A Burkholderia pseudomallei toxin inhibits helicase activity of translation factor eIF4A

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.     

Proteoglycan-specific molecular switch for RPTPσ clustering and neuronal extension

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.     

N-linked glycan profiling of mature human milk by high-performance microfluidic chip liquid chromatography time-of-flight tandem mass spectrometry

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.     

Microbial community composition and carbon cycling within soil microenvironments of conventional, low-input, and organic cropping systems

This study coupled stable isotope probing with phospholipid fatty acid analysis (¹³C-PLFA) to describe the role of microbial community composition in the short-term processing (i.e., C incorporation into microbial biomass and/or deposition or respiration of C) of root- versus residue-C and, ultimately, in long-term C sequestration in conventional (annual synthetic fertilizer applications), low-input (synthetic fertilizer and cover crop applied in alternating years), and organic (annual composted manure and cover crop additions) maize-tomato (Zea mays - Lycopersicum esculentum) cropping systems. During the maize growing season, we traced ¹³C-labeled hairy vetch (Vicia dasycarpa) roots and residues into PLFAs extracted from soil microaggregates (53-250 μm) and silt-and-clay (<53 μm) particles. Total PLFA biomass was greatest in the organic (41.4 nmol g⁻¹ soil) and similar between the conventional and low-input systems (31.0 and 30.1 nmol g⁻¹ soil, respectively), with Gram-positive bacterial PLFA dominating the microbial communities in all systems. Although total PLFA-C derived from roots was over four times greater than from residues, relative distributions (mol%) of root- and residue-derived C into the microbial communities were not different among the three cropping systems. Additionally, neither the PLFA profiles nor the amount of root- and residue-C incorporation into the PLFAs of the microaggregates were consistently different when compared with the silt-and-clay particles. More fungal PLFA-C was measured, however, in microaggregates compared with silt-and-clay. The lack of differences between the mol% within the microbial communities of the cropping systems and between the PLFA-C in the microaggregates and the silt-and-clay may have been due to (i) insufficient differences in quality between roots and residues and/or (ii) the high N availability in these N-fertilized cropping systems that augmented the abilities of the microbial communities to process a wide range of substrate qualities. The main implications of this study are that (i) the greater short-term microbial processing of root- than residue-C can be a mechanistic explanation for the higher relative retention of root- over residue-C, but microbial community composition did not influence long-term C sequestration trends in the three cropping systems and (ii) in spite of the similarity between the microbial community profiles of the microaggregates and the silt-and-clay, more C was processed in the microaggregates by fungi, suggesting that the microaggregate is a relatively unique microenvironment for fungal activity.     

Sustainable Management of Landfill Leachate by Irrigation

Leachate from domestic landfills is a significant environmentalhazard. In the urban environment, irrigation of recreational turf and parkland with nitrogen-rich landfill leachate providesboth low-cost treatment that minimises pollution of surroundingwaters and a valuable water resource. Of particular interest isthe capacity of the turf-soil system to ameliorate the ammonium-rich leachate. To address this issue, a two-year field trial was completed at the Newington Landfill irrigating with saline,ammonium-rich leachate. The field trial suggested that in situ bioremediation is sustainable provided that management strategies such as dilution of leachate to reduce solution electrical conductivity to 3.6 dS m^-1 are adopted. Furthermore, pollution due to leaching of nitrogen can be minimised by managing the soil to enhance in situ denitrification of applied nitrogen. The management regimes adopted during the Newington field trial enabled nitrogen application rates in excess of 1400 kg NH₄ ⁺ha^-1 yr^-1. However, the capacity of the system to ameliorate the leachate appears limited by soil salinity and sodicity rather than the control of nitrogen leaching by denitrification,suggesting that rates of up to 3500 kg NH₄ ⁺ha^-1 yr^-1 may be viable if the salinity hazard can be effectively managed.     

Mercury Concentrations in Largemouth BASS (Micropterus Salmoides) from Five South Carolina Reservoirs

Mercury concentrations in largemouth bass (Micropterus salmoides) bass were compared among five reservoirs in South Carolina. Three of these reservoirs (Lake Russell, Lake Thurmond, and Lake Marion) are accessible to the public and two (L-Lake and Par Pond) are located on the U.S. Department of Energy's Savannah River Site (SRS), which is closed to public access. Age-adjusted mercury concentrations were significantly higher in SRS bass compared to the offsite reservoirs. In addition, mercury concentrations were significantly higher in bass from Par Pond compared to L-Lake and in bass from Lakes Russell and Thurmond compared to Lake Marion. No mercury concentrations in excess of the U.S. Food and Drug Administration action level (1.0 mg?kg^?1) were found in any bass from the public-accessible reservoirs. However, the majority of fish from these reservoirs had mercury concentrations that fall into or exceed the U.S. Environmental Protection Agency consumption category of “no more than one per week". In addition, most fish from these reservoirs had mercury levels in excess of those believed to be detrimental to sensitive avian species.     

Nutritive evaluation of the seed,germinated seed,and string bean of Erythrina americana and the detoxification of the material by boiling

The nutritive and toxicological values of the dry seeds, germinated seeds, and string beans of Erythrina americana were studied using raw and boiled samples. Raw germinated seeds had a higher protein content and lower fiber content than dry seeds. The whole string bean had lower protein content and higher fiber content. However, the seeds of the green pod showed the same protein concentration as the dry seeds (dry basis). Boiling and elimination of broth was beneficial in diminishing the alkaloid concentration in all the samples. The trypsin inhibitors, lectins, and tannins were also diminished as was expected. The raw string bean showed the lowest LD(50). Although the total essential amino acids content of the boiled germinated seeds was increased, the quality of protein, protein efficiency ratio (PER), was lower than in boiled dry seeds, and in these, the PER was similar to the control (casein). The present results suggest that for the protein quality and low alkaloid content, the boiled dry seeds and string beans could be used for animal feeding. It could be interesting to test the raw string beans in ruminants since in this stage E. americana showed the lowest toxicity.     

Pathogenesis and clinical signs of equine herpesvirus-1 in experimentally infected ponies in vivo.

Equine herpesvirus-1 (EHV-1) causes respiratory disease, neonatal death, abortion and neurologic disease. The main purpose of this study was to identify viral antigen in respiratory tract samples by immunoperoxidase staining. Six pony foals were selected on the basis of demonstrating seronegativity to EHV-1 by virus neutralization and housed in isolation. They were infected experimentally by administering EHV-1 nebulized ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals had febrile responses, nasal discharge, and enlarged submandibular lymph nodes. Sporadic coughing was also heard. EHV-1 was isolated from nasopharyngeal swabs of 4/6 ponies and seroconversion was demonstrated in all foals. Bronchoscopic examination of the large airways revealed hyperemia. The incidence of recovery of Actinobacillus suis from nasopharyngeal swabs increased initially, with recovery of Streptococcus zooepidemicus isolates predominating at 3 wk post-infection. Cytology brushes were used to sequentially sample the respiratory tract of the infected ponies at the nasopharynx, mid-trachea and the mainstem bronchus. Bronchoalveolar lavage provided lung cells. Immunocytochemistry techniques were applied to both types of samples to locate EHV-1 antigen. Indirect immunoperoxidase staining of samples utilizing monoclonal antibodies specific for EHV-1 demonstrated viral antigen associated with cellular debris, primarily in the nasopharyngeal samples on days 3-9 post-infection.     

Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis

Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.     

Review of methodologies and a protocol for the <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of wheat

Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.