The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds

The temporal and spatial distribution of cysteine proteinases (CPRs) was analyzed immunologically and by in situ hybridization to identify the CPRs involved in the initiation of storage-globulin degradation in embryonic axes and cotyledons of germinating vetch (Vicia sativa L.). At the start of germination several CPRs were found in protein bodies in which they might have been stored in the mature seeds. Cysteine proteinase 1 was predominantly found in organs like the radicle, which first start to grow during germination. Cysteine proteinase 2 was also present at the start of germination but displayed a less-specific histological pattern. Proteinase B was involved in the globulin degradation of vetch cotyledons as well. The histological pattern of CPRs followed the distribution of their corresponding mRNAs. The latter were usually detected earlier than the CPRs but the in situ hybridization signals were histologically not as restricted as the immunosignals. Proteolytic activity started in the radicle of the embryonic axis early during germination. Within 24 h after imbibition it had also spread throughout the whole shoot. At the end of germination, newly synthesized CPRs might have supplemented the early detectable CPRs in the axis. In the cotyledons, only the abaxial epidermis and the procambial strands showed proteinase localization during germination. Both CPR1 and CPR2, as well as the less common proteinase B, might have been present as stored proteinases. Three days after imbibition, proteolytic activity had proceeded from the cotyledonary epidermis towards the vascular strands deeper inside the cotyledons. The histochemical detection of the CPRs was in accordance with the previously described histological pattern of globulin mobilization in germinating vetch [Tiedemann J, et al. (2000)]. A similar link between the distribution of CPRs and globulin degradation was found in germinating seeds of Phaseolus vulgaris L. The coincidence of the histological patterns of globulin breakdown with that of the CPRs indicates that at least CPR1, CPR2 and proteinase B are responsible for bulk globulin mobilization in the seeds of the two legumes. Received: 14 February 2000 / Accepted: 16 August 2000     

Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization

Type 2 diabetes mellitus affects 6% of western populations and represents a major risk factor for the development of skin complications, of which impaired wound healing, manifested in e.g. "diabetic foot ulcer", is most prominent. Impaired angiogenesis is considered a major contributing factor to these non-healing wounds. At present it is still unclear whether diabetes-associated wound healing and skin vascular dysfunction are direct consequences of impaired insulin/IGF-1 signaling, or secondary due to e.g. hyperglycemia. To directly test the role of vascular endothelial insulin signaling in the development of diabetes-associated skin complications and vascular function, we inactivated the insulin receptor and its highly related receptor, the IGF-1 receptor, specifically in the endothelial compartment of postnatal mice, using the inducible Tie-2CreERT (DKO(IVE)) deleter. Impaired endothelial insulin/IGF-1 signaling did not have a significant impact on endothelial homeostasis in the skin, as judged by number of vessels, vessel basement membrane staining intensity and barrier function. In contrast, challenging the skin through wounding strongly reduced neo-angiogenesis in DKO(IVE) mice, accompanied by reduced granulation tissue formation reduced. These results show that endothelial insulin/IGF signaling is essential for neo-angiogenesis upon wounding, and imply that reduced endothelial insulin/IGF signaling directly contributes to diabetes-associated impaired healing.     

Redirection of pigment biosynthesis to isocoumarins in Fusarium

Colonies of Fusarium species often appear red due to production of pigments, such as aurofusarin or bikaverin. The primary compounds in these biosynthetic pathways are YWA1 and pre-bikaverin, respectively, catalyzed by two multidomain polyketide synthases (PKSs), which both have a claisen-type cyclase domain (CLC) in their N terminal. Disruption of the CLC domains has been shown to result in formation of the lactones citreoisocoumarin and SMA93 instead of YWA1 and pre-bikaverin. In the present study we have discovered a medium with low nitrogen content which partially redirects the aurofusarin and bikaverin pathways to produce citreoisocoumarin and SMA93, respectively. This is first time that SMA93 is identified in a fungus and we suggest that it is renamed bikisocoumarin, as it is derived from the bikaverin pathway. The redirection of the aurofusarin and bikaverin biosynthetic pathways was reverted by adding inorganic nitrogen to the medium, whereas organic nitrogen in form of arginine or glutamine stimulated isocoumarin production. This suggests that nitrogen source can influence isocoumarin production. Production of isocoumarin was also repressed by alkaline conditions, which suggests that nitrogen supply is not the sole regulatory factor in the pathway. The redirection was observed in all producers of aurofusarin (6) and bikaverin (2), suggesting the presence of a conserved regulatory mechanism.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

GDNF prevents TGF-β-induced damage of the plasma membrane in cerebellar granule neurons by suppressing activation of p38-MAPK via the phosphatidylinositol 3-kinase pathway

Transforming growth factor-β (TGF-β) and glial-cell-line-derived neurotrophic factor (GDNF) have been shown to synergize in several paradigms of neuronal survival. We have previously shown that cerebellar granule neurons (CGN) degenerate in low potassium via ERK1/2 (extra-cellular-regulated kinase)-dependent plasma membrane (PM) damage and caspase-3-dependent DNA fragmentation. Here, we have investigated the putative synergistic function of GDNF and TGF-β in CGN degeneration. GDNF alone prevents low-potassium-induced caspase-3 activation and DNA fragmentation but does not affect either low-potassium-induced ERK activation or PM damage. TGF-β alone does not affect low-potassium-induced DNA fragmentation but potentiates low-potassium-induced PM damage. This effect of TGF-β is independent of ERK1/2 activation but dependent on p38-MAPK (mitogen-activated protein kinase) activation. When co-applied with TGF-β, GDNF paradoxically antagonizes TGF-β-induced potentiation of PM damage by inhibiting TGF-β-induced p38-MAPK activation. In addition, PI3K (phosphatidylinositol 3-kinase) inhibitors abolish the GDNF effect. This study thus demonstrates a differential mechanism of action of GDNF and TGF-β on CGN degeneration. GDNF inhibits caspase-3-dependent DNA fragmentation but does not affect ERK-dependent PM damage. However, GDNF can attenuate TGF-β-induced p38-MAPK-dependent PM damage via the PI3K pathway. This work was supported by the Deutsche Forschungsgemeinschaft (STR 616/1–2) and by a fellowship (Young Investigator Award) from the Medical Faculty, University of Heidelberg, Germany to S. Subramaniam.     

Methane yield and species diversity dynamics of perennial wild plant mixtures established alone,under cover crop maize (Zea mays L.), and after spring barley (Hordeum vulgare L.)

The cultivation of perennial wild plant mixtures (WPMs) in biogas cropping systems dominated by maize (Zea mays L.) restores numerous ecosystem functions and improves both spatial and temporal agrobiodiversity. In addition, the colorful appearance of WPM can help enhance landscape beauty. However, their methane yield per hectare (MYH) varies greatly and amounts to only about 50% that of maize. This study aimed at decreasing MYH variability and increasing accumulated MYH of WPM by optimizing the establishment method. A field trial was established in southwest Germany in 2014, and is still running. It tested the effects of three WPM establishment procedures (E1: alone [without maize, in May], E2: undersown in cover crop maize [in May], E3: WPM sown after whole‐crop harvest of spring barley [Hordeum vulgare L.] in June) on both MYH and species diversity of two WPMs [S1, S2]). Mono‐cropped maize and cup plant (Silphium perfoliatum L.) were used as reference crops. Of the WPM treatments tested, S2E2 achieved the highest (19,296 , 60.5% of maize) and S1E1 the lowest accumulated MYH (8,156 , 25.6% of maize) in the years 2014–2018. Cup plant yielded slightly higher than S2E2 (19,968 , 62.6% of maize). In 2014, the WPM sown under maize did not significantly affect the cover crop performance. From 2015 onward, E1 and E2 had comparable average annual MYH and average annual number of WPM species. With a similar accumulated MYH but significantly higher number of species (3.5–10.2), WPM S2E2 outperformed cup plant. Overall, the long‐term MYH performance of WPM cultivation for biogas production can be significantly improved by undersowing with maize as cover crop. This improved establishment method could help facilitate the implementation of WPM cultivation for biogas production and thus reduce the trade‐off between bioenergy and biodiversity.     

Uptake of 24Mg by excised pine roots: A preliminary study

Uptake of ²⁴Mg by excised roots of Pinus sylvestris L. during up to 4 h long incubations in 99.9 atom % ²⁴Mg (50 M) was measured by ICP-MS. A rapid initial uptake phase (30 min) was followed by a slower uptake. This was interpreted as a shift from a phase dominated by saturable ion exchange (free space uptake), to a non-saturable phase, during which the rate of uptake was 0.077±0.0.012 mol Mg g^–1 (d.wt.) h^–1. The metabolic uncoupler DNP (2,4-dinitrophenol) at 50 M decreased the Mg uptake rate by 35% only, but the effect of DNP was significant (p<0.01). Several problems related to a high variability in the experimental material were encountered, and further refinement of this approach in studies of plant Mg uptake is suggested.