Structure and mechanism of the lactose permease of Escherichia coli

Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.     

Development of A dormancy release technology: A review

Tuber dormancy can be released immediately in many commercially important potato cultivars by brief treatment (1-2 days) with bromoethane (BE) vapor at room temperature. The development of a large scale technology for BE application and safe removal through a capturing technique is necessary for successful application of this dormancy release method. Ideally, BE treatment of seed tubers would occur in a closed environment that would capture BE vapor in an unaltered form and allow controlled release for treatment of subsequent tuber lots. Results of screening studies for adsorbents indicate that the medium capacity activated carbon adsorbent, YAO has: i) a high capacity for BE; ii) a low capacity for water; and, iii) adsorbs and de-adsorbs BE quickly and easily. A plausible design of a large scale, dormancy release facility is presented. The proposed facility should meet present goals of the seed potato industry in an environmentally responsible manner.     

Assessment of bovine mammary chemokine gene expression in response to lipopolysaccharide, lipotechoic acid + peptidoglycan, and CpG oligodeoxynucleotide 2135

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-α and IL-1β. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 μg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-α, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.     

Adrenocorticotropic hormone stimulation tests in healthy foals from birth to 12 weeks of age

The purpose of this study was to investigate total baseline plasma cortisol and adrenocorticotropic hormone (ACTH) concentrations, and ACTH-stimulated cortisol concentrations in foals from birth to 12 wk of age. Plasma (baseline) cortisol and ACTH concentrations were measured in 13 healthy foals at birth and at 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 42, 56, and 84 d of age. Each foal received cosyntropin (0.1 μg/kg) intravenously. Plasma cortisol concentrations were measured before (baseline), and 30, and 60 min after cosyntropin administration at birth and at 3, 5, 7, 10, 14, 21, 28, 42, 56, and 84 d of age. Compared with baseline, cortisol concentration increased significantly 30 min after administration of cosyntropin on all days. Cortisol concentration was highest at birth, measured at 30 and 60 min after cosyntropin administration, compared with all other days. With the exception of birth measurements, cortisol concentration was significantly higher on day 84, measured at 30 and 60 min after cosyntropin administration, when compared with all other days. Baseline plasma ACTH was lowest at birth when compared with concentrations on days 2, 3, 4, 5, 7, 10, 14, 42, 56, and 84. Administration of 0.1 μg/kg of cosyntropin, IV, reliably induces cortisol secretion in healthy foals. Differences in the magnitude of response to cosyntropin are observed depending on the age of the foal. These data should serve as a reference for the ACTH stimulation test in foals and should be useful in subsequent studies to evaluate the hypothalamic-pituitary-adrenal axis in healthy and critically ill foals.     

Elimination kinetics of chlorhexidine in milk following intramammary infusion to stop lactation in mastitic mammary gland quarters of cows

OBJECTIVE: To evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in cows. DESIGN: Prospective study. ANIMALS: 6 cows. PROCEDURE: The study was performed in 2 phases. Three cows were studied in each phase. All cows were treated with chlorhexidine suspension by infusion into a mastitic mammary gland quarter after 2 milkings 24 hours apart. Foremilk samples (100 mL) were collected from treated and untreated (controls) mammary gland quarters of each cow. Chlorhexidine was extracted from raw milk, and residue concentrations were quantified by use of high-performance liquid chromatography. Foremilk samples from days 2, 5, and 8 were analyzed in phase I, and samples from time 0 and days 3, 7, 14, 21, 28, 35, and 42 were analyzed in phase II. RESULTS: In phases I and II, there was no quantifiable transference of chlorhexidine to milk in untreated mammary gland quarters. Measurable chlorhexidine residues were found in milk from treated mammary gland quarters of 2 cows throughout the 42-day sample period in phase II. Estimated mean elimination half-life for chlorhexidine in milk was 11.5 days. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the long elimination half-life of chlorhexidine in milk from treated mammary gland quarters, the lack of human dietary exposure data to suggest a food tolerance for chlorhexidine in food products, and the Food and Drug Administration's published zero tolerance for chlorhexidine in uncooked edible calf tissues, we do not recommend extralabel use of chlorhexidine suspension as a treatment to stop lactation in mastitic mammary gland quarters of cows.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

The efficacy of monepantel against naturally acquired inhibited and developing fourth-stage larvae of Teladorsagia circumcincta in sheep in the United Kingdom

The inhibition of Teladorsagia and other nematode genera at the early fourth-stage is a biological process that allows the parasites to survive in their host in a dormant state when prevailing conditions may otherwise kill them or prevent their progeny from surviving in the external environment. A study was conducted in Scotland to evaluate the efficacy of monepantel, an amino-acetonitrile derivative, against natural infections of inhibited fourth-stage Teladorsagia spp. larvae. At necropsy it was determined that the untreated control sheep were additionally infected with developing fourth-stage Teladorsagia spp. larvae and this is the first published evidence on the efficacy of monepantel against natural infections of this parasite and stage. The study sheep, which had grazed on naturally contaminated pastures since birth, were transferred to indoor housing after a subset of animals was examined to confirm the presence of inhibited larvae within the study population prior to the experiment. After 14 days of housing, monepantel was orally administered at 2.5 mg/kg to half of the animals. The sheep were necropsied seven days later and their parasite burdens recovered for the determination of efficacy, which was 99.7% for the inhibited stages and 99.3% for the developing fourth-stages. In conclusion, monepantel dosed orally at 2.5 mg/kg is a highly effective treatment against naturally acquired infections of inhibited and developing fourth-stage larvae of Teladorsagia spp.     

Prevalence and patterns of antimicrobial resistance of fecal Escherichia coli among pigs on 47 farrow-to-finish farms with different in-feed medication policies in Ontario and British Columbia

The main objectives of this study were to determine the prevalence and patterns of antimicrobial resistance in pigs on farms that medicated swine ration and those that did not. A total of 940 isolates of Escherichia coli from 188 pooled fecal samples obtained from weaner and finisher pigs on 47 farrow-to-finish swine farms (34 farms used in-feed medication and 13 did not) were tested for susceptibility to 21 antimicrobials using a breakpoint concentration method. The prevalence of resistance varied widely (0.0% to 81.3%) among the antimicrobials tested. Ninety percent of all the isolates tested were resistant to one or more antimicrobials. The most common multi-drug resistance patterns were to 2 to 6 antimicrobials. Resistance was significantly more frequent (P < 0.01) on farms that used in-feed medication compared to those that did not, and significantly more frequent (P < 0.01) in weaner pigs compared to finisher pigs. These findings indicate that resistance to a broad range of antimicrobials was prevalent among fecal E. coli isolates of pigs on study farms, and that this constitutes a potential reservoir for resistance genes that could spread to pathogens. The findings also provide further evidence that use of medication in swine rations provides selective pressure for antimicrobial resistance in E. coli in pigs.