Identification of biofilm matrix-associated proteins from an acid mine drainage microbial community

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ～2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.     

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes, oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed.     

Nutritional influences on the gut microbiota and the consequences for gastrointestinal health

The human colonic microbiota degrades dietary substrates that are indigestible in the upper GIT (gastrointestinal tract), releasing bacterial metabolites, some of which are important for gut health. Advances in molecular biology techniques have facilitated detailed analyses of the composition of the bacterial community resident in the lower GIT. Such analyses have indicated that more than 500 different bacterial species colonize an individual, and that, although there is much functional consistency in the resident bacterial groups, there is considerable inter-individual variation at the species/strain level. The bacterial community develops during early childhood until it reaches an adult-like composition. Whereas colonization and host factors influence the species composition, dietary factors also have an important impact, with specific bacterial groups changing in response to specific dietary interventions. Since bacterial species have different metabolic activities, specific diets have various consequences for health, dependent on the effect exerted on the bacterial population.     

Genetic diversity and gene flow within and between two different habitats of Primula merrilliana (Primulaceae), an endangered distylous forest herb in eastern China

Understanding whether and how different habitats shape population genetics is a fundamental question and a specific goal for evolutionary and conservation biology research. This study examined genetic diversity and gene flow within and between mountain and foothill habitats of Primula merrilliana, an endangered distylous forest herb in eastern China. Eleven population characteristics, including area, size and density variation, from the two habitats were also investigated. Mountain populations had significantly higher mean genetic diversity than foothill populations, which may be explained by stronger self‐incompatibility breeding system, more opportunity to use elevational shifts to track suitable sites under conditions of climate change and more heterogeneous environments in the former habitat, rather than by the differences of population size, gene flow and genetic drift intensity between them. Genetic analysis revealed that two distinct lineages, corresponding to the two habitats, diverged at China's ‘Last Glaciation’ (11 700–67 500 yr BP), suggesting this divergence was probably triggered by warmer climates during inter‐ (or post‐) glacial periods. Low unidirectional gene flow from mountain to foothill habitats, chiefly by seed dispersal, played a more important role in overall gene flow between habitats than within‐habitat gene flow. Within habitats, pollen contributes more substantially to gene flow than seed dispersal, especially in foothill habitats, possibly due to higher individual density and larger population sizes. These results have implications for the conservation in this and similar landscape areas and indicate the need to protect suitable habitats with wide elevational spans and sufficient size to permit ecological and elevational shifts in response to climatic changes. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 172–189.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation

The ability of Kluyveromyces marxianus to convert lactose into ethyl acetate offers a chance for an economic reuse of whey. Former experiments with K. marxianus DSM 5422 proved limitation of growth by iron (Fe) or copper as a precondition for significant ester synthesis. Several aerobic batch and chemostat cultivations were done with whey-borne media of a variable Fe content for exploring the effect of Fe on growth, the Fe content of biomass, and metabolite synthesis. At low Fe doses, Fe was the growth-limiting factor, the available Fe was completely absorbed by the yeasts, and the biomass formation linearly depended on the Fe dose governed by a minimum Fe content in the yeasts, x _Fe,min. At batch conditions, x _Fe,min was 8.8???g/g, while during chemostat cultivation at D?=?0.15?h^?1, it was 23???g/g. At high Fe doses, sugar was the growth-limiting factor, Fe was more or less absorbed, and the formed biomass became constant. Significant amounts of ethyl acetate were only formed at Fe limitation while high Fe doses suppressed ester formation. Analysis of formed metabolites such as glycerol, pyruvate, acetate, ethanol, ethyl acetate, isocitrate, 2-oxoglutarate, succinate, and malate during chemostat cultivation allowed some interpretation of the Fe-dependent mechanism of ester synthesis; formation of ethyl acetate from acetyl-SCoA and ethanol is obviously initiated by a diminished metabolic flux of acetyl-SCoA into the citrate cycle and by a limited oxidation of NADH in the respiratory chain since Fe is required for the function of aconitase, succinate dehydrogenase, and the electron-transferring proteins.     

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1α,25-(OH)₂D₃ (1,25) and 24R,25-(OH)₂D₃ (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1,25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKCα and ζ as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [γ³²P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A, (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25, but not 1,25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase. © 1996 Wiley-Liss, Inc.     

Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis

Fucoidans are sulfated fucosylated polymers from brown algae cell wall that exhibit some heparin/heparan sulfate properties. We previously demonstrated that these polysaccharides were able in vitro to stimulate dermal fibroblast proliferation and extracellular matrix deposition. Here, we investigated the action of a 16kDa fucoidan fraction on parameters involved in connective tissue breakdown. This fucoidan is able to inhibit gelatinase A secretion and stromelysin 1 induction by interleukin-1beta on dermal fibroblasts in culture. Furthermore, we observed that fucoidan increases the rate of association of MMPs with their specific inhibitors namely TIMPs. Using tissue sections of human skin in ex vivo experiments, we evidenced that this polysaccharide was able to minimize human leukocyte elastase activity resulting in the protection of human skin elastic fiber network against the enzymatic proteolysis due to this serine proteinase. These results suggested that fucoidan could be used for treating some inflammatory pathologies in which uncontrolled extracellular matrix degradation takes place.     

A rat homeobox gene, rNKx-2.5, is a homologue of the tinman gene in Drosophila and is mainly expressed during heart development

 We report the cloning of a rat homeobox-containing gene, rNkx-2.5, and investigation of its expression in adult tissues and during embryonic development. The rNkx-2.5 gene is a homologue of the tinman gene in Drosophila. Both genes share an identical TN domain (tinman-like amino-terminal decapeptide) and about 66.7% sequence identity within their homeodomain sequences. In vertebrates, the rNKx-2.5 gene is most closely related to the mouse NKx-2.5 (mNKx-2.5) gene. Coding sequences for both NKx-2.5 genes have 94.1% sequence identity, including three identical conserved domains, the TN, homeo and NK-2 domains (NK-2 specific domain, carboxy-terminal to the homeodomain in vertebrate tinman homologues). The rat NKx-2.5 gene is encoded by a 1.4-kb mRNA and in adult tissues is mainly expressed in heart, with weaker expression in testis, spleen and lung. During embryonic development, expression of rNKx-2.5 is strongly observed in developing heart, specifically in the myocardium of both atrial and ventricular chambers. In addition, rNKx-2.5 expression marks other developing tissues, including tongue, thyroid, stomach, spleen and pulmonary veins. These data show that rNKx-2.5 is expressed in a pattern similar but not identical to that previously observed for mNKx-2.5. Received: 24 February 1997 / Accepted: 23 June 1997     

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them

Applied Microbiology and Biotechnology - Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called...     

Active and passive contributions to joint kinetics during walking in older adults

The objectives of this study were to characterize the active and passive contributions to joint kinetics during walking in healthy young and older adults, and assess whether isokinetic ankle strength is associated with ankle power output during walking. Twenty healthy young (18–35 years) and 20 healthy older (65–85 years) adults participated in this study. We measured subject-specific passive-elastic joint moment–angle relationships in the lower extremity and tested maximum isokinetic ankle strength at 30 deg/s. Passive moment–angle relationships were used to estimate active and passive joint moment, power, and work quantities during walking at 80%, 100% and 120% of preferred walking speed. There were no significant differences in walking speed, step length, or cadence between the older and young adults. However, the older adults produced significantly more net positive work at the hip but less net positive work at the ankle at all walking speeds. Passive contributions to hip and ankle work did not significantly differ between groups, inferring that the older adults generated the additional hip work actively. Maximum isokinetic ankle strength was significantly less in the older adults, and correlated with peak positive plantar-flexor power at both the preferred and fast walking speeds. The results of this study suggest that age-related shifts in joint kinetics do not arise as a result of increased passive hip joint stiffness, but seem to be reflected in plantar-flexor weakness.