The Differentiation of Infection Structures as a Result of Recognition Events between some Biotrophic Parasites and their Hosts

Most uredospores of rust fungi develop infection structures in a typical pattern so that they can infect the host plant. The function of these infection structures is divided into the following three phases:

• 1 In the recognition phase, the germ tube recognizes the cuticle and the stoma. This process may occur independently from the host plant since copies of the cuticle induce similar reactions of the fungus. During fungal growth on the epidermis, unspecific stress responses of the plant are triggered.
• 2 In the signal phase, the fungal substomatal vesicle and infection hypha(e) contact the host cells within the leaf parenchyma. A signal from the host induces further development of the fungus. Haustorium mother cell differentiation is effected and haustorium formation is initiated. At the same time, the fungus suppresses the synthesis of stress metabolites by the plant.
• 3 In the parasitic phase, the fungus penetrates the host cell and complex interactions between host and parasite begin. A highly specialized interface around the haustorium develops presumably in order to allow a more efficient nutrient transfer from host to parasite. Eventual defence reactions of the plant, generally on the race-cultivar level, fail to be evoked or are suppressed in compatible combinations.

     

Toxicity and pharmacokinetics of insect growth regulators and other novel insecticides on pupae of Hyposoter didmator (Hymenoptera: Ichneumonidae), a parasitoid of early larval instars of lepidopteran pests

Susceptibility of the lepidopteran parasitoid Hyposoter didymator (Thunberg) to seven modern insecticides, azadirachtin, diflubenzuron, halofenozide, methoxyfenozide, pyriproxyfen, tebufenozide, and spinosad, was tested in the laboratory. Pupae were exposed to different doses of each compound by direct topical application. At the field recommended doses, methoxyfenozide and tebufenozide had no effect on H. didymator. Halofenozide had a low effect on both adult emergence and adult survival but the progeny size and parasitism capacity were not affected. Diflubenzuron was moderately toxic to the parasitoid, while azadirachtin, pyriproxyfen and spinosad were very toxic, affecting all its life parameters. In the pyriproxyfen and spinosad treatments, no progeny was obtained. As a second approach of this study, we determined the rate of penetration through the pupal cocoon and absorption in the parasitoid body as pharmacokinetic parameters important for toxicity. Most of the radioactivity was retained in the silken cocoon, indicating a low accumulation in the parasitoid body. Among all compounds tested, diflubenzuron exhibited the highest absorption in the parasitoid body, followed by pyriproxyfen. For halofenozide, methoxyfenozide and tebufenozide, low absorption (<2%) was found. In addition, we tested for the presence of molting hormone receptors in Hyposoter tissues using a monoclonal antibody 9B9. Our data suggest that the use of diflubenzuron azadirachtin, pyriproxyfen, halofenozide, and spinosad in combination with H. didymator in integrated pest management (IPM) programs should be carefully evaluated. Methoxyfenozide and tebufenozide could be considered safe for this parasitoid.     

Disulfide-linked heterodimeric clusterin is a component of the chicken eggshell matrix and egg white.

Clusterin is a widely expressed secretory glycoprotein which is found in mammals as a disulfide-bonded alpha/beta heterodimer generated by cleavage of the single-chain precursor. In contrast, clusterin occurs in the chicken mainly as an intracellular single-chain form and is not observed in serum. The present report identifies chicken clusterin as a component of the eggshell. This extracellular clusterin originates in the uterine fluid, where it is a disulfide-bonded heterodimer derived from the precursor polypeptide by proteolytic cleavage at the same site as in mammals. Clusterin message expression in the oviduct was measured by real time RT-PCR, and levels were found to be highest in magnum and uterus. Western blotting using protein extracts of oviduct tissues indicated major clusterin production in the magnum, while immunostaining of the oviduct identified clusterin in the tubular glands of the uterus and the magnum. In addition, clusterin was detected in egg white by Western blotting. In the decalcified eggshell, immunofluorescence and colloidal-gold immunocytochemistry revealed that clusterin was predominantly localized in the palisade and mammillary layers, but also in the mantle and core of the inner and outer shell membranes. It has been suggested recently that clusterin acts as an extracellular chaperone. Thus clusterin could function in the uterine fluid to prevent the premature aggregation and precipitation of eggshell matrix components before and during their assembly into the rigid protein scaffold necessary for ordered mineralization.     

Synthesis of D-ring-substituted (5'R)- and (5'S)-17β-pyrazolinylandrostene epimers and comparison of their potential anticancer activities

Various steroidal benzylidenes were synthetized from pregnenolone with benzaldehyde and p-substituted benzaldehydes. The resulting 17β-chalconyl derivatives of pregnenolone were reacted with hydrazine hydrate in acetic acid solution. Regardless of the starting material, the ring-closure reaction afforded (in contrast with the literature data) a mixture of two steroidal pyrazoline epimers. The epimers were critical isomer pairs, which could be separated only in their acetylated form; their structures were investigated by NMR techniques. The in vitro inhibition of rat testicular C(17,20)-lyase activity and the antiproliferative effects on four human cancer cell lines were measured, and the results obtained from the two epimer series were compared.     

The immune response to melanoma is limited by thymic selection of self-antigens

The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE(-/-) mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4(+) and MAA-specific CD8(+) T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8(+) T cell frequencies in AIRE(-/-) mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8(+) T cells were found in both AIRE(-/-) and AIRE(+/+) mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Histological grade in breast cancer: association with clinical and biological features in a series of 229 patients

In order to study the association of histological grade (HG) with specific clinical and biological parameters which may influence the clinical behavior of infiltrating ductal carcinomas of the breast (IDC), we analyzed in 229 tissue samples the cytosolic concentrations of estrogen receptor (ER), progesterone receptor (PR), pS2, cathepsin D, hyaluronic acid (HA) and tissue-type plasminogen activator (t-PA), as well as those of the erbB2 oncoprotein, epidermal growth factor receptor (EGFR), HA, CD44v5 and CD44v6 in the cell membrane fraction. Likewise, we considered size, ploidy, S-phase fraction and axillary node involvement as variables of the study. The transition from HG1 to HG2 and from HG2 to HG3 was accompanied by a number of common features: global increase in size, greater number of tumors >2.0 cm, decrease in membrane hyaluronic acid concentrations, increased cell proliferation (S-phase >7%) and greater aneuploidy. Other events observed during the transition from HG2 to HG3 were a decrease in ER, PR, t-PA and cytosolic hyaluronic acid. These results led us to consider that HG is associated with certain clinical-biological changes that may help explain its value as a prognostic factor in breast carcinomas.     

Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Induction of cytoplasmic respiratory deficient mutants in yeast by the folic acid analogue, methotrexate

Summary The folic acid analogue, methotrexate, was found to be an effective inducer of the cytoplasmic petite mutation in different strains of S. carlsbergensis and S. cerevisiae. The induction varies considerably from strain to strain and also with growth conditions under which the experiments are carried out. Determination of mitochondrial protein synthesis in the presence of methotrexate shows a considerable decrease in incorporation of radioactive leucine before any significant induction of petites has taken place.These results and a comparison with results from other laboratories obtained with different drugs provide the basis for a proposed mechanism of petite induction by methotrexate.     

Characterization of the citrate/acetate antiporter CitW of Klebsiella pneumoniae

The genome of Klebsiella pneumoniae contains at least three different genes encoding citrate transporters. Recently, a third and hitherto unknown gene encoding a citrate transport system ( citW) was identified. Escherichia coli transformed with a plasmid expressing citW was able to grow on citrate as sole carbon and energy source, identifying CitW as a citrate carrier. In this report, we provide evidence that further specifies CitW as a Na(+)-independent citrate/citrate and citrate/acetate exchanger. Kinetic analysis of citrate uptake at different pH values identified Hcitrate(2-) as the transported citrate species, with a K(m) of 25 microM. Since citW is expressed under anoxic conditions and acetate is the main end-product of citrate fermentation in K. pneumoniae, citrate/acetate exchange might be its in vivo function. Sequence similarity searches identified CitW (454 amino acids, 48.15 kDa) as a member of the 2-hydroxycarboxylate transporter family (TC 2.A.24). The substrate specificity seems to partially contradict this phylogenetic classification, but appears logical with respect to the putative functional role of CitW in the citrate fermentation pathway of K. pneumoniae.