Vesicle docking in regulated exocytosis

In electron micrographs, many secretory and synaptic vesicles are found 'docked' at the target membrane, but it is unclear why and how. It is generally assumed that docking is a necessary first step in the secretory pathway before vesicles can acquire fusion competence (through 'priming'), but recent studies challenge this. New biophysical methods have become available to detect how vesicles are tethered at the target membrane, and genetic manipulations have implicated many genes in tethering, docking and priming. However, these studies have not yet led to consistent working models for these steps. In this study, we review recent attempts to characterize these early steps and the cellular factors to orchestrate them. We discuss whether assays for docking, tethering and priming report on the same phenomena and whether all vesicles necessarily follow the same linear docking–priming–fusion pathway. We conclude that most evidence to date is consistent with such a linear pathway assuming several refinements that imply that some vesicles can be nonfunctionally docked ('dead-end' docking) or, conversely, that the linear pathway can be greatly accelerated (crash fusion).     

The mechanisms of refilling of xylem conduits and bleeding of tall birch during spring

Seasonal variations in osmolality and components of xylem sap in tall birch trees were determined using several techniques. Xylem sap was extracted from branch and trunk sections of 58 trees using the very rapid gas bubble-based jet-discharge method. The 5-cm long wood pieces were taken at short intervals over the entire tree height. The data show that large biphasic osmolality gradients temporarily exist within the conducting xylem conduits during leaf emergence (up to 272 mosmol x kg(-1) at the apex). These gradients (arising mainly from glucose and fructose) were clearly held within the xylem conduit as demonstrated by (1)H NMR imaging of intact twigs. Refilling experiments with benzene, sucrose infusion, electron and light microscopy, as well as (1)H NMR chemical shift microimaging provided evidence that the xylem of birch represents a compartment confined by solute-reflecting barriers (radial: lipid linings/lipid bodies; axial: presumably air-filled spaces). These features allow transformation of osmolality gradients into osmotic pressure gradients. Refilling of the xylem occurs by a dual mechanism: from the base (by root pressure) and from the top (by hydrostatic pressure generated by xylem-bound osmotic pressure). The generation of osmotic pressure gradients was accompanied by bleeding. Bleeding could be observed at a height of up to 21 m. Bleeding rates measured at a given height decreased exponentially with time. Evidence is presented that the driving force for bleeding is the weight of the static water columns above the bleeding point. The pressure exerted by the water columns and the bleeding volume depend on the water-filling status of (communicating) vessels.     

FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells

Background

During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards.

Methodology/Principal Findings

The experiments reported here were designed to test this hypothesis by direct measurements of the hemoglobin concentration in live, infected red cells. We developed a novel, non-invasive method to quantify the hemoglobin concentration in single cells, based on Förster resonance energy transfer between hemoglobin molecules and the fluorophore calcein. Fluorescence lifetime imaging allowed the quantitative mapping of the hemoglobin concentration within the cells. The average fluorescence lifetimes of uninfected cohorts was 270±30 ps (mean±SD; N = 45). In the cytoplasm of infected cells the fluorescence lifetime of calcein ranged from 290±20 ps for cells with ring stage parasites to 590±13 ps and 1050±60 ps for cells with young trophozoites and late stage trophozoite/ early schizonts, respectively. This was equivalent to reductions in hemoglobin concentration spanning the range from 7.3 to 2.3 mM, in line with the model predictions. An unexpected ancillary finding was the existence of a microdomain under the host cell membrane with reduced calcein quenching by hemoglobin in cells with mature trophozoite stage parasites.

Conclusions/Significance

The results support the predictions of the colloid-osmotic hypothesis and provide a better understanding of the homeostasis of malaria-infected red cells. In addition, they revealed the existence of a distinct peripheral microdomain in the host cell with limited access to hemoglobin molecules indicating the concentration of substantial amounts of parasite-exported material.     

Decomposition of plant residues at low temperatures separates turnover of nitrogen and energy rich tissue components in time

Carbon and nitrogen loss patterns from stems and leaves from Elephant grass (Miscanthus × ogiformis Honda cv. Giganteus), and five commonly used cover crop species: Hairy vetch (Vicia villosa Roth), Italian ryegrass (Lolium multiflorum L.), Crimson clover (Trifolium incarnatum L.), Rye (Secale cereale L.), and Radish (Raphanus sativus L.) were examined at 3 and 9 °C. The stratified incubation system allowed `dry' recovery of the decomposing plant residues with minimal soil contamination and without loss of soluble substances. The recovered materials were characterized biochemically and by light and scanning electron microscopy. When the data was analysed across all treatments and sampling dates, there was no significant effect of temperature on N loss, whereas C loss was significantly affected (P<0.0001) by temperature. Decomposition at 3 °C led to wider C-to-N ratios in the plant residues. At 3 °C there was no net immobilization of N, whereas at 9 °C net immobilization was strong in the L. multiflorum and M. × ogiformis treatments. The biochemical and microscopic evidence supports that microbial growth and macro-polymer utilization was reduced at 3 °C. It was apparent that the dicot materials leaked substantially more carbon during the early phase of decomposition, whereas in the monocot materials and especially in the M. × ogiformis treatment the microbial growth and substrate utilization must have been contained within the decomposing tissues. Based on this evidence, we propose that the decomposition of intracellular low molecular substances and proteins can be viewed as a process separate from the decomposition of macro-polymers in cell walls. At higher temperatures these processes coincide and thus the distinctiveness is blurred, whereas at low temperatures they may occur more separated in time as well as space due to leaking.     

Sterilization and preservation influence the biophysical properties of human amnion grafts.

The introduction of amniotic membrane (AM) transplantation in ophthalmic surgery holds great promise and in many clinical situations it offers an alternative to existing management options. The purpose of this study was to examine the influence of established sterilization and preservation procedures on biophysical and histological properties of AM grafts. Amnion was sterilized by peracetic acid/ethanol sterilization [PES] and preserved by air-drying (sterile laminar flow) [AD] or in glycerol [GLYC]. Unsterilized AM were preserved at -80 degrees C [-80 degrees C] and served as an experimental control. Amnion allografts were characterized by the determination of their thickness, moisture vapour permeability (MVP), oxygen permeability (OPERM), tensile strength and sulphur content. Immunostaining for tissue-specific and basement membrane-related proteins was performed. Differences in biophysical parameters were found between the unsterilized allografts and the sterilized, air-dried or glycerol-preserved allografts. [PES/AD] showed the highest MVP and OPERM, the highest tensile strength and the lowest sulphur content and thickness. [PES/GLYC] exhibited the lowest OPERM and the highest thickness compared to [-80 degrees C] and [PES/AD]. Collagen types V and VII were preserved the best in the control group. Sterilization and preservation affect biophysical properties important for the use of AM as allogenic grafts. It has to be determined if any change, as noted, has a clinical impact.     

Phylogeny of the Heelwalkers (Insecta: Mantophasmatodea) based on mtDNA sequences, with evidence for additional taxa in South Africa

We examined the phylogeny of Mantophasmatodea from southern Africa (South Africa, Namibia) using approx. 1300 bp of mitochondrial DNA sequence data from the genes encoding COI and 16S. The taxon sample comprised multiple specimens from eight described species (Namaquaphasma ookiepense, Austrophasma rawsonvillense, A. caledonense, A. gansbaaiense, Lobatophasma redelinghuysense, Hemilobophasma montaguense, Karoophasma botterkloofense, K. biedouwense) and four undescribed species of Austrophasmatidae; three specimens of Sclerophasma paresisense (Mantophasmatidae); and two specimens of Praedatophasma maraisi and one of Tyrannophasma gladiator (not yet convincingly assigned to any family). For outgroup comparison a broad selection from hemimetabolous insect orders was included. Equally weighted parsimony analyses of the combined COI+16S data sets with gaps in 16S scored as a fifth character state supported Austrophasmatidae and all species and genera of Mantophasmatodea as being monophyletic. Most species were highly supported with 98-100% bootstrap/7-39 Bremer support (BS), but K. biedouwense had moderate support (87/4) and A. caledonense low support (70/1). Mantophasmatodea, Austrophasmatidae, and a clade Tyrannophasma gladiator+Praedatophasma maraisi were all strongly supported (99-100/12-25), while relationships among the two latter clades and Mantophasmatidae remain ambiguous. Concerning the relationships among genera of Austrophasmatidae, support values are moderately high for some nodes, but not significant for others. We additionally calculated the partitioned BS values of COI and 16S for all nodes in the strict consensus of the combined tree. COI and 16S are highly congruent at the species level as well as at the base of Mantophasmatodea, but congruence is poor for most intergeneric relationships. In forthcoming studies, deeper relationships in the order should be additionally explored by nuclear genes, such as 18S and 28S, for a reduced sample of specimens.     

Colonization of overlaying water by bacteria from dry river sediments

We studied the diversity, community composition and activity of the primary microbial colonizers of the water above freshly re-wetted sediments from a temporary river. Dried sediments, collected from Mulargia River (Sardinia, Italy), were covered with sterile freshwater in triplicate microcosms, and changes of the planktonic microbial assemblage were monitored over a 48 h period. During the first 9 h bacterial abundance was low (1.5 x 10(4) cells ml(-1)); it increased to 3.4 x 10(6) cells ml(-1) after 28 h and did not change thereafter. Approximately 20% of bacteria exhibited DNA de novo synthesis already after 9 h of incubation. Changes of the ratios of (3)H-leucine to (3)H-thymidine incorporation rates indicated a shift of growth patterns during the experiment. Extracellular enzyme activity showed a maximum at 48 h with aminopeptidase activity (430.8 +/- 22.6 nmol MCA l(-1) h(-1)) significantly higher than alkaline phosphatase (98.6 +/- 4.3 nmol MUF l(-1) h(-1)). The primary microbial colonizers of the overlaying water - as determined by 16S rRNA gene sequence analysis - were related to at least six different phylogenetic lineages of Bacilli and to Alphaproteobacteria (Brevundimonas spp. and Caulobacter spp.). Large bacterial cells affiliated to one clade of Bacillus sp. were rare in the dried sediments, but constituted the majority of the planktonic microbial assemblage and of cells with detectable DNA-synthesis until 28 h after re-wetting. Their community contribution decreased in parallel with a rise of flagellated and ciliated protists. Estimates based on cell production rates suggested that the rapidly enriched Bacillus sp. suffered disproportionally high loss rates from selective predation, thus favouring the establishment of a more heterogenic assemblage of microbes (consisting of Proteobacteria, Actinobacteria and Cytophaga-Flavobacteria). Our results suggest that the primary microbial colonizers of the water above dried sediments are passively released into the plankton and that their high growth potential is counteracted by the activity of bacterivorous protists.     

Resistance training induces qualitative changes in muscle morphology, muscle architecture, and muscle function in elderly postoperative patients.

Although the negative effects of bed rest on muscle strength and muscle mass are well established, it still remains a challenge to identify effective methods to restore physical capacity of elderly patients recovering from hospitalization. The present study compared different training regimes with respect to muscle strength, muscle fiber size, muscle architecture, and stair walking power in elderly postoperative patients. Thirty-six patients (60-86 yr) scheduled for unilateral hip replacement surgery due to hip osteoarthritis were randomized to either 1) resistance training (RT: 3/wk x 12 wk), 2) electrical stimulation (ES: 1 h/day x 12 wk), or 3) standard rehabilitation (SR: 1 h/day x 12 wk). All measurements were performed at baseline, at 5 wk and 12 wk postsurgery. After 12 wk of resistance training, maximal dynamic muscle strength increased by 30% at 60 degrees /s (P < 0.05) and by 29% at 180 degrees /s (P < 0.05); muscle fiber area increased for type I (+17%, P < 0.05), type IIa (+37%, P < 0.05), and type IIx muscle fibers (+51%, P < 0.05); and muscle fiber pennation angle increased by 22% and muscle thickness increased by 15% (P < 0.05). Furthermore, stair walking power increased by 35% (P < 0.05) and was related to the increase in type II fiber area (r = 0.729, P < 0.05). In contrast, there was no increase in any measurement outcomes with electrical stimulation and standard rehabilitation. The present study is the first to demonstrate the effectiveness of resistance training to induce beneficial qualitative changes in muscle fiber morphology and muscle architecture in elderly postoperative patients. In contrast, rehabilitation regimes based on functional exercises and neuromuscular electrical stimulation had no effect. The present data emphasize the importance of resistance training in future rehabilitation programs for elderly individuals.     

Antifouling enzymes and the biochemistry of marine settlement

Antifouling coatings are used extensively on marine vessels and constructions, but unfortunately they are found to pose a threat to the marine environment, notably due to content of metal-based biocides. Enzymes have repeatedly been proposed as an alternative to traditional antifouling compounds. In this review, the enzymes claimed to hold antifouling activity are classified according to catalytic functions. The enzyme functions are juxtaposed with the current knowledge about the chemistry of settlement and adhesion of fouling organisms. Specific focus will be on bacteria, microalgae, invertebrate larvae and macroalgae zoospores. Two main concepts in enzyme-based antifouling are identified: breakdown of adhesive components and catalytic production of repellent compounds in-situ. The validity of the various modes of action is evaluated and the groups of enzymes with the highest potential are highlighted.     

Double quantum filtering homonuclear MAS NMR correlation spectra: a tool for membrane protein studies

13C homonuclear correlation spectra based on proton driven spin diffusion (PDSD) are becoming increasingly important for obtaining distance constraints from multiply labeled biomolecules by MAS NMR. One particular challenging situation arises when such constraints are to be obtained from spectra with a large natural abundance signal background which causes detrimental diagonal peak intensities. They obscure cross peaks, and furthermore impede the calculation of a buildup rates matrix which may be used to derive distance constraints, as carried out in "NMR crystallography". Here, we combine double quantum (DQ) filtering with 13C-13C dipolar assisted rotational resonance (DARR) experiments to yield correlation spectra free of natural abundance contributions. Two experimental schemes, using DQ filtering prior to evolution (DOPE), and after mixing (DOAM), have been evaluated. Diagonal peak intensities along the spectrum diagonal are removed completely, and crosspeaks close to the diagonal are easily identifiable. For DOAM spectra with negligible mixing times, it is possible to carry out 'assignment walks' which simplify peak identification substantially. The method is demonstrated on 13C-cys labeled proteorhodopsin, a 27 kDa membrane protein. The magnetization transfer characteristics were studied using buildup curves obtained on uniformly 13C labelled crystalline tripeptide MLF. Our data show that DQ filtered DARR experiments pave the way for obtaining through space constraints for structural studies on ligands, bound to membrane receptors, or on small fragments within large proteins.