Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium

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Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins

Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3–10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudo-monas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.     

Ants as egg predators of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Australian cotton crops

Abstract Helicoverpa armigera is a major pest of Australian cotton crops. To assess the impact of ant predation on H. armigera populations, the behaviour of four common ant taxa was observed in cotton crops in northern New South Wales over the 1999−2000 and 2001−02 seasons. Areas of cotton were artificially stocked with H. armigera eggs prior to observation. Pheidole spp. were the most frequently observed ants within the crop canopy in 1999−2000 and took the most H. armigera eggs. Iridomyrmex spp. were more frequently observed than Pheidole spp. in 2001−02 and also took some H. armigera eggs. Neither Paratrechina spp. nor Rhytidoponera metallica (Smith) took any H. armigera eggs, although both were seen in the crop canopy. Irrigation, cultivation and insecticide application disrupted foraging ants and limited their impact on H. armigera populations.     

Early events in host-pathogen interactions.

Research focused on early events in host-pathogen interactions has provided new insights into fundamental aspects of microbial pathogenicity and plant responses. Considerable progress has been made in understanding regulation of the delivery of pathogenicity determinants from bacteria into plant cells, signal cascades involved in fungal pathogenicity, the co-ordinating role of the plant cytoskeleton in plant defence and calcium flux as a primary signalling function during the hypersensitive reaction.     

Neutrophil thrombospondin receptors are linked to GTP-binding proteins

The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP-binding proteins (G-proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP-mediated motility. Both PT and CT inhibited TSP-mediated chemotaxis and substrate-associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 μM) nor 8-bromo-cAMP (300 μM) significantly affected TSP-mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 ± 0.3 to 14.2 ± 0.1 pmol/10⁶ cells). Although we could not test the PT sensitivity of TSP priming for FMLP-mediated chemotaxis (as PT inhibits FMLP-mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP-dependent priming of FMLP-mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G-proteins was obtained by examining the effect of TSP on α-subunit ADP-ribosylation, GTPase activity, and GTPγS binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT-dependent ribosylation of G_iα2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G-proteins. However, TSP did not block the CT-dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of G_iα2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 ± 2.7% of control values). In addition, GTPγS binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 ± 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G-proteins, a subpopulation of which also associates with FMLP receptors. © 1996 Wiley-Liss, Inc.     

Comparative Pathobiology of Macaque Lymphocryptoviruses

Lymphocryptoviruses (LCVs) have been identified as naturally occurring infections of both Old and New World nonhuman primates. These viruses are closely related to Epstein–Barr virus (EBV, Human herpesvirus 4) and share similar genomic organization and biological properties. Nonhuman primate LCVs have the ability to immortalize host cells and express a similar complement of viral lytic and latent genes as those found in EBV. Recent evidence indicates that nonhuman primate LCVs can immortalize B cells from genetically related species, suggesting a close evolutionary relationship between these viruses and their respective hosts. Early work with EBV in tamarins and owl monkeys revealed that cross species transmission of lymphocryptoviruses from the natural to inadvertent host may be associated with oncogenesis and the development of malignant lymphoma. Moreover, simian LCVs have the ability to induce malignant lymphomas in immunodeficient hosts and have been associated with posttransplantation lymphoproliferative disease in cynomolgus macaques undergoing solid organ transplantation. This review will focus on the comparative pathobiology of lymphocryptoviral infection and discuss the derivation of specific pathogen-free animals.Abbreviations: EBER, EBV-encoded small RNA; EBNA, Epstein–Barr nuclear antigen; EBV, Epstein–Barr virus; LCV, lymphocryptovirus; LMP, latent membrane protein; NHL, non-Hodgkin lymphoma; PTLD, posttransplantation lymphoproliferative disease; RhLCV, rhesus LCV; SHIV, simian–human immunodeficiency virus; sVCA, small viral capsid antigenThe herpesviridae family shares a number of genetic and biologic properties and is composed of 3 subfamilies: the alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae. Regardless of subfamily, herpes virions have similar ultrastructural morphology, which comprises an envelope, a capsid with icosahedral symmetry, and a centrally located core containing a linear genome of double-stranded DNA 100 to 200 kb in length. Productive replication of herpesviruses occurs within the host cell nucleus, resulting in cell lysis, and histopathologic diagnosis of these infections often is aided by the presence of intranuclear inclusion bodies, which consist of viral protein complexes. As a group, herpesviruses have large, complex genomes and often include acquired cellular homolog genes that manipulate host immunologic and cellular responses, allowing these viruses to persist for the life of the host. Pathogen–host adaptation and coevolution has resulted in, for the most part, infections of low virulence. However, these viruses often do not have strict host specificity, and there are numerous examples of severe disease resulting from cross-species transmission. An early example of this phenomenon was the experimental transmission of 2 gammaherpesvirinae, Epstein–Barr virus (EBV, Human herpesvirus 4) and herpesvirus saimiri (Saimiriine herpesvirus 2), to tamarins and owl monkeys, resulting in malignant lymphoma within several weeks of inoculation.^1,6,39,48,53The gammaherpesvirinae subfamily contains a number of important human and animal pathogens and is subdivided into the lymphocryptovirus (γ1 herpesvirus) and rhadinovirus (γ2 herpesvirus) genera.⁷⁵ The rhadinovirus genus contains Kaposi sarcoma-associated herpesvirus (Human herpesvirus 8), rhesus rhadinovirus (Cercopithecine herpesvirus 17), and retroperitoneal fibromatosis-associated herpes virus and is discussed separately in this issue.⁹³ The γ1 herpesvirus genus contains EBV and the nonhuman primate lymphocryptoviruses.²⁸ Viruses from this genus have been isolated from many species of both Old and New World nonhuman primates, and although the isolates show considerable genomic and biologic similarity, they tend to have restricted ability for immortalizing B cells from all but closely related species.^{21,37,56,70,73} This review will examine the comparative pathobiology of primate lymphocryptoviruses and explore the derivation of macaque colonies that are specific pathogen-free of these agents.     

Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses

Development, abiotic and biotic stress each affect the physical architecture and chemical composition of the plant cell wall, making maintenance of cell-wall integrity an important component of many plant processes. Cellulose biosynthesis inhibition (CBI) was employed to impair the functional integrity of the cell wall, and the plant's response to this specific stress was characterized in an Arabidopsis seedling model system. CBI caused changes in the expression of genes involved in mechanoperception, the response to microbial challenge, and lignin and cell-wall polysaccharide biosynthesis. Following CBI, activation of a UDP- d -xylose 4-epimerase gene correlated with increases in arabinose and uronic acid content in seedling cell walls. Activation of pathogen response genes, lignin deposition and lesion formation were dependent on externally supplied sugars and were suppressed by osmotic support. Lignin deposition in the root elongation zone caused by CBI was reduced in atrbohd (NADPH oxidase) mutant seedlings but increased in jasmonic acid resistant1 ( jar1-1 ) mutant seedlings. Phytohormone measurements showed that CBI-induced increases in jasmonic (JA) and salicylic acids were dependent on sugar availability and prevented by osmotic support. We show that CBI activates responses commonly attributed to both abiotic and microbial challenges. Glucose/sucrose and turgor pressure are critical components in maintenance of cell-wall integrity and the regulation of induced responses, including JA biosynthesis. Lignin deposition induced by CBI is regulated by JAR1-1 and NADPH oxidase-dependent signalling processes. Our results identify components of the mechanism that mediates the response to impairment of cell-wall integrity in Arabidopsis thaliana .     

Are age-related impairments in change-in-support balance reactions dependent on the method of balance perturbation?

Rapid “change-in-support” (stepping or grasping) balance-recovery reactions play a critical role in preventing falls. Studies investigating age-related impairments in these reactions using differing perturbation methods have shown contradictory results. The discrepancies could be due to the different mechanical and sensory stimuli provided by the different perturbation methods, but could also be due to other confounding factors (e.g. differences in perturbation predictability). This study compared two commonly used perturbation methods: weight-drop cable-pulls (CPs) and motor-driven surface-translations (STs). For each perturbation method, effects of aging on the change-in-support reactions were established by comparing 10 young (22–28 years) and 30 older (64–79 years) adults, using large unpredictable multi-directional perturbations similar to those used in previous studies showing age-related differences. Age-related differences in the pattern and spatio-temporal features of the limb movements were examined for stepping and grasping reactions evoked by antero-posterior perturbation of stance, as well as stepping reactions evoked by lateral perturbations delivered while subjects walked “in-place”. Although age-group effects were almost always more pronounced for ST perturbations, the direction of the effect was always the same for both perturbation methods; hence, the perturbation-dependent differences in mechanical and sensory stimuli did not seem to be a critical factor. Perturbation waveform appeared to be a more important factor. For the perturbation methods used here, the ST perturbations were more destabilising than the CP perturbations (leading to a more rapid rise in perturbatory ankle-torque and greater centre-of-mass motion prior to the onset of the postural reaction), and were consequently more effective in revealing age-related deficiencies.