Measuring ß-galactosidase activity in opaque dairy solutions under optimum conditions for galactooligosaccharide synthesis by isothermal titration calorimetry.

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate (thermal power) from enzyme-catalyzed reactions as a function of time. This study used ITC to measure ß-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with 2 ß-galactosidase (Enzyme Commission number 3.2.1.23) isozymes derived from Aspergillus oryzae and Kluyveromyces lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the 2 homologues differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties.

Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

Oropharyngeal airway diameter during sedation in children with and without developmental delay.

STUDY OBJECTIVE: To determine whether children with developmental delay would have closer apposition of upper airway tissues during sedation, perhaps because of poor coordination of upper airway musculature. DESIGN: Case-control and retrospective chart review. SETTING: Tertiary-care pediatric teaching hospital. PATIENTS: 40 children 3 to 6 years of age, with and without a diagnosis of developmental delay. MEASUREMENTS: Subjects received only pentobarbital sedation by a protocol. Magnetic resonance imaging (MRI) scans of the head were reviewed, and transverse airway diameters at the soft palate and tongue were determined from midline sagittal images. MAIN RESULTS: Age, weight, sedative dose, MRI window level, and window width were not different between patients with and without developmental delay. We found the airway diameter at the level of the soft palate was decreased 40% in children with developmental delay compared with those children without delay, 3 mm (1.4, 5.5 interquartile range) versus 5 mm (3, 8); p = 0.035, power 76%. CONCLUSIONS: The anteroposterior oropharyngeal airway diameter was smaller in children with developmental delay than in those without developmental delay, in static MRI images. It is possible that children with developmental delay are at higher risk for airway obstruction during sedation.

Anionic facial amphiphiles from cholic acid.

[structure:see text] Anionic facial amphiphiles have been prepared from cholic acid. These compounds offer antipodes of recently reported cationic amphiphiles derived from cholic acid. The synthesis of the anionic amphiphiles was accomplished in few steps from a common intermediate. In contrast to many other anionic facial amphiphiles, the cholic acid derived amphiphiles appeared to aggregate at relatively low concentration.

Heat generation and dissipation in plants: can the alternative oxidative phosphorylation pathway serve a thermoregulatory role in plant tissues other than specialized organs?

A number of hypothetical physiological roles have been proposed for the cyanide-insensitive alternative pathway in plants (Palmer, 1976; Laties, 1982; Meeuse, 1984; Purvis and Shewfelt, 1994; Wagner and Krab, 1995). The calorimetric observations of Raskin and co-workers (Ordentlich et al., 1991; Nevo et al., 1992; Moynihan et al., 1995) are significant contributions showing an interesting metabolic, chilling-induced response of the alternative pathway activity and differences in the low-temperature response among species adapted to different climates. Since different oxidative pathways do not have large differences in enthalpy, and observed heat rate increases are insufficient to cause significant temperature increases of physiological importance in nonthermogenic plants, other explanations must be developed for the relationship between the partitioning of electron flow and physiological conditions such as low temperature. The induction and engagement of the alternative respiratory pathway is involved in metabolic stasis, maintaining proper balance between carbon flow, ATP-ADP ratio, and electron flow during fluctuating or extreme temperature conditions. The alternative oxidase is engaged when ATP requirements are adequately met, as discussed by Palmer (1976), Meeuse (1983), Lambers (1985), and Wagner and Krab (1995). The expression and kinetic activity of the alternative oxidase are regulated by concentrations of key metabolites (Day and Wiskich, 1995; Siedow and Umbach, 1995; Wagner and Krab, 1995; Day et al., 1996). Dynamic partitioning of electron flow between Cyt oxidase and the alternative oxidase depends on the kinetic behavior of the two oxidases and the substrate dehydrogenases (Day and Wiskich, 1995; Siedow and Umbach, 1995; Wagner and Krab, 1995; Day et al., 1996). Furthermore, Moynihan et al. (1995) found that Episces cupreata Hook, adapted to the tropics, has very little alternative oxidase activity compared with wheat (Nevo et al., 1992), adapted to a large range of temperature climates. This results is consistent with the general relation between the apparent alternative oxidase activity and the climate of origin of the species.

Interactions of thionin from Pyrularia pubera with dipalmitoylphosphatidylglycerol large unilamellar vesicles.

The peptide toxin thionin from Pyrularia pubera binds to dipalmitoylphosphatidylglycerol (DPPG) large unilamellar vesicles as shown by an increase in the intensity and blue-shift of the fluorescence emission spectrum of the single tryptophan residue of the protein. The magnitude of these fluorescence changes increased with temperature near the thermotropic phase transition of DPPG (about 40 degrees C). Fluorescent probes sensitive to the structure and dynamics of the membrane were used to assess the effect of thionin binding on bilayer properties. The fluorescence emission spectra of Prodan, Patman, and Laurdan all showed spectral changes consistent with an increase in bilayer polarity at temperatures below the DPPG phase transition but a decrease in polarity at higher temperatures. Fluorescence polarization experiments and the ratio of monomer-to-excimer fluorescence of the probe 1,3-bis(1-pyrene)propane suggested that thionin increases the bilayer order above the transition temperature. Differential scanning calorimetry revealed that thionin broadens the transition and either increases or decreases the melting temperature depending on the concentration of the peptide. Taken together, the data are consistent with at least three distinct interactions of thionin with the bilayer: (1) thionin bound electrostatically to the bilayer surface; (2) tryptophan of the bound thionin inserted into the bilayer; (3) high-order aggregates of thionin-bound vesicles.

Pressure effects on metabolism in tissues from mice (Mus muscalis) and freshwater mussel (Elliptio complanata).

Metabolic rates of tissue sections from freshwater mussel gills and mouse brain and lung tissue were measured by calorimetry in ampules pressurized with gas mixtures. Increasing partial pressure of oxygen or total pressure with constant partial pressure of O2 does not affect the respiratory quotient but increases rates of tissue metabolism. Changes in metabolic activity occur over pressure and Po2 ranges commonly encountered by humans engaged in SCUBA diving.

Effects of temperature and glycerides on the enhancement of Agkistrodon piscivorus piscivorus phospholipase A2 activity by lysolecithin and palmitic acid.

The effect of temperature and various glycerides to modulate the ability of lysolecithin and fatty acid to promote high phospholipase A2 activity was studied using dipalmitoylphosphatidylcholine large unilamellar vesicles as substrate. The length of the lag phase prior to the accumulation of sufficient hydrolysis products (lysolecithin and fatty acid) to support high phospholipase activity was shortest at temperatures near the thermotropic phase transition of the phospholipid substrate. A reduction in the lag phase correlated with a reduction in the requirement for hydrolysis products at the phase transition temperature, where the bilayer exists in a state of fluctuating domains of gel and liquid crystal. Dipalmitoylglycerol and tripalmitoylglycerol also reduced the length of the lag phase. This reduction was both concentration-dependent and temperature-dependent relative to the phase transition in the presence of the glycerides. As with the effect of temperature, the ability of di- and triglycerides to decrease the lag time correlated with a decrease in the amount of reaction products necessary to promote high phospholipase activity. This effect coincided with the tendency of the glycerides to form domains in the bilayer. Glycerides that did not form domains either had no effect (monopalmitoylglycerol) or increased the length of the lag phase (dicaprylglycerol). These data suggest that the effect of the reaction products to increase phospholipase A2 activity is aided by the presence of fluctuations in lipid domains within the bilayer.

Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry.

This study describes methods and equipment for measurement of metabolic heat rates of cells and tissues under conditions that provide simultaneous determinations of the flux rates of both O2 and CO2. Isothermal measurement of metabolic heats are conducted in a sealed ampule. A trapping solution is employed to absorb metabolic CO2. Absorption of CO2 produces heat at a rate proportional to the rate of CO2 production. Under these conditions, O2 consumption by the tissue results in a decrease in the partial pressure of O2 within the sealed ampule. The decrease in pressure can be monitored with a pressure sensor and related to O2 consumption rates. The combined measurements of heat rates, CO2, and O2 fluxes provide important information on bioenergetic efficiency of cell metabolism. These data can also suggest possible shifts in metabolic pathways or substrate sources as cells develop, or are exposed to effectors, inhibitors, and environmental factors.

Time-temperature responses of tomato cells during high- and low-temperature inactivation.

Precise time and temperature dependences of the decrease of metabolism of cultured cells of tomato (Lysopersicon esculentum (L.) Mill. L. peruvianum (L.) Mill.) resulting from exposures to high and low temperatures were determined. Equations of the form Ln (activity)= C +1 [A+(T-Tm)(N)+B] describe thermal inactivation and allow prediction of activity loss following any thermal excursion beyond limits of temperature stability. The experimental parameters A, B, C and N derived from these equations allow precise comparison of temperature sensitivities of cells. Analysis of metabolic heat rates, O2-consumption rates and CO2-evolution rates demonstrated simultaneous shifts in metabolic pathways and metabolic activities towards more anaerobic metabolism below about 12° C and at high temperatures that stress growth of tomato cells.

The determination of nicotine and cotinine by ion pair reversed-phase chromatography.

An ion chromatographic method is described for the determination of nicotine and cotinine in aqueous solutions. This method is based on a type of reversed-phase chromatography involving ion pair formation of protonated nicotine, cotinine, pyridine, and pyridine derivatives. Detection is accomplished by measuring the UV absorption at 262 nm. Detection limits for nicotine and cotinine are 8 ng/mL and 2 ng/mL, respectively. Analyses of environmental samples and spiked environmental samples by both this ion chromatographic method and a previously reported gas chromatographic method have been used to demonstrate the accuracy and precision of this technique. The results of the analyses of both sets of samples by the two methods are in excellent agreement with a linear correlation coefficient of 0.97.

Kinetics of drug decomposition by heat conduction calorimetry.

The application of heat conduction calorimetry to the determination of decomposition mechanisms and rates for drugs is shown to be a rapid and generally useful method. The application of the method to determine the nature of the decomposition reaction, sources of systematic errors in the method, the equations relating the calorimetric signal to the kinetics of the reaction, and some examples of results are presented and discussed.

Effects of NaCl on metabolic heat evolution rates by barley roots.

The effect of salinity stress on metabolic heat output of barley (Hordeum vulgare L.) root tips was measured by isothermal microcalorimetry. Several varieties differing in tolerance to salinity were compared and differences quantified. Two levels of inhibition by increasing salt were found. Following the transition from the initial rate of the first level, inhibition remained at about 50% with further increases in salt concentration up to 150 millimolar. The concentration of salt required to inhibit to this level was cultivar dependent. At highter concentrations (>150 millimolar) of salt, metabolism was further decreased. This decrease was not cultivar dependent. The decreased rate of metabolic heat output at the first transition could be correlated with decreases in uptake of NO3-, NH4+, and Pi that occurred as the salt concentration was increased. The high degree of dependence of the inhibition of metabolic heat output on NaCl concentration points to a highly cooperative reaction responsible for the general inhibition of metabolism and nutrient uptake. The time required to attain the first level of salt inhibition is less than 20 minutes. Inhibition of root tips was not reversible by washing with salt free solutions. In addition to revealing these features of salt inhibition, isothermal microcalorimetry is a promising method for convenient and rapid determination of varietal differences in response to increasing salinity.

Acute toxicity of ammonium metavanadate in mice.

Acute toxicity of ammonium metavanadate solutions in normal saline (pH 6.7) or 0.1 M Tris-HCl-NaCl buffers (pH 7.2 or pH 7.8) was studied in BALB/c mice at 20 mg V/kg. Animals receiving these solutions subcutaneously started to show severe clinical signs 10-15 min postinjection and high mortality rates (45-73%) during the first 3 d. Animals dying because of vanadium toxicity did so only within the first 3 d after injection. NH4VO3-treated animals showed a tendency to increase their liver and spleen weights as compared to those receiving control solutions. Severe necrosis in lymphoid tissues (thymus, spleen, lymph nodes, and Peyer's patch), pulmonary hemorrhage, and renal acute tubular necrosis were commonly demonstrated in vanadium-treated animals. Toxicity of NH4VO3 solution in 0.1 M Tris-HCl-NaCl buffer (pH 7.8) was greatly reduced upon acidification with HCl to pH 6.1 or following boiling for 15 min (final pH of 7.7). Acidification of the solution reduced the mortality rate to 20 from 68%; however, the clinical signs were still severe. Boiling of the solution reduced the mortality rate to zero and moderated the severity of the clinical signs.