Clean conversion of cellulose into fermentable glucose

We studied the process of conversion of microcrystalline-cellulose into fermentable glucose in the formic acid reaction system using cross polarization/magic angle spinning ¹³C-nuclear magnetic resonance, X-ray diffraction and Fourier transform infrared spectroscopy. The results indicated that formic acid as an active agent was able to effectively penetrate into the interior space of the cellulose molecules, thus collapsing the rigid crystalline structure and allowing hydrolysis to occur easily in the amorphous zone as well as in the crystalline zone. The microcrystalline-cellulose was hydrolyzed using formic acid and 4% hydrochloric acid under mild conditions. The effects of hydrochloric acid concentration, the ratio of solid to liquid, temperature (55–75 °C) and retention time (0–9 h), and the concentration of glucose were analyzed. The hydrolysis velocities of microcrystalline-cellulose were 6.14 × 10^− 3 h^− 1 at 55 °C, 2.94 × 10^− 2 h^− 1 at 65 °C, and 6.84 × 10^− 2 h^− 1 at 75 °C. The degradation velocities of glucose were 0.01 h^− 1 at 55 °C, 0.14 h^− 1 at 65 °C, 0.34 h^− 1 at 75 °C. The activation energy of microcrystalline-cellulose hydrolysis was 105.61 kJ/mol, and the activation energy of glucose degradation was 131.37 kJ/mol.     

Graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose and study of its swelling, metal ion sorption and flocculation behaviour

The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10⁻²–28.0 × 10⁻² mol dm⁻³), potassium peroxymonosulphate (4.0 × 10⁻³–12.0 × 10⁻³ mol dm⁻³), thiourea (1.2 × 10⁻³–4.4 × 10⁻³ mol dm⁻³), sulphuric acid (2.0 × 10⁻³–10.0 × 10⁻³ mol dm⁻³), sodium carboxymethylcellulose (0.2–1.8 g dm⁻³) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Characteristics and seasonal variation in the semen of Markhoz bucks in western Iran

The aims of the present study were to evaluate if seasonality in semen characteristics and plasma testosterone concentrations exist in Markhoz male goats. Ten Markhoz (Angora) bucks were housed and fed according to standard recognized practices. During the observation period, semen was collected monthly with the aid of an electro-ejaculator and examined microscopically immediately after collection. Physical parameters of semen and the semen index were recorded. Blood samples were also taken monthly throughout the observation period and the plasma testosterone concentration monitored. Bucks demonstrated a higher semen quality (P < 0.05) in autumn and summer (semen index of 965 × 10⁶ and 752 × 10⁶ ml⁻¹, respectively), compared to spring and winter (semen index of 606 × 10⁶ and 512 × 10⁶, respectively). This coincided with a higher (P < 0.05) plasma testosterone concentration in autumn and summer (8.1 and 10.1 ng ml⁻¹, respectively), compared to that obtained in spring (3.0 ng ml⁻¹) and winter (2.5 ng ml⁻¹). During autumn and summer, the ejaculate volume (average of 1.2 and 1.0 ml), sperm output (1159 × 10⁶ and 1005 × 10⁶ sperm ml⁻¹), sperm mass motility (4.2 and 4.3), sperm progressive motility (83.9 and 82.0%) and percentage live sperm (90.7 and 88.2%, respectively) of the bucks were higher (P < 0.05) than in the spring (0.6 ml, 880 × 10⁶ sperm ml⁻¹, 3.3, 71.5% and 80.2%) and winter (0.7 ml, 863 × 10⁶ sperm ml⁻¹, 4.0, 71.5% and 84.9%, respectively). During autumn and summer, the percentage of sperm abnormalities (5.0 and 9.2%) was significantly lower than that in spring (12.9%) and winter (11.2%). The semen pH was slightly alkaline being significantly (P < 0.05) lower in the autumn (7.1) than in spring (7.3). Data showed season of the year to influence all semen parameters evaluated—indicating that optimal buck performance may be obtained in late summer and autumn. It can thus be said that Markhoz bucks have distinct seasonal spermatogenic activity, with poorer semen characteristics being recorded during winter and spring. This may be a critical obstacle when implementing an intensive breeding system of three kidding seasons in 2 years, with natural mating being implemented.     

The impacts of re-introducing Mississippi River water on the hydrologic budget and nutrient inputs of a deltaic estuary

Most wetlands of the Mississippi deltaic plain are isolated from riverine input due to flood control levees along the Mississippi River. These levees have altered hydrology and ecology and are a primary cause of massive wetland loss in the delta. River water is being re-introduced into coastal basins as part of a large-scale ecological engineering effort to restore the delta. We quantified freshwater, nitrogen, and phosphorus inputs to the Breton Sound Estuary for three climatically different years (2000, 2001, and 2002). Water budgets included precipitation, potential evapotranspiration, the diversion, stormwater pumps, and groundwater. Precipitation contributed 48–57% of freshwater input, while the diversion accounted for 33–48%. Net groundwater input accounted for less than 0.05% of freshwater inputs. Inputs of ammonium (NH₄-N), nitrate (NO₃-N), total nitrogen (TN), and total phosphorus (TP) were determined for each of the water sources. Atmospheric deposition was the most important input of NH₄-N (57–62% or 1.44 × 10⁵–2.32 × 10⁵ kg yr⁻¹) followed by the diversion. The diversion was the greatest source of NO₃-N (67–83%, 7.78 × 10⁵–1.64 × 10⁶ kg yr⁻¹) and TN (60–71%). The diversion contributed 41–60% of TP input (1.17 × 10⁵–2.32 × 10⁵ kg yr⁻¹). Annual loading rates of NH₄-N and NO₃-N were 0.17–0.27 and 1.2–2.3 g N m⁻² yr⁻¹, respectively, for the total basin indicating strong retention of nitrogen in the basin. Nitrogen retention through denitrification and burial was estimated for the upper basin.     

Phenol-mediated improved performance of active biomass for treatment of distillery wastewater

Activated sludge from a wastewater treatment plant consists of a consortium of microbes that utilize various organic molecules including persistent organic pollutants for their survival. Phenolic compounds and their derivatives along with dibenzofuran (DBF) are found as dominating pollutants in distillery waste. The acclimatization process leads to selective enrichment of the microbial community; and in this study, we report the acclimatizing effect of phenol on improving the treatment efficiency of two different distillery sludges—sludge from conventional aeration tanks (CAT), and from an extended aeration tank (EAT). The adaptation-dependent performance of activated biomass was studied by monitoring the increase in colony-forming units (CFUs) on mineral media and the utilization pattern for phenol (300×10³ and 530×10³ CFU for CAT and EAT sludge, respectively) and DBF (260×10³ and 430×10³ CFU for CAT and EAT sludge, respectively). The study showed that the acclimatization process remarkably improved the performance sludge for treatment of distillery wastewater. There was an improvement in chemical oxygen demand (COD) removal efficiency from 19% (unacclimatized sludge) to 31% in the case of acclimatized sludge (raw wastewater), which improved further to 82% and 87% with dilution of wastewater by 10 times (0.1×) and by 50 times (0.02×), respectively. Highest growth yields were observed with 0.1× wastewater (0.324 and 0.308 g g⁻¹ d⁻¹ for CAT and EAT sludges, respectively), while lower values are reported for the remaining two forms of wastewater. The study proposes that acclimatization step could be included as part of a treatment plant where the activated biomass could be intermittently metabolically charged by exposing it to selected molecules to increase treatment efficiency.     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

Membrane Permeability Characteristics of Metaphase II Mouse Oocytes at Various Temperatures in the Presence of Me2SO

In this study, the hydraulic conductivity (L_p), Me₂SO permeability ( _Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me₂SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me₂SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me₂SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me₂SO were plotted against time. Mean (means ± SEM) L_pvalues in the presence of Me₂SO ( ^Me₂SO_p) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10⁻²± 0.60 × 10⁻², 5.29 × 10⁻²± 0.40 × 10⁻², and 3.69 × 10⁻²± 0.30 × 10⁻²μm/min/atm, respectively. The _Me2SOvalues were 3.69 × 10⁻³± 0.3 × 10⁻³, 1.07 × 10⁻³± 0.1 × 10⁻³, 2.75 × 10⁻⁴± 0.15 × 10⁻⁴, 7.83 × 10⁻⁵± 0.50 × 10⁻⁵, 5.24 × 10⁻⁵± 0.50 × 10⁻⁵, and 3.69 × 10⁻⁵± 0.40 × 10⁻⁵cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (E_a) for ^Me₂SO_p, _Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.     

A comparison between freezing methods for the cryopreservation of stallion spermatozoa

The effects of sperm freezing concentration (40 × 10⁶ mL⁻¹ vs. 400 × 10⁶ mL⁻¹), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam^® box vs. programmable freezing machine) were evaluated in a 2 × 2 × 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 × 10⁶ mL⁻¹ was higher than for spermatozoa frozen at 400 × 10⁶ mL⁻¹. No significant differences were observed in the semen parameters assessed after cryopreservation in either 0.25 or 0.5 mL straws. However, the programmable freezer provided a more consistent and reliable freezing rate than liquid nitrogen vapour. We conclude that an effective protocol for the cryopreservation of stallion spermatozoa at low concentrations would include concentrations of 40 × 10⁶ mL⁻¹ in 0.25 mL straws using a programmable freezer. This freezing protocol would be suitable for emerging sperm technologies such as sex-preselection of stallion spermatozoa as the sorting process yields only low numbers of spermatozoa in a small volume available for either immediate insemination or cryopreservation.     

Pine nut peroxidase immobilized on chitosan crosslinked with citrate and ionic liquid used in the construction of a biosensor

A biosensor based on the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI·Tf₂N) and a novel source of peroxidase (tissue from the pine nuts of Araucaria angustifolia) was constructed. This enzyme was immobilized on chitosan crosslinked with citrate and the biosensor used for the determination of rosmarinic acid by square-wave voltammetry. The peroxidase in the presence of hydrogen peroxide catalyzes the oxidation of rosmarinic acid to quinone and the electrochemical reduction of the product was obtained at a potential of +0.15 V vs. Ag/AgCl. Different analytical parameters influencing the biosensor response, that is, peroxidase units, pH, hydrogen peroxide concentration and parameters for the square-wave voltammetry (frequency, pulse amplitude and scan increment), were investigated. The best performance was observed for the biosensor under the following conditions: 1000 units mL⁻¹ peroxidase, pH 7.0 and 8.3 × 10⁻⁴ mol L⁻¹ hydrogen peroxide with a frequency of 30 Hz, pulse amplitude of 100 mV and scan increment of 5.0 mV. The biosensor gave a linear response to rosmarinic acid over the concentration range of 9.07 × 10⁻⁷ to 4.46 × 10⁻⁶ mol L⁻¹ with a detection limit of 7.25 × 10⁻⁸ mol L⁻¹. The recovery of rosmarinic acid in plant extracts ranged from 97.0% to 109.6% and the determination of this substance in these samples using the biosensor compared favorably with that using the capillary electrophoresis method.     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

A nano-Ni based ultrasensitive nonenzymatic electrochemical sensor for glucose: Enhancing sensitivity through a nanowire array strategy

Highly ordered Ni nanowire arrays (NiNWAs) were synthesized for the first time using a template-directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template, and their morphological characterization were examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). A NiNWAs based electrode shows very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium, which has been utilized as the basis of the fabrication of a nonenzymatic biosensor for electrochemical detection of glucose. The biosensor can be applied to the quantification of glucose with a linear range covering from 5.0 × 10⁻⁷ to 7.0 × 10⁻³ M, a high sensitivity of 1043 μA mM⁻¹ cm⁻², and a low detection limit of 1 × 10⁻⁷ M. The experiment results also showed that the sensor exhibits good reproducibility and long-term stability, as well as high selectivity with no interference from other oxidable species.     

Synthesis, characterization and applications of graft copolymer (Chitosan-g-N,N-dimethylacrylamide)

An unreported graft copolymer of N,N-dimethylacrylamide (DMA) with chitosan has been synthesized under nitrogen atmosphere using peroxymonosulphate/mandelic acid redox pair. The effect of reaction conditions on grafting parameters i.e. grafting ratio, efficiency, conversion, add on and homopolymer has been studied. Experimental results show that maximum grafting has been obtained at 1.0 g dm⁻³ concentration of chitosan, 30 × 10⁻² mol dm⁻³ concentration of N,N-dimethylacrylamide and 7.0 × 10⁻³ mol dm⁻³ concentration of hydrogen ion. It has also been observed that grafting ratio, add on, conversion and efficiency increase upto 3.2 × 10⁻³ mol dm⁻³ of mandelic acid, 12.0 × 10⁻³ mol dm⁻³ of potassium peroxymonosulphate, 150 min of time and 40 °C of temperature. Grafted polymer has been characterized by FTIR spectroscopy and thermogravimetric analysis. Water swelling capacity of chitosan-g-N,N-dimethylacrylamide has been determined. It has been observed that the graft copolymer is thermally more stable than parent backbone.     

Peroxidase catalyzed phenolic compounds oxidation in presence of surfactant Dynol 604: A kinetic investigation

The kinetics of fungal peroxidase-catalyzed phenolic compounds (PCs) oxidation was investigated in presence of acetylenic-based surfactant Dynol 604 at pH 5.5 and 25 °C. It was shown that the presence of ppm concentrations of surfactant did not influence initial rate of PCs oxidation. The calculated apparent bimolecular rate constants were (1.8 ± 0.2) × 10⁵ M⁻¹ s⁻¹, (1.4 ± 0.4) × 10⁷ M⁻¹ s⁻¹, (1.30 ± 0.06) × 10⁷ M⁻¹ s⁻¹ and 1.1 × 10⁸ M⁻¹ s⁻¹ for phenol, 1-naphthol, 2-naphthol and 1-hydroxypyrene, respectively.During an extensive substrates conversion Dynol 604 showed diverse action for different PCs. The oxidation of phenol practically did not change, whereas the surfactant enhanced the conversion of 1- and 2-naphthol and 1-hydroxypyrene in dose response manner. The results accounted by a scheme, which contains a stadium of enzyme inhibition by oligomeric PC oxidation products. The action of the surfactant was explained by avoidance the enzyme active center clothing with the oligomers. The results acquired demonstrate a remarkable increase of substrates conversion in the presence of Dynol 604.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷ g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Biochemical characterization of arabidopsis developmentally regulated G-proteins (DRGs)

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as several medical conditions. Individual subfamilies within the G-protein superfamily possess unique nucleotide binding and hydrolysis rates that are intrinsic to their cellular function, and so characterization of these rates for a particular G-protein may provide insight into its cellular activity. We have produced recombinant active DRG protein using a bacterial expression system and refolding, and performed biochemical characterization of their GTP binding and hydrolysis. We show that recombinant Arabidopsis thaliana atDRG1 and atDRG2a are able to bind GDP and GTP. We also show that DRGs can hydrolyze GTP in vitro without the assistance of GTPase-activating proteins and guanine exchange factors. The atDRG proteins hydrolyze GTP at a relatively slow rate (0.94 × 10⁻³ min⁻¹ for DRG1 and 1.36 × 10⁻³ min⁻¹ for DRG2) that is consistent with their nearest characterized relatives, the Obg subfamily. The ability of DRGs to bind nucleotide substrates without assistance, their slow rate of GTP hydrolysis, heat stress activation and domain conservation suggest a possible role as a chaperone in ribosome assembly in response to stress as it has been suggested for the Obg proteins, a different but related G-protein subfamily.     

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1–400 × 10⁻⁸ M) and time periods (1–5 h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100 × 10⁻⁸ M) and GnRH (0.1 and 1 × 10⁻⁹ M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4 h of NEI incubation. NEI (100 × 10⁻⁸ M) induced a significant LH secretion after 2 h of stimulus, reaching a maximum response 4 h later. A rapid and remarkable LH release was induced by NEI (400 × 10⁻⁸ M) 1 h after stimulus, attaining its highest level at 2 h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10 × 10⁻⁸ M), caused no action when used alone, its co-incubation with GnRH (1 × 10⁻⁹ M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.     

A sugar-aza-crown ether-based fluorescent sensor for Hg and Cu

A fluorescent sensor, 5, based upon the sugar-aza-crown ether structure with two anthracenetriazolymethyl groups was prepared and its fluoroionophoric properties toward transition metal ions were investigated. In methanol, the sensor exhibits highly selective recognition of Cu²⁺ and Hg²⁺ ions among a series of tested metal ions. The association constant for 5·Cu²⁺ and 5·Hg²⁺ in methanol was calculated to be 4.0 × 10⁵ M⁻¹ and 1.1 × 10⁵ M⁻¹, respectively. The detection limits for the sensing of Cu²⁺ and Hg²⁺ ions were 1.39 × 10⁻⁶ M and 1.39 × 10⁻⁵ M, respectively.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.