Antiphytovirale Aktivität von Rhamnolipid aus Pseudomonas aeruginosa

A rhamnolipid released by Pseudomonas aeruginosa 196 Aa into the culture medium reduced the number of local lesions induced by tobacco mosaic virus on leaves of the hypersensitive host Nicotiana glutinosa L. by up to 90%. The content of potato virus X in the systemically infected host Nicotiana tabacum L. ‘Samsun’ is decreased in inoculated as well as in secondarily infected leaves by up to 50%. In a smaller degree red clover mottle virus is influenced in the systemic host Pisum sativumconvar.speciosum (Dierb.) Alef ‘Nadja’.     

The interaction of nucleotides and yeast hexokinase

The kinetics of ethenoadenosine triphosphate (?ATP) as the phosphate donor in the phosphoryl transfer reaction of hexokinase were examined to obtain the K_m′s, V's, and K_α's for the nucleotide and sugar. Dissociation constants for eATP and ?ADP with hexokinase were obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates were used as phosphate donors in the hexokinase reaction and their kinetic constants were obtained. Reactions were also performed using two nucleotides simultaneously as phosphorylating substrates for the hexokinase reaction in an attempt to find the individual dissociation constants, K_m′s and K_i′s. These were compared with the K_m′s obtained from using the nucleotides separately in the hexokinase reaction. From these kinetic and fluorescence binding studies, evidence is presented supporting the postulate that the K_m′s are primarily dissociation constants in a random bi-bi mechanism. Analysis of the K_m values provides additional evidence to support the importance of the amino group in position 6 on the purine ring as a hydrogen-bond acceptor during binding. It was found that ?CTP was a much better hexokinase substrate than CTP. These observations suggest that the V for this reaction is highly dependent upon the size of the nucleotide.     

Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.     

Expression of human asparagine synthetase in Escherichia coli

Human asparagine synthetase was expressed in Escherichia coli. Synthesis of the enzyme was demonstrated by immunoblotting and by complementation of asparagine auxotrophy in E. coli. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. Compared to asparagine synthetase isolated from beef pancreas, the one expressed in E. coli migrated at a slightly slower rate on a denaturing protein gel. In contrast with previous reports, the data obtained here strongly suggest that the active enzyme is a homodimer. The production of soluble and active enzyme was shown to be highly temperature-dependent. Expression at 37 degrees C yielded no soluble enzyme, whereas growth at 30 and 21 degrees C favored the production of soluble asparagine synthetase. The incubation temperature was also important for complementation of asparagine auxotrophy in E. coli, as growth in the absence of asparagine occurred at 30 degrees C and not at 37 degrees C.     

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.     

Obtaining a High Percentage of Explants with Negative Serological Reactions against viruses by Combining Potato Meristem Culture with Antiphytoviral Chemotherapy

A high percentage of plantlets, in which PVX, PVM, and PVS were no longer detectable by precipitin tests was obtained from 100% virus-infected stocks of the potato varieties Rosa and Priobskij rannija by combining meristem (tip) culture with antiphytoviral chemotherapy. When 2,4 - dioxohexahydro 1,3,5-triazinc (DHT) was added to the nutrient media, no virus was detectable serologically in 72% of the corresponding explants of the variety Rosa and in 66% of that of the variety Priobskij rannija, respectively. Contrary to this, all untreated control explants proved virusinfected. Treatment with 2-thiouracil resulted in 46.6%, with cyanoguanidine in 28.6% of plantlets, in which no virus was detectable serologically.     

Neurospora crassa mutants deficient in asparagine synthetase

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.