The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

EFFECTS OF FERTILIZATION ON EPICUTICULAR WAX MORPHOLOGY OF NEEDLE LEAVES OF DOUGLAS FIR,PSEUDOTSUGA MENZIESII (PINACEAE)

Fertilized stands of Pseudotsuga menziesii were found to have glaucous needles. We investigated the morphological and quantitative characteristics of the epicuticular waxes of needles of fertilized and control trees. Glaucousness was caused by ornate tubular epicuticular wax. Dipping needles in chloroform, which dissolves waxes, eliminated the glaucous appearance. Based on cryostage scanning electron microscopic observations, the epicuticular waxes in the nonstomatal region were much more ornate on the needles of the fertilized trees (experimental needles) than in unfertilized trees (control needles). The stomatal region in both experimental and control needles showed similarly ornate waxes. Quantities of waxes were similar in experimental and control needles. The glaucousness was not the result of greater quantities of wax; rather, fertilization altered wax morphology in the nonstomatal regions.     

PAEDOMORPHOSIS IN AMBYSTOMA TALPOIDEUM: MAINTENANCE OF POPULATION VARIATION AND ALTERNATIVE LIFE-HISTORY PATHWAYS

A “common garden” experiment using artifical ponds was performed to test if differences in frequency of paedomorphosis and metamorphosis among six natural populations of the salamander Ambystoma talpoideum resulted from the drying regime of the aquatic habitat acting as an agent of selection. Our experiment supports the hypothesis of genetic differentiation in the propensity to metamorphose among the populations, but gave mixed evidence that pond-drying regime is the selective force directing evolution of this trait. Some populations appear to have evolved phenotypic plasticity whereas others may have a genetic polymorphism in their propensity to metamorphose as ponds dry.     

Ethnobotanical value and conservation of sacred groves of the Kpaa Mende in Sierra Leone

Sacred groves in the Moyamba District of Sierra Leone were assessed for their value to local herbalists and traditional folk medicine practitioners of the Kpaa Mende people. Herbalists (tufablaa) collecting in 23 sacred groves were interviewed regarding their general knowledge of medicinal plants, and their perceptions regarding changes in the occurrence of medicinal plants. Over 75 medicinal plant remedies are currently in use among the Kpaa Mende, with some plants reported here for the first time in terms of their medicinal uses in Sierra Leone. A significant feature of Kpaa Mende ethnobotany is the employment of 2 or more kinds of plants in combination as a remedy for particular afflictions. The discovery of rare and uncommon plants in the groves and their medicinal uses are discussed in terms of the role of the sacred groves in medicinal plant conservation.     

Vasomotor instability preceding tilt-induced syncope: does respiration play a role?

Lipsitz, Lewis A., Raymond Morin, Margaret Gagnon, DanKiely, and Aharon Medina. Vasomotor instability precedingtilt-induced syncope: does respiration play a role? J. Appl. Physiol. 83(2): 383-390, 1997.This studyaimed to determine whether alterations in cardiovascular dynamicsbefore syncope are related to changes in spontaneous respiration.Fifty-two healthy subjects underwent continuous heart rate (HR),arterial blood pressure (BP), and respiratory measurements during10-min periods of spontaneous and paced breathing (0.25 Hz) in thesupine and 60° head-up tilt positions. Data were evaluated by powerspectrum and transfer function analyses. During tilt, 27 subjectsdeveloped syncope or presyncope and 25 remained asymptomatic. Subjectswith tilt-induced syncope had significantly greater increases inlow-frequency (0.04-0.15 Hz) systolic BP, diastolic BP, and HRpower during tilt than the asymptomatic subjects(P  0.01). This difference waspresent during spontaneous but not paced breathing. However, averagetidal volume, respiratory rate, minute ventilation, proportion ofbreaths below 0.15 Hz, and low-frequency respiratory power during tilt did not differ between syncopal and nonsyncopal subjects. Transfer magnitudes between low-frequency respiration and BP, and between BP andinterbeat interval, were also similar between groups. Thus vasomotorinstability before syncope is not related to alterations in respirationor the cardiovagal baroreflex but may reflect oscillating centralsympathetic outflow to the vasculature.

     

Rate and composition of sweat fluid losses are unaltered by hypohydration during prolonged exercise in horses

Kingston, Janene K., Raymond J. Geor, and Laura JillMcCutcheon. Rate and composition of sweat fluid losses areunaltered by hypohydration during prolonged exercise in horses.J. Appl. Physiol. 83(4):1133-1143, 1997. Rate and ionic composition of sweat fluid losses and partitioning of evaporative heat loss into respiratory and cutaneous components were determined in six horses during three 15-km phases of exercise at ~40% of maximalO₂ uptake. Pattern of change insweat rate (SR) and composition was similar during each phase. SRincreased rapidly for the first 20 min of exercise but remained at~24-28ml · m² · min¹during the remainder of each phase. Similarly, the concentrations of Naand Cl in sweat increased until 30 min of exercise but were unchangedthereafter. Sweat osmolality and concentrations of Na and Cl werepositively correlated with SR. Sweat K concentration decreased duringexercise but was not correlated with SR. Fluid losses were 33.8 ± 1.5 liters, resulting in decreases of ~21% in plasma volume and~11% in total body water. The ~6% hypohydration was notassociated with an alteration in SR, sweat composition, or heatstorage. Respiratory and cutaneous evaporative heat loss represented~23 and 70%, respectively, of the total heat dissipated, and thepartitioning of heat loss was similar in each exercise phase. Weconclude that SR and the relative proportions of respiratory andcutaneous evaporative heat loss are unchanged in horses during prolonged low-intensity exercise despite moderate hypohydration.

     

Immobilization of tyrosinase for use in nonaqueous media: Enzyme deactivation phenomena

Summary We have investigated features for minimizing the inactivation of tyrosinase (E.C. 1.14.18.1) that is caused by immobilization on glass beads and Celite^®. The degree of inactivation is dependent on the enzyme loading and the carrier's surface area. Addition of a sacrificial protein during the immobilization procedure offers a protective effect with increased residual activity on the basis of comparable enzyme loading.     

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria

In a recent communication Lehninger and co-workers (Costa, L.E., Reynaferje, B., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 4802-4811) reported values approaching 8 for the H+/O ratio of vectorial proton ejection from rat liver mitochondria respiring with succinate. Here we present a rigorous analysis of these measurements which reveals that they may significantly overestimate the true H+/O stoicheiometry.