Subversion of actin dynamics by EPEC and EHEC

During the course of infection, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) subvert the host cell signalling machinery and hijack the actin cytoskeleton to tighten their interaction with the gut epithelium, while avoiding phagocytosis by professional phagocytes. Much progress has been made recently in our understanding of how EPEC and EHEC regulate the pathways leading to local activation of two regulators of actin cytoskeleton dynamics, the Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex. A recent highlight is the unravelling of functions for effector proteins (particularly Tir, TccP, Map and EspG/EspG2) that are injected into the host cell by a type III secretion system.     

Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5′ and 3′ sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.     

Quinazolinone-based rhodanine-3-acetic acids as potent aldose reductase inhibitors: Synthesis,functional evaluation and molecular modeling study

A series of quinazolinone-based rhodanine-3-acetic acids was synthesized and tested for in vitro aldose reductase inhibitory activity. All the target compounds displayed nanomolar activity against the target enzyme. Compounds 3a, 3b, and 3e exhibited almost 3-fold higher activity as compared to the only marketed reference drug epalrestat. Structure-activity relationship studies indicated that bulky substituents at the 3-phenyl ring of the quinazolinone moiety are generally not tolerated in the active site of the enzyme. Insertion of a methoxy group on the central benzylidene ring was found to have a variable effect on ALR-2 activity depending on the nature of peripheral quinazolinone ring substituents. Removal of the acetic acid moiety led to inactive or weakly active target compounds. Docking and molecular dynamic simulations of the most active rhodanine-3-acetic acid derivatives were also carried out, to provide the basis for further structure-guided design of novel inhibitors.     

Focus: Zoonotic Disease: Zoonotic Ocular Onchocercosis by Onchocerca lupi

The parasitic filarioid Onchocerca lupi causes ocular disease characterized by conjunctivitis and nodular lesions. This nematode was first described in 1967 in a wolf from Georgia, and since then cases of infection from dogs and cats with ocular onchocercosis and sporadically from humans also with subcutaneous and cervical lesions caused by O. lupi have been reported from the Middle East, Europe, and North America. Due to its zoonotic potential, this parasitic infection has gained attention in the past 20 years. Phylogenetic studies have highlighted the recent divergence of O. lupi from other Onchocerca spp. and the importance of domestication in the evolutionary history of this worm. Moreover, the finding of an O. lupi genotype associated with subclinical and mild infection in the Iberian Peninsula, raises important questions about the pathogenicity of this presently enigmatic parasite.     

Immunoliposomes bearing lymphocyte activation gene 3 fusion protein and P5 peptide: A novel vaccine for breast cancer

LAG3-Ig as an immune adjuvant has elicited potent anti-tumor immune responses in several preclinical and clinical studies, but the full potential immunostimulatory of LAG3-Ig has yet to be achieved. We hypothesized that by anchoring LAG3-Ig to the surface of liposomes, the adjuvant activity of LAG3-Ig could be improved. We also investigated the immunotherapy by co-delivery of liposome-coupled LAG3-Ig and P5 tumor antigen in mice model of TUBO breast cancer. We prepared and characterized novel PEGylated liposomes bearing surface conjugated LAG3-Ig and P5. Consistent with our hypothesis, liposomes-conjugated LAG3-Ig via multivalent binding to MHC class II molecules exerted immunostimulatory of LAG3-Ig and markedly induced maturation of dendritic cells more efficiently than free LAG3-Ig. LAG3-Ig-P5-immunoliposomes effectively elicited protective anti-tumor responses more than locally injected soluble LAG3-Ig + P5. The higher percentage of CD4⁺ and CD8⁺ T cells in the spleen and more rapid and pronounced infiltration of these effector cells into the site of the tumor were seen following immunoliposome therapy. Finally, anti-tumor immunity induced by LAG3-Ig-P5-immunoliposomes translated into the more tumor regression and prolonged survival of treated mice, compared to soluble immunotherapy. Taken together, our findings suggest that LAG3-Ig-P5-immunoliposomes can be considered as a valuable candidate for developing a liposome-based therapeutic cancer vaccine in treating HER2/ neu⁺ breast cancer patients.     

An enclosed-chamber labeling system for the safe <Superscript>14</Superscript>C-enrichment of phytochemicals in plant cell suspension cultures

Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generale ¹⁴C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied with ¹⁴C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O₂ replenishment and complete capture of respired ¹⁴CO₂ throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [¹⁴C]sucrose as the carbon source, demonstrated a 20–23% efficiency of ¹⁴C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.     

Antifreeze proteins in overwintering plants: a tale of two activities

Antifreeze proteins are found in a wide range of overwintering plants where they inhibit the growth and recrystallization of ice that forms in intercellular spaces. Unlike antifreeze proteins found in fish and insects, plant antifreeze proteins have multiple, hydrophilic ice-binding domains. Surprisingly, antifreeze proteins from plants are homologous to pathogenesis-related proteins and also provide protection against psychrophilic pathogens. In winter rye (Secale cereale), antifreeze proteins accumulate in response to cold, short daylength, dehydration and ethylene, but not pathogens. Transferring single genes encoding antifreeze proteins to freezing-sensitive plants lowered their freezing temperatures by approximately 1 degrees C. Genes encoding dual-function plant antifreeze proteins are excellent models for use in evolutionary studies to determine how genes acquire new expression patterns and how proteins acquire new activities.     

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.