DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

Summary The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)₄, (GTG)₅, (CA)₈, and (TCC)₅, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.     

Deletion of the Cochliobolus heterostrophus mating-type (MAT ) locus promotes the function of MAT transgenes

Cochhobolus heterostrophus has alternate genes (MAT-1 andMAT-2) at its mating-type locus. Transformants of aMAT-1 or aMAT-2 strain carrying a transgene of opposite mating type can self and are dual maters; the transgene, however, promotes development of pseudothecia only, not ascospores. To determine if the resident gene interferes with the function of the transgene, transformation vectors were designed to delete different amounts (2.5 kb, 5.7 kb, and 6.3 kb) of DNA at theMAT locus. Deletions occurred at a higher frequency (about 90% of transformants) with linearized plasmid than with circular plasmid (about 15% of transformants), and all three vectors were equally efficient at gene replacement. BothMAT-1 andMAT-2 could be deleted with the same set of vectors. Re-transformation of deletion strains (regardless of deletion size) with a wild-type copy ofMAT restored full mating ability, indicating that the residentMAT gene interferes with function of theMAT transgene. Moreover, sexual development was normal whether theMAT transgene integrated at the homologous or at an ectopic site.     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.     

External oxidation pathway in nerve tissue

Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. V. Val'dman.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 4, pp. 431–433, April, 1989.     

分泌性中耳炎中耳积液和血清中sIL－2R水平的初步研究

应用酶联免疫吸附法（ＥＬＩＳＡ）对３０例正常人血清（对照组）和６０例分泌性中耳炎患者（ＳＯＭ组）中耳积液和血清中可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）进行了检测。结果示ＳＯＭ组血清ｓＩＬ－２Ｒ水平明显高于对照组，ＭＥＦ中其含量明显高于血清；鼻咽癌组血清及ＭＥＦ中ｓＩＬ－２Ｒ水平明显高于─般ＳＯＭ组；粘液组高浆液组；慢性组高于急性组（均Ｐ＜０．０１）。提示：血清及ＭＥＦ中ｓＩＬ－２Ｒ水平的测定有助于对ＳＯＭ患者免疫状态的评估；ＭＥＦ中ｓＩＬ－２Ｒ可能主要由局部中耳粘膜产生；ＭＥＰ中高浓度的ｓＩＬ－２Ｒ存在可能是ＳＯＭ迁延不愈的─个原因。     

The molecular immunology of human susceptibility to fungal diseases: lessons from single gene defects of immunity

Introduction: Fungal diseases are a threat to human health. Therapies targeting the fungus continue to lead to disappointing results. Strategies targeting the host response represent unexplored opportunities for innovative treatments. To do so rationally requires the identification and neat delineation of critical mechanistic pathways that underpin human antifungal immunity. The study of humans with single-gene defects of the immune system, i.e. inborn errors of immunity (IEIs), provides a foundation for these paradigms.

Areas covered: A systematic literature search in PubMed, Scopus, and abstracts of international congresses was performed to review the history of genetic resistance/susceptibility to fungi and identify IEIs associated with fungal diseases. Immunologic mechanisms from relevant IEIs were integrated with current definitions and understandings of mycoses to establish a framework to map out critical immunobiological pathways of human antifungal immunity.

Expert opinion: Specific immune responses non-redundantly govern susceptibility to their corresponding mycoses. Defining these molecular pathways will guide the development of host-directed immunotherapies that precisely target distinct fungal diseases. These findings will pave the way for novel strategies in the treatment of these devastating infections.     

长程体外反搏对高胆固醇血症猪高级氧化蛋白产物和高敏C反应蛋白的影响

目的探讨长程体外反搏对高胆固醇血症猪血清中高级氧化蛋白产物(AOPP)和高敏C反应蛋白(hs-CRP)的影响.方法 18头雄性乳猪随机分为正常饲养组(n=6)、高脂饲养组(n=6)及高脂饲养+体外反搏组(n=6).后2组通过高脂饲养复制高胆固醇血症猪模型并对高脂饲养+体外反搏组进行36 d共36 h的长程增强型体外反搏.分别于分组饲养前、反搏前、反搏中期和反搏结束时留取3组动物静脉血,采用分光光度法检测血清AOPP浓度,采用乳胶凝集反应法检测血清hs-CRP浓度.结果高脂饲养组和高脂饲养+体外反搏组经高脂饲养后血清总胆固醇和低密度脂蛋白明显升高(P<0.05).血清AOPP和hs-CRP浓度在分组饲养前组差异无统计学意义(P>0.05).反搏前、反搏中期和反搏结束时,高脂饲养组与高脂饲养+体外反搏组血清AOPP和hs-CRP浓度较正常饲养组同时期均有显著增高(P<0.05);而反搏中期和反搏结束时高脂饲养+体外反搏组血清AOPP浓度较高脂饲养组显著降低[(95.38±12.66)μmol/L比(128.46±12.55)μmol/L;(85.78±10.33)μmol/L比(158.22±16.32)μmoL/L,P<0.05];且反搏中期和反搏结束时高脂饲养+体外反搏组血清hs-CRP浓度较高脂饲养组也有显著降低[(0.47±0.14)mg/L比(0.62±0.32)mg/L;(0.47±0.16)mg/L比(0.59±0.43)mg/L,P<0.05].结论 AOPP和hs-CRP参与了高胆固醇血症猪的发病过程.长程体外反搏可能通过减轻机体体内氧化应激和微炎性反应过程,从而阻止高胆固醇血症的病理生理进程.     

保留部分上鼓室外侧壁鼓室成形术临床初探

目的 :探讨保留部分上鼓室外侧壁鼓室成形术的临床价值。方法 :对 4例慢性化脓性中耳炎患者行保留部分上鼓室外侧壁鼓室成形术的临床资料进行回顾性分析。结果 :经随访半年 ,听力提高 10～ 2 0dB ,鼓膜外形正常 ,全部干耳。结论 :保留部分上鼓室外侧壁鼓室成形术能有效提高听力和干耳率 ,且手术安全     

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl₂ protoplast fusion protocol. Hygromycin B-resistant (HygB^R) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygB^R progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.     

The central projection of muscle afferent fibres to the lower medulla and upper spinal cord: an anatomical study in the cat with the transganglionic transport method

The projection of muscle afferent fibres to the medulla oblongata and upper spinal cord was studied in the cat by using transganglionic transport of wheat germ agglutinin-horseradish peroxidase conjugate. The results demonstrate a precise, musculotopic termination pattern in the external cuneate nucleus; thus, fibres from the intrinsic muscles of the paw terminate medially; those from forearm, arm, and shoulder muscles terminate progressively more laterally; and those from neck and thoracic muscles terminate in the ventrolateral and dorsolateral parts, respectively. Muscle afferent fibres to the main cuneate nucleus terminate in the ventral "reticular" region of the nucleus, with a sparse projection also to the ventral part of the rostral and caudal regions, including the base of the dorsal horn. Fibres from the neck muscles terminate slightly more laterally in the ventral region than do those from the limb muscles, but otherwise, and thus contrary to the case in the external cuneate nucleus, no topographic organization was detected. In the spinal cord, projection was found to laminae I and V, and from the musculature of the back of the neck to the central cervical nucleus.