Purification strategy for recombinant Phl p 6 is applicable to the natural allergen and yields biochemically and immunologically comparable preparations

The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6 x His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.     

Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 μM. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.     

Pinealectomy blocks modulation of active avoidance by central vasopressin application in rats

The inter-relationship between central vasopressin and the pineal gland in the modulation of active avoidance behavior was investigated. In sham-operated (SO) rats, intracerebroventricular (i.c.v) application of 10 ng arginine vasopressin (AVP) after both the last acquisition and the first extinction trials prolonged the extinction of the active avoidance response; application of 50 ng of the V1 antagonist, d(CH2)5Tyr(Me)AVP (AAVP) was without effect in both experiments. In contrast to the SO in pinealectomized (PX) rats neither AVP nor AAVP influenced the extinction of the avoidance response. Intraseptal infusion of 200 pg AVP or 5 ng AAVP either after the last acquisition or the first extinction trial was without effect in both SO and PX rats. Comparison of the acquisition trials revealed no differences between SO and PX rats.     

Tubulin diversity in trophozoites of<Emphasis Type="Italic"> Giardia lamblia</Emphasis>

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.     

The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking

The Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome-lysosome fusion in vivo, and it is sufficient to inhibit endosome-endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST-SpiC pulled down Hook3 from murine macrophages, and anti-Hook3 antibodies precipitated SpiC from the cytosol of Salmonella-infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant-negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome-lysosome fusion.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999     

Physiological Characterization of a Bacterial Consortium Reductively Dechlorinating 1,2,3- and 1,2,4-Trichlorobenzene

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO₂, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.Chlorobenzenes are widespread pollutants and accumulate in the food chain due to their hydrophobicity and strong persistence against chemical and microbial degradation (34). Anaerobic reductive dechlorination of chlorinated benzenes was demonstrated for enrichment cultures from biofilm reactors, sewage sludge, river sediment, and soil (3, 4, 15, 16, 22, 31, 37). Dechlorination pathways for all multiply chlorinated benzenes were elucidated (4, 15). Some dechlorination patterns can be rationalized by thermodynamic considerations (3, 13), but little is known about the microorganisms participating in chlorobenzene dechlorination.Anaerobic bacteria transforming chlorobenzoates and/or chlorophenols have been isolated in pure cultures (5, 7, 18, 27, 39, 40, 45, 48). Desulfomonile tiedjei (12), strain 2CP-1 (7), Desulfitobacterium chlororespirans (39), and Desulfitobacterium sp. strain PCE1 (18) grow anaerobically by chlororespiration. So far, it has not been possible to evaluate whether the anaerobic dechlorination of chlorobenzenes proceeds via a similar mechanism, since pure cultures are not available.While the effect of oxygen and nitrate on the dechlorination of chloroaromatics is reported to be negative for most cultures (32), the effect of sulfur oxyanions is controversial. Some reports stated an inhibitory role of sulfate in the reductive dehalogenation of various chlorinated or fluorinated aromatics (17, 19, 25, 26); other studies found only slight inhibition (24), no inhibition (14), or even a stimulated rate of dechlorination (17, 23). For one mixed culture, the mineralization of chlorophenols was concomitantly coupled to the reduction of sulfur oxyanions (20, 21). With pure cultures of D. tiedjei, it could be shown that sulfite and thiosulfate inhibited the dechlorination of 3-chlorobenzoate in growing cells, nongrowing cells, and cell extracts, while sulfate inhibited dechlorination only in growing cells (46).The high toxicity (22) and the low solubility of chlorobenzenes in water prevented the successful isolation of bacteria with chlorobenzenes as electron acceptors. It is therefore essential to study alternative electron acceptors that could be used by chlorobenzene-dechlorinating bacteria and that could substitute for chlorobenzenes during enrichment and isolation. Information about reductive dechlorination of chlorobenzenes in the presence of other electron acceptors is also needed for the evaluation of dechlorination processes at natural sites and for in situ remediation projects. To our knowledge, detailed studies of the effects of alternative electron acceptors on the dechlorination of chlorobenzenes have not been reported so far.The aim of the present study was to describe the physiological properties of a mixed culture effectively dechlorinating trichlorobenzenes and to determine the effects of various specific inhibitors and alternative electron acceptors. For these experiments, we used a stable, sediment-free mixed consortium growing in a defined, synthetic mineral medium. This consortium has been established in our laboratory from a fluidized bed bioreactor (1, 33) and reductively dechlorinates 1,2,3-trichlorobenzene to 1,3-dichlorobenzene and 1,2,4-trichlorobenzene to 1,4- and 1,3-dichlorobenzene. By inhibiting the activity of methanogenic bacteria using the specific inhibitor bromoethanesulfonate (BES), we showed that dechlorination occurs independently from methanogenic bacteria (1), as has also been shown for other enrichment cultures dechlorinating chlorobenzenes (22, 31).