Asp-863 is a key residue for calcium-dependent activity of Escherichia coli RTX toxin alpha-haemolysin

alpha-Haemolysin is a protein toxin secreted by pathogenic strains of Escherichia coli and requires sub-millimolar Ca(2+) for optimum lytic activity. As a member of the so-called RTX toxin family it contains a Gly-rich, Asp-rich Ca(2+)-binding domain, consisting of a series of nonapeptides repeated in tandem. Asp-863 is located immediately after the last-but-one nonapeptide. A mutant in which Asp-863 has been substituted by Gly displays a requirement for Ca(2+) that is 100-fold higher than the wild-type. Membrane lytic activity, as well as a conformational change revealed through an increase in intrinsic fluorescence, and the appearance of Ca(2+)-bound protein monomers resolvable by fast protein liquid chromatography, are all three dependent on Ca(2+) concentrations in the 2-20 mM range. Most RTX toxins have an Asp or Glu residue located at a position homologous to Asp-863, thus the key role of this residue for Ca(2+) requirements of alpha-haemolysin may be a general feature of this family of toxins.     

Polar lipids and fatty acids of three wild cyanobacterial strains of the genus<Emphasis Type="Italic">Chroococcidiopsis</Emphasis>

The occurrence of n-saturated, branched, and unsaturated fatty acids of 3 wild terrestrial strains of the genus Chroococcidiopsis (Order Chroococcales): C. supralittoralis, C. umbratilis, and C. versatilis collected from Lake Kinneret, Dead Sea, and Ein Kerem (Jerusalem) was investigated and individual compounds identified by gas chromatography-mass spectrometry. Polar lipids also were examined. Among polar lipids (studied using two-dimensional thin-layer chromatography) were as major glycolipids isolated: monogalactosyl-diacylglycerols, digalactosyl-diacylglycerols, 6-sulfoquinovosyl-diacylglycerols and phosphatidylglycerol. Nonphosphorus betaine-containing lipid, viz. N,N,N-trimethylhomoserin-4-O-yl-diacylglycerol, was found for the first time in cyanobacterial species.     

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.     

Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).     

AmfS, an extracellular peptidic morphogen in Streptomyces griseus

The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus. The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.     

Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.     

Influence of the red fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>, Linnaeus 1758) on the distribution and number of breeding birds in an intensively used farmland

This study tested the hypothesis that small birds at their nest sites avoid areas around dens of the red fox (Vulpes vulpes, Linneaus 1758) in an intensively used farmland. Birds were counted at 18 points (radius 100m) located near dens, as well as at 18 control points that were located at least 600m away from the nearest den. These two types of points did not differ with respect to the number of recorded bird species. However, a negative effect of the proximity of fox dens on the total density of the bird community was observed. This effect was also recorded for the most abundant bird species, the skylark (Alauda arvensis, Linneaus 1758). In agreement with our expectations, these results indicate a negative impact of fox presence on a breeding bird community in an open farmland.     

His-859 is an essential residue for the activity and pH dependence of Escherichia coli RTX toxin alpha-hemolysin

Escherichia coli alpha-hemolysin (HlyA) is a toxin protein that, in common with other members of the RTX family, contains a calcium-binding domain consisting of a number of Gly- and Asp-rich nonapeptides (17 in this case) repeated in tandem. Amino acid number 6 in these nonapeptides is almost invariably Asp, and occasionally Asn, but HlyA contains a His residue (number 859 in the chain) in position 6 of the last-but-one nonapeptide. HlyA mutants have been prepared, by site-directed mutagenesis, in which His-859 has been replaced by an Asn (H859N) or by Asp (H859D). HlyA exists in aqueous media in an aggregate-monomer equilibrium, but only the monomer containing bound Ca(2+) (HlyA.Ca) appears to be competent to achieve target membrane insertion and subsequent lysis. In mutant H859N, equilibrium appears to be shifted toward the aggregate, therefore the protein does not exchange Ca(2+) with the aqueous environment, no HlyA.Ca monomers are detected, and the protein lacks any membrane lytic activity. Mutant H859D in turn is almost indistinguishable from the wild-type regarding its calcium binding and membrane lytic activity, however, it differs significantly in its pH dependence. Wild-type HlyA activity decreases sigmoidally with pH, following rather closely the protonation curve of a His residue (apparent pK(a) approximately 6.5). With mutant H859D activity decreases almost linearly with pH and to a smaller extent. It can be concluded that His-859 plays a critical role in several aspects of HlyA activity, namely self-aggregation properties, calcium binding, hemolysis, and pH dependence.