Dioxygen binds end-on to mononuclear copper in a precatalytic enzyme complex

Copper active sites play a major role in enzymatic activation of dioxygen. We trapped the copper-dioxygen complex in the enzyme peptidylglycine-alphahydroxylating monooxygenase (PHM) by freezing protein crystals that had been soaked with a slow substrate and ascorbate in the presence of oxygen. The x-ray crystal structure of this precatalytic complex, determined to 1.85-angstrom resolution, shows that oxygen binds to one of the coppers in the enzyme with an end-on geometry. Given this structure, it is likely that dioxygen is directly involved in the electron transfer and hydrogen abstraction steps of the PHM reaction. These insights may apply to other copper oxygen-activating enzymes, such as dopamine beta-monooxygenase, and to the design of biomimetic complexes.     

Characterization and plant expression of a glyphosate-tolerant enolpyruvylshikimate phosphate synthase

BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials. CONCLUSION: These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.     

Infiltrative lipoma in three dogs

Infiltrative lipoma was diagnosed in 3 dogs. The invasive clinical and benign microscopic characteristics of the lipomas were considered in relation to other adipose tissue tumors. Treatment consisted of wide excision or surgical debulking when tumor margins were indistinct. In spite of the aggressive appearance of the lipoma, its benign nature dictated a conservative course of surgical therapy. Accurate and early diagnosis was needed to ensure adequate excision. The tumor recurred if excision was incomplete.     

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.     

Species-specific PCR assay for the detection of Babesia odocoilei

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.     

Quantification of benzoxazinone derivatives in wheat (Triticum aestivum) varieties grown under contrasting conditions in Denmark

Three varieties of winter wheat (Triticum aestivum) were grown in both conventional and organic farming systems. The contents of the benzoxazinone derivatives 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), 2-beta-d-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc), 6-methoxybenzoxazolin-2-one (MBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA), benzoxazolin-2-one (BOA), and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) were analyzed at five growth stages (BBCH 9-10, 12, 21, 31, and 53). Major differences were found between the varieties, with Stakado exhibiting the highest contents. In contrast, only minor and erratic differences were found between the two farming systems, suggesting that the inherent differences in the content of benzoxazinone derivatives of the varieties were not significantly affected by the use of pesticides and synthetic fertilizers. The concentration of benzoxazinone derivatives in the foliage was considerably higher at the early growth stages than later in the growing season, with DIMBOA being the most abundant of the benzoxazinone derivatives. An increase in the concentration was observed in early spring compared to late autumn, suggesting that plants synthesized benzoxazinone derivatives at the commencement of growth in early spring. The concentrations in the roots were considerably lower than in the foliage at the early growth stages but remained relatively constant over time, resulting in a higher concentration than in the foliage at the late growth stages. The results are discussed in relation to previous findings that predominantly originate from experiments done under controlled conditions in either growth cabinets or greenhouses.     

Herbicidal effects of soil-incorporated wheat

The hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and the benzoxazolinones benzoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-2-one (MBOA) have been identified as important allelochemicals in wheat. This study examines the possibility of exploiting the allelopathic properties of wheat as a weed control strategy by cultivating wheat as a precrop and incorporating plant residues into the soil before the next crop is sown. Different wheat varieties were cultivated in field plots during two seasons in both conventional and organic farming systems. Plants were sampled at various growth stages, and their contents of DIMBOA, MBOA, and BOA were determined by chemical analyses. The wheat samples were incorporated into soil, and the effect on germination and growth of 12 different weed species was examined in pot experiments under controlled conditions. In some cases significant effects were obtained, but the results were inconsistent and the effects were not correlated to the content of DIMBOA, MBOA, and BOA in the incorporated wheat plants. ED50 doses of the pure compounds were estimated in dose-response experiments in Petri dishes, and these turned out to be much higher than the predicted maximum concentrations of DIMBOA, MBOA, and BOA in the soil water following incorporation. The study shows that a prerequisite for exploiting the incorporation of wheat residues as a weed control strategy is the development of wheat varieties with an increased content of allelochemicals.     

THE INFLUENCE OF DATE OF SOWING AND OF STRAIN ON HEAD PRODUCTION IN TIMOTHY

Heading behaviour of eight strains of timothy was observed in the sowing and first harvest years, after sowing on a series of dates between March and August.
All strains produced heads in the sowing year. Only a very few plants failed to head when sown between 18 March and 29 April. With subsequent sowing dates the proportion of heading plants progressively declined, most markedly in S.50 Phleum nodosum , least in the early-heading P. pratense strains. No heading occurred in the sowing year after the mid-August sowing.
The environment to which the later sowings are exposed differs from that of the earlier sown lots in that (a) the initial temperatures and daylengths are higher and (b) the decline in daylength occurs earlier in their development. One or both of these factors must be responsible for failure of heading among plants of the later sowings.
The sequence of heading dates among the strains in the sowing year differed considerably from that in the same plants after overwintering, suggesting the action of some factor other than photo-period in determining heading date in the sowing year.
Variability in heading date within the strains was much greater in the sowing year than after overwintering, and increased with lateness of sowing.