The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction.

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

Albedo and atmospheric constraints of dwarf planet Makemake from a stellar occultation.

Pluto and Eris are icy dwarf planets with nearly identical sizes, comparable densities and similar surface compositions as revealed by spectroscopic studies. Pluto possesses an atmosphere whereas Eris does not; the difference probably arises from their differing distances from the Sun, and explains their different albedos. Makemake is another icy dwarf planet with a spectrum similar to Eris and Pluto, and is currently at a distance to the Sun intermediate between the two. Although Makemake's size (1,420 ± 60 km) and albedo are roughly known, there has been no constraint on its density and there were expectations that it could have a Pluto-like atmosphere. Here we report the results from a stellar occultation by Makemake on 2011 April 23. Our preferred solution that fits the occultation chords corresponds to a body with projected axes of 1,430 ± 9 km (1σ) and 1,502 ± 45 km, implying a V-band geometric albedo p(V) = 0.77 ± 0.03. This albedo is larger than that of Pluto, but smaller than that of Eris. The disappearances and reappearances of the star were abrupt, showing that Makemake has no global Pluto-like atmosphere at an upper limit of 4-12 nanobar (1σ) for the surface pressure, although a localized atmosphere is possible. A density of 1.7 ± 0.3 g cm(-3) is inferred from the data.

Chlamydia pneumoniae Induces Interferon Gamma Responses in Peripheral Blood Mononuclear Cells in Children with Allergic Asthma.

Respiratory infections caused by Chlamydia pneumoniae have been associated with exacerbations of asthma. Cell-mediated immunity (CMI) is critical for maintaining immunity. We compared interferon (IFN)-γ responses in C. pneumoniae-infected peripheral blood mononuclear cells (PBMC) in paediatric patients ± asthma. Presence of C. pneumoniae was tested from asthma patients (N = 17) and non-asthmatic controls (N = 16) (PCR). PBMC were infected for 1 h ± C. pneumoniae AR-39 (MOI = 0.1) and cultured for 48 h. IFN-γ levels were measured in supernatants (ELISA). C. pneumoniae-IgG antibodies in serum were determined (MIF). All subjects tested negative for C. pneumoniae (PCR). C. pneumoniae-induced IFN-γ production in vitro was more prevalent in asthma compared with non-asthma; levels of IFN-γ were higher in asthma compared with non-asthma (P = 0.003). There was no association between recent respiratory infection and positive IFN-γ responses. These data show that C. pneumoniae modulates IFN-γ responses in patients with asthma, even in absence of active infection.

The Bingo Card Sign as an Early Symptom of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized pathologically by irreversible protein misfolding-induced neuronal loss, and clinically by progressive impairments in memory, judgment, decision making, literacy and numeracy. We report a patient referred to Memory Clinic with a single complaint, "reduced capacity to play bingo." We suggest that the capacity to successfully play bingo may afford clinical clues indicating an early symptom of dementia and inquiries about bingo participation may be a useful when assessing individuals for dementia. Bingo requires the use of multiple cognitive skills which are impaired by AD, including number recognition, letter recognition, short term memory and concentration. With the game of bingo steadily gaining in popularity it may become an easily utilized line of questioning to detect indications of dementia prior to the development of currently recognized clinical symptoms.

Overabundant FANCD2, alone and combined with NQO1, is a sensitive marker of adverse prognosis in breast cancer.

BACKGROUND: Defective DNA repair is central to the progression and treatment of breast cancer. Immunohistochemically detected DNA repair markers may be good candidates for novel prognostic and predictive factors that could guide the selection of individualized treatment strategies. PATIENTS AND METHODS: We have analyzed nuclear immunohistochemical staining of BRCA1, FANCD2, RAD51, XPF, and PAR in relation to clinicopathological and survival data among 1240 paraffin-embedded breast tumors, and additional gene expression microarray data from 76 tumors. The antioxidant enzyme NQO1 was analyzed as a potential modifier of prognostic DNA repair markers. RESULTS: RAD51 [hazard ratio (HR) 0.81, 95% confidence interval (CI) 0.70-0.94, P = 0.0050] and FANCD2 expression (HR 1.50, 95% CI 1.28-1.76, P = 1.50 × 10(-7)) were associated with breast cancer survival. High FANCD2 expression correlated with markers of adverse prognosis but remained independently prognostic in multivariate analysis (HR 1.27, 95% CI 1.08-1.49, P = 0.0043). The FANCD2-associated survival effect was most pronounced in hormone receptor positive, HER2-negative tumors, and in tumors with above-median NQO1 expression. In the NQO1-high subset, patients belonging to the highest quartile of FANCD2 immunohistochemical scores had a threefold increased risk of metastasis or death (HR 3.10, 95% CI 1.96-4.92). Global gene expression analysis indicated that FANCD protein overabundance is associated with the upregulation of proliferation-related genes and a downregulated nucleotide excision repair pathway. CONCLUSION: FANCD2 immunohistochemistry is a sensitive, independent prognostic factor in breast cancer, particularly when standard markers indicate relatively favorable prognosis. Taken together, our results suggest that the prognostic effect is linked to proliferation, DNA damage, and oxidative stress; simultaneous detection of FANCD2 and NQO1 provides additional prognostic value.

Odorant receptors of a primitive hymenopteran pest, the wheat stem sawfly.

The wheat stem sawfly, Cephus cinctus, is an herbivorous hymenopteran that feeds exclusively on members of the Graminae family. Synanthropically, it has become one of the most important insect pests of wheat grown in the northern Great Plains region of the USA and Canada. Insecticides are generally ineffective because of the wheat stem sawfly's extended adult flight period and its inaccessible larval stage, during which it feeds within the wheat stems, making it virtually intractable to most pest management strategies. While research towards integrated pest management strategies based on insect olfaction has proved promising, nothing is known about the molecular basis of olfaction in this important pest species. In this study we identified 28 unique odorant receptor (Or) transcripts from an antennal transcriptome. A phylogenetic analysis with the predicted Ors from the honey bee and jewel wasp genomes revealed at least four clades conserved amongst all three Hymenoptera species. Antennal expression levels were analysed using quantitative real-time PCR, and one male-biased and five female-biased Ors were identified. This study provides the basis for future functional analyses to identify behaviourally active odours that can be used to help develop olfactory-mediated pest management tools.

Estimating farm to catchment nutrient fluxes using dynamic simulation modelling--can agri-environmental BMPs really do the job?

A dynamic model of Phosphorus (P) movement through the Peel-Harvey catchment in South Western Australia was developed using system dynamics modelling software. The model was developed to illustrate watershed P flux and to predict future P loss rates under a range of management scenarios. Model input parameters were sourced from extensive surveys of local agricultural practices and regional soil testing data. Model P-routing routines were developed from the known interactions between the various watershed P compartments and fluxes between the various P stores. Phosphorus-retention characteristics of a variety of management practices were determined from local field trials where available and published values where not. The model simulated a 200 year time frame to reflect 100 years to the present day since initial land development, and forecast 100 years into the future. Although the catchment has an annual P-loss target of 70 tonnes per annum (tpa), the measured (and modelled) present-day loss is double this amount (140 tpa) and this is projected to rise to 1300 tpa if current land management practices continue. Broad implementation of neither "biological" BMPs such as perennial pastures and managed riparian zones, or of "chemical" BMPs such as reduced water solubility fertilisers and P-retentive soil amendments, produces reductions in P-loss from present-day levels. Even if broad-scale implementation of the large suite of BMPs tested in this research occurs, catchment P-losses are likely to increase from the present level of 140 tpa to approximately 200 tpa over the next 100 years. This has significant implications for both future land use and subsequent water quality in the catchment as well as questioning the wisdom and perceptions of efficacy of past and future BMP implementation strategies.

Graph 'texture' features as novel metrics that can summarize complex biological graphs.

Objective.Image texture features, such as those derived by Haralicket al, are a powerful metric for image classification and are used across fields including cancer research. Our aim is to demonstrate how analogous texture features can be derived for graphs and networks. We also aim to illustrate how these new metrics summarize graphs, may aid comparative graph studies, may help classify biological graphs, and might assist in detecting dysregulation in cancer.Approach.We generate the first analogies of image texture for graphs and networks. Co-occurrence matrices for graphs are generated by summing over all pairs of neighboring nodes in the graph. We generate metrics for fitness landscapes, gene co-expression and regulatory networks, and protein interaction networks. To assess metric sensitivity we varied discretization parameters and noise. To examine these metrics in the cancer context we compare metrics for both simulated and publicly available experimental gene expression and build random forest classifiers for cancer cell lineage.Main results.Our novel graph 'texture' features are shown to be informative of graph structure and node label distributions. The metrics are sensitive to discretization parameters and noise in node labels. We demonstrate that graph texture features vary across different biological graph topologies and node labelings. We show how our texture metrics can be used to classify cell line expression by lineage, demonstrating classifiers with 82% and 89% accuracy.Significance.New metrics provide opportunities for better comparative analyzes and new models for classification. Our texture features are novel second-order graph features for networks or graphs with ordered node labels. In the complex cancer informatics setting, evolutionary analyses and drug response prediction are two examples where new network science approaches like this may prove fruitful.

Cisplatin benefit is predicted by immunohistochemical analysis of DNA repair proteins in squamous cell carcinoma but not adenocarcinoma: theranostic modeling by NSCLC constituent histological subclasses.

BACKGROUND: Most non-small-cell lung cancer (NSCLC) patients receive cisplatin-based chemotherapy though clinical response is restricted to a subset of patients. DNA repair protein levels are possible surrogates for cisplatin-induced DNA adduct (and subsequent cell death) repair efficiency and thus molecular determinants of therapeutic efficacy. The International Adjuvant Lung Trial (IALT)-Bio study previously suggested ERCC1 and MSH2 as predictive of cisplatin-based therapeutic benefit. PATIENTS AND METHODS: DNA repair protein expression (XPF, BRCA1, ERCC1, MSH2, p53, PARP1, and ATM) was assessed by immunohistochemistry on a large subset of patients (N = 769) from the IALT trial. Tissue Microarray slides were digitally scanned and signal quantified by user-defined macros. Statistical analyses (univariate and multivariate) of 5-year disease-free survival (DFS) and 5-year overall survival used binary cut-offs (H score low/high expression). RESULTS: In patients with squamous cell carcinoma (SCC), ATM, p53, PARP1, ERCC1, and MSH2 displayed significant (borderline) predictive values, mainly on DFS with chemotherapy efficacy limited to low marker levels. Adenocarcinoma (ADC) results were not significant. BRCA1 and XPF were not significant for predictive modeling in either SCC or ADCs. CONCLUSION: Here predictive utility of DNA repair enzymes co-segregates with SCC histology, focusing their predictive value to this histological subclass of NSCLC. Distinct mechanisms of chemotherapeutic response or resistance might exist among histological subclasses of solid tumors.

Charon's size and an upper limit on its atmosphere from a stellar occultation.

Pluto and its satellite, Charon (discovered in 1978; ref. 1), appear to form a double planet, rather than a hierarchical planet/satellite couple. Charon is about half Pluto's size and about one-eighth its mass. The precise radii of Pluto and Charon have remained uncertain, leading to large uncertainties on their densities. Although stellar occultations by Charon are in principle a powerful way of measuring its size, they are rare, as the satellite subtends less than 0.3 microradians (0.06 arcsec) on the sky. One occultation (in 1980) yielded a lower limit of 600 km for the satellite's radius, which was later refined to 601.5 km (ref. 4). Here we report observations from a multi-station stellar occultation by Charon, which we use to derive a radius, R(C) = 603.6 +/- 1.4 km (1sigma), and a density of rho = 1.71 +/- 0.08 g cm(-3). This occultation also provides upper limits of 110 and 15 (3sigma) nanobar for an atmosphere around Charon, assuming respectively a pure nitrogen or pure methane atmosphere.

Zinc alters conformation and inhibits biological activities of nerve growth factor and related neurotrophins.

A role for Zn2+ in a variety of neurological conditions such as stroke, epilepsy and Alzheimer's disease has been postulated. In many instances, susceptible neurons are located in regions rich in Zn2+ where nerve growth factor (NGF) levels rise as a result of insult. Although the interaction of Zn2+ with this neurotrophin has previously been suggested, the direct actions of the ion on NGF function have not been explored. Molecular modeling studies predict that Zn2+ binding to NGF will induce structural changes within domains of this neurotrophin that participate in the recognition of TrkA and p75NTR. We demonstrate here that Zn2+ alters the conformation of NGF, rendering it unable to bind to p75NTR or TrkA receptors or to activate signal transduction pathways and biological outcomes normally induced by this protein. Similar actions of Zn2+ are also observed with other members of the NGF family, suggesting a modulatory role for this metal ion in neurotrophin function.

Runoff and leaching of dissolved phosphorus in streams from a rainfed mixed cropping and grazing catchment under a Mediterranean climate in Australia.

Managing phosphorus (P) is a global priority for environmental water quality due to P lost from agricultural land through leaching, runoff and subsurface flow. In Western Australia (WA), following decades of P fertiliser application to crops and pastures in low rainfall regions, questions have been raised about this region's contribution to environmental P loss. This study was conducted on the Fitzgerald River catchment in the south Western Australia (WA) with mixed cropping and grazing land uses and a Mediterranean climate with low mean rainfall (~350 mm yr-1). Phosphorus forms were monitored continuously over a three-year period in five separate streams, each draining a defined sub-catchment. The P concentrations in streams consistently exceeded Australian and New Zealand Environment Conservation Council (ANZECC) trigger values throughout the monitoring period. Of the measured total P concentration, ~75% was dissolved P (DRP; <0.45 µm) and 80% of that fraction was in the filterable reactive form (FRP). These water quality measurements and other independent soil investigations at this site, suggest that transport of dissolved P rather than erosion of sediment-bound P was dominant in this environment. Based on extractable soil P (Colwell P) and the P buffering index (PBI), predicted concentrations of dissolved reactive P (DRP) in soil solution in topsoils (0-10 cm) across this catchment, generally exceeded ANZECC's values of 0.07 mg PL-1. The level of exceedance was spatially variable. Streams draining areas with the lowest predicted DRP concentrations also had the lowest measured FRP concentrations. Elsewhere stream water FRP concentrations depended on both DRP concentration and the PBI of the land being drained. Our findings suggest that deployment of practices that physically filter runoff, for example riparian vegetation, would be ineffective in restricting P transport into stream in this environment. This conclusion is consistent with previous findings of the ineffectiveness of riparian buffers on coarse textured sandy soils in higher rainfall areas of southwest WA. A reduction in DRP losses without yield loss could be achieved by following evidence-based fertiliser advice from soil testing to limit losses of legacy P".

Site specificity in vimentin-membrane interactions: intermediate filament subunits associate with the plasma membrane via their head domains.

Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.

Putative melatonin receptors in a human biological clock.

In vitro autoradiography with 125I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific 125I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific 125I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific 125I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

Activation of 2-aminofluorene by cultured plant cells.

Cultured tobacco plant cells activated 2-aminofluorene to an agent mutagenic to Salmonella typhimurium strain TA98. The plant activation of 2-aminofluorene is heat-inactivated and may not involve solely cytochrome P-450. The kinetics of activation demonstrated both time- and concentration-dependent responses.

Interacting molecular loops in the mammalian circadian clock.

We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

Mutations in JARID1C are associated with X-linked mental retardation, short stature and hyperreflexia.

BACKGROUND: Mutations in the JARID1C (Jumonji AT-rich interactive domain 1C) gene were recently associated with X-linked mental retardation (XLMR). Mutations in this gene are reported to be one of the relatively more common causes of XLMR with a frequency of approximately 3% in males with proven or probable XLMR. The JARID1C protein functions as a histone 3 lysine 4 (H3K4) demethylase and is involved in the demethylation of H3K4me3 and H3K4me2. METHODS: Mutation analysis of the JARID1C gene was conducted in the following cohorts: probands from 23 XLMR families linked to Xp11.2, 92 males with mental retardation and short stature, and 172 probands from small XLMR families with no linkage information. RESULTS: Four novel mutations consisting of two missense mutations, p.A77T and p.V504M, and two frame shift mutations, p.E468fsX2 and p.R1481fsX9, were identified in males with mental retardation. Two of the mutations, p.V504M and p.E468fsX2, are located in the JmjC domain of the JARID1C gene where no previous mutations have been reported. Additional studies showed that the missense mutation, p.V504M, was a de novo event on the grandpaternal X chromosome of the family. Clinical findings of the nine affected males from the four different families included mental retardation (100%), short stature (55%), hyperreflexia (78%), seizures (33%) and aggressive behaviour (44%). The degree of mental retardation consisted of mild (25%), moderate (12%) and severe (63%). CONCLUSION: Based on the clinical observations, male patients with mental retardation, short stature and hyperreflexia should be considered candidates for mutations in the JARID1C gene.

Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses.

The pineal hormone melatonin regulates seasonal reproductive function and modulates circadian rhythms in mammals. We now report the cloning and characterization of a high affinity receptor for melatonin from the sheep and human. The receptor cDNAs encode proteins that are members of a newly discovered group within the G protein-coupled receptor family. Expression of the sheep and human receptors in COS-7 cells results in high affinity 2-[125I]iodomelatonin binding and pharmacological characteristics similar to endogenous high affinity receptors. Functional studies of NIH 3T3 cells stably expressing the sheep receptor show that the mammalian melatonin receptor is coupled to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism. In situ hybridization studies of melatonin receptor mRNA in several mammals reveal hybridization signals in the hypophyseal pars tuberalis and hypothalamic suprachiasmatic nucleus. The cloned high affinity receptor likely mediates the reproductive and circadian actions of melatonin in mammals.

Melatonin receptors are for the birds: molecular analysis of two receptor subtypes differentially expressed in chick brain.

Two receptors (CKA and CKB) of the G protein-coupled melatonin receptor family were cloned from chick brain. CKA encodes a protein that is 80% identical at the amino acid level to the human Mel1a melatonin receptor and is thus designated the chick Mel1a melatonin receptor. CKB encodes a protein that is 80% identical to the Xenopus melatonin receptor and defines a new receptor subtype, the Mel1c melatonin receptor, which is distinct from the Mel1a and Mel1b melatonin receptor subtypes. A melatonin receptor family consisting of three subtypes is supported by PCR cloning of distinct melatonin receptor fragments from Xenopus and zebrafish. Expression of CKA and CKB results in similar ligand binding and functional characteristics. The widespread distribution of CKA and CKB mRNA in brain provides a molecular substrate for the profound actions of melatonin in birds.

Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain.

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.