Substrate specificity of alpha-chymotrypsin-catalyzed esterification in organic media.

11 amino acid derivatives were tested as alpha-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. alpha-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determined. All the amino acid derivatives tested were esterified, and the highest values of kcat/KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the alpha-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.     

Shape transitions and shape stability of giant phospholipid vesicles in pure water induced by area-to-volume changes.

Shape transformations of vesicles of dimyristoylphosphatidylcholine (= DMPC) and palmitoyloleylphosphatidylcholine (= POPC) in ion-free water were induced by changing the area-to-volume ratio via temperature variations. Depending on the pretreatment we find several types of shape changes for DMPC (in pure water) at increasing area-to-volume ratio: (a) budding transitions leading to the formation of a chain of vesicles at further increase of the area-to-volume ratio, (b) discocyte-stomatocyte transitions, (c) reentrant dumbbell-pear-dumbbell transitions, and (d) spontaneous blebbing and/or tether formation of spherical vesicles. Beside these transitions a more exotic dumbbell-discocyte transition (e) was found which proceeded via local instabilities. Pears, discocytes, and stomatocytes are stable with respect to small temperature variations unless the excess area is close to values corresponding to limiting shapes of budded vesicles where temperature variations of less than or equal to 0.1 degree C lead to spontaneous budding to the inside or the outside. For POPC we observed only budding transitions to the inside leading either to chains of vesicles or to distributions of equally sized daughter vesicles protruding to the inside of the vesicle. Preliminary experiments concerning the effect of solutes are also reported. The first three types of shape transitions can be explained in terms of the bilayer coupling model assuming small differences in thermal expansivities of the two monolayers. This does not hold for the observed instabilities close to the limiting shapes.     

Taurine release associated to volume regulation in rabbit lymphocytes.

Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4 degrees C, but was unchanged at 15 degrees C or 25 degrees C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested.     

Effect of stobadine on indomethacin- and ethanol-induced stomach lesions and gastric secretion.

Stobadine was found to inhibit the ulcerogenic activity of indomethacin in relation to the dose but was ineffective against the direct necrotizing action of ethanol. It also inhibited gastric acid secretion when administered intraduodenally. Although stobadine is considered to be a scavenger of free radicals, our results indicate that, under the given experimental conditions, it is rather the inhibition of gastric acid secretion that is responsible for its antiulcerogenic effect. The preliminary results do not allow the exclusion of other mechanisms for explaining its antiulcerogenic effect.     

Expression of Rhizobium galegae common nod clones in various backgrounds.

The cosmid clone pRg30, carrying common nodulation genes of Rhizobium galegae HAMBI 1174, and pRg33, a subclone of pRg30 that contains a 5.7-kb ClaI insert carrying nodDABC were conjugated into various Rhizobium nod- mutant strains and into a Ti plasmid-cured Agrobacterium tumefaciens. Complementation and expression of the nodABC genes of R. galegae were studied by following microscopically the infection process and the nodulation on different test plants. The nodABC genes of R. galegae complemented the nod- strains of other Rhizobium species. The presence of extra copies of common nod genes in the homologous R. galegae nodABC- strain induced an increased nodulation on Galega orientalis. However, the inserts of R. galegae in pRg30 and pRg33 do not carry sufficient genetic information for normal nodulation of test plants in an Agrobacterium background, because the Agrobacterium transconjugants induced root hair deformation on Galega plants, but no infection threads were detected and nodulelike structures developed only at low frequency. The Agrobacterium carrying the nodDABC of R. galegae did not cause the root hairs of Medigo sativa to deform.     

Benthic-pelagic flux rates on mussel beds: tunnel and tidal flume methodology compared

Material flux rates in an intertidal mussel bed were measured synchronously over two tidal cycles in June 1989 with Benthic Ecosystem Tunnels and a double lane flume. The tunnels enclosed the near bottom water, whereas the flume canalized the total water column. One tunnel was set up in a mussel bed and another one in an adjacent sand bottom as a control. The flume enclosed a mussel lane and a sand lane. In the tunnel and in the flume the mussel bed revealed ammonium and phosphate discharge. At the same time, phytoplankton, dominated byPhyaeocystis globosa, was taken up intensively. These flux rates showed the same tendency but they were higher in the flume than in the tunnel. Different tendencies and flux rates for oxygen and particulate organic matter (POC, PN) were found in flume and tunnel. These differences demonstrate the importance of water column processes regarding the material exchange of a mussel bed. Tunnels enclose smaller bodies of water and are therefore expected to detect even small effects of the benthos on the passing water. In flumes, benthic influence may be diluted over the entire water column but conditions are more natural. The use of flumes is restricted to shallow waters while tunnels have the potential to be used at any depth.     

Specific and sensitive quantitation of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication

A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid—liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.     

Members of the alpha-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma

We have identified the major antigens or IgE binding components from wheat flour. Thirty-five sera from patients with baker's asthma were used to analyze the reaction with wheat salt-soluble proteins. We found a 15 kDa SDS-PAGE band which reacted with all sera tested. Purified members of the alpha-amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two-dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low-molecular-weight allergens.     

Stir-up effect of wind on a more-or-less stratified shallow lake phytoplankton community,Lake Balaton,Hungary

Microstratification of phytoplankton in the large shallow Lake Balaton (Hungary) was studied during a 24 h period. Dissolved O₂ showed biological stratification; flagellates exhibited a definite circadian rhythm. In the middle of the investigation a heavy storm broke out which was followed by the disappearance of differences between different layers of water. Storm-induced destratification is described by cluster-analysis. Abundances of dominant species changed differently in connection with the storm. Numbers of Nitzschia sp. increased due to stirring up from the sediment surface. Numbers of single-celled or colony-forming species (Cyclotella comta, Crucigenia quadrata, Coelosphaerium kuetzingianum) practically did not change. Numbers of all the three dominant filamentous species (Aphanizomenon fos-aquae f. klebahnii, Lyngbya limnetica, Planctonema lauterbornii) significantly decreased, which might be attributed to an unknown loss process and was followed by a competitive displacement by algae of small cell size.