流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).     

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Summary Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several A-T-like genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open.     

Pseudocyphellaria dissimilis: a desiccation-sensitive,highly shade-adapted lichen from New Zealand

Summary Pseudocyphellaria dissimilis, a foliose, cyanobacterial lichen, is shown not to fit into the normal ecological concept of lichens. This species is both extremely shade-tolerant and also more intolerant to drying than aquatic lichens previously thought to be the most desiccation-sensitive of lichens. Samples of P. dissimilis from a humid rain-forest site in New Zealand were transported in a moist state to Germany. Photosynthesis response curves were generated. The effect of desiccation was measured by comparing CO₂ exchange before and after a standard 20-h drying routine. Lichen thalli could be equilibrated at 15° C to relative humidities (RH) from 5% to almost 100%. Photosynthesis was saturated at a photosynthetically active radiation (PAR) level of 20 mol m^-2 s^-1 (350 bar CO₂) and PAR compensation was a very low 1 mol m^-2 s^-1. Photosynthesis did not saturate until 1500 bar CO₂. Net photosynthesis was relatively unaffected by temperature between 10° C and 30° C with upper compensation at over 40° C. Temporary depression of photosynthesis occurred after a drying period of 20 h with equilibration at 45–65% relative humidity (RH). Sustained damage occurred at 15–25% RH and many samples died after equilibration at 5–16% RH. Microclimate studies of the lichen habitat below the evergreen, broadleaf forest canopy revealed consistently low PAR (normally below 10–20 mol m^-2 s^-1) and high humidities (over 80% RH even during the day time). The species shows many features of an extremely deep shade-adapted plant including low PAR saturation and compensation, low photosynthetic and respiratory rates and low dry weight per unit area.     

Investigation of the neutral filter elution technique I. Effects of pore density and pore diameter on elution rate at pH 9.6

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E. coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns. Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca. 10(8) Da appears to be valid quantitatively.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

Purification and characterization of the antisecretory factor: a protein in the central nervous system and in the gut which inhibits intestinal hypersecretion induced by cholera toxin

The antisecretory factors (ASF) are hormone-like proteins which inhibit cholera toxin-induced intestinal hypersecretion. Although ASF concentrations in young control rats were low, those in old control rats and toxin-treated rats were high. Toxin-treated rats had 200 ED50 units/g wet weight of ASF in the pituitary gland, while their intestinal mucosa, bile and milk contained 3, 0.5 and 0.5 units/g. In adult man and in 8-9-month-old pig the pituitary level was about 20 units/g. The isoelectric points of ASF from pig and rat were 4.8 and 5.0, respectively, while the molecular size as determined by gel filtration on Bio-Gel P-150 was the same in both cases (Kav 0.43). The molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 60,000 for ASF from porcine pituitary gland. One ED50 unit of the purified porcine ASF corresponded to about 10(-13) mol (1-5 ng) of protein. There were two different ASF from human pituitary gland: pI 5.2, Kav 0.43; and pI 4.5, Kav 0.6. Since antibodies against porcine ASF failed to neutralize the latter protein, it may be unrelated to porcine ASF; the human pI 5.2-protein and rat ASF were both neutralized, but less effectively than was porcine ASF. All the ASF molecules attached to agarose gel, from which they dissociated again in methyl alpha-D-glucose: porcine and rat ASF were eluted at 0.3-0.9 M methyl alpha-D-glucose, human pI 5.2-ASF at 0.1-0.9 M, and human pI 4.5-ASF at 0.1-1.5 M methyl alpha-D-glucose.     

Secretion of lipoprotein lipid and synthesis of fatty acids, cholesterol and triacylglycerol by hepatocytes of fasted-refed rats

Synthetic rates of fatty acid, cholesterol and triacylglycerols, and contents and secretion of lipoprotein lipids, were determined in hepatocytes of rats fed ad libitum a fat-containing stock diet or of rats fasted for 48 h and then refed for 24 or 48 h with stock diet or with a glucose-rich fat-free diet. When compared with the values for the ad libitum-fed rats, fatty acid synthesis was lower in fasted rats, slightly increased in rats refed with the stock diet, but several-fold elevated after refeeding the glucose-rich fat-free diet. Cholesterol synthesis was decreased in the fasted cells, and restored to the control level upon refeeding either diet. Triacylglycerol synthesis from exogenous oleate was greatly stimulated in the cells of fasted-refed rats above the rate in cells of the ad libitum-fed rats, the increase being considerably higher after refeeding the glucose-rich fat-free diet than the stock diet. The amount of triacylglycerol secreted by the cells was also elevated by the fasting-refeeding treatment, but the difference between the two diets was much less pronounced than seen for the lipids' synthetic rates. This imbalance may underlie the huge accumulation of this lipid observed in the heptatocytes after refeeding the rats for 48 h with the glucose-rich fat-free diet.     

Control of protein synthesis by amino acid supply. The effect of asparagine deprivation on the translation of messenger RNA in reticulocyte lysates

The enzyme asparaginase, which hydrolyses asparagine to aspartic acid, inhibited cell-free protein synthesis by reticulocyte lysates. The inhibition was rapid and complete when sufficient enzyme was added but could be prevented or reversed by the addition of asparagine. The initial effect of asparaginase appears to be a block in polypeptide chain elongation due to asparagine deprivation, but there are some indications that prolonged incubation under these conditions may give rise to a secondary decrease in initiation of protein synthesis.