｏｌｉｕｍ和８－溴－ｃＡＭＰ影响．提示缺血３０ｍｉｎ,胞外ＧＬＵ的堆积受钙内流和ＰＫＣ双重调控。     

Microbial oxidation of cumene by octane-grown cells

Previously, we reported that eight glucose-grown microbial cultures out of 1229 screened oxidize the alkyl side-chain of 2-phenylpropane (cumene) stereospecifically. Now, we have adapted these cultures to grow on n-octane and found that their cumene oxidation activities increased more than 30 times. We also found an additional 11 cultures (ten bacteria, one actinomycete) that oxidized cumene when grown on octane but not on glucose. In general, octane-grown cells were more active in cumene oxidation than glucose-grown cells. Rhodococcus rhodochrous NRRL B-2153 showed the best conversion yield (2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.5%) at 25°C, pH 8.0, 250 rpm, and 12 h of reaction. Structures of the reaction products were confirmed by gas chromatography (GC)/mass spectrometry and GC/infrared analyses. Products contained 84% ee (enantiomeric excess) of the R(–) isomer, as analyzed with a GC cyclodextrin chiral column. Strain B-2153 oxidized alkylbenzenes in the following order of reaction rate: ethylbenzene >amylbenzene > butylbenzene > cumene > propylbenzene > sec-butylbenzene. tert-Butylbenzene was not oxidized.     

Characteristics of microwave evoked body movements in mice

Microwave evoked body movements were studied in mice. A resonant cavity was used to provide head and neck exposure of the mouse to pulsed and gated continuous wave (CW) 1.25 GHz microwaves. No difference in response to pulsed and gated CW stimuli of equal average power was found. The incidence of the microwave evoked body movements increased proportionally with specific absorption (dose) when the whole-body average specific absorption rate was at a constant level (7300 W/kg). Under a constant average specific absorption rate, the response incidence reached a plateau at 0.9 kJ/kg. For doses higher than 0.9 kJ/kg, response incidence was proportional to the specific absorption rate and reached a plateau at 900 W/kg. Body movements could be evoked by a single microwave pulse. The lowest whole-body specific absorption (SA) tested was 0.18 kJ/kg, and the corresponding brain SA was 0.29 kJ/kg. Bulk heating potentials of these SAs were less than 0.1 °C. For doses higher than 0.9 kJ/kg, the response incidence was also proportional to subcutaneous temperature increment and subcutaneous heating rate. The extrapolated absolute thresholds (0% incidence) were 1.21 °C temperature increment and 0.24 °C/s heating rate. Due to high subcutaneous heating rates, these microwaves must be perceived by the mouse as an intense thermal sensation but not a pain sensation because the temperature increment was well below the threshold for thermal pain. Results of the present study should be considered in promulgation of personnel protection guideline against high peak power but low average power microwaves. © 1994 Wiley-Liss, Inc.     

Dielectric properties of human red blood cells in suspension at radio frequencies

Dielectric properties of human red blood cells (RBCs) in suspension (hematocrit 50%) from 243 healthy persons (120 males, 123 females) were measured at 25 °C in a frequency range of 1–500 MHz, with a coaxial transmission line reflection method (one-side measurement). The measuring system, controlled by an IBM-PC computer, was composed of a network analyzer (HP4195A), an impedance test adapter (HP41951-61001), a coaxial line sensor, and a temperature-controlling set. The data measured revealed a statistically significant age dependence, with a critical age of about 49 years, above which permittivity and conductivity of human RBCs in suspension decreased significantly. © 1994 Wiley-Liss, Inc.     

Identification of amino acid residues of Bacillus thuringiensis delta-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori.

Alanine substitution (A3) or deletion (D3) of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type). The loss of larvicidal activity in the mutants was not caused by increased sensitivity to larval gut enzymes but could be attributed to significantly reduced binding to B. mori brush border membrane vesicles. Some or all of the affected amino acid residues may interact directly or indirectly with the B. mori membrane receptor(s). Such receptor binding appears to be directly correlated with insect toxicity.     

Overproducing the Bacillus subtilis mother cell sigma factor precursor, Pro-sigma K, uncouples sigma K-dependent gene expression from dependence on intercompartmental communication.

During sporulation of Bacillus subtilis, proteolytic activation of pro-sigma K and ensuing sigma K-dependent gene expression normally require the activity of many sporulation gene products. We report here that overproducing pro-sigma K at the onset of sporulation substantially uncouples sigma K-dependent gene expression from its normal dependency. Overproducing pro-sigma K in strains with a mutation in spoIIIG, spoIIIA, spoIIIE, or spoIVB partially restored sigma K-dependent gene expression in the mother cell and resulted in accumulation of a small amount of polypeptide that comigrated with sigma K, but these mutants still failed to form spores. In contrast, sporulation of spoIVF mutants was greatly enhanced by pro-sigma K overproduction. The products of the spoIVF operon are made in the mother cell and normally govern pro-sigma K processing, but overproduction of pro-sigma K appears to allow accumulation of a small amount of sigma K, which is sufficient to partially restore mother cell gene expression and spore formation. This spoIVF-independent mechanism for processing pro-sigma K depends on sigma E, an earlier-acting mother cell-specific sigma factor. The spoIIID gene, which encodes a mother cell-specific DNA-binding protein that is normally required for pro-sigma K production, was shown to be required for efficient pro-sigma K processing as well. bof (bypass of forespore) mutations bypassed this requirement for spoIIID, suggesting that SpoIIID is less directly involved in pro-sigma K processing than are spoIVF gene products. However, bof spoIIID double mutants overproducing pro-sigma K still failed to sporulate, indicating that SpoIIID serves another essential role(s) in sporulation in addition to its multiple roles in the production of sigma K.     

Mouse neurovirulence determinants of poliovirus type 1 strain LS-a map to the coding regions of capsid protein VP1 and proteinase 2Apro.

Poliovirus type 1 strain LS-a [PV1(LS-a)] is a OV variant adapted to mice by multiple passages through mouse and monkey tissues. To investigate the molecular basis underlying mouse neurovirulence of PV1(LS-a), a cDNA of the viral genome containing nucleotides 112 to 7441 was cloned, and the nucleotide sequence was determined. Compared with that of the mouse avirulent progenitor PV1(Mahoney), 54 nucleotide changes were found in the genome of the PV1(LS-a) virus, resulting in 20 amino acid substitutions in the virus polyprotein. Whereas the nucleotide changes were scattered throughout the genome, the amino acid substitutions were largely clustered in the capsid proteins and, to a certain extent, in the virus proteinase 2Apro. By in vitro mutagenesis, PV1(LS-a)-specific capsid mutations were introduced into a cDNA clone of PV1(Mahoney). We show that neither the individual amino acid mutations nor combinations of mutations in the region encoding VP1 conferred to PV1(Mahoney) the mouse-adapted phenotype of PV1(LS-a). Chimeric cDNA studies demonstrated that a recombinant type 1 virus containing the PV1(LS-a) sequence from nucleotide 2470 to nucleotide 3625 displayed a neurovirulent phenotype in mice. Further dissection of this region revealed that mouse neurovirulence of PV1(LS-a) was determined by multiple mutations in regions encoding both viral proteinase 2Apro and capsid protein VP1. The mouse neurovirulent viruses, PV1(LS-a), W1-M/LS-Pf [nucleotides 496 to 3625 from PV1(LS-a)], and W1-M/LS-NP [nucleotides 2470 to 3625 from PV1(LS-a)], showed increased sensitivity to heat treatment at 45 degrees C for 1 h. Surprisingly, the thermolabile phenotype was also displayed by a recombinant of PV1(Mahoney) carrying a PV1(LS-a) DNA fragment encoding the N-terminal portion of 2Apro. This suggests that base substitutions in the region encoding 2Apro affected capsid stability, thereby contributing to the neurovirulence of the virus in mice.