Skerotin-Komponenten in den Vitellocyten von Microdalyellia fairchildi (Turbellaria)

Zusammenfassung 1. An Microdalyellia fairchildi (Turbellaria, O. Neorhabdocoela) wird im Hinblick auf die chemische Natur der Eischalen das Vorkommen der Grundkomponenten des Sklerotins (Phenole, NH₂-reiche Proteine, Phenoloxidase) histo- und cytochemisch überprüft.2. An Totalpräparaten, histologischen Schnitten und isolierten Vitellocyten werden alle drei Komponenten in sogenannten Schalenvesikeln der Dotterzellen nachgewiesen, die hier neben anderen nährstoffhaltigen Vesikeln vorliegen. Für andere Gewebe und Organe, insbesondere die sogenannten Schalendrüsen, sind die ausgeführten Tests im wesentlichen negativ. Die Sklerotin-Natur der Eischalen ist damit nach entsprechenden Befunden an zahlreichen Trematoden und einigen Cestoden auch für eine Turbellarien-Gruppe erstmals erwiesen.3. Die Verwendung von H3-Tyrosin zeigt eine beträchtliche Anreicherung dieser Aminosäure in den Vitellarien sowie eine starke Beteiligung am Aufbau der Eischalen. Die Vermutung, daß die Phenolkomponente aus Tyrosin hervorgeht und an der Brückenbildung der Proteinketten beteiligt ist, bleibt zu erweisen.4. Bemerkenswert ist, daß sich die Aktivität der Phenoloxidase der Vesikel zwar auf Brenzcatechin, nicht aber auf Dopa (3,4-Dihydroxyphenylalanin) erstreckt; hierin weicht sie von den biochemischen Befunden extrahierter Phenoloxidasen verschiedener Herkunft ab. Mit verschiedenen Schwermetallkomplexbildnern (Natriumdiäthyldithiocarbaminat, D-Penicillamin) ist Inhibition möglich.

---

Sclerotin precursors in the vitelline cells of Microdalyellia fairchildi (Turbellaria)

Summary 1. In Microdalyellia fairchildi (Turbellaria, O. Neorhabdocoela) the occurrence of sclerotin precursors (phenolic substance, phenoloxidase, NH₂-rich protein) is histo- and cytochemically tested.2. In whole mount preparations, histological sections and isolated vitelline cells the three components could be demonstrated in special vesicles of the vitelline cells, which are present here along with nutrient vesicles. For other tissues and organs the tests were generally negative, especially for the so-called shell glands. After several corresponding results in many trematodes and in some cestodes the sclerotin nature of egg capsules seems now to be well-founded for a group of turbellaria too.3. Tritium-labeled tyrosine, which is ingested with food, is markedly enriched in the vitellaria and shown to be an essential ingredient of the egg-shell material. But the idea that this tyrosine is the precursor of the phenolic substance and thus possibly participates in cross-linking the protein chains of sclerotin remains to be confirmed.4. It is shown that the phenoloxidase of the vesicles catalyzes the oxidation of catechol but not that of Dopa (3,4-dihydroxyphenylalanine), a finding different from several biochemical investigations of the enzyme taken from other sources. Inhibition of the enzyme with some heavy metal complex-binding compounds (sodium diethyldithiocarbaminate, D-penicillamine) is demonstrated.

     

Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl‐phosphatidylglycerol hydrolase AcvB

Agrobacterium tumefaciens transfers oncogenic T‐DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl‐phosphatidylglycerol (L‐PG) hydrolase, which modulates L‐PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C‐terminal domain of AcvB (residues 232–456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10‐fold increase in L‐PG in Agrobacterium membranes and abolished T‐DNA transfer and tumor formation. Overproduction of the L‐PG synthase gene (lpiA) in wild‐type A. tumefaciens resulted in a similar increase in the L‐PG content (~7‐fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L‐PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L‐PG content by complementation with different acvB variants revealed that cellular L‐PG levels above 3% of total phospholipids interfere with T‐DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T‐DNA transfer.     

The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis

We identify a highly specific mutation (jf18) in the Caenorhabditis elegans nuclear envelope protein matefin MTF-1/SUN-1 that provides direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. The reorganization of chromatin in early meiosis is disrupted in mtf-1/sun-1(jf18) gonads, concomitant with the absence of presynaptic homolog alignment. Synapsis is established precociously and nonhomologously. Wild-type leptotene/zygotene nuclei show patch-like aggregations of the ZYG-12 protein, which fail to develop in mtf-1/sun-1(jf18) mutants. These patches remarkably colocalize with a component of the cis-acting chromosomal pairing center (HIM-8) rather than the centrosome. Our data on this mtf-1/sun-1 allele challenge the previously postulated role of the centrosome/spindle organizing center in chromosome pairing, and clearly support a role for MTF-1/SUN-1 in meiotic chromosome reorganization and in homolog recognition, possibly by mediating local aggregation of the ZYG-12 protein in meiotic nuclei.     

The low molecular weight proteome of Halobacterium salinarum

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.     

ADME investigations of unnatural peptides: distribution of a 14C-labeled beta 3-octaarginine in rats

The highly positively charged, cell-penetrating beta3-octaarginine has been prepared with a radioactive label by acetylation at the N-terminus with a doubly (14)C-labeled acetyl group ((14)CH3-(14)CO). With the radioactive compound, an ADME study (Absorption, Distribution, Metabolism, Excretion) was performed in male rats following an intravenous or oral dose of 1 mg/kg. Sampling was carried out after periods ranging from 5 min to 4 d or 7 d for blood/excretia and quantitative whole-body autoradioluminography (QWBA), respectively. After p.o. dosing, no systemic exposure to peptide-related radioactivity was observed, and the dose was completely excreted in the feces within 24 h suggesting the absence of relevant absorption; less than 3% of the i.v. dose was excreted from the animals within 4 d. Blood levels, after i.v. dosing, dropped within 4 d to less than 2% of Cmax and decreased afterwards only very slowly. No metabolites were observed in the systemic circulation. QWBA Data indicated that the distribution of the acetyl-beta-octaarginine-related radioactivity in the organs and tissues shifted over time. Notably, after 7 d, the highest concentration was measured in the lymph nodes, and the largest amount was found in the liver. A comparison with the results of two previous ADME investigations of beta-peptides (cf. Table 1) reveals that the distribution of the compounds within the animals is structure-dependent, and that there is a full range from oral availability with rather rapid excretion (of a tetrapeptide) to essentially complete lack of both oral absorption and excretion after i.v. administration (of a highly charged octapeptide). A discussion is presented about the in vivo stability and 'drug-ability' of peptides. In general, beta-peptides bearing proteinogenic side chains are compared with peptides consisting entirely of D-alpha-amino acid residues (the enantiomers of the 'natural' building blocks), and suggestions are made regarding a possible focus of future biomedical investigations with beta-peptides.     

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.