Abnormal tricarboxylic acid cycle metabolites in isovaleric acidaemia

Summary Background  Although a number of abnormal diagnostic metabolites have previously been described in the urine of patients with isovaleric acidaemia (IVA), they do not fully explain the clinical symptoms associated with this disease. Methods  On the basis of our current understanding of the TCA cycle and IVA, we predicted a number of abnormal methylated TCA cycle metabolites, initiated by methylsuccinic acid. We subsequently obtained characteristic gas chromatography–mass spectrometry elution times and mass spectra of the chemically synthesized predicted compounds and screened the urine of 6 IVA patients and 24 age-matched controls. Further proof for our findings was generated from a series of in vitro enzyme reactions using the chemically synthesized standards as substrates to their respective TCA cycle enzymes. Results  Apart from the previously described methylsuccinic and methylfumaric acid, 3-methylmalic acid, (2R,3S)- and (2R,3R)-methylcitric acid and 2-methyl-cis-aconitic acid were detected in the urine of all 6 IVA patients in increased amounts. Additionally, although not directly determined, the in vitro enzyme reaction using of 3-methylmalic acid and malate dehydrogenase, in conjunction with the detection of 2-ketobutyric acid in the urine of all 6 IVA patients, strongly suggests an additional synthesis of 3-methyloxaloacetic acid by the same cycle. Conclusion  Not only do these newly identified metabolites serve as additional diagnostic markers to those previously identified in IVA, but due to the structural arrangements of the (2R,3R)-methylcitric acid and 2-methyl-cis-aconitinic acid-derived 2-methylisocitric acid, inhibition of normal TCA cycle metabolism results at citrate synthase and isocitrate dehydrogenase, respectively. Synopsis  Methylsuccinic acid acts as the initiating substrate to a series of abnormal, potentially harmful, methylated tricarboxylic acid cycle metabolites in isovaleric acidaemia. Competing interests: None declared References to electronic databases: Isovaleric acidaemia: OMIM #243500. Isovaleryl-CoA dehydrogenase: EC 1.3.99.10.     

Up-regulation of ICH-1l protein by thromboxane A2 antagonists enhances cisplatin-induced apoptosis in non-small-cell lung-cancer cell lines

We evaluated the effect of thromboxane A₂ (TXA₂) blockade on cisplatin-induced apoptosis in non-small-cell lung cancer (NSCLC) cell lines. Cisplatin induced apoptosis in PC/9 and PC-9/CDDP in a dose-dependent manner. Treatment with specific TXA₂ antagonist, calcium 5(Z)-1R,2S,3S,4S-7-[3-phenylsul‐ fonylaminobicyclo[2.2.1]hept-2-yl]-5-heptonoate hydrate (S-1452) and 5(Z-6-{(1R,2R,3R,4S)-3-(N-4-bromoben‐ zenesulfonyl aminomethyl) bicyclo[2,2,1]heptane-2-yl}hex-5-enoic acid (ONO-NT-126), enhanced the cisplatin-induced apoptosis in each cell line. Acetyl-l-aspartyl-glutamyl-valyl-aspart-1-aldehyde (Ac-DEVD-CHO) inhibited cisplatin-induced apoptosis and enhancement of the apoptosis by TXA₂ blockade, but acetyl-l-tyrosyl-valyl-alanyl-aspart-1-aldehyde (Ac-YVAD-CHO) had no effect on the apoptosis. There was no difference in the interleukin-1β-converting enzyme (ICE) protease protein expression in either cell line. Cysteine protease p32(CPP32) protein expression was lower in PC-9/CDDP but was not changed by S-1452, cisplatin, or cotreatment with cisplatin and S-1452. Ice and Ced-3 homolog (ICH-1l) expression was significantly lower in PC-9/CDDP and was up-regulated by S-1452 or ONO-NT-126. These data suggest that ICH-1l might play a critical role in cisplatin-induced apoptosis and that TXA₂ blockade up-regulates ICH-1l protein expression. Overexpression of ICH-1l and treatment with cisplatin might result in an increase in apoptosis in NSCLC cell lines. Received: 15 September 1998 / Accepted: 21 January 1999     

The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation

The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7′E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7′E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate.     

Mechanisms Involved in the Attenuation of Intestinal Toxicity Induced by (S)-(+)-Ketoprofen in Re-Fed Rats

In addition to suppression of prostaglandins synthesis a number of factors have been implicated in nonsteroidal antiinflammatory drugs (NSAIDs) enteropathy, including oxygen radical-dependent microvascular injuries, depletion of glutathione, and food. Inflammatory cytokines such as tumor necrosis factor- regulate endothelial adhesion molecules expression and promote vascular neutrophil adherence. Racemic ketoprofen is a potent NSAID with a chiral structure existing in two enantiomeric forms. Its therapeutic effects reside almost exclusively in the (S)-(+) isomer nevertheless the potential contribution to side effects of the (R)-(–) isomer cannot be ignored. The aims of this study were to explore the role of prostaglandins depletion, tumor necrosis factor- production, and glutathione homeostasis in the comparative pathogenesis of intestinal injury induced by racemic-ketoprofen and its enantiomers in re-fed rats. Racemic ketoprofen and (R)-(–)-ketoprofen dose-dependently caused similar and multiple lesions in the mid-jejunum significantly higher than those observed with (S)-(+)-ketoprofen. All the treatments significantly decreased prostaglandins content. A significant increase of tumor necrosis factor- production and decreases in glutathione levels and glutathione reductase activity after treatment of the racemate and (R)-(–)-ketoprofen, were observed whereas the (S)-(+)-isomer did not change these parameters. In conclusion, (S)-(+)-ketoprofen possesses a better intestinal toxicity profile than the racemate and its (R)-(–)-isomer. Despite inhibiting cyclooxygenase activity, the attenuation of (S)-(+)-ketoprofen-induced intestinal toxicity could be correlated with a reduced oxidative damage characterized not only by a lack of changes in glutathione reductase activity and glutathione levels but also by an absence of up-regulation of tumor necrosis factor- production in intestinal mucosa.     

Excessive excretion of β-alanine and of 3-hydroxypropionic,R- andS-3-aminoisobutyric,R- andS-3-hydroxyisobutyric andS-2-(hydroxymethyl)butyric acids probably due to a defect in the metabolism of the corresponding malonic semialdehydes

A new metabolic disorder characterised by the excessive excretion of -alanine, 3-hydroxypropionic acid,R-andS-3-amino- and 3-hydroxyisobutyric acids andS-2-(hydroxymethyl)butyric acid is probably due to deficient activities of malonic, methylmalonic and ethylmalonic semialdehyde dehydrogenases. These dehydrogenation reactions could be mediated by one enzyme, or by enzymes with a common subunit, and bothR- andS-methylmalonic semialdehydes seem to be equally affected. The patient is now aged 4 years and has developed normally. He has a persistent gross hypermethioninaemia which is probably unrelated to the other biochemical abnormalities.     

Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.Terpenoids are a structurally diverse group of metabolites with functions in both primary and secondary (or specialized) metabolism. Primary metabolites derived from terpenoid pathway intermediates in plants include sterols, carotenoids, and the side chains of chlorophylls, tocopherols, and quinones of electron transport systems. Many plant hormones are also products of terpenoid metabolism, including abscisic acid, cytokinins, brassinosteroids, and strigolactones (1). Secondary plant metabolites of terpenoid origin can play critical defense-related roles (e.g., sesquiterpene lactones and triterpene saponins serve as antifeedants) and are dominant constituents of essential oils and resins (mono-, sesqui-, and diterpenes) (2). Terpene synthases (TPSs) convert a prenyl diphosphate of a specific chain length to the first pathway-specific (often cyclic) intermediate in the biosynthesis of each class of terpenoids. Whereas some terpene synthases are remarkably specific and only generate one product from a prenyl diphosphate precursor, others release a larger number of products from a common substrate, thus contributing to terpenoid chemical diversity (3). The genomes of plants may only contain one TPS gene [e.g., ent-kaurene (diterpene) synthase in the moss Physcomitrella patens (Hedw.) Bruch & Schimp.], but often harbor sizable families of TPS genes with more than 20 members, which is another source of terpenoid structural variety (4).All monoterpene synthases (MTSs) use either geranyl diphosphate (GPP) or its 2Z-isomer neryl diphosphate as substrate, but the sequence conservation across species is generally fairly low (2). However, MTSs share a common tertiary structure (the so-called αβ fold), with a C-terminal α-domain containing the active site and an N-terminal β-domain of as yet uncertain function (3). The active site harbors a highly conserved, l-aspartate-rich, DDxxD motif, which is also found in prenyl elongases (5) and a less conserved NSE/DTE motif. The l-aspartate residues bind a trinuclear cluster of divalent metal ions (Mg²⁺ or Mn²⁺) involved in the binding and activation of the diphosphate moiety, thereby generating characteristic carbocation intermediates (Fig. 1). Enzymes catalyzing these ionization-initiated cyclization reactions are commonly referred to as class I TPSs (as opposed to class II TPSs that initiate cyclizations by protonation) (3). The remaining course of MTS catalysis is variable and generates acyclic, monocyclic, and/or bicyclic monoterpenes.Open in a separate windowFig. 1.Proposed mechanism for (4S)-limonene synthase catalysis. OPP denotes the diphosphate moiety. The primary pathway in the wild-type enzyme leads to the formation of (−)-limonene (dark gray) and smaller amounts of bicyclic and acyclic products (light gray). Other products shown in this figure are released by mutant enzymes.Because of the widespread occurrence of (−)-(4S)-limonene throughout the plant kingdom, the relative simplicity of catalysis, the comparatively well-understood mechanism, and the availability of a crystal structure at 2.7-Å resolution (6), (4S)-limonene synthase [(4S)-LS] has become a model for understanding catalysis by class I TPSs. The (4S)-LS gene is translated into a preprotein with an N-terminal targeting sequence for transport to the plastidial envelope membrane (7). Based on results obtained with a series of truncated (4S)-LS mutants, the most likely cleavage site of the preprotein was determined to be at (or near) a tandem pair of arginines (R58 and R59 of the preprotein) (8). If truncated beyond R58, (4S)-LS shows no cyclization activity with GPP but is fully functional with linalyl diphosphate (LPP) as a substrate. Interestingly, the crystal structure of (4S)-LS, complexed with the nonhydrolyzable LPP analog 2-fluorolinalyl diphosphate (FLPP), does not provide evidence for a direct interaction of R58 or R59 with the substrate (9). However, weak interactions, in particular electrostatic interplay of R58 with E363 and hydrogen bonding between R59 and V357/Y435, appear to anchor the N-terminal strand to the outside of the active site, thereby possibly supporting the closure of the active site, while not interfering with the binding of the substrate and intermediates (9). The reaction mechanism of (4S)-LS from spearmint (Mentha spicata L.) has been studied in some detail. The catalytic cascade involves the migration of the diphosphate group to C3 of the geranyl cation (from the original C1) to afford enzyme-bound (3S)-LPP as an intermediate (10) (Fig. 1). Following C2–C3 rotation, the diphosphate is released again to generate a linalyl cation. The proximity of the C6–C7 double bond to the positive charge facilitates an anti-S_N′ cyclization to form the (−)-(4S)-terpinyl cation (11, 12) (Fig. 1). Deprotonation from the adjacent methyl group (C8 of the original GPP) yields the monocyclic olefin (−)-(4S)-limonene as the major product (96%). Side products are obtained by either premature deprotonation of the geranyl cation to generate the acyclic olefin myrcene (2%) or additional cyclization of the terpinyl cation (between C2 and C7 of the original GPP) to produce, after deprotonation, a mixture of the bicyclic olefins α- and β-pinene (2%) (10, 13) (Fig. 1).Using synthetic analogs of GPP with a chiral methyl group at C9 (carrying ¹H, ²H, and ³H), Coates et al. demonstrated that the final deprotonation occurs predominantly by re-facial anti-elimination (12). It has been hypothesized that the diphosphate anion released from the prenyl diphosphate substrate may act as the catalytic base for this deprotonation in various prenyl diphosphate synthases and terpene synthases, but no direct evidence is available to date (3, 14–17) and it seems highly unlikely in the present case due to spatial considerations. Here we present a comprehensive dataset to map the active site of spearmint (4S)-LS and evaluate residues with potential roles in stabilizing carbocation intermediates of the reaction cycle. We also discuss the broader implications of our findings for understanding catalysis and reaction termination by this fascinating class of enzymes.     

The prostacyclin analogue cicaprost inhibits metastasis of tumours of R 3327 MAT Lu prostate carcinoma and SMT 2A mammary carcinoma

Summary Investigations on mechanisms of metastatic tumour spread revealed a role for compounds that inhibit tumour dissemination at the time of hematogenous dissemination. The platelet aggregation inhibitor prostacyclin and its stable analogues were shown to inhibit tumour-cell-induced platelet interaction as well as tumour cell adhesive mechanisms. This study concentrates on the effect of the stable prostacyclin analogue cicaprost: 5-{(E)-(1S, 5S, 6S, 7R)-7-hydroxy-6-[(3S, 4S)-3-hydroxy-4-methylnona-1,6-diinyl]-bicyclo[3,3,0]octan-3-ylidene}-3-oxapentanoic acid (Schering AG), as cyclodextrin clathrate, on spontaneous tumour metastases of two different carcinomas of the rat. In Cop rats bearing spontaneously metastasizing R 3327 MAT Lu prostate carcinomas, cicaprost (1.0 mg/kg p.o. daily) inhibited the number of lung metastases by about 80%, whereas the lower doses (0.1 and 0.5 mg/kg) exhibited borderline efficacy. In female Wistar-Furth rats bearing s.c. implanted SMT 2A mammary carcinomas, spontaneously metastasizing into regional lymph nodes and lungs, cicaprost (0.1, 0.5 and 1 mg/kg) p.o. daily exhibited a dose-dependent inhibition of the number of lung metastases. Five out of ten animals treated by 1 mg/kg were free of visible lung metastases. The weight of the axillary lymph node was significantly reduced by the 1 mg/kg dose of cicaprost, whereas lower doses has no effect on the weight of the lymph nodes. The growth of the primary tumour was not influenced by cicaprost in the R 3327 MAT Lu prostate carcinoma nor in the SMT 2A mammary carcinoma in the dose range tested. In conclusion, the stable prostacyclin analogue cicaprost exhibits a strong antimetastatic action in two metastasizing tumours of the rat and interferes with the steps not only of haematogenous, but also of lymphogenous metastasis.     

ASP4000, a slow-binding dipeptidyl peptidase 4 inhibitor, has antihyperglycemic activity of long duration in Zucker fatty rats

ASP4000 ((2S)-1-{[(1R,3S,4S,6R)-6-hydroxy-2-azabicyclo[2.2.1]hept-3-yl]carbonyl}-2-pyrrolidinecarbonitrile hydrochloride) is a novel, potent and selective dipeptidyl peptidase 4 (DPP IV, EC 3.4.14.5) inhibitor (Keiko Tanaka-Amino et al. in Eur J pharmacol 59:444–449, 2008). The aim of the present study was to characterize the kinetic profile of and identify the long duration effect of the antihyperglycemic activity of ASP4000. ASP4000 was found to inhibit human recombinant DPP4 activity with a K _i of 1.05 nM, a k _on value of 22.3 × 10⁵ M⁻¹ s⁻¹, and a k _off of 2.35 × 10⁻³ M⁻¹ s⁻¹, with higher affinity than that of vildagliptin. The kinetic studies indicate that both the formation and dissociation of ASP4000/DPP4 complex were faster than those of vildagliptin, and that ASP4000 slow-bindingly inhibits DPP4 with a different mode of inhibition than vildagliptin. In addition, ASP4000 augmented the insulin response and ameliorated the glucose excursion during the oral glucose tolerance test in Zucker fatty rats at 4 h post dosing. ASP4000 is expected to be a promising, long duration DPP4 inhibitor for type 2 diabetes.     

<Superscript>1</Superscript>H NMR spectra of methylcitric acid in urine

Summary Methylcitric acid (3-carboxy-3-hydroxy-2-methyl-pentanedioic acid; MCA) is elevated in body fluids of patients with propionic acidaemia (PA; McKusick: 232000, 232050), methylmalonic aciduria (MMA; McKusick: 251000, 251120) and multiple carboxylase deficiency (McKusick: 253260, 253270). MCA molecule has two stereogenic centers so that it can occur in the form of four stereoisomers. Only two stereoisomers of MCA, (2S, 3S) and (2R, 3S), were found in human urine. They have different spectra and can be easily distinguished. We performed measurements of ¹H, ¹³C NMR and gs-HMQC (gradient-selected Heteronuclear Multiple Quantum Coherence) on the samples of unprocessed urine of 6 patients suffering from propionic acidaemia and methylmalonic aciduria and identified the protons of MCA diastereoisomers, 3-hydroxypropionic acid and methylmalonic acid. Having used NMR spectroscopy to identify stereoisomers of MCA in urine, we can conclude that this technique gives the possibility of analysing MCA diastereoisomers in body fluids. Electronic Supplementary Material Supplementary material is available for this article at Communicating editor: Marinus Duran     

Inducible nitric oxide synthase controls experimental Strongyloides infection

Infection with Strongyloides sp. induces a host immune response, predominantly the Th2 type, that is able to eliminate the parasite. However, little is known about the role of the nitric oxide (NO) mediator, induced by the enzyme nitric oxide synthase (NOS), in strongyloidiasis. Therefore, in this study, we investigated the immune response of mice genetically deficient in the enzyme inducible nitric oxide synthase (iNOS^?/?), infected with Strongyloides venezuelensis. C57BL/6 wild‐type (WT) and iNOS^?/? mice were individually inoculated by subcutaneous injection of 3000 S. venezuelensis L3 larvae. In the absence of iNOS, mice were more susceptible to the infection than WT animals, in which the parasite was completely eliminated. The overall production of cytokines and specific IgG, IgG1 or IgE antibodies against the parasite was significantly lowered in infected iNOS^?/? mice. The expression of iNOS was observed in the intestine of WT hosts but mainly in the wall of the parasite, despite the presence of iNOS in mice. Altogether, we concluded that iNOS expression may play an important role in the control of S. venezuelensis infection.     

Attachment of Chiral Functional Groups to Modify the Activity of New GPx Mimetics

A series of new chiral benzisoselenazol-3(2H)-ones and their corresponding diselenides bearing an o-amido function substituted on the nitrogen atom with various aliphatic and aromatic moieties were synthesized. All derivatives representing pairs of enantiomers or diastereoisomers were obtained to thoroughly evaluate the three-dimensional structure–activity correlation. First, bensisoselenazol-3(2H)-ones were synthesized by reacting 2-(chloroseleno)benzoyl chloride with an appropriate enantiomerically pure amine. Then, the Se–N bond was cleaved by a reduction–oxidation procedure using sodium borohydride and then air oxidation to obtain the corresponding diselenides. All derivatives were tested as antioxidants and anticancer agents. In general, the diselenides were more reactive peroxide scavengers, with the highest activity observed for 2,2′-diselenobis[N-(1S,2S)-(-)-trans-2-hydroksy-1-indanylbezamide]. The most cytotoxic derivative towards human promyelocytic leukemia HL-60 and breast cancer MCF-7 cell lines was N-[(1S,2R)-(-)-cis-2-hydroksy-1-indanyl]-1,2-benzizoselenazol-3(2H)-one. The structure–activity relationship of the obtained organoselenium derivatives was discussed.     

Analysis of the inhibitory effect of Ni ions on slow inward current in mammalian ventricular myocardium

In isolated papilary muscles of cats and guinea pigs, the inhibitory effect of Ni ions on the slow inward current (I_si) and on the I_si-medicated action potential (elicited after inactivation of the excitatory fast Na system of the membrane) was studied.

1. (1) Ni (1 m ) reduced the maximum I_si by about 50% and shifted the iv-relationship to weaker currents but left the time-course of I_si virtually unchanged.

2. (2) The maximum si-tail current declined to about 50% of the initial control value. τ_decay of the tail current being in the range between 35 ms and 50 ms was not altered by Ni ions. The voltage dependence of the steady-state activation (d∞) remained virtually constant and there was no shift of the d∞-curve along the voltage axis in presence of Ni.

3. (3) According to the decrease of , 1 m Ni reduced both overshoot and of the I_si-mediated action potential, and 2 m Ni induced a blockade of the I_si-mediated action potential. The inhibitory Ni effect is Ca dependent and became the weaker the higher the external Ca concentration was, indicating an interaction of Ni with Ca ions. The latter was proved to be of competitive type. Furthermore, in presence of Ni, the I_si-mediated action potential behaves no longer as a Ca electrode.

4. (4) In presence of 2 m Ni, the promoting action of catecholamines on both overshoot and of the I_si-mediated action potential depends strictly on the external Ca concentration. It never appeared at Ca concentration lower than 4 m and was the more pronounced the higher the Ca concentration was.

     

A novel mechanism of dasatinib-induced apoptosis in chronic myeloid leukemia; ceramide synthase and ceramide clearance genes

Sphingolipids are bioeffector molecules that control various aspects of cell growth, proliferation, apoptosis, and drug resistance. Ceramides, the central molecule of sphingolipid metabolism, are inducer of apoptosis and inhibitors of proliferation. Sphingosine-1-phosphate (S1P) and glucosyleceramide, converted from ceramides by sphingosine kinase-1 (SK-1) and glucosyleceramide synthase (GCS) enzymes, respectively, inhibit apoptosis and develop resistance to chemotherapeutic drugs. In this study, we examined the therapeutic potentials of bioactive sphingolipids in chronic myeloid leukemia (CML) alone and in combination with dasatinib in addition to investigate the roles of ceramide-metabolizing genes in dasatinib-induced apoptosis. Cytotoxic effects of dasatinib, C8:ceramide, PDMP, and SK-1 inhibitor were determined by XTT cell proliferation assay. Changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured using caspase-3 colorimetric assay and JC-1 MMP detection kit. Expression levels of ceramide-metabolizing genes were examined by qRT-PCR. Application of ceramide analogs and inhibitors of ceramide clearance genes decreased cell proliferation and induced apoptosis. Targeting bioactive sphingolipids towards generation/accumulation of ceramides increased apoptotic effects of dasatinib, synergistically. It was shown for the first time that dasatinib induces apoptosis through downregulating expression levels of antiapoptotic SK-1 but not GCS, and upregulating expression levels of ceramide synthase (CerS) genes, especially CerS1, in K562 cells. On the other hand, dasatinib downregulates expression levels of both GCS and SK-1 and upregulate apoptotic CerS2, −5 and −6 genes in Meg-01 cells. Increasing endogenous ceramide levels and decreasing prosurvival lipids, S1P, and GC, can open the way of more effective treatment of CML.     

Reduced glycogen synthase activity in skeletal muscle from obese patients with and without Type 2 (non-insulin-dependent) diabetes mellitus

Summary In order to evaluate the importance of a defect in insulin mediated non-oxidative glucose metabolism and glycogen synthase activity in skeletal muscles in obese subjects with and without Type 2 (non-insulin-dependent) diabetes mellitus we studied: 10 lean and 10 obese control subjects and 12 obese diabetic patients using the euglycaemic hyperinsulinaemic clamp technique (basal, 20 mU · (m²)^–1 · min^–1, 80mU·(m²)^–1·min^–1) in combination with indirect calorimetry. Muscle biopsies were taken from m. vastus lateralis at each insulin level. We found that non-oxidative glucose metabolism could be stimulated by insulin in all three groups (p<0.01). The values obtained at the highest insulin levels (around 140 U/ml) were lower in both obese groups compared to the lean control subjects (118±21, 185±31, 249±14 mg·(m²)^–1·min^–1 (p< 0.01)). Insulin stimulation of the glycogen synthase activity at a glucose-6-phosphate concentration of 0.1 mmol/l was absent in both obese groups, while activities increased significantly in the lean control subjects (19.6±4.2% to 45.6±6.8%, p< 0.01). Glycogen synthase activities at the highest insulin concentrations only differed significantly between lean control subjects and obese diabetic patients (45±7% and 31±5%, p< 0.05). We conclude that insulin resistance in peripheral tissues in obese subjects with and without Type 2 diabetes may be partly explained by a reduced insulin mediated non-oxidative glucose metabolism and that this abnormality might be due to an absent insulin stimulation of glycogen synthase in skeletal muscles. This enzyme defect is correlated to obesity itself.     

Expanding metabolism for biosynthesis of nonnatural alcohols

Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into Escherichia coli, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.     

Isolation, Structure, and Absolute Configuration of a New Natural Insect Juvenile Hormone from Manduca sexta

Two juvenile hormones are isolated from organ cultures of corpora allata of the tobacco hornworm moth, Manduca sexta Johannson, and are purified by high-resolution liquid chromatography. These are identified as methyl (2E, 6E)-(10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate, a new natural hormone, and methyl (2E,6E) - (10R,11S) - 10,11 - epoxy - 3,7,11 - trimethyl-2,6-tridecadienoate. [¹⁴C]Methionine is incorporated only into their methoxycarbonyl group. Details of the in vitro techniques and the chemical proof of structures are presented. The significance of the occurrence of a new juvenile hormone and the new techniques used for production, isolation, and identification are discussed.     

Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase.

Anthranilate synthase [chorismate pyruvatelyase (amino-accepting), E.C.4.1.3.27] catalyzes the formation of anthranilate (o-aminobenzoate) and pyruvic acid from chorismate and glutamine. A mutant form of the enzyme from Salmonella typhimurium accumulates a compound that we had isolated and identified as trans-6-amino-5-[(1-carboxyethenyl)-oxy]-1,3- cyclohexadiene-1-carboxylic acid, commonly called aminodeoxyisochorismate (ADIC). Here we report that ADIC is formed by a reversible, Mg(2+)-dependent ADIC synthase activity of anthranilate synthase that can be functionally uncoupled from a Mg(2+)-dependent ADIC lyase activity of the enzyme by single amino acid substitutions in the TrpE subunit of the anthranilate synthase complex of S. typhimurium. Both of the component activities of the enzyme are sensitive to feedback inhibition by L-tryptophan. Purified ADIC is quantitatively converted to anthranilate and pyruvic acid by the ADIC lyase activity of wild-type anthranilate synthase. ADIC also serves as a substrate for the formation of chorismate by the enzyme in the absence of glutamine and (NH4)2SO4. The rate of ADIC formation by the mutant enzyme and the steady-state parameters for ADIC utilization by the wild-type enzyme are consistent with a role for ADIC as an enzyme-bound intermediate that does not accumulate during the course of the anthranilate synthase reaction. The altered catalytic specificity of mutant anthranilate synthase enzymes suggests a potential role for ADIC in secondary metabolism.     

Insights into the reactivation of cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every ≈2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH^CT) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH^CT (_s-sMetH^CT) that offer further insight into the reactivation of MetH. The structure of _s-sMetH^CT with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of _s-sMetH^CT with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.     

Enantiomers of flurbiprofen can distinguish key pathophysiological steps of NSAID enteropathy in the rat

Background—Non-steroidal anti-inflammatorydrugs (NSAIDs) cause gastrointestinal damage by a non-prostaglandin(PG) dependent "topical" action and by inhibiting cyclooxygenase.
Aims—To discriminate between these two effects bystudying some key pathophysiological steps in NSAID enteropathyfollowing administration of (R)- and(S)-flurbiprofen, the racemic mixture, and an uncoupler, dinitrophenol.
Methods—The effects of dinitrophenol, racemic,(R)-, and (S)-flurbiprofen on mitochondriawere assessed in vitro and on key pathophysiological features of smallintestinal damage in vivo (ultrastructure by electron microscopy,mucosal prostanoid concentrations, intestinal permeability,inflammation, and ulcer count) in rats.
Results—All the drugs uncoupled mitochondrialoxidative phosphorylation in vitro, caused mitochondrial damage invivo, and increased intestinal permeability. Dinitrophenol and(R)-flurbiprofen caused no significant decreases inmucosal prostanoid concentrations (apart from a decrease in thromboxane(TX) B₂ concentrations following (R)-flurbiprofen) while racemic and (S)-flurbiprofen reduced mucosal prostanoids significantly (PGE,TXB₂, and 6-keto-PGF_1α concentrations by73-95%). Intestinal inflammation was significantly greater followingadministration of (S)-flurbiprofen and racemate than withdinitrophenol and (R)-flurbiprofen. No small intestinal ulcers were found following dinitrophenol or(R)-flurbiprofen while both racemic and(S)-flurbiprofen caused numerous ulcers.
Conclusions—Dinitrophenol and(R)-flurbiprofen show similarities in their actions touncouple mitochondrial oxidative phosphorylation in vitro, altermitochondrial morphology in vivo, increase intestinal permeability, andcause mild inflammation without ulcers. Concurrent severe decreases inmucosal prostanoids seem to be the driving force for the development ofsevere inflammation and ulcers.

Keywords:non-steroidal anti-inflammatory drug; enteropathy; flurbiprofen