Phosphatidylethanolamine methyltransferase: evidence for influence of diet fat on selectivity of substrate for methylation in rat brain synaptic plasma membranes

Male weanling rats were fed diets containing 20% (w/w) fat differing in fatty acid composition for 24 days. Synaptic plasma membranes were isolated from the brain and the fatty acid composition of phosphatidylethanolamine and phosphatidylcholine was determined. In vitro assays of phosphatidylethanolamine methyl-transferase activity were performed on fresh membrane samples to assess effect of dietary fat on the rate of phosphatidylethanolamine methylation for phosphatidylcholine synthesis via the phosphatidylethanolamine methyltransferase pathway. Dietary level of n-6 and ratio of n-6 to n-3 fatty acids influenced membrane phospholipid fatty acid composition and activity of the lipid-dependent phosphatidylethanolamine methyltransferase pathway. Rats fed a diet rich in n-6 fatty acids produced a high ratio of n-6/n-3 fatty acids in synaptosomal membrane phosphatidylethanolamine, and elevated rates of methylation of phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine methyltransferases, suggesting that the pathway exhibits substrate selectivity for individual species of phosphatidylethanolamine containing long-chain homologues of dietary n-6 and n-3 fatty acids (20:4(n-6), 22:4(n-6), 22:5(n-6) and 22:6(n-3). It may be concluded that diet alters the membrane content of n-6, n-3 and monounsaturated fatty acids, and that change in phosphatidylethanolamine species available for methylation to phosphatidylcholine alters the rate of product synthesis in vivo by the phosphatidylethanolamine methyltransferase pathway.     

Streptococcus mutans, an assessment of its physiological potential in relation to dental caries.

Streptococcus mutans converts low levels of sucrose to lactic acid, but at high levels favours synthesis of glucans for plaque accumulation. Thus, the continued exposure to sucrose fluxes would select microorganisms in the oral cavity (S. mutans being a prototype) with highly specialized adaptation and potential dental caries activity. The bacteria that have evolved physiological systems to function efficiently under these conditions are the lactic acid bacteria. These organisms survive in environments where carbohydrate availability is constantly changing. High tolerances to acidic environments may be an important determinant in establishing the ecology of the carious lesion. Also, the intercellular polysaccharide storgae (glycogenamylopectin) and extracellular polymer reserves (levan and soluble glucan) are important during carbohydrate depletion. Further, the formation of insoluble glucans is a prerequisite for the caries process on smooth surfaces of teeth through plaque development. These conditions could result in an increase in S. mutans and cariogenic microorganisms. As a result, this process may be best understood as a manifestation of an amphibiotic shift.     

Sourcing faecal pollution from onsite wastewater treatment systems in surface waters using antibiotic resistance analysis

AIMS: To identify the sources of faecal contamination in investigated surface waters and to determine the significance of onsite wastewater treatment systems (OWTS) as a major contributor to faecal contamination. METHODS AND RESULTS: Antibiotic resistance patterns (ARP) were established for a library of 717 known Escherichia coli source isolates obtained from human, domesticated animals, livestock and wild sources. Eight commonly used antibiotics, including amoxicillin, cephalothin, erythromycin, gentamicin, ofloxacin, chlortetracycline, tetracycline and moxalactam, at four different concentrations were used to obtain ARPs for E. coli isolates. Discriminant analysis (DA) was used to differentiate between the ARP of sources isolates. The developed ARP library was found to be adequate for discriminating human from nonhuman isolates, and was used to classify 256 enumerated E. coli isolates collected from monitored surface water locations. CONCLUSIONS: The resulting ARP DA indicated that a majority of the faecal contamination in more rural areas was nonhuman; however, the percentage of human isolates increased significantly in urbanized areas using OWTS for wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study signifies the feasibility of using ARP for source tracking faecal contamination in surface waters, and linking faecal contamination to OWTS. The information will enable regulatory authorities to implement appropriate management practices to reduce the contamination of water resources caused by high densities and failing OWTS.     

Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay

The recent discovery of several nonpeptide CGRP antagonists have led to significant advances in our understanding of CGRP receptor pharmacology. Specifically, these antagonists have demonstrated a clear species selectivity with >100-fold greater affinity for human CGRP receptor compared to receptors from other species, such as rat, rabbit and guinea pig. Therefore, nonhuman primate models are required to accurately assess the in vivo activity of these antagonists. The commonly used model in marmosets involves electrical stimulation of the trigeminal ganglia and is a technically difficult and terminal procedure. In this report, we describe a noninvasive pharmacodynamic model in which topical application of capsaicin is utilized to induce the release of endogenous CGRP and a vasodilatory response which can be measured using laser Doppler imaging. Using the potent and selective CGRP antagonist Compound 3, which is an analog of the well-characterized compound BIBN4096BS, we demonstrated 62% inhibition with 300 microg/kg, i.v., in the rat. When tested in the rhesus monkey, only 30 microg/kg of Compound 3 was needed to produce complete inhibition, suggesting that the rhesus CGRP receptor shares a pharmacological profile similar to marmoset and human receptors. Two separate measurements were obtained in this model to provide an indication of both the acute inhibitory effect as well as the prophylactic effect of the CGRP antagonist. At the doses studied, Compound 3 was equally effective on both the acute and prophylactic inhibition of CGRP-mediated vasodilation in rat and rhesus. In conclusion, this is the first report to describe and validate a noninvasive model in nonhuman primates that allows rapid evaluation of CGRP antagonist activity against endogenous CGRP.     

Do floral syndromes predict specialization in plant pollination systems? An experimental test in an “ornithophilous” African<Emphasis Type="Italic"> Protea</Emphasis>

We investigated whether the ornithophilous floral syndrome exhibited in an African sugarbush, Protea roupelliae (Proteaceae), reflects ecological specialization for bird-pollination. A breeding system experiment established that the species is self-compatible, but dependent on visits by pollinators for seed set. The cup-shaped inflorescences were visited by a wide range of insect and bird species; however inflorescences from which birds, but not insects, were excluded by wire cages set few seeds relative to open-pollinated controls. One species, the malachite sunbird (Nectarinia famosa), accounted for more than 80% of all birds captured in P. roupelliae stands and carried the largest protea pollen loads. A single visit by this sunbird species was enough to increase seed set considerably over unvisited, bagged inflorescences. Our results show that P. roupelliae is largely dependent on birds for pollination, and thus confirm the utility of floral syndromes for generating hypotheses about the ecology of pollination systems.     

Inter- and intramolecular determinants of the specificity of single-stranded DNA binding and cleavage by the F factor relaxase

The TraI protein of conjugative plasmid F factor binds and cleaves a single-stranded region of the plasmid prior to transfer to a recipient. TraI36, an N-terminal TraI fragment, binds ssDNA with a subnanomolar K(D) and remarkable sequence specificity. The structure of the TraI36 Y16F variant bound to ssDNA reveals specificity determinants, including a ssDNA intramolecular 3 base interaction and two pockets within the protein's binding cleft that accommodate bases in a knob-into-hole fashion. Mutagenesis results underscore the intricate design of the binding site, with the greatest effects resulting from substitutions for residues that both contact ssDNA and stabilize protein structure. The active site architecture suggests that the bound divalent cation, which is essential for catalysis, both positions the DNA by liganding two oxygens of the scissile phosphate and increases the partial positive charge on the phosphorus to enhance nucleophilic attack.     

Soil environmental variables affecting the flux of methane from a range of forest, moorland and agricultural soils

Measurements of the net methane exchange over a range of forest, moorland, and agricultural soils in Scotland were made during the period April to June 1994 and 1995. Fluxes of CH₄ ranged from oxidation –12.3 to an emission of 6.8 ng m^–2 s^–1. The balance between CH₄ oxidation and emission depended on the physical conditions of the soil, primarily soil moisture. The largest oxidation rates were found in the mineral forest soils, and CH₄ emission was observed in several peat soils. The smallest oxidation rate was observed in an agricultural soil. The relationship between CH₄ flux and soil moisture observed in peats (Flux_{CH 4} = 0.023 × %H₂O (dry weight) – 7.44, p > 0.05) was such that CH₄ oxidation was observed at soil moistures less than 325%( ± 80%). CH₄ emission was found at soil moistures exceeding this value. A large range of CH₄ oxidation rates were observed over a small soil moisture range in the mineral soils. CH₄ oxidation in mineral soils was negatively correlated with soil bulk density (Flux_{CH 4} = –37.35 × bulk density (g cm^–3) + 48.83, p > 0.05). Increased nitrogen loading of the soil due to N fixation, atmospheric deposition of N, and fertilisation, were consistently associated with decreases in the soil sink for CH₄, typically in the range 50 to 80%, on a range of soil types and land uses.