Effects of cadmium accumulation on growth and respiration of a cadmium-sensitive strain of Bacillus subtilis and a selected cadmium resistant mutant

The effects of cadmium on the growth and respiration of two strains of Bacillus subtilis are compared to the accumulation of Cd by viable and cyanide-killed cells, protoplasts and cell fractions of the strains. Growth and respiration of strain 1A1 were significantly inhibited at 10g Cd²⁺/ml while the growth and respiration of strain 1A1R, a selected mutant of 1A1, were only slightly affected. Similarly, 1A1R protoplasts were more resistant to Cd than were 1A1 protoplasts. The differential resistance of the strains correlates with the accumulation of Cd by the two strains, with 1A1 accumulating approximately 10 times the level of Cd after a 4 h exposure to 1 g Cd²⁺/ml. The distributions of Cd throughout the cells, however, were similar between strains. Based on the accumulation of Cd by cyanide-killed protoplasts, uptake of Cd by 1A1 appears to be an active process, while for 1A1R, Cd accumulation is independent of protoplast viability.Non-standard abbreviations SMM Subtilis Minimal Medium - AAS Atomic Absorption Spectrophotometry - TSA Trypticase Soy Agar - PCA Plate Count Agar - INT 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-2H tetrazolium chloride - dd H₂O double distilled demineralized water - OD Optical Density     

Cell cycle re-entry sensitizes podocytes to injury induced death

Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy.     

Taking into account water use impacts in the LCA of biofuels: an Argentinean case study

Purpose  

The assessment of biofuels has until now mainly focused on energy demand and greenhouse gas emissions. Only little attention has been given to other impacts, although the general importance of water use for the life cycle assessment (LCA) of agricultural products has been recognized in recent publications. The aim of this work is to assess in detail the water consumption along a biofuel production chain taking into account irrigation efficiencies, levels of water scarcity, and type of feedstock, and to integrate those results in a full LCA. Furthermore, we compare the results for biofuels from various feedstocks and regions with conventional petrol.     

Structural and functional characterization of the Wnt inhibitor APC membrane recruitment 1 (Amer1)

Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing β-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of β-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the β-catenin destruction complex at the plasma membrane by recruiting β-catenin, adenomatous polyposis coli, and Axin/Conductin. Deletion or specific mutations of the membrane binding domain of Amer1 abolish its membrane localization and abrogate negative control of Wnt signaling, which can be restored by artificial targeting of Amer1 to the plasma membrane. In line, a natural splice variant of Amer1 lacking the plasma membrane localization domain is deficient for Wnt inhibition. Knockdown of Amer1 leads to the activation of Wnt target genes, preferentially in dense compared with sparse cell cultures, suggesting that Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts Wnt-induced degradation of Axin, which requires membrane localization of Amer1. The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by acting as a scaffold protein for the β-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.     

IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcgammaRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.     

Exospore formation in Methylosinus trichosporium.

Formation of exospores in Methylosinus trichosporium was examined by electron microscopy; serial sectioning was used to visualize the shape and location of the developing exospore in relation to the vegetative cell. The initial stage was the formation of a budlike enlargement on one end of the vegetative cell. The enlargement was surrounded by the exospore capsule, and the cell wall was continuous around both the cell and the developing exospore. A constriction occurred in the area where the budlike structure was attached to the vegetative cell, and the constriction continued to form until the immature exospore was detached from the vegetative cell. The cup-shaped immature exospore was surrounded by the exospore capsule, which appeared to hold the exospore close to the vegetative cell. After separation from the vegetative cell, the immature exospore developed further by forming the exospore wall and by becoming spherical.     

Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function

In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit. Comparison with known sequences revealed that the carboxyl-terminal half of the polypeptide is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer arm dynein. Immunoblot analysis with a monoclonal antibody to the 74-kD species indicated a widespread tissue distribution, as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognized a 67-kD species in ram sperm flagella and pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, we predict an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with membranous organelles and kinetochores.