Active-site-directed inhibition of sucrose-phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin

Sucrose-phosphate synthase (SPS, E.C. 2.4.1.14) from spinach (Spinacia oleracea L.) was partially purified and the inhibition of the enzyme reaction by 1-deoxynojirimycin and Cibacron blue F3G-A analyzed. Cibacron blue was a high-affinity competitive inhibitor with respect to the substrate UDPglucose (K_i = 80 nM) and a mixed-type inhibitor with respect to fructose-6-phosphate. 1-Deoxynojirimycin was a mixed-type inhibitor of SPS with respect to UDPglucose [K_i(EI) = 5.8 mM] and a uncompetitive inhibitor with respect to fructose 6-phosphate. These results are discussed in relation to the mechanism of the reaction catalysed by SPS and the secondary structure of the enzyme.Abbreviations DN 1-deoxynojirimycin - Glc6P glucose-6-phosphate - Fru6P fructose-6-phosphate - SPS sucrose-phosphate synthase - UDPG1c UDPglucose We are grateful to M. Stitt (University of Heidelberg, Germany) for many helpful discussions and J. Harr and P. Bocion (both SANDOZ AGRO, Switzerland) for supporting the work.     

Characterization of novel clonal murine endothelial cell lines with an extended life span

Summary A murine endothelial cell line was recently established from microvessels that had invaded a subcutaneous sponge implant (Dong, Q. G.; Bernasconi, S.; Lostaglio, S., et al. Arterioscl. Thromb. Vasc. Biol. 17:1599–1604; 1997). From these sponge-induced endothelial (SIE) cells, we have isolated two subpopulations endowed with different phenotypic properties. Clone SIE-F consists of large, highly spread cells that have a relatively slow growth rate, form contact-inhibited monolayers, do not grow under anchorage-independent conditions, express elevated levels of thrombospondin-1 (TSP-1) and are not tumorigenic in vivo. In contrast, clone SIE-S2 consists of small, spindle-shaped cells that have a high proliferation rate, do not show contact-inhibition, grow under anchorage-independent conditions, express very low levels of TSP-1 and are tumorigenic in vivo. Both clones express the endothelial markers vascular endothelial-cadherin and vascular intercellular adhesion molecule-1, but do not express CD31 and E-selectin. In addition, SIE-S2 cells, but not SIE-F cells, express the α-smooth muscle actin isoform. SIE-S2 cells, but not SIE-F cells, are able to form branching tubes in fibrin gels. The SIE-F and SIE-S2 clones, which have properties of nontransformed and transformed cells, respectively, should provide useful tools to investigate physiological and pathological processes involving vascular endothelium.     

A Kinetic Study on the Interaction between Tazobactam (A Penicillanic Acid Sulphone Derivative) and Active-Site Serine beta-Lactamases

The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.     

Incidental and intentional flavor memory in young and older subjects

Incidental and intentional learning and memory for 2 novel flavors were compared in young and elderly subjects. Incidental and intentional learning groups rated 2 new soups on acceptability for different occasions and were tested for memory the next day. On the first day, only the intentional group was asked to memorize the stimuli. With incidental learning, elderly and young were equally good, but the young performed better with intentional than with incidental learning, whereas the elderly did not. There were no age-related differences in perceptual discrimination. When comparing perceived flavor with the memory of it, the elderly tend to overrate intensities of remembered flavor attributes, whereas the young tend to underrate them. Memory was not related to flavor pleasantness or neophobia. Like memory for taste and texture, flavor memory seems to be mainly tuned at detecting changes and based on "feelings of not knowing" rather than on precise identification and recognition of previously encountered stimuli.     

A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation

Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365-374{216-225}) of the so-called activation domain and the 170-loop (residues 313-322{170A-175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S(1) and S(3) substrate pockets, the activation pocket, and N-terminal insertion.     

A phosphatase‐centric mechanism drives stress signaling response

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase‐driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho‐proteomic response is the Endosulfine‐mediated inhibition of protein phosphatase 2A‐Cdc55 (PP2A^Cdc55), while we do not observe concurrent kinase activation. In fact, many of the stress‐induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2A^Cdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well‐established kinase‐centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re‐evaluating the impact of phosphatases on shaping the phosphorylome.     

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli

Summary A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.