Effects of human recombinant alpha-2b interferon and feline recombinant omega interferon on in vitro replication of feline herpesvirus-1

OBJECTIVE: To evaluate the effects of recombinant human interferon alpha-2b (rHuIFN-alpha2b) and recombinant feline interferon omega (rFeIFN-omega) on in vitro replication of feline herpesvirus (FHV)-1. SAMPLE POPULATION: Cultures of Crandell-Rees feline kidney (CRFK) cells. PROCEDURES: CRFK cells were treated with rFeIFN-omega or rHuIFN-alpha2b at concentrations ranging from 100 to 500,000 U/mL. Cultures were then inoculated with FHV-1. Constant concentrations of interferon products were maintained throughout the study. Reductions in the number and size of plaques were used as indicators of antiviral activity. Six plaque reduction assays were performed in duplicate. A 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay was used to detect cytotoxic effects of interferon. A 1-way ANOVA and Dunnett test were used to determine significant differences. RESULTS: Treatment with rFeIFN-omega at various concentrations resulted in significant reductions in the number of plaques (100,000 U/mL, 54.7%; and 500,000 U/mL, 59.8%) and in plaque size (100,000 U/mL, 47.5%; 250,000 U/mL, 81.0%; and 500,000 U/mL; 70.5%). Treatment with various concentrations of rHuIFN-alpha2b resulted in a significant reduction in plaque size (100,000 U/mL, 56.0%; 250,000 U/mL, 75.7%; and 500,000 U/mL, 69.0%). None of the tested concentrations of interferon caused significant cellular toxicosis. CONCLUSIONS AND CLINICAL RELEVANCE: At some of the higher concentrations, the antiviral effect of rFeIFN-omega was greater than the antiviral effect of rHuIFN-alpha2b. Reduction in plaque size appeared to be a good indicator of the antiviral activity of interferon against FHV-1.     

Investigation of the Origanum onites L. essential oil using the chorioallantoic membrane (CAM) assay

The in vivo test on the chorioallantoic membrane of the fertilized hen's egg (CAM assay) is a current method to determine antiangiogenic, antiinflammatory activity and toxic effects of individual compounds or complex plant extracts. The method is used for testing natural compounds in small amounts for revealing various modes of action and the complex mechanisms related to angiogenesis and inflammation. Furthermore, possible side effects such as membrane irritation, toxic, and anticoagulant properties of the investigated material in question can be detected. For the evaluation, the essential oil obtained by hydrodistillation of the aerial parts of Origanum onites L., a common spice and medicinal plant, was tested for its effect in the chorioallantoic membrane (CAM) assay. The essential oil composition was revealed by means of gas chromatography-mass spectrometry (GC-MS). Eighty three components were identified, representing 99.1% of the total oil. Carvacrol, thymol, p-cymene, and gamma-terpinene were found as major components and were also individually tested in the CAM assay. Along with the monoterpenes carvacrol and thymol, their methyl ether derivatives were also examined for comparison of their physiological action. Neither the essential oil nor its components showed any pronounced antiinflammatory or antiangiogenic property in the CAM assay, at 10-250 microg/pellet. However, the irritant effect of the essential oil was linked to thymol in a dose-response fashion, up to 10 microg/pellet, where it was still showing irritation.     

Restriction of the felid lentiviruses by a synthetic feline TRIM5-CypA fusion

Gene therapy approaches to the treatment of HIV infection have targeted both viral gene expression and the cellular factors that are essential for virus replication. However, significant concerns have been raised regarding the potential toxic effects of such therapies, the emergence of resistant viral variants and unforeseen biological consequences such as enhanced susceptibility to unrelated pathogens. Novel restriction factors formed by the fusion of the tripartite motif protein (TRIM5) and cyclophilin A (CypA), or "TRIMCyps", offer an effective antiviral defence strategy with a very low potential for toxicity. In order to investigate the potential therapeutic utility of TRIMCyps in gene therapy for AIDS, a synthetic fusion protein between feline TRIM5 and feline CypA was generated and transduced into cells susceptible to infection with feline immunodeficiency virus (FIV). The synthetic feline TRIMCyp was highly efficient at preventing infection with both HIV and FIV and the cells resisted productive infection with FIV from either the domestic cat or the puma. Feline TRIMCyp and FIV infection of the cat offers a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.     

Development of multiple co-inoculants of different biofertilizers and their interaction with plants

On the basis of a yield model for the three kinds of winter crop the ecological framework conditions are characterized from the yield amounts and the degree of fungus infestation. For the first time this is applied to naturally coherent environments, the so-called soil-climate-regions (Boden-Klima-Regionen (BKR)). Ecological framework conditions, which according to a recognizable rhythm change annually, are on the one hand responsible for possible alterations in annual yield amounts and on the other hand they largely determine the regionally different degrees of fungus infestation. With the help of the model-supported prognosis of the annual ecological framework conditions in individual soil-climate-regions (BKR) conclusions can be drawn for sensible ecological and economic investment strategies.     

Establishing a reproducible method for the culture of primary equine corneal cells

Objective  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
Procedures  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
Results  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogenous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
Discussion  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.     

挪威云杉及杂种落叶松稳定胚性细胞系及体胚再生系统的建立

本文报道了挪威云杉(Picea abies)和杂种落叶松(Larix deciduas× L. kaempferi)人工授粉种子诱导胚性细胞系及植株的再生.挪威云杉在德国Saxon Ore 山区被选为对空气污染有抗性的树种并种植在位于Waldsieversdorf的林木遗传育种研究所的种植园中.对挪威云杉测试了几个不同亲本组合的种子形成胚性细胞系的能力.这些云杉种子于1992-01采收,含有干燥的成熟合子胚.作为对照,相同的亲本组合云杉种子于1993-08和1998-08采收,合子胚处于成熟中.杂种落叶松则只有一个人工授粉组合,大田试验证实幼苗生长良好.分别于1995和1996年采收含有未成熟合子胚的未成熟种子.诱导愈伤形成用附加细胞分裂素(激动素和/或BAP)和生长素(NAA, 2,4-D)及有机氮源(水解酪蛋白和谷氨酰胺)的标准培养基(SPE和MSG),于黑暗下培养.尽管在建立培养体系后5～12周产生了大量胚性愈伤组织,诱导率超过25%,但1 a后只有超过1%的胚性愈伤组织稳定生长.挪威云杉稳定胚性细胞系的数量与成熟合子胚的采收季节及建立细胞系的时间(8月或1月)无关,但在8月收获的合子胚在挪威云杉5～12周后胚性愈伤组织诱导率要高些,然而能在悬浮培养中生长及随后成熟体胚能顺利再生植株的稳定胚性细胞系的数量有限.挪威云杉体胚未充分发育的根影响其移栽到土壤中的成活率.在建立的12个细胞系中只有一个在干燥保存后能从体胚再生正常植株.同时考察了影响挪威云杉形成成熟体胚的因素,如用PEG4 000(7.5%)及ABA[8mg·L-1(30.26μmol)-1]处理及ABA灭菌方式(高温高压灭菌或过滤灭菌)对成熟体胚形成的影响.总体来说,过滤灭菌的ABA增加了成熟体胚的数量.营养培养基中用淀粉部分取代蔗糖只导致2个细胞系中的一个增加了成熟体胚的数量.1995和1996年对人工授粉杂种落叶松合子胚发育与自然授粉(不套授粉袋)的相比较,发现人工授粉袋的使用延迟了合子胚的发育.我们的实验中成熟合子胚没有形成胚性细胞系,只有未成熟合子胚形成了胚性细胞系.1995年用落叶松未成熟合子胚建立的胚性细胞系没有形成体胚,而1996年建立的4个胚性细胞系得到了可以移栽到温室中的再生植株.1.5 a的连续继代培养以后,大多数的杂种落叶松和挪威云杉的胚性细胞系的再生能力都下降.导致这种现象可能有多种原因包括培养物的老化以及出现难以检测到的内生细菌.尽管一组实验从最初培养即用了抗生素,但没有收效.因此目前只得到少量的稳定胚性细胞系(<5%),长期继代后丧失再生能力及无法成功移栽到土壤都是植株再生的重要的限制因素,有待进一步研究克服这些困难.     

Studies on holocellulose and alpha-cellulose from spruce wood using cryo-ultramicrotomy

Summary Cryo-ultramicrotomy was applied to holocellulose and alpha-cellulose from spruce wood (Picea abies Karst.) for a light and electron microscopical study of the removal of lignin during chlorite delignification and the changes in swelling during delignification and alkali extraction. The swelling state of the fibre walls during each stage of treatment was well preserved, and distinct differences could be observed. Staining with uranyl acetate brought out the fine structure of the fibre walls down to the range of elementary fibrils.Submitted by Deutsche Forschungsgemeinschaft. The assistance of Dr. D. Grosser at the light microscope and of Miss U. Schwarz with the chemical analysis is greatly appreciated.