Dimerization of a flocculent protein from Moringa oleifera: experimental evidence and in silico interpretation

Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, the dimerization property of a coagulant protein (MO_2.1) from Moringa oleifera seeds was addressed through laboratory experiments, protein–protein docking studies and binding free energy calculations. The structure of MO_2.1 was predicted by homology modelling, while binding free energy and residues-distance profile analyses provided insight into the energetics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms of MO_2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.     

Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae Lz4W

Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringae Lz4W, which accumulated linear chromosomal DNA leading to cell death and growth inhibition at 4°C. RecBCD protein generally repairs DNA double‐strand breaks by RecA‐dependent homologous recombination pathway. Here we show that ΔrecA mutant of P. syringae is not cold‐sensitive. Significantly, inactivation of additional DNA repair genes ruvAB rescued the cold‐sensitive phenotype of ΔrecBCD mutant. The ΔrecA and ΔruvAB mutants were UV‐sensitive as expected. We propose that, at low temperature DNA replication encounters barriers leading to frequent replication fork (RF) arrest and fork reversal. RuvAB binds to the reversed RFs (RRFs) having Holliday junction‐like structures and resolves them upon association with RuvC nuclease to cause linearization of the chromosome, a threat to cell survival. RecBCD prevents this by degrading the RRFs, and facilitates replication re‐initiation. This model is consistent with our observation that low temperature‐induced DNA lesions do not evoke SOS response in P. syringae. Additional studies show that two other repair genes, radA (encoding a RecA paralogue) and recF are not involved in providing cold resistance to the Antarctic bacterium.     

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.     

ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature

RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5' overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNA-binding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.     

Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria

Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.     

Isolation and characterization of novel lipase gene LipHim1 from the DNA isolated from soil samples

Metagenomics is a magnificent tool to isolate genes from unknown/uncharacterized species and also from organisms that cannot be cultured. In this study, we constructed a metagenomic library from isolated DNA of soil samples collected from Palamuru University campus premises, in Mahabubnagar district of Andhra Pradesh, India. We isolated a novel lipase gene LipHim1, which has an open reading frame of 591 base pairs and encodes ～23 kDa protein consisting of 196 amino acids. The Lipase LipHim1 showed maximum 32% homology at the protein level with the extracellular Aeromonas hydrophila lipase (Class II, GDSL family) and was significantly different from all other known lipases. The isolated lipase catalyzed the hydrolysis of fatty acid esters of polyoxyethylene sorbitan such as Tween 60. Our results indicate that the isolated lipase gene is novel.     

All Three Subunits of RecBCD Enzyme Are Essential for DNA Repair and Low-Temperature Growth in the Antarctic Pseudomonas syringae Lz4W

Background

The recD mutants of the Antarctic Pseudomonas syringae Lz4W are sensitive to DNA-damaging agents and fail to grow at 4°C. Generally, RecD associates with two other proteins (RecB and RecC) to produce RecBCD enzyme, which is involved in homologous recombination and DNA repair in many bacteria, including Escherichia coli. However, RecD is not essential for DNA repair, nor does its deletion cause any growth defects in E. coli. Hence, the assessment of the P. syringae RecBCD pathway was imperative.

Methodology/Principal Findings

Mutational analysis and genetic complementation studies were used to establish that the individual null-mutations of all three genes, recC, recB, and recD, or the deletion of whole recCBD operon of P. syringae, lead to growth inhibition at low temperature, and sensitivity to UV and mitomycin C. Viability of the mutant cells dropped drastically at 4°C, and the mutants accumulated linear chromosomal DNA and shorter DNA fragments in higher amounts compared to 22°C. Additional genetic data using the mutant RecBCD enzymes that were inactivated either in the ATPase active site of RecB (RecB^K29Q) or RecD (RecD^K229Q), or in the nuclease center of RecB (RecB^D1118A and RecB^Δnuc) suggested that, while the nuclease activity of RecB is not so critical in vivo, the ATP-dependent functions of both RecB and RecD are essential. Surprisingly, E. coli recBCD or recBC alone on plasmid could complement the defects of the ΔrecCBD strain of P. syringae.

Conclusions/Significance

All three subunits of the RecBCD^Ps enzyme are essential for DNA repair and growth of P. syringae at low temperatures (4°C). The RecD requirement is only a function of the RecBCD complex in the bacterium. The RecBCD pathway protects the Antarctic bacterium from cold-induced DNA damages, and is critically dependent on the helicase activities of both RecB and RecD subunits, but not on the nuclease of RecBCD^Ps enzyme.     

Nuclear localization of Munc18-1 (p67) in the adult rat brain and PC12 cells

Munc18-1, also referred to as p67, co-purifies with Cdk5 and has an important role in neurotransmitter release. The role of Munc18-1 for functional connectivity of the nervous system was demonstrated by gene knockout experiments in mice, wherein accumulation of neurotransmitter and silencing of synaptic activity was observed. Our earlier studies have shown that both Munc18-1 and Cdk5 co-purify and co-localize with cytoskeletal components, implying that apart from having a regulatory role in vesicle docking and fusion, Munc18-1 could also affect the dynamics of neuronal cytoskeleton. In the present study we have shown the presence of Munc18-1 in nuclear rich fraction from rat brain and confirmed the nuclear localization of this protein in PC12 cells and adult rat brain neurons by immunofluorescence and immunoelectron microscopy. We also demonstrate the binding of Munc18-1 to double stranded (ds) DNA. The ability of Munc18-1 to bind dsDNA, albeit the lack of DNA binding domains, suggests that the binding may be mediated through protein-protein interaction through some other DNA-binding proteins. The presence of both nuclear import and export signals in Munc18-1 primary structure corroborates its nuclear localization and makes it a putative shuttle protein between nuclear and cytoplasmic compartments, the precise physiological relevance of which needs to be elucidated.     

Altered circulating levels of matrix metalloproteinases and inhibitors associated with elevated type 2 cytokines in lymphatic filarial disease

Background

Infection with Wuchereria bancrofti can cause severe disease characterized by subcutaneous fibrosis and extracellular matrix remodeling. Matrix metalloproteinases (MMPs) are a family of enzymes governing extracellular remodeling by regulating cellular homeostasis, inflammation, and tissue reorganization, while tissue-inhibitors of metalloproteinases (TIMPs) are endogenous regulators of MMPs. Homeostatic as well as inflammation-induced balance between MMPs and TIMPs is considered critical in mediating tissue pathology.

Methods

To elucidate the role of MMPs and TIMPs in filarial pathology, we compared the plasma levels of a panel of MMPs, TIMPs, other pro-fibrotic factors, and cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag−) active infection to those with clinically asymptomatic infections (INF) and in those without infection (endemic normal [EN]). Markers of pathogenesis were delineated based on comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag− compared to EN).

Results and Conclusion

Our data reveal that an increase in circulating levels of MMPs and TIMPs is characteristic of the filarial disease process per se and not of active infection; however, filarial disease with active infection is specifically associated with increased ratios of MMP1/TIMP4 and MMP8/TIMP4 as well as with pro-fibrotic cytokines (IL-5, IL-13 and TGF-β). Our data therefore suggest that while filarial lymphatic disease is characterized by a non-specific increase in plasma MMPs and TIMPs, the balance between MMPs and TIMPs is an important factor in regulating tissue pathology during active infection.     

Molecular insights into the ecology of a psychrotolerant Pseudomonas syringae

Low temperatures constrain cellular life due to reductions in nutrient uptake, enzyme kinetics, membrane permeability, and function of other biomacromolecules. This has implications for the biophysical limits of life on Earth, and the plausibility of life in extraterrestrial locations. Although most pseudomonads are mesophilic in nature, isolates such as the Antarctic Pseudomonas syringae Lz4W exhibit considerable psychrotolerance, with an ability to grow even between 4 and 0°C. In this review, we explore the molecular traits and characteristic phenotypes of P. syringae Lz4W that enable life at low temperatures. We describe adaptations that enhance membrane fluidity; examine genes involved in cellular function and survival in the cold; assess capability for energy generation at low temperature; and detail the mechanics of DNA repair and RNA processing at low temperature, and speculate that P. syringae Lz4W can also synthesize glycerol to maintain flexibility of macromolecular systems. In the range 4 to 0ºC, there are considerable changes in the properties and behaviour of water. Specifically, density can have adverse impacts on plasma-membrane functions, cytoplasmic viscosity, protein behaviour, and other essential properties of cellular system. We identified a combination of adaptations that may be peculiar to cold-tolerant P. syringae, including increase of unsaturated fatty acids in the plasma membrane; a RNA polymerase able to function at 0°C; RecBCD- and RuvAB-dependent reestablishment of replication fork; and efficiencies of degradosome machinery and RNA processing by RNaseR at low temperature. Several unresolved questions are discussed in the context of astrobiology, and further work needed on the psychrotolerance of P. syringae.