Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.

Imaging of Neuromodulation and Surgical Interventions for Epilepsy.

About one-third of epilepsy cases are refractory to medical therapy. During the past decades, the availability of surgical epilepsy interventions has substantially increased as therapeutic options for this group of patients. A wide range of surgical interventions and electrophysiologic neuromodulation techniques are available, including lesional resection, lobar resection, thermoablation, disconnection, multiple subpial transections, vagus nerve stimulation, responsive neurostimulation, and deep brain stimulation. The indications and imaging features of potential complications of the newer surgical interventions may not be widely appreciated, particularly if practitioners are not associated with comprehensive epilepsy centers. In this article, we review a wide range of invasive epilepsy treatment modalities with a particular focus on their postoperative imaging findings and complications. A state-of-the-art treatment algorithm provides context for imaging findings by helping the reader understand how a particular invasive treatment decision is made.

Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis.

Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and (S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by (S)-aspartate semi-aldehyde was observed for the B. anthracis enzyme (K(si(ASA))=5.4+/-0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis.

Combination peroxisome proliferator-activated receptor gamma and alpha agonist treatment in Type 2 diabetes prevents the beneficial pioglitazone effect on liver fat content.

AIMS: Peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-alpha agonists individually reduce intra-organ triglyceride content and improve insulin sensitivity. However, the precise effects of combined PPAR-gamma and PPAR-alpha therapy on intra-organ triglyceride content and insulin sensitivity in subjects with Type 2 diabetes have not yet been determined. METHODS: Diet-controlled Type 2 subjects (n = 9) were studied before and after 16 weeks of combined PPAR-gamma [pioglitazone (PIO), 45 mg daily] and PPAR-alpha [bezafibrate (BEZA), modified release 400 mg daily] agonist therapy. Glucose metabolism and endogenous glucose production were measured following a standard liquid test meal. Liver and muscle triglyceride levels were measured by (1)H magnetic resonance spectroscopy. RESULTS: Combined PIO and BEZA therapy reduced mean fasting (7.5 +/- 0.5 vs. 6.5 +/- 0.2 mmol/l, P = 0.04) and peak postprandial plasma glucose (15.3 +/- 1.1 vs. 11.7 +/- 0.6 mmol/l, P = 0.007). No significant change in hepatic or muscle triglyceride content was observed. Postprandial suppression of endogenous glucose production remained similar on both study days. Both subcutaneous and visceral fat content increased following therapy. CONCLUSIONS: Combined PIO and BEZA therapy in Type 2 diabetes does not decrease intrahepatic triglyceride content or postprandial endogenous glucose production. This study demonstrates an unexpected adverse interaction of PPAR-alpha with PPAR-gamma agonist therapy.

Ultrastructure of clots during isometric contraction.

We explored the retraction or contraction of platelet-fibrin clots under isometric conditions. In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0.1 to 0.2 g/min per cm2 (initial cross-sectional area). Electron microscopy of clots fixed after attaining a force of 1.6 g/cm2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of tension. The development of tension could not be explained simply on the basis of platelet-platelet association and interaction alone. First, factor XIII-dependent cross-linking of fibrin fibers was critical to normal isometric contraction. Second, tension decreased linearly, rather than exponentially, when the platelet count in the platelet-fibrin clot was decreased, suggesting that platelets must be interacting with another component (i.e. fibrin). Thrombasthenic platelets, deficient in fibrinogen receptors, failed to develop tension or to align fibrin strands or pseudopods in the clot. Platelet-fibrin clots treated with vincristine to disassemble microtubules or cytochalasin B to disrupt microfilaments failed to develop tension and relaxed if these agents were added after tension had developed. Relaxation under these conditions, however, was not associated with loss of orientation of fibrin strands. Our findings suggest that platelet-fibrin interaction in clots under isometric conditions leads to orientation of fibrin strands and platelets in the direction of force generation. Tension develops as platelets simultaneously attach to and spread along fibrin strands, and contract. The contraction draws some fibrin into platelet-fibrin clumps and aligns other strands in the long axis of tension. The achievement and maintenance of maximum tension appears to depend on the development of platelet-fibrin attachments and extension of platelet bodies and long pseudopods containing bundles of microfilaments and microtubules along the oriented fibrin fibers.

Reciprocal transmembranous receptor-cytoskeleton interactions in concanavalin A-activated platelets.

Concanavalin A (Con A) has been used to activate platelets, inducing a specific interaction between the glycoprotein IIb-IIIa complex and the cytoskeleton of the activated platelet. In agreement with this, we have shown that Con A activates human platelets, initiating phosphorylation, secretion, and cytoskeletal formation. Con A and cytochalasin B were used to demonstrate a reciprocal interaction of the glycoprotein complex with the platelet cytoskeleton. Additionally, we have shown that a similar reciprocity is provided by the multivalent fibrin-fibrinogen platelet interaction found in the thrombin-induced clot. Con A differs from other activators in precipitating an apparent cytoskeletal core despite a complete inhibition of platelet activation by prostaglandin E1. We suggest, from this result, that Con A may be cross-linking a membrane-associated cytoskeletal complex present in the unactivated platelet.

Epinephrine reduction of heme: implication for understanding the transmission of an agonist stimulus.

Alpha-adrenergic agonists that promote platelet aggregation were found to reduce ferric heme to ferrous heme. Agents that bind iron in heme inhibited epinephrine-induced platelet aggregation. It is proposed that epinephrine first binds to its receptor and then reduces an adjacent heme group to transmit its agonist stimulus.

Histamine is an intracellular messenger mediating platelet aggregation.

Inhibition of human platelet aggregation by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl (DPPE), a novel antagonist of histamine binding, suggested that histamine might serve a critical role in cell function. Phorbol-12-myristate-13-acetate (PMA) or collagen was found to increase platelet histamine content in parallel with promotion of aggregation. Inhibitors of histidine decarboxylase (HDC) suppressed both aggregation and the elevation of histamine content, whereas DPPE inhibited aggregation only. In saponin-permeabilized platelets, added histamine reversed the inhibition by DPPE or HDC inhibitors on aggregation induced by PMA or collagen. The results indicate a role for histamine as an intracellular messenger, which in platelets promotes aggregation.

Is it ethical for a general practitioner to claim a conscientious objection when asked to refer for abortion?

Abortion is one of the most divisive topics in healthcare. Proponents and opponents hold strong views. Some health workers who oppose abortion assert a right of conscientious objection to it, a position itself that others find unethical. Even if allowance for objection should be made, it is not clear how far it should extend. Can conscientious objection be given as a reason not to refer when a woman requests her doctor to do so? This paper explores the idea of the general practitioner (GP) who declines to make a direct referral for abortion, asking the woman to see another GP instead. The purpose is to defend the claim that an appeal to conscientious objection in this way can be reasonable and ethical.

Hyperactivity of neutrophil leukotactic responses during active bacterial infection.

To determine if changes in neutrophil leukocyte function occur during active bacterial infection, the neutrophils of 25 patients with active bacterial infection and 25 age-matched controls were compared for leukotactic activity, random mobility, and nitroblue tetrazolium reduction. The neutrophil leukocytes of patients with bacterial infection were hyperactive in unidirectional movement toward a chemotactic stimulus as measured in the leukotactic assay and usually had increased nitroblue tetrazolium reduction. The mean leukotactic index was 165+/-56 in patients with bacterial infection and 70+/-11 in controls (P < 0.001). After 7-10 days of appropriate therapy with clinical and bacteriological response, leukotactic activity returned to normal values. A hyperactive leukotactic response continued, however, in patients with persisting bacterial infection. The hyperactive leukotactic response of circulating neutrophils appears to be an early and sensitive event in the inflammatory cycle stimulated by bacterial infection and may aid in the localization of invading bacteria.

The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.

The levels and expression of the proteins CD63 and granulophysin in platelets from control and from a Hermansky-Pudlak syndrome subject (a condition characterized by dense granule and lysosomal deficiencies and the accumulation of ceroid-like material in reticuloendothelial cells) were examined. Immunofluorescence studies indicated that anti-CD63 and anti-granulophysin antibodies recognized similar numbers of granules; coapplication of antibodies did not identify more granules than the individual antibodies. Significantly fewer granules were recognized in Hermansky-Pudlak syndrome platelets than in control using either antibody. Immunoblotting studies demonstrated that anti-CD63 and anti-granulophysin antibodies apparently recognize the same protein, which was deficient in Hermansky-Pudlak platelets. Analysis by fluorescence-activated cell sorter (FACS) showed biphasic expression of CD63 and granulophysin after thrombin stimulation of control but not Hermansky-Pudlak platelets. Anti-CD63 effectively blocked detection of the protein by anti-granulophysin using immunofluorescence, ELISA, immunoblotting, and FACS analysis. Amino-terminal sequencing over the first 37 amino acids revealed that granulophysin was homologous to CD63, melanoma antigen ME491, and pltgp40. These results suggest that granulophysin and CD63 are possibly identical proteins. This is the first report of a protein present in platelet dense granules, lysosomes, and melanocytes, but deficient in a patient with Hermansky-Pudlak syndrome.

The chemiluminescence response of human platelets.

Human platelets and platelet particulate fractions were found to emit a burst of chemiluminescence during incubation with arachidonic acid. The magnitude of light emission was directly related to the number of platelets in the reaction mixture and varied little for the same individual from day to day. The chemiluminescence response of platelets was localized to the particulate fraction and was almost totally oxygen dependent. In addition to arachidonate, seven other polyunsaturated fatty acids, including several that are not prostaglandin precursors, also induced platelet chemiluminescence.A correlation was sought between chemiluminescence and platelet prostaglandin synthesis. Platelets incubated in low concentrations of aspirin, or platelets from subjects who had ingested aspirin, had markedly decreased arachidonic acid-induced chemiluminescence. Salicylic and sulfosalicylic acid had no inhibitory effect. A time-response curve of aspirin inhibition of arachidonate-induced chemiluminescence closely paralleled a time-response curve of aspirin inhibition of malondialdehyde production. Linoleic acid-induced platelet chemiluminescence was also markedly inhibited using aspirin-incubated platelets or platelets from subjects who had ingested aspirin. These studies implicate activation of the enzyme prostaglandin synthetase in the arachidonate-induced platelet chemiluminescence. They provide evidence that linoleic acid may also specifically activate platelet cyclooxygenase to produce electronically excited species capable of light emission.

Biochemical studies of two patients with the gray platelet syndrome. Selective deficiency of platelet alpha granules.

The biochemistry of platelets from two unrelated patients with the gray platelet syndrome, a deficiency of platelet alpha-granules, has been evaluated. Ultrastructural studies of their platelets revealed the number of alpha-granules to be less than 15% of normal, whereas the number of dense bodies was within normal limits. Platelets from both patients had severe deficiencies of platelet factor 4 and beta-thromboglobulin (less than 10% of normal). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a marked deficiency of thrombin-sensitive protein in both patients. Analysis of the platelet-derived growth factor in one patient showed it was also markedly reduced. Levels of lysosomal enzymes, adenine nucleotides, serotonin, and catalase, and conversion of arachidonic acid by the lipoxygenase and cyclo-oxygenase enzymes, were within normal limits. The results provide important evidence to define the contents of alpha-granules and to differentiate these contents from the contents of lysosomal granules, dense bodies, and peroxisomes. Functional studies of these platelets showed deficiencies in ADP, thrombin, and collagen aggregation. The results suggest that alpha-granules or their contents make a contribution to normal platelet aggregation.

Inhibiting protein-protein interactions as an emerging paradigm for drug discovery.

Association of proteins into homo- and hetero-oligomers plays an important role in a plethora of biological phenomena. Inhibition of these interactions is increasingly recognized as a valuable new direction in drug design. In this mini-review we consider inhibition of protein misfolding and aggregation, molecules that disrupt enzyme quaternary structure, and signaling inhibitors, as emerging drugs.

Growth-promoting activity of desmopressin in murine leukemia cells treated in vitro.

The synthetic vasopressin analogue, desmopressin (dDAVP), has been shown to influence membrane transport of melphalan in murine L5178Y lymphoblasts. Accordingly, the effect of dDAVP on the cytocidal activity of melphalan in L5178Y cells was evaluated. dDAVP did not affect the cytocidal activity of melphalan in these cells, but significantly affected the cloning efficiency of stationary phase or slowly dividing L5178Y cells over a range of concentrations. In particular, stationary phase cells showed an increase in cloning efficiency from 4.3 +/- 0.5% in control cells to 7.0 +/- 0.3% in cells treated with 25 nM dDAVP (P less than 0.001), whereas cells doubling every 26 h showed an increase from 10.8 +/- 1.2% in control cells to 21.0 +/- 2.0% in cells treated with 150 nM dDAVP (P less than 0.001). This phenomenon was associated with significant elevations of 1,2[3H] diacylglycerol after incubation with dDAVP for 9 min (P less than 0.01) and of total [3H]diacylglycerols after incubation for both 3 min (P less than 0.05) and 9 min (P less than 0.02). Within 10 s of treatment with 100 nM dDAVP, there was a marked decrease in the levels of inositol 1,4,5-trisphosphate and inositol 1-phosphate, but subsequently no change was observed for up to 9 min after treatment. We postulate that the increase of diacylglycerol content produced by dDAVP might be primarily from a phosphatidylcholine source and that the growth-promoting activity of desmopressin may be a consequence of activation of protein kinase C.

Correlation of the antiproliferative action of diphenylmethane-derivative antiestrogen binding site ligands with antagonism of histamine binding but not of protein kinase C-mediated phosphorylation.

The nonestrogen receptor-mediated antiproliferative action of antiestrogen binding site (AEBS) ligands, including triphenylethylene antiestrogens and phenothiazines, has been linked to their ability to inhibit protein kinase C (PKC). Recent studies indicate that some diphenylmethane derivatives inhibit growth, are potent AEBS ligands, and antagonize histamine binding at an AEBS-related histamine site different from H1 and H2. Three novel diphenylmethane derivatives, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCI (DPPE), 4-decanoyl-DPPE (dec-DPPE), and 4-benzylphenyl decanoate (BPD) were studied in an attempt to determine whether PKC or histamine interactions best correlate with their antiproliferative effects. Platelet aggregation and the phosphorylation of a platelet Mr 47,000 protein (p47) induced by phorbol-12-myristate-13-acetate (PMA) represent two processes mediated by PKC. DPPE inhibits PMA-induced aggregation [50% inhibitory concentration (IC50) = 31.2 +/- 2.4 (SEM) x 10(-6) M] but does not significantly inhibit either PMA-induced phosphorylation of Mr 47,000 protein (IC50 greater than 500 x 10(-6) M), or binding of [3H]phorbol dibutyrate to platelets. dec-DPPE is a more potent inhibitor of PMA-induced platelet aggregation (IC50 = 18.8 +/- 0.7 x 10(-6) M), a weak inhibitor of Mr 47,000 phosphorylation (IC50 = 80-200 x 10(-6) M), but is without effect on [3H]phorbol dibutyrate binding. BPD, which lacks the alkylaminoethoxy side chain necessary for binding to the AEBS/DPPE site, is devoid of anti-PMA effects. These results are compared to the inhibition of [3H]histamine binding in rat cortex membranes (Ki value for DPPE = 0.83 +/- 0.62 x 10(-6) M; Ki value for dec-DPPE = 6.6 +/- 3.5 x 10(-6) M; BPD is inactive) and growth inhibition of MCF-7 cells (IC50 value for DPPE = 4.5 x 10(-6) M; IC50 value for dec-DPPE = 1.5 x 10(-5) M; BPD is ineffective at all concentrations tested). Thus, while dec-DPPE is a more potent inhibitor of PKC-mediated phosphorylation, DPPE is a more potent inhibitor of histamine binding and is correspondingly more antiproliferative than dec-DPPE. The results support a relationship between antagonism of histamine binding and growth inhibition but argue against an association between the antiproliferative effects of DPPE and dec-DPPE and inhibition of PKC. The findings for DPPE suggest that platelet response to PMA, antagonized by diphenylmethane-type AEBS-ligands, may be mediated, at least in part, by mechanisms other than activation of protein kinase C-dependent phosphorylation.

Independence of firing correlates of anatomically proximate hippocampal pyramidal cells.

In neocortex, neighboring neurons frequently exhibit correlated encoding properties. There is conflicting evidence whether a similar phenomenon occurs in hippocampus. To assess this quantitatively, a comparison was made of the spatial and temporal firing correlations within and between local groups of hippocampal cells, spaced 350-1400 microm apart. No evidence of clustering was found in a sample of >3000 neurons. Moreover, cells active in two environments were uniformly interspersed at a scale of <100 microm, as assessed by the activity-induced gene Arc. Independence of encoding characteristics implies uncorrelated inputs, which could enhance the capacity of the hippocampus to store arbitrary associations.

Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin.

Platelets, when stirred with 3 U thrombin/10(9) platelets, produced significant quantities of palmitoyllysophosphatidic acid (2.17 ng/10(9) platelets), stearoyllysophosphatidic acid (2.11 ng/10(9) platelets), and arachidonoyllysophosphatidic acid (1.06 ng/10(9) platelets). When platelets were pretreated with 100 microM of the phospholipase A2 inhibitor U10029A, there was a significant decrease in thrombin-stimulated production of stearoyllysophosphatidic acid (to 0.16 ng/10(9) platelets), while arachidonoyllysophosphatidic acid production was unchanged. U10029A concomitantly increased thrombin-stimulated production of stearoyl-containing phosphatidic acid species (primarily stearoylarachidonoylphosphatidic acid) from 5.99 to 9.71 ng/10(9) platelets. The results are consistent with the concept that stearoyllysophosphatidic acid production in platelets occurs via phospholipase A2 degradation of phosphatidic acid.

Lysophosphatidic acid can activate platelets without increasing 32P-labelling of phosphatidic acid.

Stimulation of platelets by thrombin produced a rise in [32P]phosphatidic acid labelling of platelets which was greater in medium without added calcium than in medium with 2.5 mM calcium. A rise in [32P]lysophosphatidic acid was also seen in platelets stimulated by thrombin in the presence of 2.5 mM extracellular calcium, though it was of lesser magnitude (average 35%) than the rise in phosphatidic acid. In platelets resuspended without added calcium no change in [32P]lysophosphatidic acid was seen in response to thrombin. Lysophosphatidic acid can itself induce platelet aggregation. Similarly to the calcium ionophore A23187, lysophosphatidic acid produced minimal change (in medium with no added calcium) to no change (in medium with 2.5 mM external calcium) in [32P]lysophosphatidic acid. The endoperoxide analog U46619 produced changes in 32P-labelling of platelet phosphatidic and lysophosphatidic acid similar to those produced by thrombin but of lesser magnitude. The results of these studies show that the action of lysophosphatidic acid on platelets differs from the action of thrombin, U46619 and platelet-activating factor, which produce a rapid rise in [32P]phosphatidic acid, and suggests that lysophosphatidic acid, like A23187, largely bypasses the initial receptor-coupled breakdown of phosphoinositides leading to formation of diacylglycerols and phosphatidic acid.

Differential effects of spermine on aggregation, inositol phosphate formation and protein phosphorylation in human platelets in response to thrombin, arachidonic acid and lysophosphatidic acid.

Platelet aggregation stimulated by thrombin, arachidonic acid or lysophosphatidic acid is associated with rapid phosphorylation of two platelet proteins, myosin light chain and a 47 kDa protein. The polyamine, spermine, inhibited platelet aggregation stimulated by all three agents. Spermine inhibited thrombin-stimulated phosphorylation of myosin light chain and the 47 kDa proteins as well as thrombin-induced production of the inositol phosphates and phosphatidic acid. In contrast, spermine did not inhibit phosphorylation of either protein or the formation of inositol phosphates and phosphatidic acid in response to arachidonic acid or lysophosphatidic acid. Although spermine has been demonstrated to inhibit both phosphatidylinositol-specific phospholipase C and calcium-dependent protein kinases in cell free systems, these results suggest that, in the intact platelet, spermine does not directly inhibit these enzymes. Inhibition of aggregation stimulated by arachidonic acid and lysophosphatidic acid is secondary to interference with platelet-platelet interaction but not with platelet activation. In contrast, spermine inhibits thrombin-induced platelet activation. This thrombin-specific inhibition may be related to interference with the binding of thrombin to its receptor or to its catalytic substrate on the cell surface.