Gene flow between populations of two invertebrates in springs

1. Using allozymes, we analysed genetic structure of the freshwater gastropod Bythinella dunkeri and the freshwater flatworm Crenobia alpina. The two species are habitat specialists, living almost exclusively in springs. The sampled area in Hesse (Germany) covers a spatial scale of 20 km and includes two river drainages. From the biology of the two species we expected little dispersal along rivers. However, the possibility exists that groundwater provide suitable pathways for dispersal. 2. In B. dunkeri heterozygosity decreased from west to east. For some alleles we found clines in this geographic direction. These clines generated a positive correlation between geographic distance and genetic differentiation. Furthermore patterns of genetic variation within populations suggested that populations may have been faced with bottlenecks and founder effects. If populations are not in population genetic equilibrium, such founder effects would also explain the rather high amount of genetic differentiation between populations (10%). 3. For C. alpina the mean number of alleles decreased with increasing isolation of populations. Genetic differentiation between populations contributed 19% to the total genetic variation. Genetic differentiation was not correlated to geographic distance, but compared with B. dunkeri variability of pairwise differentiation between pairs of populations was higher in C. alpina. 4. Overall B. dunkeri appears to be a fairly good disperser, which may use groundwater as dispersal pathway. Furthermore populations seem to be not in equilibrium. In contrast C. alpina forms rather isolated populations with little dispersal between springs and groundwater seems to play no important role for dispersal.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

M89V Sialic Acid Acetyl Esterase (SIAE) and All Other Non-Synonymous Common Variants of This Gene Are Catalytically Normal

Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.     

Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells

Summary Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP dimethylallylpyrophosphate - iP isopentenyladenine - iPA isopentenyladenosine - iPMP isopentenyladenosine 5-monophosphate - IPT isopentenyltransferase - trans-Z trans-zeatin     

Phloem translocation of Fe,Cu, Mn,and Zn in Ricinus seedlings in relation to the concentrations of nicotianamine,an endogenous chelator of divalent metal ions,in different seedling parts

During the first 8 days of germination the Ricinus seedling is supplied with all nutrients by the endosperm via phloem transport. In 4- to 8-days-old seedlings the concentrations and contents of Fe, Cu, Mn and Zn, and nicotianamine (NA) in the endosperm, cotyledons, hypocotyl and roots were estimated. From the data obtained translocation rates and flow profiles for the metals were established. The main sink for Fe, Mn and Zn were the cotyledons whereas Cu was mainly imported into the hypocotyl. Maximum flow rates occurred between days 5 and 7, for Zn between days 6 and 8.The time kinetics of NA and divalent metal ion concentrations and contents are interpreted as co-transport. The role of NA as transport vehicle of micronutrients in the sieve tubes is discussed.     

The effect of ozone exposure on the release of eicosanoids in guinea-pig BAL fluid in relation to cellular damage and inflammation

The observed effects after ozone exposure strongly depend on ozone concentration and exposure time. We hypothesized that depending on the O3 exposure protocol, mainly either an oxidant damage or an inflammation will determine the O3 toxicity. We compared two different ozone exposure protocols: an acute exposure (3 ppm 2 h) for studying the oxidant damage and an exposure (1 ppm 12 h) where an inflammatory component is also probably involved. We measured LDH activity and protein and albumin exudation as markers for cellular damage. After the acute exposure an increase in LDH activity was measured and after exposure to 1 ppm ozone for 12 h the exudation of protein and albumin was also enhanced. The histological examinations showed a neutrophilic inflammatory response only after exposure to 1 ppm ozone for 12 h. The acute exposure protocol resulted in an increased release of PGE2, PGD2, PGF2alpha and 6-ketoPGF1alpha whereas exposure to 1 ppm ozone for 12 h led to an additional release of LTB4. No effects were measured on the release of TxB2 and LTC4/D4/E4. These changed amounts of eicosanoids will probably contribute to the ozone-induced lung function changes.     

Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.     

Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

The ATP-dependent insertion of Mg²⁺ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg²⁺ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity.     

The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

Indicator selection in life cycle assessment to enable decision making: issues and solutions

Purpose

With an ever increasing list of indicators available, life cycle assessment (LCA) practitioners face the challenge of effectively communicating results to decision makers. Simplification of LCA is often limited to an arbitrary selection of indicators, use of single scores by using weighted values or single attribute indicators. These solutions are less attractive to decision makers, since value judgments are introduced or multi-indicator information is lost. Normalization could be a means to narrow the list of indicators by ranking indicators vs. a reference system. This paper shows three different normalization approaches that produce very different ranking of indicators. It is explained how normalization helps maintain a multi-indicator approach while keeping the most relevant indicators, allowing effective decision making.

Methods

The approaches are illustrated on a hand dishwashing case study, using ReCiPe as the impact assessment method and taking the European population (year 2000) as the reference situation. Indicators are ranked using midpoint normalization factors, and compared to the ranking from endpoint normalization broken down by midpoint contribution.

Results and discussion

Endpoint normalization shows Resources as the most relevant area of protection for this case, closely followed by Human Health and Ecosystem. Broken down by their key driving midpoints, fossil depletion, climate change and, to a lesser extent, particulate matter formation and metal depletion, are most relevant. Midpoint normalization, however, indicates Freshwater Eutrophication, Natural Land Transformation and Toxicity indicators (marine and freshwater ecotoxicity and human toxicity) are most relevant.

Conclusions

A three-step approach based on endpoint normalization is recommended to present only the most relevant indicators, allowing more effective decision making instead of communicating all LCA indicators. The selection process breaks out the normalized endpoint results into the most contributing midpoints (relevant indicators) and reports results with midpoint level units. Bias due to lack of data completeness is less of an issue in the endpoint normalization process (compared to midpoint normalization), while midpoint results are less subject to uncertainty (compared to endpoint results). Focusing on the relevant indicators and key contributing unit processes has proven to be effective for non-LCA expert decision makers to understand, use, and communicate complex LCA results.