Salinity tolerance and morphology of the osmoregulation organs in Cladocera with special reference to Cladocera from the Aral sea

The hyperosmotic regulation of adult Cladocera is determined mainly by the amount of salts consumed with the food and by reabsorption of salts in cells of the nuchal (neck) organ. The hypoosmotic regulation both in adults and embryos is determined mainly by excretion of salts in special epipodite cells or in cells of the nuchal (neck) organ. The salinity of the Aral sea for the last 30 years increased from 8–10 to 26–28, which led to changes in the Cladocera fauna. At present only 4 species of Cladocera inhabit the Aral sea instead of 14 species that were previously found. These changes are in agreement with osmoregulation capacities of Cladocera. Note added in proof. Since this paper was accepted for publication, all Cladocera have disappeared from the Aral Sea. This happened when salinity reached 30–32. This disappearance was predicted by and agrees with earlier laboratory experiments with Aral Sea Cladocera (Aladin, 1982b).     

Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus.

The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.     

Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.     

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.     

Physical mapping of the human carbonic anhydrase gene cluster on chromosome 8

A cluster of genes encoding the three cytoplasmic carbonic anhydrase isozymes CAI, CAII, and CAIII lie on the long arm of chromosome 8 (8q22) in humans. These genes have been mapped using pulsed-field gel electrophoresis. The genes lie in the order CA2, CA3, CA1. CA2 and CA3 are separated by 20 kb and are transcribed in the same direction, away from CA1. CA1 is separated from CA3 by over 80 kb and is transcribed in the direction opposite to CA2 and CA3. The arrangement of the genes is consistent with proposals that the duplication event which gave rise to CA1 predated the duplication which gave rise to CA2 and CA3. The order of these three genes differs from that suggested for the mouse based on recombination frequency.     

How can a catalytic lesion be offset? The energetics of two pseudorevertant triosephosphate isomerases

The reaction energetics of four triosephosphate isomerase mutants are compared with those of the wild-type enzyme. The two primary mutants, E165D and H95N, contain site-specific alterations of active site residues. In one case the active site base has been altered (E165D), and in the other, an active site electrophile has been removed (H95N), yet the major effect in each case is the relative destabilization of the transition states for the two chemical (enolization) steps that constitute the catalytic reaction. When the genes encoding each of these sluggish mutant isomerases were subjected to random mutagenesis using chemical reagents and a selection for isomerases of increased catalytic potency was performed, pseudorevertant enzymes with dramatic increases in activity were found. Remarkably, the same second-site suppressor locus partially corrects each lesion. The E165D,S96P pseudorevertant is a 20-fold better catalyst than the E165D mutant from which it is derived, and the H95N,S96P pseudorevertant is about 60 times more active than its H95N parent. The S96P substitution thus increases the catalytic activity in each of two different contexts, H95N and E165D. The energetic consequences of the S96P change are suprisingly similar in each pseudorevertant. The H95N,S96P enzyme is more effective than H95N at stabilizing the intermediate enediol(ate) phosphate and its flanking transition states. The E165D,S96P enzyme likewise stabilizes the transition states for enolization better than E165D, and this pseudorevertant also forms a tighter enzyme-dihydroxyacetone phosphate complex than its parent. These data show how, in these two cases, the catalytic potency of sluggish mutant enzymes can be improved by second-site changes. The results thus provide the beginnings of a detailed understanding of the kinetic refinement of enzyme catalysts.     

Lipid-protein interactions in stacked and destacked thylakoid membranes and the influence of phosphorylation and illumination. Spin label ESR studies

The effects of membrane destacking, protein phosphorylation, and continuous illumination have been studied in pea thylakoid membranes using ESR spectroscopy of an incorporated spin-labelled phosphatidylglycerol. This spin-labelled analogue of an endogenous thylakoid lipid has previously been shown to exhibit a selectivity of interaction with thylakoid proteins. Neither destacking, phosphorylation nor illumination was found to change the ESR spectra appreciably, suggesting that for phosphatidylglycerol at least, neither the number of protein-associated membrane lipids nor their pattern of selectivity was altered. The redistribution of the thylakoid protein complexes in the membrane, under these various conditions, therefore takes place with conservation of the properties of the lipid/protein interface.     

Targeted point mutation that creates a unique Eco RI site within the signal codons of the beta-lactamase gene without altering enzyme secretion or processing

A method has been developed for constructing site-specific mutations by using a strongly selectable marker on which to "piggy-back" a desired mutation that may be phenotypically silent. Using this approach, a new unique Eco RI restriction site has been generated at the beginning of the signal codons of the beta-lactamase gene of the plasmid pBR322. The consequential alteration of the second amino acid of the signal from Ser to Arg has no effect on either the transport or the processing of the beta-lactamase.     

Contribution of bacterial cell nitrogen to soil humic fractions

Summary Living cells ofSerratia marcescens, uniformly labelled with¹⁵N, were added to samples of maple (Acer saccharum) and black spruce (Picea mariana) forest soils. After different periods of incubation from zero time to 100 days, the soils were subjected to alkali-acid and phenol extraction to provide humic acid, fulvic acid, humin and humoprotein fractions. Significant amounts of the cell nitrogen were recovered in the humic and fulvic acids immediately after addition. After incubation, less cell, nitrogen appeared in the humic acid and more in the fulvic acid. The amount of cell nitrogen recovered in the humin fraction increased with incubation. Roughly 5 to 10 per cent of the added cell nitrogen was found as amino acid nitrogen from humoprotein in a phenol extract of the humic acid. The data are consistent with the occurrence of co-precipitation of biologically labile biomass nitrogen compounds with humic polymers during the alkaline extraction procedure involved in the humic-fulvic fractionation.