BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression

For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer.     

Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with Phylogenetic Analyses Based on Small Subunit rRNA Gene Sequences

ABSTRACT. The identification of Favella ehrenbergii, a marine planktonic ciliate, has largely been based on its lorica features. This approach is potentially problematic given the polymorphic lorica during this organism's life cycle. We isolated a population of F. ehrenbergii from the coastal waters of Incheon, Korea, and revealed its infraciliature using the protargol staining method. Phylogenetic analysis based on small subunit rRNA gene sequences was also performed. Results showed that this population possessed 16 collar membranelles (CM) and about 100 somatic kineties. These features are highly conserved, even in later dividers. As such, the number of CM and somatic kineties can be used as key characteristics for identification of Favella species.     

Burkholderia species are ancient symbionts of legumes

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia . Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia . The symbiosis-related genes formed deep Burkholderia -specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.     

Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.The rumen is a complex microbial ecosystem that is composed of an immense variety of bacteria, protozoa, fungi, and viruses (5). Among these microorganisms, bacteria are the most investigated population and have a significant effect on the animal''s performance. However, our understanding of how rumen bacteria change and adapt to different ruminal environments is in its infancy.In the feedlot cattle industry, when animals on a forage diet are directly put on a high-grain diet, a decrease in ruminal pH due to lactate production has been observed (23, 31, 42), which leads to the possibility of digestive disorders, which can cause a decrease in the animal''s performance (23, 45). Therefore, feeding programs have been implemented to adapt feedlot cattle from a high-forage diet to a high-concentrate diet by gradually increasing the concentration of grain in the diet and decreasing the fiber content (2, 35). During this adaptation to high-grain diets, significant changes in the ruminal environment and rumen bacterial population structure have been reported (17, 46, 48). However, the microbial changes that occur during this transition phase are poorly understood (17, 21, 26, 46). Studies performed to date have utilized culture-based techniques or have looked at the fluctuation of a few indicator bacteria (48, 47) to evaluate bacterial population changes. Due to limitations in culturing rumen bacteria, the use of culture-based techniques to evaluate bacterial populations substantially underestimates the diversity of microorganisms within the rumen. In this study, we have utilized culture-independent approaches to evaluate bacterial population structure and diversity using terminal restriction fragment length polymorphisms (T-RFLPs) and sequence analysis of 16S rRNA gene libraries to compare the rumen bacterial population structure in animals on prairie hay against that in animals adapting to a high-concentrate (high-grain) diet. We have also quantified the fluctuations in the populations of previously reported indicator bacterial species using quantitative real-time PCR (qRT-PCR) to assess the role of these organisms during adaptation to a high-concentrate diet.     

Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Background

Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results

Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions

The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.     

Endogenous 3,4-Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: LINKS TO MITOCHONDRIALLY DERIVED OXIDATIVE STRESS VIA HYDROXYL RADICAL*

Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.Generation of reactive oxygen species (ROS)¹ and reactive nitrogen species is a normal consequence of aerobic metabolism that, in excess, results in oxidative stress that further leads to oxidative modification of proteins, lipids, and DNA, events that may lead to altered cellular function and even cell death (1, 2). Chronic oxidative stress is well recognized as having a central role in disease and is responsible for both direct alteration of biomolecular structure-function and compensatory changes in cellular processes (1–4). It is increasingly recognized that oxidative modifications of proteins can serve as potential biomarkers indicative of the physiological states and changes that occur during disease progression. Thus, the ability to quantitatively measure specific protein oxidation products has the potential to provide the means to monitor the physiological state of a tissue or organism, in particular any progression toward pathology. Given Parkinson disease (PD) as an example, a number of oxidative modifications on proteins pertinent to PD have been identified, further supporting the potential importance of oxidative modifications to disease pathogenesis (5).Many oxidative modifications on specific amino acid residues, such as protein carbonylation (6), cysteine S-nitrosylation (7–9), cysteine oxidation to sulfinic or sulfonic acid (10–12), methionine oxidation (13, 14), and tyrosine nitration (15–21) within complex protein mixtures, have been detected by MS-based proteomics; however, their low abundance levels within complex proteomes often hinder confident identification of these potentially significant modifications (22). For example, tyrosine nitration is a well studied post-translational modification mediated by peroxynitrite (ONOO⁻) or nitrogen dioxide (^·NO₂), which commonly occur in cells during oxidative stress and inflammation; however, only a small number of nitrotyrosine proteins have been identified from a given proteome sample because of insufficient analytical sensitivity and the chance of incorrect peptide assignments (19, 23). With recent advances in high resolution MS that provide high mass measurement accuracy, the ability to confidently identify modified peptides has been significantly enhanced (24).Hydroxyl radical (HO^·) is one of the most reactive and major species generated under aerobic conditions in biological systems (1, 25, 26). Among several HO^·-mediated oxidative modifications, the protein tyrosine modification 3,4-dihydroxyphenylalanine (DOPA) has been reported as a major product and index of HO^· attack on tyrosine residues in proteins (Fig. 1) (27, 28). DOPA is also formed on protein tyrosine residues via controlled enzymatic pathways through enzymes such as tyrosinase or tyrosine hydroxylase (28). Once formed, protein-bound DOPA has the potential to initiate further oxidative reactions through binding and reducing transition metals or through redox cycling between catechol and quinone (dopaquinone) forms (29, 30). Recent studies have suggested that protein-bound DOPA is involved in triggering antioxidant defenses (30) and mediating oxidative damage to DNA (31). Moreover, elevated levels of protein-bound DOPA have been reported in several diseases, including atherosclerosis, cataracts, and myocardial disease, and in PD patients undergoing levodopa therapy (26, 32–36). However, the specific DOPA-modified proteins, which could provide mechanistic knowledge of the progression of these diseases, have not been identified (27, 28). The ability to identify site-specific protein modifications should lead to a better understanding of the role of DOPA modification in disease pathologies as well as new molecular signatures or therapeutic targets for diseases.Open in a separate windowFig. 1.DOPA and dopaquinone formation from tyrosine.Therefore, in this study, we demonstrate the ability to identify site-specific DOPA and dopaquinone (DQ) modifications on protein tyrosine residues in normal mouse brain and heart tissues and their relative stoichiometries that are present in vivo under non-stressed conditions. Such endogenous protein modifications were detected using LC-MS/MS. The results from this global proteomics survey suggests that HO^· in tissues under normal conditions is generated largely from the mitochondria and metal-binding proteins where the resulting DOPA/DQ modifications have the potential to disrupt mitochondrial respiration as well as alter tyrosine phosphorylation signaling pathways such as 14-3-3-mediated signaling in brain tissue.     

Mechanisms of non-apoptotic programmed cell death in diabetes and heart failure

Programmed cell elimination is an important pathological mediator of disease. Multiple pathways to programmed cell death have been delineated, including apoptosis, autophagy and programmed necrosis. Cross-talk between the signaling pathways mediating each process has made it difficult to define specific mechanisms of in vivo programmed cell death. For this reason, many “apoptotic” diseases may involve other death signaling pathways. Recent advances in genetic complementation using mouse knockout models are helping to dissect apoptotic and necrotic cell death in different pathological states. The current state of research in this area is reviewed, focusing upon new findings describing the role of programmed necrosis induced by the mitochondrial permeability transition in mouse models of heart failure and diabetes.Key words: apoptosis, necrosis, mitochondrial permeability transition pore     

Daily and seasonal vertical migration rhythms in Daphnia

1. Clones of Daphnia longispina (O.F.M.), collected from ponds with different levels of predation, were monitored using video techniques in order to test the effects of temperature and visual cues on vertical migration behaviour.
2. Endogenous rhythms were found to have a part in the control of vertical migration in some clones but responses were highly variable.
3. Low temperature caused a marked increase in the amplitude of vertical migration in D. longispina , consistent with field studies.
4. Clones from ponds with fish predators had larger vertical migration amplitudes than clones from ponds with no fish, providing further evidence for the genetic basis of this mechanism.     

Ranking Alaska Moose Nutrition: Signals to Begin Liberal Antlerless Harvests

Abstract: We focused on describing low nutritional status in an increasing moose (Alces alces gigas) population with reduced predation in Game Management Unit (GMU) 20A near Fairbanks, Alaska, USA. A skeptical public disallowed liberal antlerless harvests of this moose population until we provided convincing data on low nutritional status. We ranked nutritional status in 15 Alaska moose populations (in boreal forests and coastal tundra) based on multiyear twinning rates. Data on age-of-first-reproduction and parturition rates provided a ranking consistent with twinning rates in the 6 areas where comparative data were available. Also, short-yearling mass provided a ranking consistent with twinning rates in 5 of the 6 areas where data were available. Data from 5 areas implied an inverse relationship between twinning rate and browse removal rate. Only in GMU 20A did nutritional indices reach low levels where justification for halting population growth was apparent, which supports prior findings that nutrition is a minor factor limiting most Alaska moose populations compared to predation. With predator reductions, the GMU 20A moose population increased from 1976 until liberal antlerless harvests in 2004. During 1997-2005, GMU 20A moose exhibited the lowest nutritional status reported to date for wild, noninsular, North American populations, including 1) delayed reproduction until moose reached 36 months of age and the lowest parturition rate among 36-month-old moose (29%, n = 147); 2) the lowest average multiyear twinning rates from late-May aerial surveys (x = 7%, SE = 0.9%, n = 9 yr, range = 3-10%) and delayed twinning until moose reached 60 months of age; 3) the lowest average mass of female short-yearlings in Alaska (x̄ = 155 ± 1.6 [SE] kg in the Tanana Flats subpopulation, up to 58 kg below average masses found elsewhere); and 4) high removal (42%) of current annual browse biomass compared to 9-26% elsewhere in boreal forests. When average multiyear twinning rates in GMU 20A (sampled during 1960-2005) declined to <10% in the mid- to late 1990s, we began encouraging liberal antlerless harvests, but only conservative annual harvests of 61-76 antlerless moose were achieved during 1996-2001. Using data in the context of our broader ranking system, we convinced skeptical citizen advisory committees to allow liberal antlerless harvests of 600-690 moose in 2004 and 2005, with the objective of halting population growth of the 16,000-17,000 moose; total harvests were 7-8% of total prehunt numbers. The resulting liberal antlerless harvests served to protect the moose population's health and habitat and to fulfill a mandate for elevated yield. Liberal antlerless harvests appear justified to halt population growth when multiyear twinning rates average ≤10% and ≥1 of the following signals substantiate low nutritional status: <50% of 36-month-old moose are parturient, average multiyear short-yearling mass is <175 kg, or >35% of annual browse biomass is removed by moose.     

Contributions of <Emphasis Type="Italic">I</Emphasis><Subscript>h</Subscript> to feature selectivity in layer II stellate cells of the entorhinal cortex

Stellate cells (SCs) of the entorhinal cortex generate prominent subthreshold oscillations that are believed to be important contributors to the hippocampal theta rhythm. The slow inward rectifier I _h is expressed prominently in SCs and has been suggested to be a dominant factor in their integrative properties. We studied the input-output relationships in stellate cells (SCs) of the entorhinal cortex, both in control conditions and in the presence of the I _h antagonist ZD7288. Our results show that I _h is responsible for SCs’ subthreshold resonance, and contributes to enhanced spiking reliability to theta-rich stimuli. However, SCs still exhibit other traits of rhythmicity, such as subthreshold oscillations, under I _h blockade. To clarify the effects of I _h on SC spiking, we used a generalized form of principal component analysis to show that SCs select particular features with relevant temporal signatures from stimuli. The spike-selected mix of those features varies with the frequency content of the stimulus, emphasizing the inherent nonlinearity of SC responses. A number of controls confirmed that this selectivity represents a stimulus-induced change in the cellular input-output relationship rather than an artifact of the analysis technique. Sensitivity to slow features remained statistically significant in ZD7288. However, with I _h blocked, slow stimulus features were less predictive of spikes and spikes conveyed less information about the stimulus over long time scales. Together, these results suggest that I _h is an important contributor to the input-output relationships expressed by SCs, but that other factors in SCs also contribute to subthreshold oscillations and nonlinear selectivity to slow features. Action Editor: Xiao-Jing Wang