Signal-peptide-peptidase-like 2a (SPPL2a) is targeted to lysosomes/late endosomes by a tyrosine motif in its C-terminal tail

Signal-peptide-peptidase-like 2A (SPPL2a), an aspartyl intramembrane protease, has been implicated in the proteolysis of TNF-alpha, Fas Ligand and Bri2. Here, we show that endogenous SPPL2a - in agreement with overexpression studies - is localised in membranes of lysosomes/late endosomes. Furthermore, we have analysed the molecular determinants for lysosomal sorting of SPPL2a by creating chimaeric constructs between SPPL2a and its plasma membrane localised homologue SPPL2b. Lysosomal transport of SPPL2a critically depends on its cytosolic carboxyterminal tail. A canonical tyrosine-based sorting motif of the YXX? type at position 498 is sufficient to direct SPPL2a to lysosomal/late endosomal compartments. This motif accounts for the differential localisation of the homologous proteases SPPL2a and SPPL2b and thereby influences the access to substrates and biological function of SPPL2a.     

Childhood Trauma Questionnaire (CTQ) in Brazilian Samples of Different Age Groups: Findings from Confirmatory Factor Analysis

The Childhood Trauma Questionnaire (CTQ) is internationally accepted as a key tool for the assessment of childhood abuse and neglect experiences. However, there are relative few psychometric studies available and some authors have proposed two different factor solutions. We examined the dimensional structure and internal consistency of the Brazilian version of the CTQ. A total of 1,925 participants from eight different clinical and non-clinical samples including adolescents, adults and elders were considered in this study. First, we performed Confirmatory Factor Analysis to investigate the goodness of fit of the two proposed competitive factor structure models for the CTQ. We also investigated the internal consistency of all factors. Second, multi-group analyses were used to investigate measurement invariance and population heterogeneity across age groups and sex. Our findings revealed that the alternative factor structure as opposed to the original factor structure was the most appropriate model within adolescents and adults Brazilian samples. We provide further evidence for the validity and reliability of the CTQ within the Brazilian samples and report that the alternative model showed an improvement in fit indexes and may be a better alternative over the original model.     

Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia

Introduction

Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.

Methods

All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca²⁺]_o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca²⁺]_o in the presence or absence of NPS-2143, ATF936 or AXT914.

Results

All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca²⁺]_o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.

Conclusion

The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.     

Biodiversity on the brink: an assessment of conservation strategies for North American freshwater mussels

Fish and zooplankton populations of nine Ethiopian freshwater lakes were quantitatively sampled along a North–South gradient. Differences in altitude and latitude resulted in a temperature gradient from North to South. We tested three hypotheses: (1) the degree of zooplanktivory decreases with water temperature, i.e. from North to South; (2) the degree of zooplanktivory increases with the abundance of large-bodied zooplankton; and (3) the pattern of zooplanktivory in eutrophic Ethiopian water bodies differs from other tropical and temperate water bodies. Proportions of zooplanktivory in the fish communities did not show a geographical trend, but mainly depended on fish species, zooplankton density and the availability of large-bodied cladocerans. The degree of zooplanktivory in eutrophic Ethiopian water bodies differs from other eutrophic water bodies, both temperate and tropical. In Ethiopia, the degree of zooplanktivory can be both low and high, in contrast with other tropical water bodies where zooplanktivory is generally low and with temperate eutrophic water bodies where it is generally high. As a result, predation pressure on zooplankton by fish varies dramatically amongst Ethiopian water bodies.     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.     

Neural networks in the cockpit of the fly

Flies have been buzzing around on earth for over 300 million years. During this time they have radiated into more than 125,000 different species (Yeates and Wiegmann 1999), so that, by now, roughly every tenth described species is a fly. They thus represent one of the most successful animal groups on our planet. This evolutionary success might, at least in part, be a result of their acrobatic maneuverability, which enables them, for example, to chase mates at turning velocities of more than 3000 degrees s(-1) with delay times of less than 30 ms (Land and Collett 1974; Wagner 1986). It is this fantastic behavior, which has initiated much research during the last decades, both on its sensory control and the biophysical and aerodynamic principles of the flight output (Dickinson et al. 1999, 2000). Here, we review the current state of knowledge about the neural processing of visual motion, which represents one sensory component intimately involved in flight control. Other reviews on this topic have been published with a similar (Hausen 1981, 1984; Hausen and Egelhaaf 1989; Borst 1996) or different emphasis (Frye and Dickinson 2001; Borst and Dickinson 2002). Because of space limitations, we do not review the extensive work that has been done on fly motion-sensitive neurons to advance our understanding of neural coding (Bialek et al. 1991; Rieke et al. 1997; de Ruyter et al. 1997, 2000; Haag and Borst 1997, 1998; Borst and Haag 2001). Unless stated otherwise, all data presented in the following were obtained on the blowfly Calliphora vicina which we will often casually refer to as 'the fly'.     

On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.     

Are Brazil nut populations threatened by fruit harvest?

Harvest of Brazil nuts from the large, iconic tree Bertholletia excelsa generates substantial income for smallholders, providing a strong incentive to conserve the mature forests where it grows. Although much previous work has focused on the impact of nut harvest on new seedling recruits into B. excelsa populations, the connection between harvest rates and long‐term population stability is still unclear. Moreover, there is additional uncertainty for Brazil nut management in terms of population response to climate change and other anthropogenic influences. We drew on 14 years of research in two sites in Acre, Brazil with different B. excelsa nut harvest intensities (39% and 81%), to produce stochastic and deterministic matrix population models which incorporated parameter uncertainty in vital rates. Adult abundance was projected to remain close to the current observed abundance or higher through the next 50 years. Elasticity analyses revealed that the asymptotic population growth rate (λ) was most sensitive to stasis vital rates in sapling, juvenile, and adult stages. Deterministic transition matrices calculated using diameter growth rates dependent on rainfall yielded average λ values around 1.0 under extreme high, extreme low, and average annual rainfall. While sustained high rates of Brazil nut harvest and climate change could potentially negatively impact B. excelsa populations, changes in human use of the forested landscape are more immediate concern. To reduce the risk of population decline, smallholders and managers of B. excelsa rich forests should focus on conservation of pre‐mature and mature individuals.     

Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae Tü2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation.

A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae Tü2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C.