alpha-conotoxins PnIA and [A10L] PnIA stabilize different states of the alpha 7-L247T nicotinic acetylcholine receptor

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [ A10L] PnIA and alanine scan derivatives of [ A10L] PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L] PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nM, respectively. Rates of onset of inhibition were similar for PnIA and [ A10L] PnIA; however, the rate of recovery was slower for [ A10L] PnIA, indicating that the increased potency of [ A10L] PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [ A10L] PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [ A10L] PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nM. In contrast, [ A10L] PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [ A10L] PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [ A10L] PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

Structure of the HERG K+ channel S5P extracellular linker - Role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp(585)-Ile(593) and Gly(604)-Tyr(611) of the channel. The Trp(585)-Ile(593) helix has distinct hydrophilic and hydrophobic surfaces. The Gly(604)-Tyr(611) helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.

Production of proteinases by psychrotrophic bacteria in raw milk stored at low temperature

Raw milk samples from two different sources were stored at 2degreesC, 4degreesC and 7degreesC for 10 days and the growth of psychrotrophic bacteria, production of proteinase and proteolysis in the milks were measured during storage. Peptide analyses by the fluorescamine method and RP-HPLC were used in determination of proteolysis and proteinase activity. The average times taken for the psychrotroph counts to reach 10(7) cfu/mL at 2degreesC, 4degreesC and 7degreesC were approximately 9, 7 and 4 days, although there was considerable variation in growth rates in the different milks. There was little correlation between psychrotroph counts and either proteolysis or proteinase activity levels. At 2degreesC, no milk stored showed significant proteolysis by the fluorescamine method after 10 days' storage, but significant proteinase activity could be measured in some of these milks at 8 and 10 days. RP-HPLC analysis was a more sensitive means of detecting peptides than the fluorescamine method.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

A comparison of the effects of propofol and thiopental on tear production in dogs

The tear film plays an important role in maintaining the integrity of the ocular suface. During general anesthesia, tear production is considerably reduced, which requires care to prevent adverse effects that result in diseases of these structures. Studies comparing the effects of induction of anaesthesia with thiopental and propofol on tear production have not been carried out yet. Because these drugs are used in veterinary medicine, we decided to evaluate the tear production in 30 dogs undergoing experimental surgery as well as routine procedures at the veterinary hospital of Federal University of Viçosa. Patients were divided into two groups of equal number. All animals were sedated with clorpromazine and maintained with isoflurane in diluted oxygen. Group 1 was induced with thiopental whereas group 2 with propofol. Schirmer tear test 1 was performed before sedation (T0), 15 minutes after sedation (T1) and 10 minutes after induction of anesthesia (T2) with the drug chosen for one of the groups. There was a significant decrease in tear production for both drugs, but no significant statistical differences were found between them. Thus, considering the results and the way in which the study was conducted, we suggest protecting the cornea and conjunctiva of patients during anesthesia using any of the drugs here evaluated.

Processing and characterization of alpha-elastin electrospun membranes

Elastin isolated from fresh bovine ligaments was dissolved in a mixture of 1,1,1,3,3,3-Hexafluoro-2-propanol and water and electrospun into fiber membranes under different processing conditions. Fiber mats of randomly and aligned fibers were obtained with fixed and rotating ground collectors and fibrils were composed by thin ribbons whose width depends on electrospinning conditions; fibrils with 721 nm up to 2.12 m width were achieved. After cross-linking with glutaraldehyde, -elastin can uptake as much as 1700 % of PBS solution and a slight increase on fiber thickness was observed. The glass transition temperature of electrospun fiber mats was found to occur at ~ 80 ºC. Moreover, -Elastin showed to be a perfect elastomeric material, and no mechanical hysteresis was found in cycle mechanical measurements. The elastic modulus obtained for oriented and random fibers mats in a PBS solution was 330 ± 10 kPa and 732 ± 165 kPa, respectively. Finally, the electrospinning and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Cell culture results showed good cell adhesion and proliferation in the cross-linked elastin fiber mats.

Influence of the N/K ratio on the production and quality of cucumber in hydroponic system

The objective of this work was to evaluate the quality of fruits and the nutritional status of cucumber CV. Aodai cultivated in nutrient solutions with different N:K ratios. The hydroponic cultivation was initially performed, during the vegetative growth, in nutrient solution with 1:2.0 mmol L-1 N:K, and, later, during fruit setting, in four different nutrient solutions with N:K (w/w) at the ratios 1:1.4, 1:1.7, 1:2.0 and 1:2.5. An additional treatment with a nutrient solution containing the ratio 1:2.2 (w/w) N:K during the vegetative growth and N:K 1:1.4 (w/w) during fruit setting, both with 10% ammonium (NH4+) was included. The treatments were arranged in a randomized design with six replicates. Irrigation was carried out with deionized water until seed germination, and then with nutrient solution until 30 days after germination, when plants were transplanted. Plants in the hydroponic growing beds were irrigated with the solutions for vegetative growth, and, after 21 days, the solutions were replaced by solutions for fruit setting. At 45 and 60 days after transplanting, the fresh weight, length, diameter, volume and firmness of the fruit were evaluated, and, at 45 days after transplanting, the macronutrient concentrations in the leaves were determined. The use of different N:K ratios during fruit setting influenced the cucumber production. The ratio of 1.0:1.7 N: K (w/w), with 10% of N in the form of ammonia, is recommended for the whole cycle.

Production of cabbage grown in pots containing legumes' root and shoot

Roots effect is not generally considered in studies assessing the performance of crops in response to green manuring. However, such effect can contribute to a better understanding of crop rotation. The aim of this study was to assess the effect of root and shoot of two legumes on the production of cabbage. The experiment was conducted in pots of 10 liters containing substrate of 2:1 soil/sand. The experiment was arranged in a factorial scheme (2x3 + 2) in a randomized block design with five replicates using two legume species (Crotalaria juncea L. and Canavalia ensiformis L), three plant parts (root, shoot, or whole plant), and two additional treatments (mineral fertilization with 100% and 50% of the recommended dose of N for growing cabbage). Pots with legume treatments received mineral fertilizer with 50% of the recommended dose of N for growing cabbage. The experimental plot consisted of a pot containing one plant of cabbage. Legumes were grown in pots and harvested at 78 days. The root biomass was determined in extra pots. Production was assessed using head fresh and dry weight. The application of the whole plant of both legume species reduced cabbage production. However, root or shoot of both legume species was equivalent to 50% of mineral N fertilization required for the cultivation of cabbage.

Production of basic potato seed minitubers in substrate and different nitrogen rates

The optimal dose of nitrogen (N) in potato crop depends on the production system. The objective of this study was to determine the optimal dose of N for the production of basic potato seed minitubers and evaluate the effect of N rates on physiological and nitrogen indices in the youngest fully developed leaf (fourth leaf) and in the oldest leaf of the plants at 60 days after planting. The experiment was conducted in a greenhouse at the Departamento de Fitotecnia da Universidade Federal de Viçosa. The treatments consisted of five N rates (0, 45, 90, 180 and 360 mg dm-3), with 10% of each dose applied at planting and the remainder through irrigation water, daily, for 30 days. The nitrogen rates positively influenced the physiological indices (length, width, leaf area, number of leaves, fresh mass and dry mass) and nitrogen (level and content of N and N-NO³ in the dry mass and SPAD) both in the fourth leaf and in the oldest leaf. Likewise, the N rates positively influenced the number and mass of harvested tubers. The largest number (5.44 tubers/plant) and the maximum mass of tubers (243.5 g/plant) were obtained with 360.0 and 332.9 mg N dm-3, respectively. Therefore, the mass and number of tubers were not optimized by the same N rate. The critical SPAD index was 38.8 in the fourth leaf, which was more sensitive to the effect of N rates than the oldest leaf.

Comparison of pupil diameter and tear production in dogs treated with acepromazine, tramadol and their combination

Some ophthalmic surgeries require induction of mydriasis, however, drugs traditionally used for this purpose significantly reduces tear production. To evaluate the effect of acepromazine and tramadol, used alone or in combination, on pupil diameter, tear production, heart and respiratory rate, systolic blood pressure and rectal temperature, these drugs were administered to seven clinically normal dogs divided into three experimental groups (G1 - acepromazine; G2 - tramadol; G3 - tramadol + acepromazine) that differed only in the sedation protocol. Parameters were measured in four experimental moments. Miosis occurred in G1, in addition to reduced tear production and respiratory rate. No significant changes were found in the parameters assessed in G2, whereas in G3, there was decrease in tear production of the right eye, decrease in the respiratory rate and rectal temperature. Tramadol proved to be a drug suitable for pre-anesthetic procedures that require the maintenance of pupil diameter and keeps the tear production within normal parameters. However, the use of acepromazine alone or in combination with tramadol requires protection of the patient's eye surface to prevent the occurrence of undesirable ophthalmic changes.

Effect of N and K doses in nutritive solution on growth, production and coffee bean size

An adequate supply of nutrients is essential for obtaining high yields of coffee. The objective of this study was to evaluate the effect of N, K and the N:K ratio on vegetative and reproductive growth of coffee. For this purpose, coffee plants were grown in nutrient solution containing K in the concentrations of 1.08; 2.15; 3.23 and 5.38 mmol L-1 combined with a dose of 6 mmol L-1 N, resulting in the N:K ratios (w/w): 1:0.5; 1:1; 1:1.5 and 1:2.5. The control treatment consisted of the doses 3 and 1.61 mmol L-1 of N and K respectively, resulting in the N:K ratio (w/w) 1.0:1.5. The following variables were evaluated: height, stem diameter, number of nodes of the eighth plagiotrofic branch (index branch), pairs of plagiotrofic branches and number of nodes in the orthotropic branch every three weeks from the beginning of the experiment. Additionally, it was evaluated the chemical composition of processed beans and leaves between the flowering and the rapid expansion stage of the cherry beans, production of cherry beans per plant and classification of beans according to the size. N influenced mainly the characteristics of vegetative growth and K influenced mainly the reproductive growth evaluated by the production. The lowest production resulted in the highest percentages of beans retained on sieves with holes larger than 16/64", while the highest production promoted an increase in the percentage of beans retained on sieves with holes smaller than 16/64".

Zinc supplementation, production and quality of coffee beans

Besides its importance in the coffee tree nutrition, there is almost no information relating zinc nutrition and bean quality. This work evaluated the effect of zinc on the coffee yield and bean quality. The experiment was conducted with Coffea arabica L. in "Zona da Mata" region, Minas Gerais, Brazil. Twelve plots were established at random with 4 competitive plants each. Treatments included plants supplemented with zinc (eight plots) and control without zinc supplementation (four plots). Plants were subjected to two treatments: zinc supplementation and control. Yield, number of defective beans, beans attacked by berry borers, bean size, cup quality, beans zinc concentration, potassium leaching, electrical conductivity, color index, total tritable acidity, pH, chlorogenic acids contents and ferric-reducing antioxidant activity of beans were evaluated. Zinc positively affected quality of coffee beans, which presented lower percentage of medium and small beans, lower berry borer incidence, lower potassium leaching and electrical conductivity, higher contents of zinc and chlorogenic acids and higher antioxidant activity in comparison with control beans.

Effect of cattle manure on sunflower production and water use in two types of soil

The aim of the present study was to evaluate water consumption, use efficiency and yield components of sunflower variety Embrapa 122 V/2000 cultivated in two types of soil (Fluvissol and Haplic Luvisol) subjected to increasing doses of cattle manure. The experiment was carried out in a greenhouse at Universidade Estadual da Paraíba. The experimental design was completely randomized in a factorial scheme. The irrigation was performed every other day, replacing the water absorbed by the plants. The water consumption and the use efficiency were evaluated, being the use efficiency determined by the ratio of the total dry mass of sunflower and the amount of water used to produce it in each treatment. Plants were harvested at 95 days after sowing when the following parameters were evaluated: number of seeds per plant, weight of seeds per plant, weight of 1000 seeds and the outer diameter of the capitulum (head). The results showed that the sunflower was positively affected by cattle manure application, increasing the production components and the water use efficiency, regardless of the type of soil. Excepting for the 1000 seeds weight and the water use efficiency, the type of soil affected significantly the water use, the number and weight of seeds per plant. The plants cultivated in Haplic Luvisol had a better performance.

Efficiency and response of conilon coffee clones to phosphorus fertilization

Studies on nutritional efficiency of phosphorus in conilon coffee plants are important tools to unravel the high limitation that natural low levels of this nutrient in soil impose to these species cultivars. Therefore, this study aimed at evaluating the nutritional efficiency and the response to phosphorus of conilon coffee clones. Plants were managed during 150 days in pots containing 10 dm³ of soil, in greenhouse. A factorial scheme 13 x 2 was used, with three replications, being the factors: 13 clones constituting the clonal cultivar "Vitória Incaper 8142" and two levels of phosphate fertilization (0% and 150% of the P2O5 usualy recommended), in a completely randomized design (CRD). The results indicate a differentiated response of dry matter production and of phosphorus content on each level of phosphate fertilization for the conilon coffee clones and that CV-04, CV-05 and CV-08 clones are nutritionally efficient and responsive to the phosphate fertilization.