Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.     

HeatMapper: powerful combined visualization of gene expression profile correlations, genotypes, phenotypes and sample characteristics

Background  

Accurate interpretation of data obtained by unsupervised analysis of large scale expression profiling studies is currently frequently performed by visually combining sample-gene heatmaps and sample characteristics. This method is not optimal for comparing individual samples or groups of samples. Here, we describe an approach to visually integrate the results of unsupervised and supervised cluster analysis using a correlation plot and additional sample metadata.     

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (approximately 5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane.     

Survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG in the Human Gastrointestinal Tract with Daily Consumption of a Low-Fat Probiotic Spread

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Therefore, probiotic strains should be able to survive passage through the human gastrointestinal tract. Human gastrointestinal tract survival of probiotics in a low-fat spread matrix has, however, never been tested. The objective of this randomized, double-blind, placebo-controlled human intervention study was to test the human gastrointestinal tract survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. Forty-two healthy human volunteers were randomly assigned to one of three treatment groups provided with 20 g of placebo spread (n = 13), 20 g of spread with a target dose of 1 × 10⁹ CFU of L. reuteri DSM 17938 (n = 13), or 20 g of spread with a target dose of 5 × 10⁹ CFU of L. rhamnosus GG (n = 16) daily for 3 weeks. Fecal samples were obtained before and after the intervention period. A significant increase, compared to the baseline, in the recovery of viable probiotic lactobacilli in fecal samples was demonstrated after 3 weeks of daily consumption of the spread containing either L. reuteri DSM 17938 or L. rhamnosus GG by selective enumeration. In the placebo group, no increase was detected. The results of selective enumeration were supported by quantitative PCR, detecting a significant increase in DNA resulting from the probiotics after intervention. Overall, our results indicate for the first time that low-fat spread is a suitable carrier for these probiotic strains.The human intestinal microflora or microbiota constitutes a metabolically active microbial environment. This community is relatively stable in the guts of healthy individuals (20). Some of the microbial groups harbor species that are potentially harmful, whereas others, such as the bifidobacteria and lactobacilli, are regarded as beneficial (8). Specific members of the genera Lactobacillus and Bifidobacterium are being applied in functional foods as probiotics (25). Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (9). The current scientific consensus is that probiotics should be alive to exert their beneficial effect in the human gastrointestinal (GI) tract (6). Consequently, probiotics should remain alive in the product, such that the daily effective dose per serving is still present at the end of the shelf life (14). Food matrices, production processes, or product usages that involve heating can affect the viability of probiotics (24).Typically, those members selected for probiotic application are chosen for their resistance to passage through the upper GI tract and thus are able to transiently colonize the gut (25). Human GI tract survival of probiotics should lead to shedding of live cells in fecal samples. GI tract survival is, however, dependent on both the strain and the food matrix involved (27). Fecal recovery of several probiotic strains has been demonstrated in different food matrices, including fermented milk and yoghurt (10, 26, 29), fruit drinks (21), a cereal bar (22), supplements (13, 17, 27), and infant formula (29).For this study, we have selected two well-established probiotic strains to test the suitability of a low-fat spread as a probiotic carrier, namely, Lactobacillus reuteri DSM 17938 (BioGaia, Sweden) and Lactobacillus rhamnosus GG (ATCC 53103; Valio, Finland). L. reuteri DSM 17938 was derived from L. reuteri ATCC 55730 by curing of two plasmids harboring antibiotic resistance genes (23). A series of in vitro experiments confirmed the retention of the functional properties of the daughter strain, as no differences in colony morphology, fermentation patterns, production of reuterin, generation time, mucus-binding ability, or tolerance to bovine bile were found between L. reuteri ATCC 55730 and DSM 17938. The daughter strain is somewhat more resistant to low pH and grows to a higher density in vitro (23). Several studies have been published which provide data on the survival of L. reuteri ATCC 55730 in the human GI tract at doses of 4 × 10⁸ to 1 × 10¹⁰ CFU/day in freeze-dried matrices and chewable tablets (32-34). Furthermore, L. reuteri DSM 17938 was demonstrated to survive human GI tract passage in the same way as L. reuteri ATCC 55730 (23).L. rhamnosus GG has been isolated from a healthy human intestinal flora by Goldin et al. (10). L. rhamnosus GG is relatively resistant to acid and bile, adheres in vitro to epithelial cells, and can produce an antimicrobial substance (10, 15). A wide range of studies have been published which provide data on the survival of L. rhamnosus GG in the human GI tract (3, 4, 10, 18, 19, 27-30), as well as transient colonization of the intestinal microbiota in healthy adults in various formats, including freeze-dried powder, capsules, and tablets or via fermented milk drinks, yoghurt, or fruit juice. Saxelin et al. (28) evaluated the dose-response effect of orally administered L. rhamnosus GG in powder form on fecal colonization in healthy adults, which indicated that consumption of approximately 10¹⁰ to 10¹¹ CFU/day was required to reach detectable levels in fecal samples from volunteers. This was also the case when L. rhamnosus GG was administered in gelatin capsules (29). Additionally, Saxelin et al. (27) observed that milk, but possibly also other protective compounds in food, can improve the survival of L. rhamnosus GG. Fecal recovery of L. rhamnosus GG in milk-based products was shown at dose levels of around 2 × 10⁹ CFU/day.It is, however, not known whether probiotics can survive passage through the human GI tract after the consumption of a low-fat spread. The objective of this randomized, double-blind, placebo-controlled human intervention study was therefore to test the human GI tract survival of L. reuteri DSM 17938 and L. rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. The primary outcome parameter of this study was a significant change from the baseline in the number of probiotic bacteria of the respective strains in fecal samples.     

A plant's perspective of extremes: terrestrial plant responses to changing climatic variability

We review observational, experimental, and model results on how plants respond to extreme climatic conditions induced by changing climatic variability. Distinguishing between impacts of changing mean climatic conditions and changing climatic variability on terrestrial ecosystems is generally underrated in current studies. The goals of our review are thus (1) to identify plant processes that are vulnerable to changes in the variability of climatic variables rather than to changes in their mean, and (2) to depict/evaluate available study designs to quantify responses of plants to changing climatic variability. We find that phenology is largely affected by changing mean climate but also that impacts of climatic variability are much less studied, although potentially damaging. We note that plant water relations seem to be very vulnerable to extremes driven by changes in temperature and precipitation and that heatwaves and flooding have stronger impacts on physiological processes than changing mean climate. Moreover, interacting phenological and physiological processes are likely to further complicate plant responses to changing climatic variability. Phenological and physiological processes and their interactions culminate in even more sophisticated responses to changing mean climate and climatic variability at the species and community level. Generally, observational studies are well suited to study plant responses to changing mean climate, but less suitable to gain a mechanistic understanding of plant responses to climatic variability. Experiments seem best suited to simulate extreme events. In models, temporal resolution and model structure are crucial to capture plant responses to changing climatic variability. We highlight that a combination of experimental, observational, and/or modeling studies have the potential to overcome important caveats of the respective individual approaches.     

Limited decadal growth of mountain birch saplings has minor impact on surrounding tundra vegetation

Temperatures over the Arctic region are increasing at three times the rate of the global average. Consequently, Arctic vegetation is changing and trees are encroaching into the tundra. In this study, we examine the establishment and growth of mountain birch (Betula pubescens ssp. tortuosa), which forms the treeline in subarctic Europe, and its impact on community composition across the treeline ecotone nearby Abisko, Sweden. Birch advancement along elevational gradients was studied by comparing data collected in 2016 with data collected 10 and 15 years previously. Species identity, cover, and phylogenetic relatedness were used to assess the impact of birch encroachment on community composition. Our results show that birch occurrence above the treeline did not affect plant community composition, probably owing to the observed lack of significant growth due to herbivore browsing, nitrogen limitation, or a reduction in snow cover. Independent of birch performance, the tundra community structure shifted toward a novel community dissimilar from the forest plant community found below the treeline. Taken together, our findings are explained by species‐specific responses to climate change, rather than by a linear forest advance. Future treeline advancements are likely more restricted than previously expected.     

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.     

Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010. © 2010 Wiley‐Liss, Inc.     

Simulating cellular galectin networks by mixing galectins in vitro reveals synergistic activity

BackgroundEven though members of the family of adhesion/growth-regulatory galectins are increasingly detected to be co-expressed, they are still being routinely tested separately. The recent discovery of heterodimer formation among galectins-1, -3, and -7 in mixtures prompts further study of their functional activities in mixtures.MethodsCell agglutination, galectin binding to cells, as well as effects on cell proliferation, onset of apoptosis and migration were determined in assays using various cell types and mixtures of galectins-1, -3, and -7.ResultsEvidence for a more than additive increases of experimental parameters was consistently obtained.ConclusionTesting galectins in mixtures simulates the situation of co-expression in situ and reveals unsuspected over-additive activities. This new insight is relevant for analyzing galectin functionality in (patho)physiological conditions.     

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 ± 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the β-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F₁F₀-ATPase and the lysosomal V-type ATPase.