Surface phenotypic characteristics and virulence of Spanish isolates of Aeromonas salmonicida after passage through fish.

Eleven strains of Aeromonas salmonicida were passaged twice by intraperitoneal injection through rainbow trout and reisolated from the kidney of moribund fish. The surface characteristics and virulence of the strains changed following passage through fish. None of the in vitro tests used could effectively predict the in vivo virulence.     

Lethality and mutagenicity inHalobacterium mediterranei caused by N-methyl-N′-nitro-N-nitrosoguanidine

Compared withEscherichia coli, Halobacterium mediterranei was highly resistant to the lethal effect of N-methyl-N-nitro-N-nitrosoguanidine (nitrosoguanidine), but it was sensitive to the mutagenic action of this chemical agent. Nitrosoguanidine at 500 g ml^–1 gave a cell survival level between 1% and 10%, and this allowed us to obtain more Josamycin-resistant mutants compared with lower concentrations, which gave higher survival rates but fewer mutants. The efficiency of the mutagenicity obtained with the nitrosoguanidine treatment was examined under a variety of conditions. The optimal conditions for obtaining Josamycinresistant mutants were achieved by exposing, in darkness and without shaking, a suspension of about 10⁸ log-phase cells to 500 g nitrosoguanidine in 1 ml of 50 mM modified saline Tris-maleate buffer at pH 7.5, or in 1 ml of 5 mM modified saline Tris-citrate-maleate for 30 min at 37°C.     

Dihydroisocoumarins and phthalide from wood samples infested by fungi

Wood samples, infested by fungi during storage, were shown to contain, besides the known 5-methyl-mellein, additional (3R)-8-hydroxy-3-methyl-3,4-dihydroisocoumarins substituted by 7-methyl, 5-formyl, 5-carboxy, 5-hydroxy, 5-methoxy, 6-methoxy-5-methyl and 6,7-dimethoxy-5-methyl groups, as well as 6-formyl-7-hydroxy-5-methoxy-4-methylphthalide. Several 2-methylchromanones were synthesized in order to show that this class of compounds can be distinguished from 3-methyl-3,4-dihydroisocoumarins by MS.     

Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine. Involvement of a pertussis toxin-sensitive G protein and protein kinase C.

The phospholipase C-mediated hydrolysis of phosphatidylcholine has been shown recently to be activated by a number of agonists. Muscarinic receptors, which trigger various signal transduction mechanisms including inhibition of adenylate cyclase through Gi, have been shown to be potent stimulants of this novel phospholipid degradative pathway. We demonstrate here, by exogenous addition of Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C, that phosphatidylcholine breakdown mimics the ability of carbachol to inhibit adenylate cyclase. This effect is sensitive to pertussis toxin and is entirely dependent on the presence of protein kinase C. This kinase is also required for the inhibition by carbachol of adenylate cyclase. These results suggest that the activation of phosphatidylcholine breakdown by phospholipase C may play an important role linking or favoring the coupling muscarinic receptors to Gi. Results presented here also show that phospholipase C-mediated hydrolysis of phosphoinositides by exogenous addition of Bacillus thuringiensis phosphoinositide-hydrolyzing phospholipase C does not affect adenylate cyclase, despite the fact that protein kinase C is translocated to an extent similar to that produced by the hydrolysis of phosphatidylcholine. According to the results shown here, both phospholipases also differ in their ability to down-regulate protein kinase C as well as to phosphorylate p80 and to transmodulate the binding of epidermal growth factor, two well established effects of protein kinase C in Swiss 3T3 fibroblasts. This emphasizes the complexity, from a functional point of view, of protein kinase C activation "in vivo."     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.     

Tau-related protein present in paired helical filaments has a decreased tubulin binding capacity as compared with microtubule-associated protein tau

We have isolated, after exhaustive detergent treatments, a 33 kDa tau-related protein isolated from paired helical filaments from Alzheimer's disease patient brains. The N-terminal sequence of the 33 kDa protein begins at residue 71 of the sequence described for human fetal tau protein. This truncated form of tau is not the consequence of the translation of a tau RNA lacking a region at its 5' end, as measured by primer extension analyses, suggesting that the 33 kDa protein must be generated by proteolysis of previously synthesized tau. This tau-related protein has only one blocked cysteine residue and also has a decreased tubulin binding capacity as compared with that of tau protein.     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.