G proteins as regulators in ethylene-mediated hypoxia signaling

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O₂ concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.Key words: submergence, hypoxia, ethylene, G protein, reactive oxygen species, H₂O₂     

Synthesis and characterization of high affinity inhibitors of the H+/peptide transporter PEPT2.

In this study, we describe the rational synthesis and functional analysis of novel high affinity inhibitors for the mammalian peptide transporter PEPT2. Moreover, we demonstrate which structural properties convert a transported compound into a non-translocated inhibitor. Starting from Lys[Z(NO(2))]-Pro (where Z is benzyloxycarbonyl), which we recently identified as the first competitive high affinity inhibitor of the intestinal peptide transporter PEPT1, a series of different lysine-containing dipeptide derivatives was synthesized and studied for interaction with PEPT2 based on transport competition assays in Pichia pastoris yeast cells expressing PEPT2 heterologously and in renal SKPT cells expressing PEPT2. In addition, the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. Synthesis and functional analysis of Lys-Lys derivatives containing benzyloxycarbonyl or 4-nitrobenzyloxycarbonyl side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by PEPT2 with K(i) values as low as 10 +/- 1 nm. This is the highest affinity of a ligand of PEPT2 ever reported. Moreover, based on the structure-function relationship, we conclude that the spatial location of the side chain amino protecting group in a dipeptide containing a diaminocarbonic acid and its intramolecular distance from the Calpha atom are key factors for the transformation of a substrate into an inhibitor of PEPT2.     

Characterization of the AINV gene and the encoded invertase from the dimorphic yeast Arxula adeninivorans

The invertase-encoding of AINV gene Arxula adeninivorans was isolated and characterized. The gene includes a coding sequence of 2700 bp encoding a putative 899 amino acid protein of 101.7 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of alpha-glucosidases from different sources. The gene activity is regulated by carbon source. In media supplemented with sucrose induction of the AINV gene and accumulation of the encoded invertase in the medium was observed. In addition the extracellular enzyme level is influenced by the morphological status of the organism, with mycelia secreting the enzyme in titres higher than those observed in budding yeasts. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AINV gene under control of the strong A. adeninivorans -derived TEF1 promoter. For both proteins a molecular mass of 600 kDa was determined, a pH optimum at pH 4.5 and a temperature optimum at 55 degrees C. The preferred substrates for the enzyme included the ss-D-fructofuranosides sucrose, inulin and raffinose. Only a weak enzyme activity was observed for the alpha-D-glucopyranosides maltotriose, maltose and isomaltose. Thus the invertase primarily is a ss-fructosidase and not an alpha-glucosidase as suggested by the homology to such enzymes.     

The<Emphasis Type="Italic"> diageotropica</Emphasis> mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor

The diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H⁺-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.Abbreviations ABP auxin-binding protein - CCD charge-coupled device - 2,4-D 2,4-dichlorophenoxyacetic acid - dgt diageotropica - FC fusicoccin     

Intracellular transport of a heterologous membrane protein,the human transferrin receptor,in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae. The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the plasma membrane (PM). The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar compartment. Degradation is further dependent on favorable transport conditions to this compartment. The main bottleneck of transport seems to be the transition from the ER to the PM. The chaperone Cne1p, which is involved in quality control in the ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein. This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins. Electronic Publication     

Microencapsulation of hybridomas by cellulose sulfate-polydimethyldiallylammonium chloride procedure

Two hybridoma cell lines of human origin and two of murine origin were encapsulated using the cellulose sulfate-polydimethyldiallylammonium chloride procedure. All lines could be cultivated in capsules, but the time of growth and product synthesis were limited. The membranes formed were impermeable to albumin and transferrin over a period of 240 minutes. Therefore, the stop of cell proliferation is assumed to be caused by the lack of at least these two proteins. The cell densities inside the capsules reached a maximum of 4.1 × 10⁶/ml (human line) and 3.9 × 10⁷/ml (murine line). The maximum product concentration in the capsules measured by IgG- and IgM-ELISA was 0.17 mg/ml (human line) and 7 mg/ml (murine line). The calculated corresponding retention was 93% (human IgM) and 52% (murine IgG). The characterization of the product showed that the immunoglobulin was not intact and might have been destroyed during harvest. Using the same cell lines the method was compared with the alginate-PLL procedure originally developed by LIM and SUN [1].     

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.