Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells

Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex?, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex? (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.     

Balloon Valvuloplasty for Treatment of Pulmonic Stenosis in English Bulldogs with an Aberrant Coronary Artery

Background: English Bulldogs (EB) with pulmonic stenosis (PS) sometimes have an aberrant coronary artery (CA) type R2A encircling the pulmonary artery (PA). Balloon valvuloplasty (BV) is treatment of choice for severe PS, but is considered to be contraindicated in dogs with aberrant CA. Hypothesis: Conservative BV in EB with aberrant CA is safe and improves clinical signs and quality of life. Animals: Four client‐owned EB with severe PS were retrospectively reviewed/analysed. Methods: Retrospective study: Case records, echocardiography, BV, and follow‐up investigations of EB diagnosed with severe PS and treated with BV were reviewed. The ratios of PA to aortic (Ao) velocity time integral (VTI) were calculated to assess progression/improvement of PS. Results: An aberrant CA was confirmed on angiography in all EB. Conservative BV was performed, using a balloon of the size of the PA annulus or smaller (0.6–1 × PA annulus size). All dogs survived the procedure, but only a mild reduction in pressure gradient was achieved. There was an improvement in PA to Ao VTI in 3 of 4 dogs (P≤ .017), which were free of evidence of congestive heart failure (CHF) 5, 10, and 15 months after BV. One dog that had right‐sided CHF when BV was performed died due to progressive right‐sided CHF within 3 months. Conclusions: Conservative BV in EB might be safe and might improve quality and quantity of life.     

DDGS和酵母培养物对奶牛生产性能和乳成分的影响

选择16头泌乳天数为127±52d经产荷斯坦泌乳牛用于评价酵母培养物(达农威益康XP)和玉米酒糟及其可溶物(DDGS)在试验日粮中含有限草料纤维数量(草料中的中性洗涤纤维含量为19.3%,干物质基础)之情况下对产奶量和乳成分的影响及其它们之间的互作效应。本次试验采用4重复4×4拉丁方设计,试验时间为4个星期。处理组均按照2×2因子排列而设定,其中:1)日粮中不含酵母培养物和DDGS(对照组);2)日粮中含有20%DDGS,无酵母培养物(DDGS组);3)日粮中含有60g/d的酵母培养物益康XP(XP组);4)日粮中含有20%DDGS和60g/d的益康XP(XP+DDGS组)。本次试验结果表明:各处理组之间对奶牛体重和体况评分无显著的影响。日粮的处理并不影响干物质采食量(DMI),在使用草料纤维含量较低且添加20%DDGS的日粮情况下乳脂率会降低。添加益康XP的确能够提高产奶量及乳蛋白产量,但却不能够完全阻止因DDGS所造成的乳脂率下降的趋势。无论日粮中是否含有DDGS,益康XP都能够提高奶牛的生产性能。     

Seroprevalence of Toxoplasma gondii in American free-ranging or captive pumas (Felis concolor) and bobcats (Lynx rufus)

Toxoplasma gondii is a major zoonotic agent infecting a wide range of mammals, including wild felids. Like domestic cats, wild felids are involved in the complete infective cycle of T. gondii, as they can host in their gastrointestinal tract sexually mature parasites and shed infective oocysts in their feces. In order to evaluate the importance of this wildlife reservoir, 438 serum samples collected between 1984 and 1999 from 438 pumas (Felis concolor) and from 58 bobcats (Lynx rufus) from North America, Central America and South America were screened for antibodies to T. gondii. The overall prevalence of T. gondii antibodies was 22.4% in pumas and 51.7% in bobcats, with regional variations. Adults were more likely to be seropositive than juveniles and kittens (prevalence ratio (PR) = 2.61; confidence interval (CI) = 1.15, 4.04). In the US, pumas from the southwestern states (Arizona, California and New Mexico) were more likely to be seropositive for T. gondii ( PR = 2.61; 95% CI = 1.32-5.18 ) than pumas from the northwestern and mountain states (Colorado, Idaho, Oregon, Utah and Wyoming). Male pumas from the US were more likely to be seropositive than females (PR = 2.08; 95% CI = 1.11-3.92), whereas female pumas from Mexico, Central America and South America were more likely to be seropositive than female pumas from Canada and the US (PR = 2.49; 95% CI = 1.09-5.69). Captive pumas were also more likely to be seropositive (21.7%, 29/92) for T. gondii than free-ranging animals (19.9%, 69/346) (PR = 1.85; 95% CI = 1.06, 3.17).     

Management of cor triatriatum dexter by balloon dilatation in three dogs

Two dogs, one immature and one adult, were presented with a history of progressive ascites. In a third, immature dog, increasing exercise intolerance had been noted. Echocardiography demonstrated a partition in the right atrium (cor triatriatum dexter) and echocontrast studies documented normal flow from the cranial vena cava into the right atrium and ventricle. A saphenous vein contrast study demonstrated flow from the caudal vena cava into an accessory right atrial chamber (sinus venarum). The sinus venarum communicated with the true right atrium via a small defect in the atrial membrane in one dog, and additionally with the left atrium via a right-to-left shunting foramen ovale in the other dogs. All defects were visualised on angiographic studies by selective catheterisation of the caudal vena cava via the femoral vein. Balloon dilatation of the defect was then performed using a small followed by a larger balloon angioplasty catheter to enlarge the defect in the atrial membrane. Clinical signs improved within days and were sustained in the long-term in all cases.     

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

Objectives To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis .
Design Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates.
Procedure DNAs from 35 Australian isolates of M para-tuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared.
Results The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales.
Conclusion Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis , and could be used to study the transmission of strains in Australia.