Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action

TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1-7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1-6). Unlike TOT1-3 (ELP1-3) and TOT5-7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.     

Kinetic Analysis of the Guanine Nucleotide Exchange Activity of TRAPP, a Multimeric Ypt1p Exchange Factor

TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5′-(β,γ-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (∼ 10^− 4 s^− 1) and accelerated > 1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg²⁺-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by < 80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic.     

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.     

Biaryl cannabinoid mimetics--synthesis and structure-activity relationship

Synthesis, in vitro biological evaluation, and structure-activity relationships of a biaryl cannabinoid mimetic 2 are reported. Variations in the substitution pattern yielded a number of agonists with low nanomolar affinity. Replacing the phenol group by a methyl morpholino acetate group led to compound 28, a 500-fold selective CB(2) receptor agonist.     

Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding

PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P₂-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P₂ with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding.     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Provenance- and life-history stage-specific responses of the dwarf shrub Calluna vulgaris to elevated vapour pressure deficit

Climate change may alter microscale-effective ecosystem properties such as atmospheric water vapour pressure, but consequences for plant growth are insufficiently understood. Within a northwest German heathland an open-top chamber experiment was established to analyse the effects of elevated vapour pressure deficit (eVPD) on growth responses of Calluna vulgaris considering both plant origin (Atlantic (AP), sub-Atlantic (SAP), sub-Continental (SCP)) and life-history stage (1-year vs. 10-year old plants). We hypothesised that the plants’ sensitivity to eVPD decreases (i) from AP to SCP and (ii) with progressing life-history stage. Elevated VPD caused a provenance-specific decrease of shoot increment whilst aboveground biomass productivity remained unaffected. AP and SAP responded with increasing belowground biomass δ¹³C signatures to eVPD, whereas δ¹³C values decreased for SCP. Moreover, eVPD increased and decreased belowground biomass δ¹³C signatures of 1- and 10-year old plants, respectively. These responses to eVPD were related to differences in morphological-chemical traits and the plants’ trait plasticity in response to eVPD. SCP showed the highest aboveground tissue mass density and significantly increased tissue C:N ratios under eVPD. One-year old plants had a tenfold higher shoot:root ratio than 10-year old plants, making young plants more sensitive to eVPD. Our findings demonstrate that the atmospheric water status affects the morphology and physiology of Calluna independent of the soil water status. The results have implications for the conservation of heathlands under climate change: (i) SCP may constitute an appropriate ecotype for assisted migration-approaches, and (ii) management needs to weigh different options for heathland rejuvenation.

     

Compositional turnover and variation in Eemian pollen sequences in Europe

Vegetation History and Archaeobotany - The Eemian interglacial represents a natural experiment on how past vegetation with negligible human impact responded to amplified temperature changes...     

Genetic variation in an ephemeral mudflat species: The role of the soil seed bank and dispersal in river and secondary anthropogenic habitats

Many ephemeral mudflat species, which rely on a soil seed bank to build up the next generation, are endangered in their natural habitat due to the widespread regulation of rivers. The aim of the present study was to elucidate the role of the soil seed bank and dispersal for the maintenance of genetic diversity in populations of near‐natural river habitats and anthropogenic habitats created by traditional fish farming practices using Cyperus fuscus as a model. Using microsatellite markers, we found no difference in genetic diversity levels between soil seed bank and above‐ground population and only moderate differentiation between the two fractions. One possible interpretation is the difference in short‐term selection during germination under specific conditions (glasshouse versus field) resulting in an ecological filtering of genotypes out of the reservoir in the soil. River populations harbored significantly more genetic diversity than populations from the anthropogenic pond types. We suggest that altered levels and patterns of dispersal together with stronger selection pressures and historical bottlenecks in anthropogenic habitats are responsible for the observed reduction in genetic diversity. Dispersal is also supposed to largely prohibit genetic structure across Europe, although there is a gradient in private allelic richness from southern Europe (high values) to northern, especially north‐western, Europe (low values), which probably relates to postglacial expansion out of southern and/or eastern refugia.     

Growth forms and life-history strategies predict the occurrence of aquatic macrophytes in relation to environmental factors in a shallow peat lake complex

Hydrobiologia - Aquatic ecosystems provide vital services, and macrophytes play a critical role in their functioning. Conceptual models indicate that in shallow lakes, plants with different growth...