The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of >99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.     

鱼类LZF-IDNA指纹探针的制备

利用Ｏｌｉｇｏ１０００ＤＮＡ合成仪（Ｂｅｃｋｍａｎ）合成了长度为４５ｂｐ的寡核苷酸单链,经纯化后用同位素γ－３２Ｐ－ＡＴＰ作５′末端标记后,制备成鱼类ＬＺＦ－ＩＤＮＡ指纹探针。通过对鱼类的群体实验、亲子鉴定实验、组织细胞的稳定性实验和鱼类种类的适用范围实验后,测得：（１）ＬＺＦ－ＩＤＮＡ指纹探针属多位点寡核苷酸探针;（２）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类种群中的鉴别机率为９．２３×１０－１６;（３）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类亲子鉴定实验中的父系概率为０．９９９９６２;（４）ＬＺＦ－ＩＤＮＡ指纹探针,是一种稳定的,既具有个体识别能力,又具有一定种属特异性的、适用于鱼类ＤＮＡ指纹图研究的基因指纹探针。     

A Continuum theory for the prediction of lateral and rotational positioning of α-helices in membrane proteins: Bacteriorhodopsin

We have developed a new method for the prediction of the lateral and the rotational positioning of transmembrane helices, based upon the present status of knowledge about the dominant interaction of the tertiary structure formation. The basic assumption about the interaction is that the interhelix binding is due to the polar interactions and that very short extramembrane loop segments restrict the relative position of the helices. Another assumption is made for the simplification of the prediction that a helix may be regarded as a continuum rod having polar interaction fields around it. The polar interaction field is calculated by a probe helix method, using a copolymer of serine and alanine as probe helices. The lateral position of helices is determined by the strength of the interhelix binding estimated from the polar interaction field together with the length of linking loop segments. The rotational positioning is determined by the polar interaction field, assuming the optimum lateral configuration. The structural change due to the binding of a prosthetic group is calculated, fixing the rotational freedom of a helix that is connected to the prosthetic group. Applying this method to bacteriorhodopsin, the optimum lateral and rotational positioning of transmembrane helices that are very similar to the experimental configuration was obtained. This method was implemented by a software system, which was developed for this work, and automatic calculation became possible for membrane proteins comprised of several transmembrane helices. © 1995 Wiley-Liss, Inc.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Staining of living bacteria with rhodamine 123

Abstract It is possible to stain live bacteria with rhodamine 123 (R123). The stained fluorescent cells still keep the ability to replicate ( Staphylococcus aureus, Bordetella pertussis ) and to swim (e.g., Salmonella minnesota ). Dead cells or cells with a dissipated transmembrane potential showed markedly diminished fluorescence. Gram-negative strains were stained with different efficiency, presumably reflecting the different constitutions of the outer membrane.     

Molecular packing of cholesterol in phospholipid vesicles as probed by dehydroergosterol

Ergosta-5,7,9,22-tetraen-3-β-ol (dehydroergosterol) was synthesized and employed as a probe of cholesterol behavior in phospholipid bilayers. Circular dichroism (CD) spectra were obtained. The CD of dehydroergosterol in sonicated egg phosphatidylcholine vesicles was dependent on cholesterol concentration, while in unsonicated egg phosphatidylcholine liposomes and in vesicles obtained by oxctylglucoside dialysis, the CD observed was independent of cholesterol content. The CD of dehydroergosterol in sonicated sphingomyelin vesicles exhibited a different dependence on cholesterol content than seen in sonicated egg phosphatidylcholine vesicles. These data are interpreted in terms of differences between the packing of cholesterol in systems of large and small radii of curvature and in different interactions between dehydroergosterol and phosphatidylcholine and sphingomyelin.     

Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, , determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5–2·10^–6 cm s^–1 bar^–1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.     

A combined scanning and transmission electron microscopic study and electron probe microanalysis of human pineal acervuli

Summary Untreated, decalcified and trypsinized acervuli from human pineal bodies were studied with the scanning and transmission electron microscope as well as by electron probe microanalysis. The mulberry-like acervuli are composed of a various number of spherical lobes (135–800 m) between which clustered groups of globuli (4–14 urn in diameter) are observed. The acervular lobes are very probably formed by an aggregation of these globuli. Small round particles 125–500 Å in diameter are observed on the surface of the pineal concretions. These are not influenced by either decalcification or trypsin treatment. The acervular mineral corresponds morphologically to hydroxyapatite. The electron probe microanalysis reveals the existence of calcium and phosphorus as main components of the acervuli. Small quantities of magnesium and strontium were also detected.Dedicated to Professor Berta Scharrer on the occasion of her 70th birthdayWith the technical assistance of Mr. P.A. MilliquetThe author wishes to thank Mr. Bauer and Mr. Fryder (Nestec SA, La Tour de Peilz) for the use of the Cambridge Stereoscan electron microscope and Dr. T. Jalanti (C.M.E., Lausanne) for his help with the use of the X-ray microanalyser     

一种基于配体肽的CRP荧光检测探针的研制

C反应蛋白（C-reaction protein,CRP）是反映机体炎症的有效标志物,早期检测是判断炎症相关疾病的关键,因此,研制CRP新型检测制剂具有重要意义。利用噬菌体表面展示技术对CRP特异性亲和配体进行了筛选,采用固相肽合成技术对目标配体进行了合成,并经生物标记、高效液相色谱法（high performance liquid chromatography,HPLC）分析及质谱（mass spectrometry,MS）鉴定,成功制得检测CRP的荧光探针。经3轮筛选、ELISA检测、重组噬菌体测序及序列比对后得到1个目标配体肽：S-P-H-N-R-S-N-L-V-Q-E-L;经肽合成及生物标记获得1种CRP荧光探针：FITC-(Acp)-S-P-H-N-R-SN-L-V-Q-E-L。研究结果为CRP的有效检测提供了一种新型制剂。