Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Inhibition of yeast tRNATrp aminoacylation by 4-methyltryptophan

The effect of the tryptophan analogue 4-methyltryptophan in Saccharomyces cerevisiae has been investigated. 4-Methyltryptophan inhibits the aminoacylation of tryptophan specific transfer ribonucleic acid (tRNA^Trp). The mode of inhibition is competitive and the analogue is not charged onto tRNA^Trp. Thus 4-methyltryptophan application depletes the cells from charged tRNA^Trp. As a consequence cell growth and protein synthesis are strongly reduced. 4-Methyltryptophan is degraded efficiently in culture media inoculated with the wild type strain; the effects of 4-methyltryptophan were therefore found to be transient.     

Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.     

Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.     

Rediscovery of Nebela ansata (Amoebozoa: Arcellinida) in eastern North America: biogeographical implications

Aim The question whether free‐living protists are generally cosmopolitan is currently a matter of debate. In this study we investigate the geographical distribution of a distinctive testate amoeba species, Nebela ansata, and use our data to assess the potential for highly restricted distribution patterns in some protist species. Location Global. Methods We analysed (1) 3400 testate amoeba publications from North America and other continents, (2) unpublished slides of the Penard Collection of the Natural History Museum, London, UK, and (3) 104 Sphagnum samples from eastern North America. Non‐metric multidimensional scaling (NMDS) was used to visualize the similarities in testate amoeba community composition among 1012 North American samples, including two communities that contained N. ansata. Results We rediscovered N. ansata at a site in New Jersey located close to its type locality, and in Nova Scotia. We also report the existence of an apparently unpublished museum specimen originally collected from New Jersey. Our extensive literature survey confirmed the presence of this species only in the temperate part of eastern North America. The NMDS revealed that communities with N. ansata were less similar to each other than to communities from other parts of North America, suggesting that favourable habitats for N. ansata occur in other Sphagnum‐dominated peatlands, a habitat type that has been extensively sampled in North America and elsewhere. Main conclusions These data provide an unusually convincing case of a free‐living microorganism with a very limited distribution range in the temperate part of eastern North America. The remarkably restricted distribution of N. ansata highlights the extent of our ignorance about the natural history of free‐living microorganisms, and raises questions about the lack of attention to microbial diversity in conservation biology.     

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.     

Sexually transmitted infections, bacterial vaginosis, and candidiasis in women of reproductive age in rural Northeast Brazil: a population-based study

Population-based data on sexually transmitted infections (STI), bacterial vaginosis (BV), and candidiasis reflect the epidemiological situation more accurately than studies performed in specific populations, but such data are scarce. To determine the prevalence of STI, BV, and candidiasis among women of reproductive age from a resource-poor community in Northeast Brazil, a population-based cross sectional study was undertaken. All women from seven hamlets and the centre of Pacoti municipality in the state of Ceará, aged 12 to 49 years, were invited to participate. The women were asked about socio-demographic characteristics and genital symptoms, and thereafter examined gynaecologically. Laboratory testing included polymerase chain reaction (PCR) for human papillomavirus (HPV), ligase chain reaction (LCR) for Chlamydia trachomatis and Neisseria gonorrhoeae, ELISA for human immunodeficiency virus (HIV), venereal disease research laboratory (VDRL) and fluorescent treponema antibody absorption test (FTA-ABS) for syphilis, and analysis of wet mounts, gram stains and Pap smears for trichomoniasis, candidiasis, and BV. Only women who had initiated sexual life were included in the analysis (n = 592). The prevalences of STI were: HPV 11.7% (95% confidence interval: 9.3-14.7), chlamydia 4.5% (3.0-6.6), trichomoniasis 4.1% (2.7-6.1), gonorrhoea 1.2% (0.5-2.6), syphilis 0.2% (0.0-1.1), and HIV 0%. The prevalence of BV and candidiasis was 20% (16.9-23.6) and 12.5% (10.0-15.5), respectively. The most common gynaecological complaint was lower abdominal pain. STI are common in women in rural Brazil and represent an important health threat in view of the HIV pandemic.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Analysis of historic changes in regional ecosystem service provisioning using land use data

Land use change has a major impact on goods and services that our environment supplies for society. While detailed ecological or biophysical field studies are needed to quantify the exact amount of ecosystem service supply at local scales, such a monitoring might be unfeasible at the regional scale. Since field scale monitoring schemes for ecosystem services or ecosystem functioning are missing, proxy based indicators can help to assess the historic development of ecosystem services or ecosystem functioning at the regional scale. We show at the example of the historic development (1964–2004) in the district of Leipzig/Germany how land use/land cover data can be used to derive regional scale indicators for ecosystem functions. We focus thereby on two hypotheses: (1) the ecosystem functioning has degraded over time and (2) changes in land use configuration play an important role in this degradation. The study focuses on indicators for ecosystem functions related to (i) water purification by riparian buffer strips, (ii) pollination, (iii) food production and (iv) outdoor recreation. Each indicator builds on the analysis of land use configuration and land use composition information and is tested on sensitivity/robustness with respect to parameters which had to be estimated based on expert knowledge. We show that land use composition is an important aspect in our ecosystem service assessment. Although our study region is faced with a maximum land use change of 11% in the major land use classes between 1964 and 2004, we see a decrease of ecosystem function indicators up to 23%. The regional assessment shows an overall trend for degradation of ecosystem functioning from 1964 to 1984. This trend is reversed between 1984 and 1994 but the process slowed down until 2004 without reaching the level of 1964.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.