Comparative and Evolutionary Analysis of the Bacterial Homologous Recombination Systems

Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes.     

Oriented loading of FtsK on KOPS

In Escherichia coli, the ATP-dependent DNA translocase FtsK transports DNA across the site of cell division and activates recombination by the XerCD recombinases at a specific site on the chromosome, dif, to ensure the last stages of chromosome segregation. DNA transport by FtsK is oriented by 8-base-pair asymmetric sequences ('KOPS'). Here we provide evidence that KOPS promote FtsK loading on DNA and that translocation is oriented at this step.     

Description of a tissue culture technique using tissue from mussel (Mytilus edulis) for the preparation of chromosomes]

Explants from mantle and foot tissues of adult mussel were grown in culture tubes containing a medium composed of Eagle's Basal Medium supplemented with salts, Hepes buffer, egg yolk and antibiotics. The cultures were maintained at 18 degrees C and pH 7.50, without medium renewal. After 6-7 days, the cultures were stopped and harvested for slide preparation. Numerous metaphase spreads that were good enough for karyotyping were consistently obtained. This method may prove to be a reliable source of actively dividing cells that is a prerequisite for extensive chromosome structure analyses in the bivalves.     

Does the failure to acquire helminthic parasites predispose to Crohn's disease?

Two polarized patterns (Th1 and Th2) of cytokines regulate inflammatory responses. Each cytokine pattern inhibits production of the opposing pattern. Lymphocytes from inflamed intestine due to Crohn's disease secrete a Th1 pattern of cytokines. Crohn's disease is most prevalent in highly industrialized countries with temperate climates. It occurs rarely in tropical third world countries with poor sanitation. We propose that exposure to an environmental agent predisposes individuals to Crohn's disease. Parasitic worms (helminths) are common in tropical climates and in populations subject to crowding and poor sanitation. Children are most subject to helminthic colonization. Many helminths live within or migrate through the human gut where they interact with the mucosal immune system. The host mounts a mucosal response that includes Th2 cytokine production limiting helminthic colonization. Helminths and their eggs probably are the most potent stimulators of mucosal Th2 responses. The Th2 response provoked by parasitic worms can modulate immune reactions to unrelated parasitic, bacterial, and viral infections. Many people in developed countries now live in increasingly hygienic environments, avoiding exposure to helminths. Perhaps failure to acquire these parasites and experience mucosal Th2 conditioning predisposes to Crohn's disease, which is an overly active Th1 inflammation.     

Sedimentary development of a continuous Middle Devonian to Mississippian section from the fore-reef fringe of the Brilon Reef Complex (Rheinisches Schiefergebirge, Germany)

The Brilon-reef complex is one of the biggest Devonian carbonate buildups (~80 km²) of the Rheinisches Schiefergebirge. The Burgberg section is located in the southeastern fore-reef area of the Brilon Reef Complex and exposes a succession of strata (117 m thick), which extends from the Middle Givetian (middle varcus conodont Zone) to the Viséan (bilineatus conodont Zone). Field and microfacies observations led to the definition of nine microfacies that are integrated into a sedimentary model divided into off-reef, intermediate fore-reef, and proximal fore-reef sedimentary domains (SD). The off-reef domain (SD1) is the most distal setting observed and is characterized by fine-grained sediments, dominated by pelagic biota and the local occurrence of gravity-flow deposits. The intermediate fore-reef (SD2) is characterized by a mixture of biota and sediments coming from both deeper-water and shallow-water sources and is influenced by storm and gravity-flow currents. In this domain, Renalcis mound-like structures developed locally. Finally, the proximal fore-reef (SD3) corresponds to the most proximal setting that is strongly influenced by gravity-flow currents derived from the Brilon Reef Complex. The temporal evolution of microfacies in the fore-reef setting of the Burgberg section show five main paleoenvironmental trends influenced by the onset, general development, and demise/drowning of the Brilon Reef Complex. Fore-reef to off-reef lithologies and their temporal changes are from the base to the top of the section: (U1)—fine-grained sediments with large reef debris, corresponding to the initial development of the reef building upon submarine volcaniclastic deposits during the Middle Givetian (middle varcus Zone) and first export of reef debris in the fore-reef setting; (U2)—high increase of reef-derived material in the fore-reef area, corresponding to a significant progradation of the reef from the Middle Givetian to the Early Frasnian (maximum extension of the Brilon Reef Complex to the south, disparilis to the falsiovalis conodont biozones); (U3)—progressive decrease of shallow-water derived material and increase of fine-grained sediments and deep-water biota into the fore-reef setting, corresponding to the stepwise withdrawal of the reef influence; from the Middle to the Late Frasnian (jamieae conodont Zone); (U4)—development of a submarine rise characterized by nodular and cephalopod-bearing limestones extending from the Late Frasnian to the Late Famennian corresponding to the demise and drowning of the Brilon Reef Complex as a result of the Late Frasnian Kellwasser events (upper rhenana and triangularis conodont biozones); (U5)—significant deepening of the Burgberg area starting in the Late Famennian, directly followed by an aggrading trend marked by pelagic shales overlying the nodular limestone deposits.     

Nebulized Fibrinolytic Agents Improve Pulmonary Fibrinolysis but Not Inflammation in Rat Models of Direct and Indirect Acute Lung Injury

Background

Critically ill patients frequently develop acute lung injury (ALI). Disturbed alveolar fibrin turnover, a characteristic feature of ALI, is the result of both activation of coagulation and inhibition of fibrinolysis. Nebulized fibrinolytic agents could exert lung–protective effects, via promotion of fibrinolysis as well as anti–inflammation.

Methods

Rats were challenged intratracheally with Pseudomonas aeruginosa, resulting in pneumonia as a model for direct ALI, or received an intravenous bolus infusion of lipopolysaccharide, as a model for indirect ALI. Rats were randomized to nebulization of normal saline (placebo), recombinant tissue plasminogen activator (rtPA), or monoclonal antibodies against plasminogen activator inhibitor–type 1 (anti–PAI–1).

Results

Nebulized rtPA or anti–PA1–1 enhanced the bronchoalveolar fibrinolytic system, as reflected by a significant reduction of PAI–1 activity levels in bronchoalveolar lavage fluid, and a consequent increase in plasminogen activator activity (PAA) levels to supranormal values. Both treatments also significantly affected systemic fibrinolysis as reflected by a significant increase in PAA levels in plasma to supranormal levels. Neither nebulized rtPA nor anti–PA1–1 affected pulmonary inflammation. Neither treatment affected bacterial clearance of P. aeruginosa from the lungs in case of pneumonia.

Conclusions

Local treatment with rtPA or anti–PA1–1 affects pulmonary fibrinolysis but not inflammation in models of direct or indirect ALI in rats.     

Dramatic changes in a phytoplankton community in response to local and global pressures: a 24‐year survey of the river Loire (France)

The impact of climate change and of other anthropogenic pressures on the structure and composition of phytoplankton communities of large European rivers remains poorly documented. Here we report the findings of a study of the changes in the phytoplankton community of the middle segment of the river Loire over the past 24 years. An attempt is made to distinguish between the impact of changes acting at the local scale and that of those acting more globally. A dramatic reduction in phytoplankton abundance was observed, particularly in the mid ‐1990s; this was concomitant with an increase in the relative proportion of cyanobacteria. At the same time, the phytoplankton community displayed increasing richness and diversity, and little change in its size structure. All these changes seem to be related to local changes, in particular to the reduction in phosphorus concentrations, as well as to changes in climate, throughout modifications in the river discharge and water temperature. Interestingly, herbicide contamination also appeared to be of particular importance in explaining the unexpected increase in the proportion of cyanobacteria in the phytoplankton community after the 1990s. These findings suggest that combinations of numerous anthropogenic pressures acting at different spatial and temporal scales have led to a mix of predictable and unpredictable changes occurring in the phytoplankton community of the river Loire, with probable consequences for the trophic networks in this river.     

Disease pathways at the Rat Genome Database Pathway Portal: genes in context-a network approach to understanding the molecular mechanisms of disease

Background

Biological systems are exquisitely poised to respond and adjust to challenges, including damage. However, sustained damage can overcome the ability of the system to adjust and result in a disease phenotype, its underpinnings many times elusive. Unraveling the molecular mechanisms of systems biology, of how and why it falters, is essential for delineating the details of the path(s) leading to the diseased state and for designing strategies to revert its progression. An important aspect of this process is not only to define the function of a gene but to identify the context within which gene functions act. It is within the network, or pathway context, that the function of a gene fulfills its ultimate biological role. Resolving the extent to which defective function(s) affect the proceedings of pathway(s) and how altered pathways merge into overpowering the system's defense machinery are key to understanding the molecular aspects of disease and envisioning ways to counteract it. A network-centric approach to diseases is increasingly being considered in current research. It also underlies the deployment of disease pathways at the Rat Genome Database Pathway Portal. The portal is presented with an emphasis on disease and altered pathways, associated drug pathways, pathway suites, and suite networks.

Results

The Pathway Portal at the Rat Genome Database (RGD) provides an ever-increasing collection of interactive pathway diagrams and associated annotations for metabolic, signaling, regulatory, and drug pathways, including disease and altered pathways. A disease pathway is viewed from the perspective of networks whose alterations are manifested in the affected phenotype. The Pathway Ontology (PW), built and maintained at RGD, facilitates the annotations of genes, the deployment of pathway diagrams, and provides an overall navigational tool. Pathways that revolve around a common concept and are globally connected are presented within pathway suites; a suite network combines two or more pathway suites.

Conclusions

The Pathway Portal is a rich resource that offers a range of pathway data and visualization, including disease pathways and related pathway suites. Viewing a disease pathway from the perspective of underlying altered pathways is an aid for dissecting the molecular mechanisms of disease.

     

Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.     

Is there a role for antioxidant carotenoids in limiting self-harming immune response in invertebrates?

Innate immunity relies on effectors, which produce cytotoxic molecules that have not only the advantage of killing pathogens but also the disadvantage of harming host tissues and organs. Although the role of dietary antioxidants in invertebrate immunity is still unknown, it has been shown in vertebrates that carotenoids scavenge cytotoxic radicals generated during the immune response. Carotenoids may consequently decrease the self-harming cost of immunity. A positive relationship between the levels of innate immune defence and circulating carotenoid might therefore be expected. Consistent with this hypothesis, we show that the maintenance and use of the prophenoloxidase system strongly correlate with carotenoid concentration in haemolymph within and among natural populations of the crustacean Gammarus pulex.