Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

Trace metals in the benthic habitat of the Maarsseveen Lakes system,The Netherlands

The cadmium, zinc, lead and copper concentrations in benthic invertebrates and sediment were determined during two consecutive winters in the Maarsseveen Lakes system. A sequential extraction procedure was applied to estimate the bioavailability of the trace metals in the sediment. Based on the trace metal analyses of organisms and sediment, it is concluded that the Maarsseveen Lakes system has background levels of cadmium, zinc, lead and copper. As the majority of metals was present in geochemically more stable sediment phases, the sequential extractions provided limited additional information on trace metal bioavailability.     

Observations on the initiation and stimulation of oviposition of theVarroa mite

FemaleVarroa mites were collected from adult bees and were classified as swollen or not swollen. After introduction of these mites into recently sealed worker brood cells the average number of offspring of reproducing swollen mites was similar to that of naturally invaded mites, but the non-swollen mites produced a significantly lower number of offspring. This suggests that the oviposition of adult mites is stimulated by a preceding stay on adult bees. When (non-swollen) mites collected from brood cells were kept for 35 days in Eppendorf test tubes containing a larva or a pupa, their reproduction was similar to that of swollen mites.Contact of young mites, collected from brood cells, with adult bees was not essential for the initiation of oviposition. However, the number of offspring of reproducing mites, even after a third or fourth introduction into brood cells, was as a rule lower than that of mites that had been in contact with adult bees.The period of artificial introduction into sealed brood cells proved to be essential for subsequent reproduction. When introduced 48–52 h or 72–76 h after cell sealing, the mites did not produce eggs. When introduced 0–4 h after cell sealing a high percentage of the mites reproduced. Contact of the mites with a spinning larva seems necessary for initiation of oviposition.     

Ultrastructural evidence for a triple structure of the lateral element of the synaptonemal complex.

This study describes composition and localization of several substructures of the synaptonemal complex (SC) using different techniques. The techniques which were used were surface spreading, critical point drying of isolated SCs, and sectioning of Lowicryl embedded testis material. The lateral elements (LEs) of the SC appear to be composed of three lateral substructures: two morphologically identical major strands and a third strand which is considerably thinner. The thinner strand is localized on the inner side of the two major strands of the lateral element. In late pachytene/early diplotene stages when the SC starts to disintegrate more than three strands can be observed in the LEs. A model is presented and the function of the different substructures is speculated upon.     

Some features of feeding,respiration and energy conversion of Dapnia Magna

Some results of studies with Daphnia magna are presented. These results can be used as background information for toxicologists, but the techniques referred to might well be used for toxicity tests. Daphnia magna is a filter-feeder. With the Coulter Counter it was shown that the feeding mechanism is aselective for size of the food particles. It was also shown that algal cells can pass the gut of Daphnia magna several times before being completely digested. The uptake of food is proportional to the food concentration up to a critical concentration. Above this concentration the food uptake is constant. Respiration is also dependent on the food concentration, and has a maximum value at food concentrations near the critical concentration of the feeding process. Growth efficiency is independent of the food concentration. The effect of temperature on the feeding process is different for low and high food concentrations. Growth efficiency is maximal at 10°C and above 22°C growth efficiency was negative, which means that the population cannot survive under the experimental conditions used, at temperatures above 22°C.     

Decline in intracellular free Mg2+ is associated with irreversible tissue injury after brain trauma

Much of the tissue damage resulting from trauma to the central nervous system appears to result from secondary, delayed biochemical changes that follow primary mechanical injury. However, the early biochemical events remain to be elucidated. In the present studies, we have used phosphorus (31P) magnetic resonance spectroscopy (MRS) to examine in vivo, the temporal changes in brain intracellular free Mg2+ concentration following fluid percussion head injury in rats. We report that injury caused a profound and rapid decrease in intracellular free Mg2+ which was significantly correlated with the severity of injury. At high levels of injury, the decrease in intracellular free Mg2+ concentration was associated with a decrease in total Mg2+ concentration as determined by atomic absorption spectrophotometry. Prophylactic treatment with MgSO4 prevented the post-traumatic decrease in intracellular free Mg2+ and resulted in a significant improvement in acute neurological outcome. Because magnesium is essential for a number of critical enzyme reactions, including those of glycolysis, oxidative and substrate level phosphorylation, protein synthesis, and phospholipid synthesis, changes in free Mg2+ after brain trauma may represent a critical early factor leading to irreversible tissue damage.     

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

Summary The purified (H⁺ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H⁺-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H⁺)ATPase molecules were closely associated with liposomes, exhibiting a high H⁺-pumping activity (150,000% quenching· min^–1·mg^–1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H⁺)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H⁺)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (inside-out orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H⁺)ATPase could also functionally insert into bilayer from PCPEPG or PCPEPI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H⁺)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H⁺)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation     

Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.