Oligosaccharide biotechnology: an approach of prebiotic revolution on the industry

Applied Microbiology and Biotechnology - Oligosaccharides are polymers with two to ten monosaccharide residues which have sweetener functions and sensory characteristics, in addition to exerting...     

Taraxerol 4‐Methoxybenzoate,an in vitro Inhibitor of Photosynthesis Isolated from Pavonia multiflora A. St‐Hil. (Malvaceae)

A phytochemical study of Pavonia multiflora A. St ‐Hil . (Malvaceae) led to the isolation through chromatographic techniques of 10 secondary metabolites: vanillic acid ( 1 ), ferulic acid ( 2 ), p‐hydroxybenzoic acid ( 3 ), p‐coumaric acid ( 4 ), loliolide ( 5 ), vomifoliol ( 6 ), 4,5‐dihydroblumenol A ( 7 ), 3‐oxo‐α‐ionol ( 9 ), blumenol C ( 10 ), and taraxerol 4‐methoxybenzoate ( 8 ), the latter being a novel metabolite. Their structures were identified by ¹H‐ and ¹³C‐NMR, using one‐ and two‐dimensional techniques, and X‐ray crystallography. In this work, we report the effect of compounds 5 and 8 on several photosynthetic activities in an attempt to search for new compounds as potential herbicide agents that affect photosynthesis. Both compounds inhibited the electron flow from H₂O to methyl viologen; therefore, they act as Hill reaction inhibitors. Using polarographic techniques and studies of the fluorescence of chlorophyll a, the interaction sites of these compounds were located at photosystem II.     

Edge and land use effects on dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) in Brazilian cerrado vegetation

The Edge Influence is one of the most pervasive effects of habitat fragmentation, as many forest remnants in anthropogenic landscapes are within 100 m of edges. Forest remnants may also affect the surrounding anthropogenic matrix, possibly resulting in a matrix–edge–remnant diversity gradient for some species groups. We sampled dung beetles in 15 agricultural landscapes using pitfall traps placed along transects in matrix–edge–remnant gradients. The remnants were a native savanna-like vegetation, the cerrado, and the matrix was composed of three human-dominated environments (sugarcane, eucalyptus, pasture). More species were observed in cerrado remnants than in adjacent land uses. Dung beetles were also more abundant in the cerrado than in the landscape matrix of sugarcane and eucalypt, but not of pasture. Dung beetles were severely affected by anthropogenic land uses, and notwithstanding their high abundance in some land uses such as pasture, the species richness in these areas tended to be smaller than in the cerrado remnants. We also found that the influence of the edge was evident only for abundance, particularly in landscapes with a pasture matrix. However, this land use disrupts the species composition of communities, indicating that communities located in cerrado and pasture have a distinct species composition, and that both communities are affected by edge distance. Thus, anthropogenic land uses may severely affect dung beetles, and this impact can extend to communities located in cerrado remnants as well as to those in matrices, with possible consequences for ecological processes such as decomposition and nutrient cycling.     

Patch Size,Functional Isolation,Visibility and Matrix Permeability Influences Neotropical Primate Occurrence within Highly Fragmented Landscapes

Forest fragmentation and habitat loss are among the major current extinction causes. Remaining fragments are mostly small, isolated and showing poor quality. Being primarily arboreal, Neotropical primates are generally sensitive to fragmentation effects. Furthermore, primates are involved in complex ecological process. Thus, landscape changes that negatively interfere with primate population dynamic affect the structure, composition, and ultimately the viability of the whole community. We evaluated if fragment size, isolation and visibility and matrix permeability are important for explaining the occurrence of three Neotropical primate species. Employing playback, we verified the presence of Callicebus nigrifrons, Callithrix aurita and Sapajus nigritus at 45 forest fragments around the municipality of Alfenas, Brazil. We classified the landscape and evaluated the metrics through predictive models of occurrence. We selected the best models through Akaike Selection Criterion. Aiming at validating our results, we applied the plausible models to another region (20 fragments at the neighboring municipality of Poço Fundo, Brazil). Twelve models were plausible, and three were validated, two for Sapajus nigritus (Area and Area+Visibility) and one for Callicebus nigrifrons (Area+Matrix). Our results reinforce the contribution of fragment size to maintain biodiversity within highly degraded habitats. At the same time, they stress the importance of including novel, biologically relevant metrics in landscape studies, such as visibility and matrix permeability, which can provide invaluable help for similar studies in the future and on conservation practices in the long run.     

Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsK_N) is essential for division, and the C terminus (FtsK_C) is a well characterized DNA translocase. Although the function of FtsK_N is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsK_N and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsK_N is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsK_N was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsK_N also provides an important platform for future studies on essential interactions required for this process.     

A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry

Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa subproteomes and provides a framework for identifying and studying entire pathways critical to biofilm biology in this model pathogenic organism. The identification of novel protein targets could contribute to the development of much needed antimicrobial therapies to treat the chronic infections found in patients with cystic fibrosis.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a significant cause of morbidity and mortality in immunocompromised individuals, including those with cystic fibrosis (CF).¹ Late-stage polymicrobial infections in CF patients are dominated by P. aeruginosa (1). This highly adaptable bacterium contributes to excessive pulmonary inflammation, subsequent destruction of lung tissue, and decreased lung function (2–4). The ubiquity and persistence of this pathogen are due to a combination of virulence and resistance mechanisms that allow it to adapt to environmental changes and colonize the CF host (5). A key component of its adaptation and persistence is the transition from free-living (planktonic) bacteria into organized, surface-attached communities known as biofilms (6). Importantly, biofilms are a barrier to host defenses and reduce the efficacy of antimicrobial therapies (7, 8). Attempts to understand the biochemical basis of biofilm development and resilience have consistently shown differences in protein composition in the model organism P. aeruginosa PAO1 (9–13) (versus its planktonic counterparts). However, technical limitations of these studies have left much of the biofilm proteome unresolved. Accordingly, many aspects of biofilm structure and function are still poorly understood.In this study, we aimed to establish a quantitative proteomic framework to describe P. aeruginosa cells as they progress through planktonic and biofilm lifestyles. To achieve this, we performed an in-solution digest of triplicate whole cell lysates from growth-condition-matched planktonic and biofilm cultures collected at three different time points. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using a high-performance quadrupole Orbitrap mass spectrometer (Q Exactive), which resulted in the high-confidence identification of 1884 proteins. We identified proteins unique to each growth mode and those that were differentially regulated and assigned functions according to the P. aeruginosa Community Annotation Project (PseudoCAP) (10). This work advances the growing body of P. aeruginosa planktonic subproteomes (11) and provides new knowledge about the influence of growth mode on the protein composition of both planktonic and biofilm P. aeruginosa over time. A better understanding of the key aspects of P. aeruginosa biofilm biology has the potential to reveal novel targets for antimicrobial therapies and vaccine development for CF patients (12).     

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca<Superscript>2+</Superscript>-induced cysteine relative mobility/mitochondrial permeability transition pore

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca²⁺-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys⁵⁶ relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca²⁺-induced ANT conformational change and mitochondrial swelling. ANT-Pro⁶¹ isomerization increased ANT-Cys⁵⁶ relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca²⁺ induced ANT “c” conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca²⁺-induced ANT “c” conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca²⁺ induces the ANT “c” conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.     

Bacteriocins Pep5 and Epidermin Inhibit Staphylococcus epidermidis Adhesion to Catheters

Pep5 and epidermin bacteriocins were tested on clinical strains of Staphylococcus epidermidis and S. aureus isolated from catheter-related infections. These bacteriocins were inhibitory to several isolates at a concentration of 640 activity units mL⁻¹. The ability of bacteriocins in inhibiting adhesion of S. epidermidis to silicone catheters was evaluated. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a significant decrease in the cell number of S. epidermidis adhered to silicone catheters. Bacteriocins used to decrease bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.