Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex

Picornaviruses have a peptide termed VPg covalently linked to the 5'-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C(2)-3Dpol complex that extrapolates well to all picornaviruses.     

Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation

The 5' ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3' end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3' end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed.     

Biogeographical variation in community response to root allelochemistry: novel weapons and exotic invasion

Centaurea diffusa is one of the most destructive invasive weeds in the western USA and allelopathy appears to contribute to its invasiveness ( Callaway & Aschehoug 2000 ). Here we identify a chemical from the root exudates of C. diffusa, 8‐hydroxyquinoline, not previously reported as a natural product, and find that it varies biogeographically in its natural concentration and its effect as an allelochemical. 8‐Hydroxyquinoline is at least three times more concentrated in C. diffusa‐invaded North American soils than in this weed's native Eurasian soils and has stronger phytotoxic effects on grass species from North America than on grass species from Eurasia. Furthermore, experimental communities built from North American plant species are far more susceptible to invasion by C. diffusa than communities built from Eurasian species, regardless of the biogeographical origin of the soil biota. Sterilization of North American soils suppressed C. diffusa more than sterilization of Eurasian soils, indicating that North American soil biota may also promote invasion by C. diffusa. Eurasian plants and soil microbes may have evolved natural resistance to 8‐hydroxyquinoline while North American plants have not, suggesting a remarkable potential for evolutionary compatibility and homeostasis among plants within natural communities and a mechanism by which exotic weeds destroy these communities.     

Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms

A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 μm/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

In vitro propagation of Spilanthes mauritiana DC., an endangered medicinal herb, through axillary bud cultures

Summary Spilanthes mauritiana DC., (Compositae), a East African medicinal herb containing pharmaceutically promising secondary metabolites, has successfully been raised in vitro. We have developed a clonal propagation protocol that uses juvenile plants as starting material. The addition of benzylaminopurine (BA) (1.0 μM) and naphthaleneacetic acid (NAA) (0.1 μM) to the culture medium resulted in maximum shooting response with minimal callusing. Shoots rooted best in vitro in MS medium supplemented with indole-3-acetic acid (IAA; 0.2 μM), and plants that had already developed roots showed better growth, with maximum survival rate, in the greenhouse after an initial hardening.     

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

Summary A transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d⁻¹) and esculin content (18.5gl⁻¹), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.     

Molecular characterization and mapping of ALMT1, the aluminium-tolerance gene of bread wheat (Triticum aestivum L.).

The major aluminum (Al) tolerance gene in wheat ALMT1 confers. An Al-activated efflux of malate from root apices. We determined the genomic structure of the ALMT1 gene and found it consists of 6 exons interrupted by 5 introns. Sequencing a range of wheat genotypes identified 3 alleles for ALMT1, 1 of which was identical to the ALMT1 gene from an Aegilops tauschii accession. The ALMT1 gene was mapped to chromosome 4DL using 'Chinese Spring' deletion lines, and loss of ALMT1 coincided with the loss of both Al tolerance and Al-activated malate efflux. Aluminium tolerance in each of 5 different doubled-haploid populations was found to be conditioned by a single major gene. When ALMT1 was polymorphic between the parental lines, QTL and linkage analyses indicated that ALMT1 mapped to chromosome 4DL and cosegregated with Al tolerance. In 2 populations examined, Al tolerance also segregated with a greater capacity for Al-activated malate efflux. Aluminium tolerance was not associated with a particular coding allele for ALMT1, but was significantly correlated with the relative level of ALMT1 expression. These findings suggest that the Al tolerance in a diverse range of wheat genotypes is primarily conditioned by ALMT1.     

Indigenous salt-tolerant rhizobacterium <Emphasis Type="Italic">Pantoea dispersa</Emphasis> (PSB3) reduces sodium uptake and mitigates the effects of salt stress on growth and yield of chickpea

Salt stress has multiple damaging effects on plants including physiological damage, reduced growth, and productivity. Plant growth-promoting rhizobacteria (PGPR) are one of the valuable options to mitigate the negative effects of this stress. In the present study, native bacteria from chickpea’s rhizosphere were isolated, and checked for their salt tolerance and plant growth-promoting attributes (phosphate (P) solubilization, siderophores, indole-3-acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production). One isolate, subsequently identified as Pantoea dispersa, showed appreciable production of IAA (218.3 µg/ml) and siderophores (60.33% SU), P-solubilization (3.64 µg/ml) and ACC deaminase activity (207.45 nmol/mg/h) in the presence of 150 mM NaCl, under laboratory conditions. Salt stress in uninoculated chickpea (GPF2 cultivar) plants induced high accumulation of Na⁺ ions (3.86 mg g^?1 dw) in the leaves, along with significant reduction in K⁺ uptake, membrane integrity, chlorophyll concentration, and leaf water content, thus resulting in impaired growth of the plant and yield (pods and seeds) in a salt concentration-dependent manner. The damage due to salt stress was restored significantly in plants inoculated with P. dispersa. A significant improvement in biomass (32–34%), pods number (31–34.5%), seeds number (32–35.7%), pods weight (30–32.6%), and seeds weight (27–35%) per plant occurred in salt stress-affected plants, which was associated with significant reduction in Na⁺ uptake, reduced membrane damage, significantly improved leaf water content, chlorophyll content, and K⁺ uptake. This study suggests for the first time that native P. dispersa strain PSB3 can be used to alleviate the negative effects of salt stress on chickpea plants and holds the potential to be used as a biofertilizer.     

Biotransformation of tea catechins into theaflavins with immobilized polyphenol oxidase

Theaflavins, an active antioxidant, a natural pigment and pharmacologically active molecule obtained from black tea were bioprocessed on an immobilized tea polyphenol oxidase (PPO) system by the conversion of tea catechins extracted from green tea leaves with an overall conversion efficiency of 85% about 14-fold increase over maximum achievable in normal black teas. The immobilized enzyme (IE) system consists of activated cellulose matrix on to which the freshly extracted tea leaf polyphenol oxidase was covalently linked. Cellulose as a matrix of choice was considered primarily for its non-toxic nature, natural origin, low cost and easy availability. The kinetic parameters of the IE system were; protein loading capacity 11.8 mg/g; pH optimum 5.9; temperature optimum 37.5 °C; K_m 4.76 ± 0.08 mM; V_max 20 ± 1.80 nmol/min; enzyme activity retention (83.58%) and number of batches per turnover without loss of activity was 14. The product from IE system was identified by HPLC and ESI-QTOF-MS spectrometry.