Ultrafiltrative separation of rhamnolipid from culture medium

Classic methods of biosurfactant separation are difficult and require large amounts of organic solvents, thus generate high amounts of waste. This work presents and discusses in detail an original procedure to separate rhamnolipid from fermentation broth using high performance membrane techniques. Due to the unique properties of surface active agents, such as capability of forming aggregates above the critical micelle concentration, it is possible to easily purify the biosurfactant with high efficacy using inexpensive and commonly used membranes. In this article, two-stage ultrafiltration is proposed as a method for separating and purifying rhamnolipid from the culture medium. The obtained purified rhamnolipid solution was capable of reducing surface tension of water down to 28.6 mN/m at critical micelle concentration of 40 mg/l. Separation of rhamnolipid was confirmed by HPLC; three types of rhamnolipids were identified (RL1, RL2, RL4), with considerable predominance of RL2.     

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen⁻¹). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψ_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.     

Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.     

Venturia inaequalis: the causal agent of apple scab

The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis. TAXONOMY: Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes.     

Frequency and Rates of Outdoor Activities, and Perceptions of Places to Perform these Activities by Native Americans and Caucasians Interviewed in Tennessee

Activity patterns and perceptions play a key role in human health risk, management, and planning. A sample of 233 people attending a Native American festival in Cookeville, Tennessee was interviewed to determine the types, percent participation, and outdoor activities rates, and their perceptions of the importance of characteristics of nuclear sites. Results indicate that: (1) a high percentage of respondents used outdoor environments, (2) they used them for consumptive (hunting, fishing), non-consumptive (hiking, walking, bird-watching), and religious/sacred activities, (3) a higher percentage of respondents engaged in non-consumptive than consumptive activities, (4) praying or meditating, communing with nature, and bird-watching had the highest uses rates (5) the environmental characteristics rated the highest were lack of radionuclides that presented a health risk, no visible smog, clean air, and unpolluted water, (6) the presence of people, buildings and roads were rated the lowest, and (7) Native Americans had higher outdoor participation rates, participated more frequently, and evaluated environmental characteristics higher than did Caucasians. This information can be used by managers to create and maintain outdoor habitats that fit the needs of local people. Planning and management require information on public policy, human needs and requirements, and human perceptions and evaluations of environmental characteristics.