Rubidium transport in irradiated vitamin-E-deficient bone marrow cells

We showed previously that the Rb⁺ transport rate in bone marrow cells (BMC) of vitamin-E-deficient mice is significantly lower than that in BMC of euvitaminotic mice. It is now evident that 4 h after whole-body, low-dose (0.01–1.0 Gy) gamma-irradiation of avitaminotic mice, there is an increase in the rate of Rb⁺ transport. This increase is quite pronounced, exceeding at all dose levels the rate of Rb⁺ transport in euvitaminotic mice exposed to the same radiation dose.On leave from the University of Rochester, School of Medicine and Dentistry, Department of Biophysics, Rochester, New York, USA     

Zur Wirkung von Antimycotica auf das Wachstum des Luftmycels bei einigen Pilzen

Zusammenfassung Das Luftmycel von Pilzen reagiert auf Antibiotica im allgemeinen nur schwach. Einige Basidiomyceten schränken aber das Wachstum der Lufthyphen bei geringen Konzentrationen bestimmter Hemmstoffe, vor allem Polyenantibiotica, stärker ein als das des Substratmycels. Die Reaktionsweise des Luftmycels läßt im Gegensatz zur Auffassung mancher Autoren keine Schlüsse auf das Fehlen einer Leitung von Griseofulvin in Pilzhyphen zu. Polyporus versicolor zeigt in seiner Myceldifferenzierung ausgeprägte Unterschiede bei Einwirkung gewisser strukturell verwandter Stoffe.

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Summary The aerial mycelium of fungi is only slightly inhibited by antibiotics in most cases, but in some species of Basidiomycetes little concentrations of some inhibitors, e.g. polyene antibiotics, retard the growth of aerial hyphae more than the growth of substrate mycelium. The reaction of aerial hyphae doesn't permit any conclusion about transport of griseofulvin in fungal hyphae. Polyporus versicolor shows great differences in mycelial differentiation influenced by structurally related compounds.

     

Biochemical Large-Scale Interaction Analysis of Murine Olfactory Receptors and Associated Signaling Proteins with Post-Synaptic Density 95, Drosophila Discs Large,Zona-Occludens 1 (PDZ) Domains

G protein-coupled receptors (GPCRs) constitute the largest family among mammalian membrane proteins and are capable of initiating numerous essential signaling cascades. Various GPCR-mediated pathways are organized into protein microdomains that can be orchestrated and regulated through scaffolding proteins, such as PSD-95/discs-large/ZO1 (PDZ) domain proteins. However, detailed binding characteristics of PDZ–GPCR interactions remain elusive because these interactions seem to be more complex than previously thought. To address this issue, we analyzed binding modalities using our established model system. This system includes the 13 individual PDZ domains of the multiple PDZ domain protein 1 (MUPP1; the largest PDZ protein), a broad range of murine olfactory receptors (a multifaceted gene cluster within the family of GPCRs), and associated olfactory signaling proteins. These proteins were analyzed in a large-scale peptide microarray approach and continuative interaction studies. As a result, we demonstrate that canonical binding motifs were not overrepresented among the interaction partners of MUPP1. Furthermore, C-terminal phosphorylation and distinct amino acid replacements abolished PDZ binding promiscuity. In addition to the described in vitro experiments, we identified new interaction partners within the murine olfactory epithelium using pull-down-based interactomics and could verify the partners through co-immunoprecipitation. In summary, the present study provides important insight into the complexity of the binding characteristics of PDZ–GPCR interactions based on olfactory signaling proteins, which could identify novel clinical targets for GPCR-associated diseases in the future.PDZ domain proteins comprise one of the largest families among interaction domain scaffolding proteins and are highly abundant in various multicellular eukaryotic species. These proteins fulfill important physiological functions in a broad range of different tissues and cells as they orchestrate complex protein networks. Among putative PDZ interaction partners, one important protein family is the group of GPCRs¹, constituting the largest family of membrane proteins in mammals (1). Here, signal efficiency, speed, desensitization, and internalization can be modulated by PDZ proteins (2–5). Olfactory receptors (ORs) represent a multigene family within this group of seven-transmembrane domain proteins and encompass 2% of the mammalian genome (6). Belonging to class I GPCRs, ORs share many general features of this receptor family, making them an interesting target for interactions involving PDZ proteins. Until recently, an organizing complex builder, such as the inactivation no afterpotential D (InaD) protein in the visual system of Drosophila melanogaster (7, 8), could not be clearly identified for olfactory signaling.The multiple PDZ domain protein 1, with 13 individual PDZ domains, represents the largest of the described PDZ proteins to date (9) and interacts with different GPCRs (10–12). One well-described example is its interaction with GABA_B receptors, leading to enhanced receptor stability at the plasma membrane and prolonged signaling duration (2). In previous studies, we demonstrated that PDZ domains 1 + 2 can interact with a selected subset of ORs (13). Furthermore, we showed that MUPP1 binds to a specific OR and that most of the described proteins are involved in mammalian olfactory signal transduction in the native system, making MUPP1 a promising candidate for orchestrating the olfactory system (14).Many PDZ–ligand interactions depend on classical binding motifs at the ligand''s C-terminal end, thereby building weak transient protein complexes (15, 16). However, an increasing number of PDZ interactions have emerged that seem to provide more complex binding modalities, differing from the canonical interactions (17, 18). Ligand binding seems not to be exclusively restricted to C-terminal sites, and PDZ domains cannot be distinctly classified but are evenly distributed throughout a selective space (17, 19–21). Therefore, it is of great interest to analyze OR–PDZ interactions to characterize the putative binding requirements and to further investigate the role of MUPP1 in olfactory signaling.In the present study, we characterized the binding modalities between the 13 individual PDZ domains of MUPP1 and a broad range of murine olfactory receptors in a large-scale approach, indicating that classical binding motifs were not overrepresented among the evaluated binding partners. In addition, we identified new binding partners from the murine olfactory epithelium using pull-down-based interactomics.     

Characterization of human reflex tear proteome reveals high expression of lacrimal proline‐rich protein 4 (PRR4)

In‐depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC‐ESI‐MS/MS strategy for label‐free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC‐ESI‐MS/MS and targeted‐MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline‐rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland‐specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha‐1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.     

Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.     

The two‐phase partitioning system – a powerful technique to purify integral membrane proteins of Corynebacterium glutamicum for quantitative shotgun analysis

We established a single consecutive strategy which assigned the most comprehensive number of integral membrane proteins from Gram‐positive bacteria to date. For this purpose, we adapted a biphasic partitioning system for the biotechnologically intensively used Corynebacterium glutamicum and proved for the first time that such a system is well suited for quantitative comparison. 297 integral membrane proteins were identified by our integrated approach, which depletes stringently cytosolic proteins. In combination with our previously developed SIMPLE strategy, our data comprise 61% (374 integral membrane proteins) of the entire membrane proteome, which aims towards an almost comprehensive coverage. Wild type and a production strain of C. glutamicum were compared by ¹⁵N metabolic labelling and quantitation was obtained by spectral counting and peak areas. Both quantification strategies display a consistent trend in up or downregulation of proteins. Nevertheless, spectral counting often provides results indicating a much stronger regulation compared to ProRata values. Either spectral counting seems to exaggerate protein regulation or ProRata tends to attenuate the information about the regulation level. We highlight some of the biologically relevant candidates, which prove that our approach helps to give a deeper quantitative insight towards the understanding of transport and other membrane associated processes, important for strain development of C. glutamicum.     

Differential threshold effects of habitat fragmentation on gene flow in two widespread species of bush crickets

Effects of habitat fragmentation on genetic diversity vary among species. This may be attributed to the interacting effects of species traits and landscape structure. While widely distributed and abundant species are often considered less susceptible to fragmentation, this may be different if they are small sized and show limited dispersal. Under intensive land use, habitat fragmentation may reach thresholds at which gene flow among populations of small-sized and dispersal-limited species becomes disrupted. Here, we studied the genetic diversity of two abundant and widespread bush crickets along a gradient of habitat fragmentation in an agricultural landscape. We applied traditional (G(ST), θ) and recently developed (G'ST', D) estimators of genetic differentiation on microsatellite data from each of twelve populations of the grassland species Metrioptera roeselii and the forest-edge species Pholidoptera griseoaptera to identify thresholds of habitat fragmentation below which genetic population structure is affected. Whereas the grassland species exhibited a uniform genetic structuring (G(ST) = 0.020-0.033; D = 0.085-0.149) along the whole fragmentation gradient, the forest-edge species' genetic differentiation increased significantly from D < 0.063 (G(ST) < 0.018) to D = 0.166 (G(ST) = 0.074), once the amount of suitable habitat dropped below a threshold of 20% and its proximity decreased substantially at the landscape scale. The influence of fragmentation on genetic differentiation was qualitatively unaffected by the choice of estimators of genetic differentiation but quantitatively underestimated by the traditional estimators. These results indicate that even for widespread species in modern agricultural landscapes fragmentation thresholds exist at which gene flow among suitable habitat patches becomes restricted.     

The Arabidopsis GNOM ARF-GEF mediates endosomal recycling,auxin transport,and auxin-dependent plant growth

Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.     

Presymptomatic detection of Parkinson's disease

Parkinson's disease (PD) is characterized by a progressive degeneration of mesencephalic dopaminergic neurons. More than half of these neurons are lost in a presymptomatic phase of an estimated 4-6 years duration. It is obvious that any type of treatment aimed at slowing down the disease process should preferably be applied in this presymptomatic phase. Presymptomatic detection of PD has therefore become an important goal. In a recent study in a population of 361 asymptomatic first degree relatives of PD patients, we were able to demonstrate that presymptomatic detection is possible by means of a combination of three olfactory processing tasks and [123l] beta-CIT single photon emission computed tomography (SPECT) scanning of the nigrostriatal dopaminergic system. These results are a first step towards the development of a screening strategy that may be applied in the general population. Impairments of olfactory function, however, are not specific to PD but are also associated with other neurodegenerative disorders (e.g. Alzheimer's disease) and certain lifestyle characteristics (e.g. smoking). In the next few years our research efforts will focus on two different approaches to develop a more specific screening strategy. First, olfactory processing tasks will be combined with tasks aimed at detecting subtle (visuo)motor disturbances and early cognitive impairments. In parallel, an effort will be made to define disease-specific patterns of olfactory dysfunction in neurodegenerative disorders.