The Pilin Protein FimP from Actinomyces oris: Crystal Structure and Sequence Analyses

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.     

Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.     

Analysis of T cell receptor β and γ genes from peripheral blood,regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients

Summary A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4⁺CD8^–, although some were CD4^–CD8⁺. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) and gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. ProbingHindIII-digested DNA with TCR and TCR probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing ofBamHI-digested DNA with TCR and TCR probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4⁺ T cell populations in each of several separate immune compartments in these patients.This investigation was supported by National Institutes of Health, National Research Service Award CA-08 397 from the National Cancer Institute as well as NIH CA-32 685, CA-30 688, DOE FG028 760 502 and American Cancer Society Grant ACS CH-237     

Specialists in ancient trees are more affected by climate than generalists

Ancient trees are considered one of the most important habitats for biodiversity in Europe and North America. They support exceptional numbers of specialized species, including a range of rare and endangered wood‐living insects. In this study, we use a dataset of 105 sites spanning a climatic gradient along the oak range of Norway and Sweden to investigate the importance of temperature and precipitation on beetle species richness in ancient, hollow oak trees. We expected that increased summer temperature would positively influence all wood‐living beetle species whereas precipitation would be less important with a negligible or negative impact. Surprisingly, only oak‐specialist beetles with a northern distribution increased in species richness with temperature. Few specialist beetles and no generalist beetles responded to the rise of 4°C in summer as covered by our climatic gradient. The negative effect of precipitation affected more specialist species than did temperature, whereas the generalists remained unaffected. In summary, we suggest that increased summer temperature is likely to benefit a few specialist beetles within this dead wood community, but a larger number of specialists are likely to decline due to increased precipitation. In addition, generalist species will remain unaffected. To minimize adverse impacts of climate change on this important community, long‐term management plans for ancient trees are important.     

Microtubules,chromosome movement,and reorientation after chromosomes are detached from the spindle by micromanipulation

The relationship between chromosome movement and mirotubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.     

Excitatory Amino Acid-Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.     

Intentional Weight Loss and All-Cause Mortality: A Meta-Analysis of Randomized Clinical Trials

Background

Obesity is associated with increased mortality, and weight loss trials show rapid improvement in many mortality risk factors. Yet, observational studies typically associate weight loss with higher mortality risk. The purpose of this meta-analysis of randomized controlled trials (RCTs) of weight loss was to clarify the effects of intentional weight loss on mortality.

Methods

2,484 abstracts were identified and reviewed in PUBMED, yielding 15 RCTs reporting (1) randomization to weight loss or non-weight loss arms, (2) duration of ≥18 months, and (3) deaths by intervention arm. Weight loss interventions were all lifestyle-based. Relative risks (RR) and 95% confidence intervals (95% CI) were estimated for each trial. For trials reporting at least one death (n = 12), a summary estimate was calculated using the Mantel-Haenszel method. Sensitivity analysis using sparse data methods included remaining trials.

Results

Trials enrolled 17,186 participants (53% female, mean age at randomization = 52 years). Mean body mass indices ranged from 30–46 kg/m², follow-up times ranged from 18 months to 12.6 years (mean: 27 months), and average weight loss in reported trials was 5.5±4.0 kg. A total of 264 deaths were reported in weight loss groups and 310 in non-weight loss groups. The weight loss groups experienced a 15% lower all-cause mortality risk (RR = 0.85; 95% CI: 0.73–1.00). There was no evidence for heterogeneity of effect (Cochran’s Q = 5.59 (11 d.f.; p = 0.90); I² = 0). Results were similar in trials with a mean age at randomization ≥55 years (RR = 0.84; 95% CI 0.71–0.99) and a follow-up time of ≥4 years (RR = 0.85; 95% CI 0.72–1.00).

Conclusions

In obese adults, intentional weight loss may be associated with approximately a 15% reduction in all-cause mortality.     

The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein–protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.     

New genetic markers for identifying Cronartium flaccidum and Peridermium pini and examining genetic variation within and between lesions of Scots pine blister rust in Sweden

Microsatellite markers were developed as an identification tool and for analysis of the genetic variation in the pathogens Cronartium flaccidum and Peridermium pini, causing Scots pine blister rust in Pinus spp. Six reference aeciospore samples from Finland were used to examine genetic differences between the two pathogens. Genetic variation within and between 27 lesions on Scots pines from seven locations in Sweden was also investigated. Aeciospores were collected from single aecia within the lesions. Reference samples from P. pini were homozygous for all seven microsatellite loci investigated, while the three C.?flaccidum samples contained heterozygous loci. These results confirm previous studies, where homozygous aeciospores were indicated to be characteristic for P. pini. The majority of aeciospores had two nuclei in both heterozygotic and homozygotic samples. Five of the Swedish lesions contained only homozygotic aecia, while the aecia in the remaining 22 lesions were heterozygotic. All lesions with homozygotic aecia contained only one single multilocus genotype, while many of the lesions with heterozygotic aecia contained several genotypes. The latter indicates the occurrence of multiple matings within a lesion between the resident spermogonia and alien fertilizing spermatia.     

A comparison of three methods to survey saproxylic beetles in hollow oaks

One of the most endangered assemblages of species in Europe is insectsassociated with old trees. For that reason there is a need of developing methodsto survey this fauna. This study aims at comparing three methods – windowtrapping, pitfall trapping and wood mould sampling – to assess speciesrichness and composition of the saproxylic beetle fauna in living, hollow oaks.We have used these methods at the same site, and to a large extent in the sametrees. Useful information was obtained from all methods, but they partiallytarget different assemblages of species. Window trapping collected the highestnumber of species. Pitfall trapping collected beetles associated with treehollows which rarely are collected by window traps and therefore it isprofitable to combine these two methods. As wood mould sampling is the cheapestmethod to use, indicator species should preferably be chosen among specieswhich are efficiently collected with this method.