Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-kappaB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.     

The effect of sugars on niger embryogenesis and plant regeneration in anther culture

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05–0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg’s B5 medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 μM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 μM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 μM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.     

Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels.     

Selection of Daucus carota somatic hybrids using drug resistance markers and characterization of their mitochondrial genomes

Summary Protoplasts from different Daucus carota L. cell strains carrying resistance to glyphosate, 5-methyltryptophan, sodium selenate or selenocystine were fused in three combinations using dextran. Clones were selected for both of the resistances carried by the individual parental strains in medium with both inhibitors. No doubly resistant colonies formed from unfused controls or from protoplasts from each individual parental strain alone. Suspension cultures from the selected clones contained predominantly the additive chromosome numbers of the parental strains. Apparently the four resistances used are expressed dominantly in fusion hybrids. Analysis of mitochondrial DNA showed that recombination occurred in one fusion combination since the mitochondrial DNA in the hybrid cells was different from that of either parent as shown by restriction endonuclease fragment analysis. Mitochondrial DNA in the other two somatic hybrid combinations was parental. Thus, a dominant, nuclear resistance marker system has been developed to select efficiently for somatic hybrids in which mitochondrial DNA recombination can be studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - Glp Glyphosate - mt mitochondrial - MX Murashige and Skoog (1962) medium containing 0.4 mg/l 2,4-D - 5MT 5-methyltryptophan - MXG MX containing (5% Glucose) - SC selenocystine - SS sodium selenate     

Contribution of the VA mycorrhizal hyphae in acquisition of phosphorus and zinc by maize grown in a calcareous soil

An investigation was carried out to test whether the mechanism of increased zinc (Zn) uptake by mycorrhizal plants is similar to that of increased phosphorus (P) acquisition. Maize (Zea mays L.) was grown in pots containing sterilised calcareous soil either inoculated with a mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappe or with a mixture of mycorrhizal fungi, or remaining non-inoculated as non-mycorrhizal control. The pots had three compartments, a central one for root growth and two outer ones for hyphal growth. The compartmentalization was done using a 30-m nylon net. The root compartment received low or high levels of P (50 or 100 mg kg^–1 soil) in combination with low or high levels of P and micronutrients (2 or 10 mg kg^–1 Fe, Zn and Cu) in the hyphal compartments.Mycorrhizal fungus inoculation did not influence shoot dry weight, but reduced root dry weight when low P levels were supplied to the root compartment. Irrespective of the P levels in the root compartment, shoots and roots of mycorrhizal plants had on average 95 and 115% higher P concentrations, and 164 and 22% higher Zn concentrations, respectively, compared to non-mycorrhizal plants. These higher concentrations could be attributed to a substantial translocation of P and Zn from hyphal compartments to the plant via the mycorrhizal hyphae. Mycorrhizal inoculation also enhanced copper concentration in roots (135%) but not in shoots. In contrast, manganese (Mn) concentrations in shoots and roots of mycorrhizal plants were distinctly lower, especially in plants inoculated with the mixture of mycorrhizal fungi.The results demonstrate that VA mycorrhizal hyphae uptake and translocation to the host is an important component of increased acquisition of P and Zn by mycorrhizal plants. The minimal hyphae contribution (delivery by the hyphae from the outer compartments) to the total plant acquisition ranged from 13 to 20% for P and from 16 to 25% for Zn.     

Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.)

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.     

Aspartate aminotransferase activity during early development of chicken embryo.

Aspartate aminotransferase (AAT) activity is studied, employing two different procedures, during early development stages of chicken embryo. ATT activity is steady from pre-streak to the definitive primitive streak stage after which it suddenly increases as growth proceeds. INH or IIH administration in this embryonic system led to almost instantaneous and complete disappearance of AAT activity which could be reversed to 80 percent by treatment with equimolar pyridoxal phosphate. Histochemical studies from the literature support a view that the period of intense differentiation coincides with an increased RNA content. The present study shows more AAT activity per mg wet embryo during the same development stages. Whether this is due to availability of more aspartic acid for pyrimidine is not clear due to possible presence of two AAT activities, the many competing reactions that can use aspartic acid, and in situ conditions during differentiation.     

Comparative studies of active site-ligand interactions among various recombinant constructs of human beta-amyloid precursor protein cleaving enzyme

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.     

Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.     

Volvariella-treated Eucalyptus sawdust stimulates wheat and onion growth

Conversion of sawdust, a major waste generated by the timber industry, to an agricultural supplement was explored. Beds made with a mixture of Eucalyptus sawdust and soil reduced the chlorophyll content and the growth rate of wheat seedlings. However, following treatment with Volvariella volvaceae and urea, the sawdust in growth beds enhanced both the chlorophyll content and growth of wheat seedlings. Application of treated sawdust to soil increased the biomass of both bulb and foliage of onion. This represents a small beginning in the conversion of an industrial solid waste into a soil conditioner for enhancing agricultural productivity.