Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles)

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radio-active filter technique. Trypsin treatment of vesicles resulted in a 3-fold activation of Ca2+ uptake and loss of calmodulin sensitivity. Immunoblotting experiments with an antiserum raised against the Ca2+-ATPase showed that the trypsin activation was accompanied by a decrease in the amount of intact Ca2+-ATPase (111 kD) and by successive appearances of polypeptides of 102 and 99 to 84 kD. 125I-Calmodulin overlays showed that only the intact Ca2+-ATPase bound calmodulin. Removal of the calmodulin-binding domain (about 9 kD) was not enough to obtain full activation. Trypsin proteolysis resulted in a Ca2+ concentration necessary for half-maximal activity of 0.5 [mu]M, whereas a value of about 2 [mu]M was obtained with untreated membranes in the presence of calmodulin. Without trypsin treatment or calmodulin the activity was not saturated even at 57 [mu]M free Ca2+. The data suggest that trypsin digestion and calmodulin activate the cauliflower Ca2+-ATPase by at least partly different mechanisms

Forest gardens - places for children to connect with nature in times of urbanization

Abstract identifier: 150604-1185</p

Transmembrane Electron Transport in Plasma Membrane Vesicles Loaded with an NADH-Generating System or Ascorbate

Sugar beet (Beta vulgaris L.) leaf plasma membrane vesicles were loaded with an NADH-generating system (or with ascorbate) and were tested spectrophotometrically for their ability to reduce external, membrane-impermeable electron acceptors. Either alcohol dehydrogenase plus NAD(+) or 100 millimolar ascorbate was included in the homogenization medium, and right-side-out (apoplastic side-out) plasma membrane vesicles were subsequently prepared using two-phase partitioning. Addition of ethanol to plasma membrane vesicles loaded with the NADH-generating system led to a production of NADH inside the vesicles which could be recorded at 340 nanometers. This system was able to reduce 2,6-dichlorophenolindophenol-3′-sulfonate (DCIP-sulfonate), a strongly hydrophilic electron acceptor. The reduction of DCIP-sulfonate was stimulated severalfold by the K(+) ionophore valinomycin, included to abolish membrane potential (outside negative) generated by electrogenic transmembrane electron flow. Fe(3+)-chelates, such as ferricyanide and ferric citrate, as well as cytochrome c, were not reduced by vesicles loaded with the NADH-generating system. In contrast, right-side-out plasma membrane vesicles loaded with ascorbate supported the reduction of both ferric citrate and DCIP-sulfonate, suggesting that ascorbate also may serve as electron donor for transplasma membrane electron transport. Differences in substrate specificity and inhibitor sensitivity indicate that the electrons from ascorbate and NADH were channelled to external acceptors via different electron transport chains. Transplasma membrane electron transport constituted only about 10% of total plasma membrane electron transport activity, but should still be sufficient to be of physiological significance in, e.g. reduction of Fe(3+) to Fe(2+) for uptake

Forest gardens – new opportunities for urban children to understand and develop relationships with other organisms

This case study explores a learning situation in a forest garden in Sweden. A forest garden is an edible polyculture landscape with different layers of mostly perennial vegetation. The forest garden is designed to maximize the yield of useful plants while minimizing the input of energy and resources, including human labour. Forest gardens may offer learning situations that contextualize interconnectedness and relations between organisms as well as situations that are beneficial for evaluative development (Kellert, 2002), i.e. the development of values, beliefs and moral perspectives in children. Twenty-seven seven to eight year old primary school children were followed in the first six months of a three year project in which they participated in developing a forest garden. The aim of the study is to investigate how the children reason with respect to different organisms’ dependence on and relations to each other, themselves included. Specifically: How do the children describe their own relationships with other organisms, as well as the relationships between other organisms in the forest garden? What values of nature are expressed by the children, and in relation to which situations in the forest garden? Data were collected in the form of field notes, audio and video recordings and photos from the children’s visits to the forest garden. The photos were used for stimulated recall in focus group interviews. The data were analysed using a combination of qualitative content analysis (Patton, 2002) and semi-quantitative methods. The children in the study presented a unidirectional perspective about the relationship between themselves and the organisms, especially the insects, in the forest garden. Rather than asking what these organisms can do for me/us, they pose the question: What can I/we do for the bugs/plants/ bees?   The humanistic values, expressed by the children as a willingness to help other organisms (mostly insects) are in line with the explicit aims of the former curriculum for Biology to “promote care and respect for nature”. We should note that these humanistic values are no longer explicitly stated in the current curriculum. It is striking that the anthropocentric ecosystem services perspective (introduced in the current curriculum from grade 4), is so rare in the data. The children seldom mentioned the benefits for humans from insect pollination, even though this relationship is clearly stated by the pedagogues together with humanistic values.  In observations, the children showed a great deal of curiosity for the natural environment (naturalistic value) as well as joy and enthusiasm about participating in the different activities that took place in the forest garden. Aesthetic values were expressed in relation to flowers, cones, berries, a snail’s shell etc. This study shows that forest gardens have the potential to be places where children can connect emotionally and cognitively to other organisms

Det är den digitala kompetensen som måste utvecklas

Replik till Peter Hallgrens debattinlägg "Våra barn riskerar att hamna i ett digitalt utanförskap" i Jönköpings-Posten, 2019-11-23. </p

Reconstitution and Characterization of a Calmodulin-Stimulated Ca-Pumping ATPase Purified from Brassica oleracea L

Purification and functional reconstitution of a calmodulin-stimulated Ca(2+)-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca(2+), together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca(2+) uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 mumol min(-1) (mg protein)(-1), and the Ca(2+)/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca(2+)-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca(2+)) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca(2+)-pumping ATPase in Brassica oleracea inflorescences