Novel role for surfactant protein A in gastrointestinal graft-versus-host disease

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.     

Autoproteolytic Activation of ThnT Results in Structural Reorganization Necessary for Substrate Binding and Catalysis

cis-Autoproteolysis is a post-translational modification necessary for the function of ThnT, an enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. This modification generates an N-terminal threonine nucleophile that is used to hydrolyze the pantetheinyl moiety of its natural substrate. We determined the crystal structure of autoactivated ThnT to 1.8 Å through X-ray crystallography. Comparison to a mutationally inactivated precursor structure revealed several large conformational rearrangements near the active site. To probe the relevance of these transitions, we designed a pantetheine-like chloromethyl ketone inactivator and co-crystallized it with ThnT. Although this class of inhibitor has been in use for several decades, the mode of inactivation had not been determined for an enzyme that uses an N-terminal nucleophile. The co-crystal structure revealed the chloromethyl ketone bound to the N-terminal nucleophile of ThnT through an ether linkage, and analysis suggests inactivation through a direct displacement mechanism. More importantly, this inactivated complex shows that three regions of ThnT that are critical to the formation of the substrate binding pocket undergo rearrangement upon autoproteolysis. Comparison of ThnT with other autoproteolytic enzymes of disparate evolutionary lineage revealed a high degree of similarity within the proenzyme active site, reflecting shared chemical constraints. However, after autoproteolysis, many enzymes, like ThnT, are observed to rearrange in order to accommodate their specific substrate. We propose that this is a general phenomenon, whereby autoprocessing systems with shared chemistry may possess similar structural features that dissipate upon rearrangement into a mature state.     

Spatial Ecology of the Endangered Milne-Edwards’ Sifaka (Propithecus edwardsi): Do Logging and Season Affect Home Range and Daily Ranging Patterns?

Primates often live in human-altered habitats; Malagasy lemurs are no exception. It is important to understand if habitat alteration affects primates’ space use patterns across multiple spatial and temporal scales, as this drives population density. We quantified the daily, seasonal, and annual space-use of seven groups of Milne-Edwards’ sifaka (Propithecus edwardsi) living in unlogged and logged rain forest in Ranomafana National Park, Madagascar between December 2002 and November 2003. Concurrent data showed that sifakas consumed higher quality foods in the unlogged than in logged forests; thus we explored how space use patterns were related to energy use strategies. Sifaka groups in the logged rain forest traveled 7–13% less per day than groups in the unlogged rain forest, despite their larger home ranges (median: 46.12 and 23.52 ha, in the logged and unlogged forests, respectively). Sifakas may thus use an energy-minimizing strategy at the scale of the individual day but an energy-maximizing strategy at the annual home range scale. Sifakas exhibited fidelity to the home range across seasons, but their core area of use shifted considerably with season. We found no difference in population density between sites. However, given the interannual variability in sifaka foods, a multiyear study is needed to assess if energy strategies observed in this study are consistent across longer time periods. Our findings suggest that lemurs may persist in logged habitats by altering spatial use patterns; future work should attempt to quantify the threshold level of forest regeneration from logging that will allow lemurs to persist at similar densities as in unlogged forest.     

Excitable behavior can explain the "ping-pong" mode of communication between cells using the same chemoattractant

Here we elucidate a paradox: how a single chemoattractant-receptor system in two individuals is used for communication despite the seeming inevitability of self-excitation. In the filamentous fungus Neurospora crassa, genetically identical cells that produce the same chemoattractant fuse via the homing of individual cell protrusions toward each other. This is achieved via a recently described "ping-pong" pulsatile communication. Using a generic activator-inhibitor model of excitable behavior, we demonstrate that the pulse exchange can be fully understood in terms of two excitable systems locked into a stable oscillatory pattern of mutual excitation. The most puzzling properties of this communication are the sudden onset of oscillations with final amplitude, and the absence of seemingly inevitable self-excitation. We show that these properties result directly from both the excitability threshold and refractory period characteristic of excitable systems. Our model suggests possible molecular mechanisms for the ping-pong communication.     

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.     

A review of current proteomics technologies with a survey on their widespread use in reproductive biology investigations

Proteomics is very much a technology-driven field. The ambition is to identify, quantify and to assess the state of posttranslational modification and interaction partners for every protein in the cell. The proteome is in a state of flux and is thus extremely complex. Analysis of the proteome is exacerbated by the huge dynamic concentration range of proteins in the cellular environment. The impact that mass spectrometry-based proteomics has had on the field of biology has heavily depended on dramatic improvements in mass spectrometry that have been made in recent years. We examined 1541 reports indexed in PubMed relating to proteomics and reproduction to identify trends in the field and to make some broad observations for future work. To set the scene, in the first part of the report, we give a comprehensive overview of proteomics and associated techniques and technologies (such as separations and mass spectrometry). The second part examines the field in light of these techniques and suggests some opportunities for application of these tools in the area of reproduction.     

Compared to dinoprost tromethamine, cloprostenol sodium increased rates of estrus detection, conception and pregnancy in lactating dairy cows on a large commercial dairy

Using two PGF_2α treatments 14 days apart as a way to enhance estrus detection rate following the 2nd treatment is a reproductive management tool that continues to be used on large dairy farms. In one study, in cows with a functional CL and a dominant follicle, treatment with cloprostenol vs. dinoprost resulted in greater peripheral estradiol concentrations. The objective of the present study was to determine if cloprostenol could enhance pregnancy rates of cows in a large dairy herd using a PGF_2α program for 1st artificial insemination (AI). Lactating dairy cows (n = 4549) were randomly assigned to receive two treatments of either 500 μg cloprostenol or 25 mg dinoprost 14 days apart, with the 2nd treatment on the 1st day of the voluntary waiting period (57 DIM). Cows detected in estrus within 5 days after the 2nd treatment were inseminated. There was no effect of treatment on day of estrus detection, with 78% of cows inseminated on Days 3 or 4 following treatment. Cloprostenol increased (P < 0.01) estrus detection rates in 1st parity cows compared to dinoprost, 42.4 vs. 34.0%. In cows inseminated on Days 3 or 4 after treatment, cloprostenol increased (P = 0.05) conception rates compared to dinoprost, 38.3 vs. 34.4%. When treatments and parities were combined, conception rates increased (P < 0.02) with interval after treatment (27.0, 36.4, and 44.5% for Days 1 or 2, Days 3 or 4, and Day 5, respectively). Cloprostenol increased (P = 0.02) overall pregnancy rate compared to dinoprost, 14.4 vs. 12.2%. In summary, cloprostenol increased fertility in 1st parity cows inseminated on Days 3 or 4 following treatment and subsequently enhanced pregnancy rates of 1st parity lactating dairy cows compared to dinoprost. Fertility appeared greater in cows expected to have had a young antral ovarian follicle at treatment.     

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.     

DECIPHER, a search-based approach to chimera identification for 16S rRNA sequences

DECIPHER is a new method for finding 16S rRNA chimeric sequences by the use of a search-based approach. The method is based upon detecting short fragments that are uncommon in the phylogenetic group where a query sequence is classified but frequently found in another phylogenetic group. The algorithm was calibrated for full sequences (fs_DECIPHER) and short sequences (ss_DECIPHER) and benchmarked against WigeoN (Pintail), ChimeraSlayer, and Uchime using artificially generated chimeras. Overall, ss_DECIPHER and Uchime provided the highest chimera detection for sequences 100 to 600 nucleotides long (79% and 81%, respectively), but Uchime's performance deteriorated for longer sequences, while ss_DECIPHER maintained a high detection rate (89%). Both methods had low false-positive rates (1.3% and 1.6%). The more conservative fs_DECIPHER, benchmarked only for sequences longer than 600 nucleotides, had an overall detection rate lower than that of ss_DECIPHER (75%) but higher than those of the other programs. In addition, fs_DECIPHER had the lowest false-positive rate among all the benchmarked programs (<0.20%). DECIPHER was outperformed only by ChimeraSlayer and Uchime when chimeras were formed from closely related parents (less than 10% divergence). Given the differences in the programs, it was possible to detect over 89% of all chimeras with just the combination of ss_DECIPHER and Uchime. Using fs_DECIPHER, we detected between 1% and 2% additional chimeras in the RDP, SILVA, and Greengenes databases from which chimeras had already been removed with Pintail or Bellerophon. DECIPHER was implemented in the R programming language and is directly accessible through a webpage or by downloading the program as an R package (http://DECIPHER.cee.wisc.edu).