ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.     

Chemical and microbiological changes during vermicomposting of coffee pulp using exotic (<Emphasis Type="Italic">Eudrilus eugeniae</Emphasis>) and native earthworm (<Emphasis Type="Italic">Perionyx ceylanesis</Emphasis>) species

Coffee pulp is the main solid residue from the wet processing of coffee berries. Due to presence of anti-physiological and anti-nutritional factors, coffee pulp is not considered as adequate substrate for bioconversion process by coffee farmers. Recent stringent measures by Pollution Control authorities, made it mandatory to treat all the solid and liquid waste emanating from the coffee farms. A study was conducted to evaluate the efficiency of an exotic (Eudrilus eugeniae) and a native earthworm (Perionyx ceylanesis) from coffee farm for decomposition of coffee pulp into valuable vermicompost. Exotic earthworms were found to degrade the coffee pulp faster (112 days) as compared to the native worms (165 days) and the vermicomposting efficiency (77.9%) and vermicompost yield (389 kg) were found to significantly higher with native worms. The multiplication rate of earthworms (280%) and worm yield (3.78 kg) recorded significantly higher with the exotic earthworms. The percentage of nitrogen, phosphorous, potassium, calcium and magnesium in vermicompost was found to increase while C:N ratio, pH and total organic carbon declined as a function of the vermicomposting. The plant nutrients, nitrogen (80.6%), phosphorus (292%) and potassium (550%) content found to increase significantly in the vermicompost produced using native earthworms as compared to the initial values, while the calcium (85.7%) and magnesium (210%) content found to increase significantly in compost produced utilizing exotic worms. Vermicompost and vermicasts from native earthworms recorded significantly higher functional microbial group’s population as compared to the exotic worms. The study reveals that coffee pulp can be very well used as substrate for vermicomposting using exotic (Eudrilus eugeniae) and native earthworm (Perionyx ceylanesis).     

A comparative analysis reveals weak relationships between ecological factors and beta diversity of stream insect metacommunities at two spatial levels

The hypotheses that beta diversity should increase with decreasing latitude and increase with spatial extent of a region have rarely been tested based on a comparative analysis of multiple datasets, and no such study has focused on stream insects. We first assessed how well variability in beta diversity of stream insect metacommunities is predicted by insect group, latitude, spatial extent, altitudinal range, and dataset properties across multiple drainage basins throughout the world. Second, we assessed the relative roles of environmental and spatial factors in driving variation in assemblage composition within each drainage basin. Our analyses were based on a dataset of 95 stream insect metacommunities from 31 drainage basins distributed around the world. We used dissimilarity‐based indices to quantify beta diversity for each metacommunity and, subsequently, regressed beta diversity on insect group, latitude, spatial extent, altitudinal range, and dataset properties (e.g., number of sites and percentage of presences). Within each metacommunity, we used a combination of spatial eigenfunction analyses and partial redundancy analysis to partition variation in assemblage structure into environmental, shared, spatial, and unexplained fractions. We found that dataset properties were more important predictors of beta diversity than ecological and geographical factors across multiple drainage basins. In the within‐basin analyses, environmental and spatial variables were generally poor predictors of variation in assemblage composition. Our results revealed deviation from general biodiversity patterns because beta diversity did not show the expected decreasing trend with latitude. Our results also call for reconsideration of just how predictable stream assemblages are along ecological gradients, with implications for environmental assessment and conservation decisions. Our findings may also be applicable to other dynamic systems where predictability is low.     

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na and Cl accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na⁺ and Cl⁻ accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.     

Energy estimation in protein design

The progress achieved by several groups in the field of computational protein design shows that successful design methods include two major features: efficient algorithms to deal with the combinatorial exploration of sequence space and optimal energy functions to rank sequences according to their fitness for the given fold.     

Transmembrane domains in the functions of Fc receptors

In the present study, we use a novel method, PHDhtm, to predict the exact locations and extents of the transmembrane (TM) domains of multisubunit immunoglobulin Fc-receptors. Whereas most previous studies have used single residue hydrophobicity plots for characterizing of these domains, PHDhtm utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the above. Present PHDhtm application predicts TM domains of immunoglobulin Fc-receptors that in many cases differ significantly from those derived by using earlier methods. Comparisons of helical wheel projections of the presently derived TM domains from PHDhtm with those produced earlier reveal different hydrophobic moments as well as hydrophobic and hydrophilic surfaces. These differences probably alter the character of subunit association within the receptor complexes. This new algorithm can also be used for other membrane protein complexes and may advance both understanding the principles underlying such complexes formation and design of peptides that can interfere with such TM domain association so as to modulate specific cellular responses.     

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.