Active site of the enzyme which demethylates receptors during bacterial chemotaxis

The CheB methylesterase catalyzes the hydrolysis of glutamyl methyl esters in bacterial chemoreceptor proteins. Studies with residue-specific inhibitors suggest that a cysteine residue is required. The nucleotide sequence of the cheB gene predicts a 349-amino acid protein with cysteine residues at positions 207 and 309. Oligonucleotide-directed mutagenesis was used to change each cysteine to an alanine. Whereas the Cys207-Ala mutation had essentially no effect on esterase activity, the Cys309-Ala mutation caused a complete inactivation of the enzyme. Cys309 is located adjacent to a sequence of amino acids which is characteristic of the beta-alpha-beta motif found in a number of nucleotide binding proteins associated with receptor function in vertebrate tissues. A central feature of this structure is Gly-X-Gly-X-X-Gly. Mutation of the second glycine in this region (Gly284) to a valine also caused a complete loss of esterase activity.     

Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins

The CheA protein of the Salmonella typhimurium chemotaxis system is phosphorylated by ATP. Phospho-CheA transfers its phosphoryl group to a second chemotaxis protein, CheY. Unlike phospho-CheA, phospho-CheY is relatively unstable, rapidly decaying to phosphate and CheY. We propose that phosphorylation of CheY may play a role in its function as a tumble regulator to control motor behavior in response to attractant and repellent stimuli.     

Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.     

Short-term effects of a catastrophic beaver dam collapse on a stream fish community

Synopsis We examined the short-term effects of the natural catastrophic collapse of a beaver dam on downstream benthic insect density and fish community structure in a headwater tributary of the Mississippi River. The catastrophic collapse of the dam and ensuing flash flood resulted in a dramatic (< 90%) decrease in benthic insect density in riffle and pool habitats. Sixty days after collapse of the dam, insect densities in riffles were 62% of pre-collapse densities. Insect recolonization of pools was slower than for riffles; 60 days after collapse of the dam insect densities in pools were 8% of pre-collapse levels. Collapse of the beaver dam altered the structure of the downstream fish community by causing a short-term (2–4 days) influx of pond species, resulting in a brief increase in species richness and abundance. Fish species richness and abundance then decreased for 4–60 days to levels below those prior to the collapse.     

Impacts of invading N2-fixing Acacia species on patterns of nutrient cycling in two Cape ecosystems: evidence from soil incubation studies and 15N natural abundance values

This study examines the impacts of woody, N₂-fixing invasive Acacia spp. on the patterns of nutrient cycling in two invaded ecosystems of differing nutrient status in the Cape floristic region. Patterns of soil nutrient mineralization were measured by a field incubation method while the significance of the fixation process in altering nutrient cycling was assessed by the ¹⁵N natural abundance technique. The results confirm earlier reports that invasion by woody shrubs results in organic matter and nutrient enrichment of surface soils of both ecosystems. However, patterns of nutrient availability (phosphorus and nitrogen) were not necessarily enhanced. In the more fertile strandveld both phosphorus and nitrogen (significant at P<0.10) showed trends towards enhanced annual mineralization rates upon invasion, while in the low nutrient fynbos system only phosphorus followed this trend. It is unclear whether this differential response is a consequence of plant- or soil-derived feedbacks on the decomposition processes in each system. The ¹⁵N values of the soils from the invaded sites of both ecosystems indicated a strong influence of the alien species on the soil nitrogen component. However, as with other studies of natural ecosystems, the contribution of nitrogen from fixation could not be readily quantified with the ¹⁵N natural abundance method because of problems in selecting suitable non-N₂-fixing reference plants. A technique of disrupting nodule structure and function, by fumigation with O₂, to obtain the ¹⁵N value of a non-N₂-fixing speciment of the study species was tried and found to overcome some of the problems associated with the lack of suitable reference plants. With this technique it was possible to detect the almost total dependence of A. saligna on N₂-fixation in the fynbos soils with their low nitrogen mineralization rates. In the strandveld ecosystem with much higher soil nitrogen release rates A. cyclops was only partly dependent on fixation (about half) for its nitrogen. The nutrient enrichment of both ecosystems and trends towards enhanced rates of nutrient mineralization could have profound implications on the long-term success of alien invader clearing operations and the restoration of the indigenous flora at these sites.     

Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection.

Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion.     

Evolutionary stability of the R1 retrotransposable element in the genus Drosophila

R1 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts into a specific sequence of insect 28S ribosomal RNA genes. We have previously shown that this element has been maintained through vertical transmission in the melanogaster species subgroup of Drosophila. To address whether R1 elements have been vertically transmitted for longer periods of evolutionary time, the analysis has been extended to 11 other species from four species groups of the genus Drosophila (melanogaster, obscura, testecea, and repleta). All sequenced elements appeared functional on the basis of the preservation of their open-reading frames and consistently higher rate of substitution at synonymous sites relative to replacement sites. The phylogenetic relationships of the R1 elements from all species analyzed were congruent with the species phylogenies, suggesting that the R1 elements have been vertically transmitted since the inception of the Drosophila genus, an estimated 50-70 Mya. The stable maintenance of R1 through the germ line appears to be the major mechanism for the widespread distribution of these elements in Drosophila. In two species, D. neotestecea of the testecea group and D. takahashii of the melanogaster group, a second family of R1 elements was also present that differed in sequence by 46% and 31%, respectively, from the family that was congruent with the species phylogeny. These second families may represent occasional horizontal transfers or, alternatively, they could reflect the ability of R1 elements to diverge into new families within a species and evolve independently.      

Results of Early Mobilization and Discharge after Myocardial Infarction

A total of 342 patients with acute myocardial infarction who were admitted to a coronary care unit are reviewed to assess the results of early mobilization and discharge. The mean duration of admission was 8·4 days and 89% of the survivors were discharged from hospital by the tenth day. The inpatient mortality was 15·5%. An additional 6·7% died during the six weeks'' follow-up period, giving a total mortality of 22·2%. Altogether, 7·6% of patients were readmitted. Venous thromboembolic phenomena occurred in 3·5% during the inpatient period. Of patients who were eligible 62% were back at work five months after their myocardial infarction. We think the results justify a short hospital admission period for acute myocardial infarction.