Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.

Mutation in the Caenorhabditis elegans gene osm-6 was previously shown to result in defects in the ultrastructure of sensory cilia and defects in chemosensory and mechanosensory behaviors. We have cloned osm-6 by transposon tagging and transformation rescue and have identified molecular lesions associated with five osm-6 mutations. The osm-6 gene encodes a protein that is 40% identical in amino acid sequence to a predicted mammalian protein of unknown function. We fused osm-6 with the gene for green fluorescent protein (GFP); the fusion gene rescued the osm-6 mutant phenotype and showed accumulation of GFP in ciliated sensory neurons exclusively. The OSM-6::GFP protein was localized to cytoplasm, including processes and dendritic endings where sensory cilia are situated. Mutations in other genes known to cause ciliary defects led to changes in the appearance of OSM-6::GFP in dendritic endings or, in the case of daf-19, reduced OSM-6::GFP accumulation. We conclude from an analysis of genetic mosaics that osm-6 acts cell autonomously in affecting cilium structure.     

Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient Relish^E20 flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that Relish^E20 flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.     

Comparison of Peak Cardiopulmonary Performance Parameters on a Robotics-Assisted Tilt Table,a Cycle and a Treadmill

Robotics-assisted tilt table (RATT) technology provides body support, cyclicalstepping movement and physiological loading. This technology can potentially beused to facilitate the estimation of peak cardiopulmonary performance parametersin patients who have neurological or other problems that may preclude testing ona treadmill or cycle ergometer. The aim of the study was to compare themagnitude of peak cardiopulmonary performance parameters including peak oxygenuptake (VO_2peak) and peak heart rate (HR_peak) obtainedfrom a robotics-assisted tilt table (RATT), a cycle ergometer and a treadmill.The strength of correlations between the three devices, test-retest reliabilityand repeatability were also assessed. Eighteen healthy subjects performed sixmaximal exercise tests, with two tests on each of the three exercise modalities.Data from the second tests were used for the comparative and correlationanalyses. For nine subjects, test-retest reliability and repeatability ofVO_2peak and HR_peak were assessed. AbsoluteVO_2peak from the RATT, the cycle ergometer and the treadmill was(mean (SD)) 2.2 (0.56), 2.8 (0.80) and 3.2 (0.87) L/min, respectively (p< 0.001). HR_peak from the RATT, the cycle ergometer and thetreadmill was 168 (9.5), 179 (7.9) and 184 (6.9) beats/min, respectively (p< 0.001). VO_2peak and HR_peak from the RATT vs thecycle ergometer and the RATT vs the treadmill showed strong correlations.Test-retest reliability and repeatability were high for VO_2peak andHR_peak for all devices. The results demonstrate that the RATT isa valid and reliable device for exercise testing. There is potential for theRATT to be used in severely impaired subjects who cannot use the standardmodalities.     

Cellular Functions and X-ray Structure of Anthrolysin O, a Cholesterol-dependent Cytolysin Secreted by Bacillus anthracis

Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.Cholesterol-dependent cytolysins (CDCs)⁴ are a family of pore-forming toxins from many organisms, including but not limited to the genera Archanobacterium, Bacillus, Clostridium, Listeria, and Streptococcus. Recently, work in vertebrates has revealed that CDCs and membrane attack complex/perforin superfamily domain-containing proteins share a similar fold, suggesting that vertebrates use a similar mechanism for defense against infection (1, 2). A common feature of the CDC family is the requirement of cholesterol in the membrane to form pores (3). In addition to cholesterol, certain members of the family also require a cellular receptor, such as CD59 for the toxin ILY from Streptococcus intermedius (4). The specific mechanism by which CDCs form pores is not completely resolved; however, what is generally known is that ring-shaped oligomerization at the cellular membrane is followed by large conformational changes in each unit of the oligomer, resulting in the insertion of a β-barrel into the cellular membrane (5). Pore formation results in a variety of downstream signaling effects, including but not limited to the influx of Ca²⁺ into the cell (6).A good deal is known about structures of the prepore conformation of CDCs. The crystal structures of prepore PFO, from Clostridium perfringens, and ILY have previously been elucidated (7, 8). Each structure shows a characteristic four-domain architecture, in which domain 4 (D4) is involved in membrane recognition, domain 3 (D3) is involved in β-sheet insertion, and domain 2 (D2) is the hinge region that undergoes a large conformational change (9-11). Nevertheless, despite the similarities, structural differences in D4 orientation and the conformation of a highly conserved segment named the undecapeptide region confer functional differences to PFO and ILY (8). Noting these differences, we decided to explore the structure and function of another member of the CDC family, anthrolysin O (ALO).ALO is secreted by Bacillus anthracis, the etiologic agent for anthrax. ALO is chromosomally encoded by a gene whose regulation is poorly understood, and it is highly homologous to other members of the CDC family (12). ALO has been shown to have hemolytic and cytolytic activity (13, 14). Although clinical studies have shown that B. anthracis is weakly hemolytic (15), anthrax bacteria do produce biologically relevant amounts of hemolytic ALO, although the levels of expression are under complex regulation and are dependent on the culture media and growth conditions (12, 13, 16). At lower concentrations, ALO can disrupt cell signaling (13, 14). Search for a cellular receptor of ALO has lead to the conclusion that it is a TLR4 agonist (17). However, it is not known that ALO binds to TLR4 directly and, if so, whether ALO also binds other cellular receptors.In addition to ALO, B. anthracis secrete ∼400 proteins, termed the anthrax secretome (18). Of those, two exotoxins, edema toxin (ET) and lethal toxin (LT) have been characterized in greatest detail. ET raises intracellular cAMP to pathologic levels, whereas LT impairs mitogenic and stress responses by inactivating mitogen-activating protein kinase kinase (19, 20). The complex interplay between these two toxins on various aspects of host cellular functions have been demonstrated (20-25). ALO could also work in conjunction with other anthrax virulence factors to modulate their cellular toxicity. For example, ALO and LF together induce macrophage apoptosis, whereas ALO and PLC play a redundant role in a murine inhalation anthrax model (17, 26). Interplay among anthrax secreted factors on cells relevant to anthrax infection is just beginning to be understood. This network of interactions is vital to the molecular basis of how anthrax bacteria interact with the hosts during anthrax infection.Anthrax infection initiates when B. anthracis spores enter the host through one of three routes: cutaneous, inhalational, or gastrointestinal (GI) (27, 28). All three routes of infection can lead to systemic infection and are ultimately lethal. Different from inhalational anthrax, spores are ingested and germinate on or within the epithelium of the GI tract in GI anthrax (29). This is primarily based on pathological observations that primary lesions of the GI tract are found in GI anthrax, whereas no primary lesions of the lung are found in inhalational anthrax (29). Inhalational anthrax is a disease of choice for biological weapons because of its high infectivity and mortality (30). The initiation of GI anthrax requires much higher doses of spores than inhalational anthrax, and the molecular basis for the initiation of GI anthrax remains elusive (31).Since the primary function of GI epithelia is to control the flux of material into the body, disruption of this barrier can lead to movement of bacteria into the surrounding tissue (32). The barrier is produced by a matrix of transmembrane and membrane-associated proteins. These cell to cell contacts, or tight junctions, are sometimes altered during bacterial infection to specifically disrupt the barrier function of epithelial cells. Using a functional model for the gut epithelium, human gut epithelial Caco-2 brush border expressor (C2BBE) cells, we report that ALO decreases the barrier function of C2BBE cells through disruption of tight junctions. We also show that ALO disruption of barrier function is dependent on epithelial cell polarity. We also present the crystal structure of the soluble state of ALO and compare it with the known structures of other CDCs. In addition, we show that ALO exists primarily as a monomer, in contrast to its closely related homologue PFO, which exists as a dimer. Finally, we used domain swapping to examine the structural components that confer specificity of ALO to gut epithelial cells.     

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K_m value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.Many pathogenic bacteria and fungi produce toxins that interfere with various functions of plant cells and may affect plant defense mechanisms (Durbin, 1981). Toxin production is commonly associated with disease severity and can be involved in colonization or systemic invasion by the pathogen (Schäfer, 1994). Toxin resistance has been shown in most cases to be based on the ability of the plant to metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance genes that encode proteins involved in detoxification mechanisms have been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and Birch, 1997). In many cases a relationship exists between toxin tolerance and resistance to the disease (Anzai et al., 1989; Meeley et al., 1992). The availability of toxin-resistance genes will permit a greater understanding of the mechanisms causing plant disease and will also set the stage for engineering resistance to plant disease (Keen, 1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by the ascomycete fungus Eutypa lata (Pers.: Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991). This disease is responsible for considerable loss in yield and is the most devastating disease of grapevine (Vitis vinifera) in many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The fungus infects the stock through pruning wounds and is present in the xylem and phloem of the vine trunk and branches (Moller and Kasamitis, 1978; Duthie et al., 1991). After a long incubation period, a canker forms around the infected wound. The toxin synthesized by the fungus in the trunk is believed to be transported by the sap to the herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates grapevine cells through passive diffusion and its accumulation in the cytoplasm has been explained by an ion-trapping mechanism related to the ionization state of the molecule (Deswarte et al., 1996b). In the cell the effects of eutypine include reduction of adenylated nucleotide content, inhibition of succinate dehydrogenase, uncoupling of oxidative phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to dwarfed and withered new growth of branches, marginal necrosis of the leaves, dryness of the inflorescence, and, finally, death of one or more branches (Moller and Kasamitis, 1981). The toxin appears to be an important virulence factor involved in symptom development of the disease (Deswarte et al., 1996a). However, the absence of toxin-deficient mutants of the fungus and its long incubation period in the trunk before symptom development have prevented a critical study of the toxin in vine plants. Determining the gene responsible for eutypine resistance would therefore be an important critical tool in determining the role of eutypine toxin in symptom development in the disease; and it has the potential to confer resistance to transgenic grapevines.Recently, Colrat et al. (1998) found detoxification to occur in grapevine cells through the enzymatic reduction of eutypine into its corresponding alcohol, eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have determined that this derivative of the toxin is nontoxic for grapevine tissues. Furthermore, we have established a relationship between the susceptibility of grapevine to eutypa dieback and the ability of tissues to inactivate eutypine, suggesting that the detoxification mechanism plays an important role in defense reactions. Eutypine is enzymatically detoxified in numerous plant species and, among them, we found that the tissues of mung bean (Vigna radiata), a nonhost plant for the pathogen, exhibit an efficient detoxification activity. As a prerequisite for demonstrating the involvement of eutypine toxin in eutypa dieback, we report here the purification to homogeneity and the characterization of an ERE from etiolated mung bean hypocotyls.     

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous,multidrug resistant,biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolatedfrom a tertiary care hospital in Chennai, India. Here, we report the completeannotated genome sequence of strain U25 obtained using PacBio RSII. This is the firstreport of the whole genome of K. pneumoniaespecies from Chennai. Itconsists of a single circular chromosome of size 5,491,870-bp and two plasmids ofsize 211,813 and 172,619-bp. The genes associated with multidrug resistance wereidentified. The chromosome of U25 was found to have eight antibiotic resistant genes[blaOXA-1,blaSHV-28,aac(6’)1b-cr,catB3, oqxAB,dfrA1]. The plasmid pMGRU25-001 was found to have only oneresistant gene (catA1) while plasmid pMGRU25-002 had 20 resistantgenes [strAB, aadA1,aac(6’)-Ib,aac(3)-IId,sul1,2,blaTEM-1A,1B,blaOXA-9,blaCTX-M-15,blaSHV-11, cmlA1,erm(B),mph(A)]. A mutation in the porin OmpK36was identified which is likely to be associated with the intermediate resistance tocarbapenems in the absence of carbapenemase genes. U25 is one of the few K.pneumoniaestrains to harbour clustered regularly interspaced shortpalindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 familyhelicase were identified in the genome. When compared to K.pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13was found inserted.     

Diffusion of anionic and neutral GFP derivatives through plasmodesmata in epidermal cells of Nicotiana benthamiana

Plasmodesmata (Pd) are trans-wall membrane channels that permit cell-to-cell transport of metabolites and other small molecules, proteins, RNAs, and signaling molecules. The transport of cytoplasmic soluble macromolecules is a function of the electrochemical gradient between adjacent cells, the number of Pd per interface between adjacent cells, Stokes radius (R(S)), area of the cytoplasmic annulus, and channel length. The size of the largest molecule that can pass through Pd defines the Pd size exclusion limit. However, since the shape and size of a molecule determines its capacity to diffuse through pores or tubes, R(S) is a better measure. Relatively small changes in R(S) can cause large differences in the mobility of molecular probes, particularly if the pore size is close to that of the probe. In addition, as the dimensions of a macromolecule approach that of the channel, membrane charge effects may become important. We employed quantitative tools and molecular modeling to measure the apparent coefficient of conductivity of Pd, C(Pd), for the non-targeted transport of macromolecules. This method allowed us to examine the influence of protein charge and R(S) on C(Pd) in Nicotiana benthamiana. The C(Pd) of modified green fluorescent proteins (GFPs) of different sizes but with the same charge as native GFP and of a more negatively charged derivative were determined. We found that the C(Pd) of cytoplasmic soluble GFP and cytoplasmic forms of modified GFP were the most strongly correlated with R(S) and that the apparent aberrant increase in C(Pd) of a negatively charged GFP derivative was, at least in part, the result of the charge effect on R(S).     

Development of a Soil-temperature Data Base on Meloidogyne arenaria for a Simulation Model

Models for the development rate and death rate of Meloidogyne arenaria relative to temperature and time were developed to improve the predictive performance of a computer simulator. About 74% of the eggs hatched fairly rapidly, whereas the remainder hatched at a lower rate and appeared to be uninfluenced by temperature shock and root exudates. Death rates of eggs were rapid at extremes of temperature (5 and 36 C) during the first week, because of sensitivity of younger eggs, and then declined and later increased gradually with time. Death rates varied little with time at optimum temperatures for survival.     

Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation

Ozone is a major gaseous pollutant thought to contribute to forest decline. Although the physiological and morphological responses of forest trees to ozone have been well characterized, little is known about the molecular basis for these responses. Our studies compared the response to ozone of ozone-sensitive and ozone-tolerant clones of hybrid poplar (Populus maximowizii × Populus trichocarpa) at the physiological and molecular levels. Gas-exchange analyses demonstrated clear differences between the ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although ozone induced a decrease in photosynthetic rate and stomatal conductance in both clones, the magnitude of the decrease in stomatal conductance was significantly greater in the ozone-tolerant clone. RNA-blot analysis established that ozone-induced mRNA levels for phenylalanine ammonia-lyase, O-methyltransferase, a pathogenesis-related protein, and a wound-inducible gene were significantly higher in the ozone-tolerant than in the ozone-sensitive plants. Wound- and pathogen-induced levels of these mRNAs were also higher in the ozone-tolerant compared with the ozone-sensitive plants. The different physiological and molecular responses to ozone exposure exhibited by clones 245 and 388 suggest that ozone tolerance involves the activation of salicylic-acid- and jasmonic-acid-mediated signaling pathways, which may be important in triggering defense responses against oxidative stress.     

Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat

A Cd²⁺-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species of Thlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO₃)₂ solutions, the electrode exhibited a Nernstian response in solutions with Cd²⁺ activities as low as 50 nm. Addition of Ca²⁺ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd²⁺. Addition of high concentrations of K⁺ and Mg²⁺ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd²⁺ than for any of those potentially interfering ions tested. Flux measurements along the roots of the two Thlaspi species showed no differences in the pattern or the magnitude of Cd²⁺ uptake within the time frame considered. The Cd²⁺-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.     

Biochemical Analysis of Plant Protection Afforded by a Nonpathogenic Endophytic Mutant of Colletotrichum magna

A nonpathogenic mutant of Colletotrichum magna (path-1) was previously shown to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) “plant-defense” response were investigated in anthracnose-resistant and -susceptible cultivars of cucurbit seedlings exposed to four treatments: (1) water (control), (2) path-1 conidia, (3) wild-type conidia, and (4) challenge conditions (inoculation into path-1 conidia for 48 h and then exposure to wild-type conidia). Collectively, these analyses indicated that disease protection in path-1-colonized plants was correlated with the ability of these plants to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone. Watermelon plants colonized with path-1 were also protected against disease caused by Colletotrichum orbiculare and Fusarium oxysporum. A model based on the kinetics of plant-defense activation is presented to explain the mechanism of path-1-conferred disease protection.     

Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C-terminal construct

The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C-terminus of the NMDA receptor bind to cytoskeletal proteins and facilitate membrane targeting and synaptic clustering, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C-terminal constructs in neurons to disrupt synaptic clustering via competition for binding motifs in cytoskeletal proteins and postsynaptic densities. Biolistic particle-mediated gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r)NMDA receptor subunit 1-1 (rNR1-1) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrated unique distribution patterns within the cell. Expression of the full length rNR1-1:GFP construct was cytosolic and localized to membranous patches similar to endogenous NR1-1 protein expression in sensory neurons. Expression of a construct containing only the C-terminus, GFP:C0C1C2, demonstrated nuclear and membranous localization. When the GFP:C0C1C2 construct was co-expressed with rNR1-1 in sensory neurons, membranous localization of rNR1-1 was disrupted. In contrast, co-expression of a C-terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNR1-1 did not alter the membranous distribution of rNR1-1. This observation verifies the utility of a gene transfer strategy to diminish membranous NR1-1 content by expressing a construct containing the C1 exon cassette.