Cyclotrimerization of phenylacetylene catalyzed by a cobalt half-sandwich complex embedded in an engineered variant of transmembrane protein FhuA.

An (η5-cyclopentadienyl)cobalt(i) complex was covalently incorporated into an engineered variant of the transmembrane protein ferric hydroxamate uptake protein component: A, FhuA ΔCVFtev, using a thiol-ene reaction. A CD spectrum shows the structural integrity of the biohybrid catalyst. MALDI-TOF of the segment containing the anchoring site for the cobalt complex Cys545 confirmed successful conjugation. This biohybrid catalyst catalyzed the cyclotrimerization of phenylacetylene to give a mixture of regioisomeric 1,2,4- and 1,3,5-triphenylbenzene in aqueous medium.

Metatheases: artificial metalloproteins for olefin metathesis.

The incorporation of organometallic catalyst precursors in proteins results in so-called artificial metalloenzymes. The protein structure will control activity, selectivity and stability of the organometallic site in aqueous medium and allow non-natural reactions in biological settings. Grubbs-Hoveyda type ruthenium catalysts with an N-heterocyclic carbene (NHC) as ancillary ligand, known to be active in olefin metathesis, have recently been incorporated in various proteins. An overview of these artificial metalloproteins and their potential application in olefin metathesis is given.

Impact of single-port cholecystectomy on postoperative pain.

BACKGROUND: This study compared postoperative pain following four-port laparoscopic cholecystectomy (LC) and single-port cholecystectomy (SPC). METHOD: This prospective, quasi-randomized, single-centre trial focusing on postoperative pain included 49 patients undergoing elective surgery with either a conventional LC, or SPC using a surgical glove port. Postoperative pain was evaluated using a visual analogue scale (VAS) and postoperative analgesic use as primary outcome measures. Total duration of operation, length of hospital stay, blood test results on the day after surgery and total port cost were secondary outcome measures. RESULTS: Twenty-five LCs and 24 SPCs were undertaken. The VAS score on day 1 after surgery was significantly less in the SPC group than in the LC group: median (range) 24 (12-38) versus 45 (33-57) mm (P = 0·002). Significantly fewer patients in the SPC group required analgesia (9 of 24 versus 19 of 25 in the LC group; P = 0·007). There were no significant differences in total duration of operation, length of hospital stay, and blood test results on the day after surgery. CONCLUSION: Single-port surgery using a surgical glove port reduces postoperative pain compared with conventional LC.

Insulin secretion by anomers of d-glucose.

Isolated rat islets were incubated for 5 minutes in the media containing either the alpha or beta anomer of D-glucose (2 milligrams per milliliter). The amounts of secreted insulin and changes of anomers ratio were concomitantly determined. In spite of rapid mutarotation, significantly greater stimulation of insulin secretion was observed by alpha-D-glucose as compared with beta-D-glucose.

Cell wall fraction of Enterococcus hirae ameliorates TNF-alpha-induced barrier impairment in the human epithelial tight junction.

AIMS: The evaluation of the effects of Enterococcus hirae, an intestinal bacterium in the adjacent mucosa (mucosal bacterium), on tumour necrosis factor-alpha (TNF-alpha)-induced barrier impairment in human epithelial Caco-2 cells. METHODS AND RESULTS: The filter-grown Caco-2 monolayers were used as an intestinal epithelial model system. In Caco-2 cells, heat-killed E. hirae ATCC 9790(T) suppressed the TNF-alpha-induced barrier impairment and increase in interleukin-8 (IL-8) secretion, but lipase- and mutanolysin-treated E. hirae ATCC 9790(T) did not have these effects. It was demonstrated that lipoteichoic acid (LTA) from E. hirae ATCC 9790(T) is responsible for Caco-2 cells' recovery from TNF-alpha-induced impairments. In addition, Caco-2 cells had the same response to Toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4) as they did to LTA. Increased expression of zonula occludens-1 was observed by the addition of E. hirae ATCC 9790(T) to TNF-alpha-treated Caco-2 cells, and decreased expression of myosin light chain kinase was observed by the addition of LTA and Pam(3)Cys-Ser-(Lys)(4); this, in turn, led to barrier enforcement. CONCLUSIONS: Enterococcus hirae ATCC 9790(T) cell wall fractions, such as LTA, protect against intestinal impairment by regulation of epithelial tight junction via TLR2 signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus hirae could be useful in the treatment of inflammatory bowel disease, as well as other intestinal disorders.

1,4-Dihydropyridyl complexes of magnesium: synthesis by pyridine insertion into the magnesium-silicon bond of triphenylsilyls and catalytic pyridine hydrofunctionalization.

Magnesium bis(triphenylsilyl) [Mg(SiPh3)2(THF)2]·THF (1) reacted with a stoichiometric amount of pyridine to give the magnesium 4-(triphenylsilyl)dihydropyridyl complex [Mg(NC5H5-4-SiPh3)2(THF)3] (2). Using an excess of pyridine, a mixture of magnesium dihydropyridyl [Mg(NC5H6)2(py)4] (3) and 4-(triphenylsilyl)pyridine was formed. Complex 3 underwent exchange with pyridine-d5 at 25 °C to give [Mg(NC5D5H-4)2(py-d5)4] (3-HD). Analogous reactions with Me3TACD-supported magnesium triphenylsilyls [(Me3TACD)Mg(SiPh3)] (4) and [(Me3TACD·AlEt3)Mg(SiPh3)] (6) ((Me3TACD)H = Me3[12]aneN4: 1,4,7-trimethyl-1,4,7,10-tetraazacyclododecane) with pyridine gave [(Me3TACD)Mg(NC5H5-4-SiPh3)] (5), [(Me3TACD·AlEt3)Mg(NC5H5-4-SiPh3)] (7) and a mixture of [(Me3TACD)Mg(NC5H6)] (8) and 4-(triphenylsilyl)pyridine. Complex 8 is also formed by reacting 3 with (Me3TACD)H and underwent exchange with pyridine-d5 at higher temperatures. The activation energy for the exchange is about 25 kJ mol-1 higher than that for the exchange reaction of 3 to 3-HD. Complexes 2, 3, 3-HD, 5, 7 and 8 were characterized by NMR spectroscopy and 3, 5 and 8 by single crystal structure analysis. Complex 3 was found to be slightly active in the hydrosilylation of pyridine using phenylsilane, whereas complex 8 showed no activity. Both complexes 3 and 8 were active in the hydroboration of pyridine with pinacolborane.

Formate complexes of titanium(iv) supported by a triamido-amine ligand.

The terminal formate complex [(OCHO)Ti(N3N)] (3) containing the trianionic triamido-amine ligand (Me3SiNCH2CH2)3N3- (N3N) was prepared via salt metathesis of [ClTi(N3N)] (1) with sodium formate or alternatively by treatment of the alkyl complex [nBuTi(N3N)] (2) with ammonium formate [HNEt3][OCHO]. Deprotonation of 3 with potassium hexamethyldisilazide gave a polymeric helical chain of the oxo complex {K[OTi(N3N)]}n (4). Reaction of 2 with the trityl salt [Ph3C][B(3,5-Cl2C6H3)4] or the Brønsted acid [HNEt3][B(C6F5)4] gave [(Et2O)Ti(N3N)][BR4] (6[BR4]·Et2O) with R = 3,5-Cl2C6H3 or C6F5. The diethyl ether ligand was easily replaced by other L-type donor ligands such as tetrahydrofuran, pyridine, and 4-dimethylaminopyridine to give 6[BR4]·L with L = thf, py, and dmap. Reaction of 6[BR4]·Et2O with a stoichiometric amount of CO2 gave the dimeric, dicationic bis(carbamate)-bridged complexes [Ti{N(CH2CH2NSiMe3)2(CH2CH2NSiMe3(µ-CO2-ηO:ηO'))}]2[BR4]2 (7[BR4]2) through insertion of one CO2 into one of the titanium-amido bonds. Addition of pyridine to 7[B(C6F5)4]2 formed the monomeric carbamate complex [(py)Ti{((O2C-κ2O,O')NSiMe3CH2CH2)N(CH2CH2NSiMe3)2}][B(C6F5)4] (8[B(C6F5)4]·py). The cationic formate-bridged species [(Ti(N3N))2(µ-OCHO-ηO:ηO')][BR4] (10[BR4]) readily formed when the terminal formate complex 3 was reacted with the cationic 6[BR4]. The reactivity of triamido-amine stabilized titanium(iv) complexes is shown to differ considerably from that of related titanium tris(anilide) complexes.

Molecular magnesium hydrides supported by an anionic triazacyclononane-type ligand.

Molecular magnesium hydride with a terminal metal-hydrogen bond [Mg(iPr2TACN·AliBu3)H]2 supported by a monoanionic TACN-type ligand (TACN = 1,4-di-isopropyl-1,4,7-triazacyclononane) rearranges to form the dinuclear magnesium hydride [Mg(iPr2TACN·AlHiBu2)(µ-H)]2, exhibiting a rare MgH-Al interaction.

Comparative study of the phospholipid composition of plasma membranes isolated from rat primary hepatomas induced by 3'-methyl-4-dimethylaminoazobenzene and from normal growing rat livers.

Plasma membranes (PM's) were isolated from primary hepatomas induced in Wistar rats by 3'-methyl-4-dimethylaminoazobenzene, from nonhepatomal regions of the same rat livers, and also from various normal rat livers, including the resting and regenerating livers of adult rats and developing livers of postnatal rats. Phospholipid analyses of these PM preparations revealed the following differences in the hepatoma PM in comparison with those of the PM of normal adult resting livers: (a) decrease in the content of total phospholipids; (b) large increase in plasmalogen content; (c) relative increase of sphingomyelin and ethanolamine phospholipids, and the decrease of choline phosphoglycerides, i.e., decrease of the ratios of choline phosphoglycerides to choline phosphosphingoside and of choline-containing phospholipids to ethanolamine-containing phospholipids; (d) decrease in phosphatidylserine and phosphatidylinositol. The phospholipid composition of the PM's from normal growing livers showed definite decreases in phosphatidylserine and sphingomyelin and increases in ethanolamine phospholipids; however, no significant alteration was observed in plasmalogen content in comparison to the PM of normal adult resting livers. The PM from the nonhepatomal regions of the hepatoma-bearing livers did not show those differences observed in hepatoma PM.

Exchange of free and bound coenzyme of flavin enzymes studied with [14C]FAD.

The exchange of bound FAD for free FAD was studied with D-amino acid oxidase (D-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and beta-D-glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). For a simple measurement of the reaction rate, equimolar amounts of the enzyme and [14C]FAD were mixed. The exchange occurred very rapidly in the holoenzyme of D-amino acid oxidase at 25 degrees C, pH 8.3 (half life of the exchange: 0.8 min), but slowly in the presence of the substrate or a competitive inhibitor, benzoate. It also occurred slowly in the purple complex of D-amino acid oxidase. In the case of beta-D-glucose oxidase, however, the exchange occurred very slowly at 25 degrees C, pH 5.6, regardless of the presence of the substrate or p-chloromercuribenzoate. On the basis of these findings, the turnover of the coenzymes of flavin enzymes in mammals is discussed.

Stimulatory effect of fatty acid treatment on glucose utilization in human erythrocytes.

We previously reported that treatment of human erythrocytes with bee venom phospholipase A2 increased the rate of lactate production from glucose. This increase was suggested to be mediated through liberation of free fatty acids from membrane phospholipids. So, in the present study we examined the mechanism of stimulation of glycolysis by fatty acids. Treatment of intact erythrocytes with most of the 15 fatty acids tested resulted in stimulation of lactate production from glucose. Among the fatty acids tested, myristoleic acid showed the highest stimulatory activity. The ratio of moles of lactate produced to those of glucose utilized was about 1.9 in both myristoleic acid-treated and untreated cells. Treatment of erythrocytes with myristoleic acid did not affect the amount of 2,3-bisphosphoglycerate. Lactate production from D-glyceraldehyde, which is thought to be phosphorylated to D-glyceraldehyde 3-phosphate and then metabolized in the glycolytic pathway, was not at all affected by treatment of cells with myristoleic acid. The cross-over plot of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of the 6-phosphofructokinase (PFK) step. Fatty acids incorporated into erythrocytes were found to be present predominantly in the cytoplasm rather than in the plasma membrane. The PFK activity, but not the hexokinase activity, in hemolysates was clearly increased by a set of fatty acids, and myristoleic acid was again the most potent. However, partially purified human erythrocyte PFK was not activated by the acid. We conclude that fatty acids stimulate glycolysis through activation of PFK in cooperation with some other component(s) in erythrocytes.

Glucose transport into human erythrocytes treated with phospholipase A2 or C.

Phospholipase A2 induced crenation of human erythrocytes and decreased glucose transport activity (influx rate) by 40% when 51% of phosphatidylcholine (PC) in the membrane was hydrolyzed. On the other hand, phospholipase C induced invagination of the cells and negligibly affected the glucose transport in the case of 21% hydrolysis of the PC. By altering the pH of the medium for suspending cells treated with phospholipase A2 from 7.4 to 6.0, cell shape was changed from clear crenation to slight invagination, but glucose transport activity was not affected. Cells that were treated with phospholipase A2 and then washed with albumin to remove free fatty acids produced in the cell membrane showed an almost normal cell shape and slightly higher glucose transport activity than did untreated cells. The ratios of beta-D-glucose transport rate to alpha-D-glucose transport rate in untreated cells, cells treated with phospholipase A2 and cells treated with phospholipase C were 1.13, 1.04, and 1.20, respectively. These results demonstrate that the drastic morphological change (invagination or crenation) induced by the treatment with phospholipases bears no clear relationship to the activity of glucose transport and suggest that the increase in the volume of the outer half of the lipid bilayer might reduce the rate of glucose transport across the human erythrocyte membrane and change the anomeric preference of glucose transport.

Stimulatory effect of phospholipase A2 treatment on glucose utilization in human erythrocytes.

We examined whether modification of membrane phospholipids of human erythrocytes by hydrolysis with phospholipase A2 (PLA2 from bee venom) would affect glucose utilization, chosen as a typical model of intracellular metabolism, and, if so, intended to clarify the mechanism of the alteration of glycolysis. Treatment of erythrocytes with PLA2 induced a marked shape change (i.e., crenation) and significantly increased the rate of lactate production from glucose. Available evidence indicated that there is no relevance of this cell-shape change to the alteration of glycolysis. The lack of a detectable effect of papain treatment on glycolysis in PLA2-treated cells suggested that the increase in glycolysis by PLA2 treatment might not be caused by the conformational change of band-3 protein through modulation of membrane phospholipids. The result of the measurement of lactate production in the presence and absence of ouabain did not support the idea that hydrolysis of phospholipids by PLA2 treatment makes plasma membranes leaky to Na+ and consequently enhances glycolysis through activation of Na+/K(+)-ATPase. The action of PLA2 on glycolysis was abolished by extraction of free fatty acids in the cell membrane with bovine serum albumin. Loading erythrocytes with free fatty acid (oleic acid, linoleic acid, or arachidonic acid) caused a significant increase in glycolysis. Analysis of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of 6-phosphofructokinase. The data, thus, indicate that treatment of human erythrocytes with PLA2 significantly accelerates glucose utilization and suggest that the stimulation of glycolysis is caused by activation of 6-phosphofructokinase through liberation of free fatty acids of membrane phospholipids by PLA2.

Participation of glucokinase inactivation in inhibition of glucose-induced insulin secretion by 2-cyclohexen-1-one.

We assessed our speculation that 2-cyclohexen-1-one (CHX) impairs glucose-induced insulin secretion through inactivation of glucokinase. Treatment of pancreatic islets with CHX at concentrations (0-5 mM) that caused a dose-dependent inactivation of glucokinase activity similarly inhibited glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets was little affected by CHX. CHX-induced inactivation of glucokinase was blocked by the presence of its substrates (glucose and mannose) and an inhibitor (N-acetylglucosamine), all of which also protected against the inhibitory effect of the drug on glucose-induced insulin secretion. CHX also impaired insulin secretion induced by D-glyceraldehyde and dimethyl succinate, which are believed to stimulate the release of the hormone by being directly oxidized by glyceraldehyde-3-phosphate dehydrogenase, by entering the midstream of the glycolytic pathway as glyceraldehyde 3-phosphate, or by entering the tricarboxylic acid cycle in mitochondria after intracellular hydrolysis. The inhibitory effect of CHX on glucose-induced insulin secretion, however, was far more marked than that on insulin secretion evoked by D-glyceraldehyde and dimethyl succinate at any CHX concentrations used. Our study revealed that the inhibitory action of CHX on glucose-induced insulin secretion is exerted mainly, but not solely, through inactivation of glucokinase. This conclusion supports the view that glucokinase is a key enzyme in the recognition of glucose as an insulin secretagogue in pancreatic islets.

Inhibition of glucose-induced insulin secretion through inactivation of glucokinase by glyceraldehyde.

D-Glyceraldehyde irreversibly inhibited rat liver glucokinase in a concentration-dependent manner. The inactivation of glucokinase by glyceraldehyde was blocked by the presence of its substrates such as glucose and mannose. Glucokinase was highly sensitive to glyceraldehyde compared with some other glycolytic enzymes (from animal tissues) including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The amino acid analysis of untreated and glyceraldehyde-treated glucokinase suggested that glyceraldehyde-induced inactivation of glucokinase is caused by glycation of Lys residues of the enzyme by the triose. Treatment of pancreatic islets with 6 mM glyceraldehyde for 1 h at 37 degrees C caused both inactivation of glucokinase and inhibition of glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets, however, was little affected by glyceraldehyde. In addition, glyceraldehyde did not affect the insulin secretory responses of islets to nonglucose secretagogues such as glyceraldehyde and Leu. When pancreatic islets were cultured with a lower concentration (1 mM) of glyceraldehyde for a longer time (17 h) in the presence of 10 mM glucose to mimic the in vivo conditions, both glucokinase activity and glucose-induced insulin secretion were again decreased. This study demonstrates that glucose-induced insulin secretion is impaired by glyceraldehyde through the inactivation of glucokinase. The implication of this finding in the pathophysiology of type II diabetes is discussed.

Formation of α-[KSiH3] by hydrogenolysis of potassium triphenylsilyl.

Hydrogenation of easily accessible potassium triphenylsilyl [K(Me6TREN)SiPh3] gave the hydrogen storage material α-[KSiH3] in high yields by an unusual hydrogenolytic cleavage of silicon-phenyl bonds.

Preparation and characterization of monoclonal antithyrotropin receptor antibodies obtained from peripheral lymphocytes of hypothyroid patients with primary myxedema.

Anti-TSH receptor antibodies (TSH-R Ab), which have been detected in the serum of some patients with primary myxedema, are themselves considered to induce hypothyroidism. These are termed blocking-type TSH-R Ab (TSH-R BAb), because they inhibit adenylate cyclase stimulation by TSH on thyrocytes or nonthyroidal cells transfected with TSH-R complementary DNA. We prepared monoclonal TSH-R BAb and characterized them. Peripheral lymphocytes from three patients with primary hypothyroidism and potent TSH-R BAb were transformed by Epstein-Barr virus, and the culture supernatants were screened by TSH binding inhibitor immunoglobulin (TBII) assay. Twenty positive and 7 negative lymphocyte clones were obtained; their monoclonality was confirmed by Southern blot analysis, using an immunoglobulin (Ig) JH probe. These monoclonal antibodies were then tested for TSH-R BAb activity. TSH-R BAb activity ranged from 24.1-58.5% (normal range, < 24%) in all 20 TBII-positive clones and in 2 of 7 TBII-negative clones. An enzyme-linked immunosorbent assay showed that the Ig isotypes of these clones with TBII and/or TSH-R BAb activity were IgG in 8 and IgM in 14. Another enzyme-linked immunosorbent assay and Southern blot analysis of the light chains revealed that 13 clones had kappa-chains, whereas the light chains could not be determined in the other 9 clones. To summarize, 1) we obtained 22 clones that produced monoclonal TSH-R BAb, including 8 IgG-type clones. 2) The clones exhibited dominant usage of the kappa-chain. 3) Although all TBII clones had TSH-R BAb activity, their TBII and TSH-R BAb activities were not significantly correlated, and two TSH-R BAb clones did not show TBII activity.

Changes in subcellular and zonal distribution of glucokinase in rat liver during postnatal development.

Subcellular and zonal distribution of glucokinase in rat liver during postnatal development was examined immunohistochemically. Before day 11 after birth, only some hepatocytes were immunostained, and a positive immunostaining was found in the cytoplasm but not in the nucleus. No zonal distribution of glucokinase was observed in livers of such pups. From day 15, at which time a dietary change from milk to laboratory chow begins to take place, glucokinase immunoreactivity increased; this increase was associated with increases in glucokinase activity and in glucokinase protein, and also the immunostaining was observed mainly in the nuclei. At day 21, the glucokinase immunoreactivity was found almost exclusively in the perivenous zone. At day 30, an intense immunostaining was seen both in the perivenous zone and in the periportal zone, being slightly predominant in the former. The present results indicate that dramatic changes in the distribution of glucokinase in developing rat liver may be related to dietary change.

Hemispheric asymmetry of processing temporal aspects of repetitive movement in two patients with infarction involving the corpus callosum.

Two right-handed patients with infarction involving the forebrain commissural fibres produced irregular but rapid repetitive movements with their left hands while producing normal movements with their right hands as well as having normal oral expression. The abnormality was more conspicuous when producing slow tapping patterns. When required to reproduce rapid tapping patterns of around 5 beats/sec, one of the patients produced a comparatively regular tapping pattern. Thus, we believe that both hemispheres are equipped with the faculty to produce rapid repetitive patterns and that only the left hemisphere is responsible for the temporal processes involved in producing required repetitive patterns.

Non-enzymatic reduction of alloxan by reduced nicotinamide nucleotide.

Much evidence has been reported that the diabetogenic action of alloxan is caused by the formation of cytotoxic free radicals during the autoxidation of dialuric acid, a reduction product of alloan, to alloxan. The mechanism by which alloxan is reduced in vivo to dialuric acid, however, is unknown. The non-enzymatic reaction of alloxan with NAD(P)H was studied as a possible candidate for the reduction of alloxan. The reaction was carried out at 37 degrees in 50 mM phosphate buffer (mostly at pH 7.0) and was followed by measuring the decrease in absorbance at 340 nm. NADH and NADPH were found to be stoichiometrically oxidized by alloxan to NAD and NADP respectively. When the alloxan concentration (1.0 mM) was kept constant and the concentration of NAD(P)H (0.05 to 0.2 mM) was varied, the rate of decrease in the relative concentration of NAD(P)H was almost constant, suggesting that the autoxidation of dialuric acid by O2 was rapid enough to neglect its presence in the medium. The reaction between alloxan and NAD(P)H was accelerated by decreasing the pH. Both the rate of decrease in NAD(P)H concentration and the rate of O2 consumption resulting from autoxidation of the dialuric acid formed by reduction of alloxan were not affected by the presence of 20 mM D-glucose. Ethylene formation by the reaction of methional with . OH, one of the autoxidation products of dialuric acid, was clearly reduced by the presence of alpha- or beta-D-glucose (20 mM), but there was no significant difference between the effects of the two anomers. These results with D-glucose ruled out the possibility that the protection of beta-cells by D-glucose against the diabetogenicity of alloxan can be explained either by its inhibitory action on dialuric acid formation or by its scavenging effect on . OH.