The AAA‐type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana

The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant‐negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1) under the control of the trichome‐specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant‐negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant‐negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell‐cycle regulation.     

Revision and affinities ofGalium (Rubiaceae) in Madagascar

The only representatives ofRubiaceae-Rubieae in Madagascar are five species ofGalium. G. thunbergianum andG. chloroionanthum are essentially afromontane species, ± widely distributed in both the African mainland and Madagascar. The new speciesG. andringitrense andG. ankaratrense are endemic to Madagascar but show close affinities to two afromontane—afroalpine groups from the African mainland, theG. simense—G. ruwenzoriense andG. glaciale complex respectively.G. polyacanthum (new combination) exhibits certain primitive morphological traits and appears to be an old Madagascan endemic without close allies. New chromosome counts for populations from the African mainland demonstrate thatG. chloroionanthum andG. simense are 4x (x=11). Distribution patterns and origins of theGalium species in Madagascar and their relationships with African taxa are discussed.     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.     

Phosphatidylcholine exchange protein catalyzes the net transfer of phosphatidylcholine to model membranes

2-Stearoyl spin-labeled phosphatidylcholine (PC*) has been introduced into the phosphatidylcholine exchange protein from bovine liver and its electron spin resonance (ESR) spectrum determined. The spin-labeled group in the PC*- exchange protein complex was strongly immobilized. Addition of sodium deoxycholate micelles released PC* from its binding site, producing a mobile signal. This was also observed when micelles of lysophosphatidylcholine and vesicles of phosphatidic acid were added, indicating that the exchange protein can insert its endogenous PC* into interfaces devoid of phosphatidylcholine. ESR spectroscopy was used to measure transfer of PC* from spin-labeled "donor" vesicles to unlabeled "acceptor" vesicles as described by Machida & Ohnishi [Machida, K., & Ohnishi, S. (1978) Biochim. Biophys. Acta 507, 156-164]. The donor vesicles consisted of PC* and phosphatidic acid (75:25 mol%) and the acceptor vesicles of phosphatidylethanolamine and phosphatidic acid (81:19 mol%). Addition of exchange protein catalyzed a net transfer of PC* from donor to acceptor vesicles. This transfer proceeded until the acceptor vesicles contained approximately 2 mol% of PC*. A spontaneous transfer of PC* was not observed. As for the mode of action, it appears that the exchange protein, after insertion of its endogenous PC* into the acceptor, leaves the interface without a bound phospholipid molecule yet continues to shuttle PC* from donor to acceptor.     

VARIABILITY OF CELL WALL STRUCTURE AND HYDROCARBON TYPE IN DIFFERENT STRAINS OF BOTRYOCOCCUS BRAUNII1

Different samples of Botryococcus braunii Kütz., freshly collected from nature or laboratory-grown from culture collection strains, were studied by electron microscopy and their hydrocarbon content analyzed. Although the general internal structure of the cells was rather constant, the organization of the outer walls forming the hydrocarbon-rich matrix of the colonies differed greatly from one sample to another. In the majority of cultivated strains, the colonies were rather small, the different successive external walls remained distinct and all strains contained dienic or trienic hydrocarbons. In contrast, most of the collected samples possessed large colonies with a rather compact matrix formed by the hydrocarbon-rich part of the successive closely appressed external wall layers. These samples contained polyunsaturated hydrocarbons, i.e. botryococcenes. Well defined cell caps which sheared off the cells were observed only in those strains with a compact matrix. The Austin strain and some collected samples, however, were intermediate with rather small colonies, dense matrix, definite cell caps and dienic hydrocarbons. Thus, the hydrocarbon composition did not correlate directly with the variations in wall structure; however, the occurence of dienic and botryococcene-like hydrocarbons together in one strain was never observed, although analyzed at various stages of growth. Thus, the existence of distinct strains of Botryococcus braunii, some synthesizing dienes, others botryococcenes, appears highly probable.     

The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.     

The α2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries

The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the α(2) isoform of the Na,K-pump (α(2)), Na(+), Ca(2+)-exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating α(2) in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca(2+)](i) was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 μM ouabain uncoupled them. Downregulation of α(2) reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of α(2) in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endothelium-dependent hyperpolarizing factor-like component of the response was significantly diminished in α(2)-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the α(2)-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on α(2)-downregulated arteries. In the presence of a low NE concentration the α(2)-downregulated arteries had higher [Ca(2+)](i) and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca(2+)-sensitivity. The α(2)-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K(+) was not affected. Our results demonstrate the importance of α(2) for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone.     

Effect of cheese and butter intake on metabolites in urine using an untargeted metabolomics approach

Cheese intake has been shown to decrease total cholesterol and LDL cholesterol concentrations when compared to butter of equal fat content. An untargeted metabolite profiling may reveal exposure markers of cheese but may also contribute with markers which can help explain how the intake of cheese affects cholesterol concentrations. Twenty-three subjects collected 2 × 24 h urine samples after 6 weeks of cheese and 6 weeks of butter intake with equal amounts of fat in a cross-over intervention study. The samples were analyzed by UPLC-QTOF/MS. A two-step univariate data analysis approach using linear mixed model was applied separately for positive and negative ionization mode: In the first step a total of 44 features related to treatment were identified and in the second step 36 of these features were related to total cholesterol concentrations. Cheese intake resulted in increased urinary indoxyl sulfate, xanthurenic acid, tyramine sulfate, 4-hydroxyphenylacetic acid, isovalerylglutamic acid and several acylglycines including isovalerylglycine, tiglylglycine and isobutyrylglycine when compared to butter intake of equal fat content. The biological mechanisms of action linking the metabolites to cholesterol concentrations need to be further explored.