Newly discovered polyamine, 2-hydroxyspermidine, in Pseudomonas acidovorans.

A previously unknown hydroxylated polyamine has been recovered from Pseudomonas acidovorans 29. It has been identified as 2-hydroxyspermidine, N4-(3-aminopropyl)-1,4-diaminobutane-2-ol, by its chromatographic behavior, electrophoretic mobility, and reaction with metaperiodate. It can be synthesized enzymatically from 2-hydroxyputrescine by cell-free preparations from Escherichia coli or P. acidovorans 29 which contain propylamine transferase. It is interesting to note that the naturally occurring compound is the 2-hydroxyspermidine and not the 3-hydroxyspermidine, N1-(3-aminopropyl)-1,4-diaminobutane-2-ol, indicating that the propylamine transferase reacts preferentially with the amine distal to the hydroxyl group. A mixture of 2- and 3-hydroxyspermidines and hydroxyspermine was synthesized by reacting acrylonitrile with 2-hydroxyspermidine and catalytic reduction of the products with hydrogen. N-(gamma-aminopropyl)-beta-alanine, used to help identify the hydroxyspermidines, was synthesized from N-(3-aminopropyl)-3-aminopropanenitrile by hydrolysis with 10% NaOH.     

Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.     

Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

Rat pancreatic islets contain a Ca2+-activated and thiol-dependent transglutaminase (EC 2.3.2.13) comparable in activity with that found in rat liver, lung and spleen. The Ca2+-dependence of this enzyme is such that half-maximal velocity was obtained in the region of 40 microM. Preincubation of rat islets with primary-amine substrates of transglutaminase (monodansylcadaverine, methylamine, ethylamine, propylamine and cystamine) led to an inhibition of glucose-stimulated insulin release by these amines. Kinetic analysis of the competitive substrates methylamine, monodansylcadaverine, propylamine and ethylamine for their ability to inhibit islet transglutaminase activity indicated a potency that matched their ability to inhibit glucose-stimulated insulin release. When these amines were tested for their effects on glucose-stimulated protein synthesis and glucose utilization, the most potent inhibitor of insulin release, monodansylcadaverine, had no effect on either process at 100 microM. The amines cystamine, ethylamine, methylamine and propylamine had variable effects on these metabolic processes. For ethylamine, methylamine and propylamine, concentrations were found which inhibited glucose-stimulated insulin release in a manner which was found to be independent of their effects on either glucose oxidation or protein synthesis. Primary amines may therefore inhibit insulin release through their incorporation by islet transglutaminase into normal cross-linking sites. A role for protein cross-linking in the secretory mechanism is suggested.     

Effect of primary aliphatic amines on oxidative phosphorylation in rat liver mitochondria

The dynamics of primary aliphatic amines (ethylamine, propylamine) effects on the processes of oxidative phosphorylation in rat liver mitochondria was estimated. The inhibiting action of ethylamine and propylamine on the oxidative phosphorylation processes in the rat liver mitochondria was revealed.     

Synthesis and biological evaluation of novel propylamine derivatives as orally active squalene synthase inhibitors

Squalene synthase inhibitors are potentially superior hypolipidemic agents. We synthesized novel propylamine derivatives, as well as evaluated their ability to inhibit squalene synthase and their lipid-lowering effects in rats. 1-Allyl-2-[3-(benzylamino)propoxy]-9H-carbazole (YM-75440) demonstrated potent inhibition of the enzyme derived from HepG2 cells with an IC(50) value of 63 nM. It significantly reduced both plasma total cholesterol and plasma triglyceride levels following oral dosing to rats with a reduced tendency to elevate plasma transaminase levels.     

Toward a PKB inhibitor: modification of a selective PKA inhibitor by rational design

Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.     

A high-throughput screen for detection of compound-dependent phosphodiester bond cleavage at abasic sites

We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA. Finally, we characterized the phosphodiester backbone cleavage at the abasic site using denaturing gel electrophoresis. This study provides a useful template for researchers seeking to rapidly identify new artificial nucleases.     

UK-73,093: A non-peptide neurotensin receptor antagonist

UK-73,093 was identified in a screening program as a compound able to displace [³H]-neurotensin from its bovine brain receptor. We describe the discovery of this compound, species differences in receptor affinity and its characterization as a functional neurotensin antogonist in vitro and in vivo.     

Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17β

We reported that spermidine and spermine pools in the uterus both doubled within 24h after oestradiol administration to castrated rats (Russell & Taylor, 1971). Now we have studied the enzymic synthesis of spermine (by spermidine-dependent S-adenosyl-l-methionine decarboxylase) and find that the activity of the enzyme(s) involved is elevated soon after hormone administration. Enzyme activity is increased within 4h and is five times that of controls within 24h. Cycloheximide or actinomycin D administered at the time of oestradiol injection completely blocked the increase in enzyme activity. The enzyme involved in spermine synthesis, S-adenosyl-l-methionine decarboxylase, with S-adenosyl-l-methionine and spermidine as required substrates, was partially purified on Sephadex and DEAE-cellulose columns. The decarboxylation of S-adenosyl-l-methionine could not be separated from the transfer of a propylamine moiety from the decarboxylated S-adenosyl-l-methionine to spermidine to form spermine. We were unable also to separate this system from the enzyme that formed spermidine when S-adenosyl-l-methionine and putrescine are used as substrates. Spermidine-stimulated S-adenosyl-l-methionine decarboxylase has an apparent half-life of 60min, identical with the half-life reported for putrescine-stimulated S-adenosyl-l-methionine decarboxylase. These results strongly suggest that the same enzyme(s) operate in the synthesis of both spermidine and spermine.     

Improved synthesis and pharmacologic activity of the enantiomers of a new benzofurane type antiarrhythmic compound

An improved synthesis of the enantiomers of the new benzofurane-type antiarrhythmic compound 1 is described, which makes use of the enantiomerically pure mbe-lactol. Thus, acylation of the benzofurane 4 with acetic anhydride and subsequent bromination gave the bromoacetyl-derivative 6 , which, after reduction with LiAlH₄, was protected with mbe-lactol to give a mixture of the diastereomers 8A and 8b . After separation via column chromatography assignment of absolute configuration was carried out using a well-established NMR- method. Reaction with propylamine and cleavage of the protective group gave (R)- 1 and (S)- 1 , respectively. Enantiomeric purity was confirmed using a direct HPLC method with rsp-cyclodextrine as stationary phase. Pharmacological investigations on isolated guinea pig heart muscle preparations showed that GE 68 and its two enantiomers did not significantly differ from each other with regard to their negative inotropic, negative chronotropic, and lack of β-adrenoceptor blocking action. In contrast, the reference drug propafenone was equally potent in its negative inotropic and chronotropic activity as GE 68, but additionally showed a weak β-adrenoceptor blocking activity. © 1994 Wiley-Liss, Inc.     

Isolation and identification of 5-methyl-imidazolin-4-one derivative as glyceraldehyde-derived advanced glycation end product

We isolated and identified the glyceraldehyde-derived advanced glycation product (AGE) formed from glyceraldehyde and N(alpha)-acetylarginine. A major product was identified as N(alpha)-acetyl-N(delta)-(5-methyl-imidazolin-4-one-2-yl)-ornithine. The compound has been reported as methylglyoxal-derived AGE, MG-H1. This study suggests that MG-H1 is formed through both glyceraldehyde-related and methylglyoxal-related pathways. There is a possibility that MG-H1 becomes an index of injury to glyceraldehyde and methylglyoxal-related enzymes.     

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [¹⁴C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine.When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly.The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation.Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific.Degradation of endocytosed ¹²⁵I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes.The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.     

N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily

We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.     

弯齿琵甲防御分泌物的化学成分测定及抗菌活性分析

弯齿琵甲Blaps femoralis Fischervon Waldheim是中国北方的优势土壤昆虫,其防御腺分泌物用于御敌和引起本种群体的集结,体外具有一定毒性,民间将该虫作为药用昆虫资源利用年代久远。由该虫防御腺体获得分泌物并经气相色谱-质谱联用技术(GC-MS)分析,共计获得14种小分子化合物,其中以2-甲基苯醌、1-十三碳烯和2,4-二甲基-3-呋喃乙酮含量较高,苯醌、苯二酚、3,5-二羟基甲苯和十二烷醛等小分子化合物次之。研究证实该分泌物具有较明显的抑菌能力。研究结果为今后进一步深入开展弯齿琵甲的防御腺化学分泌物天然产物的研究积累了工作。     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain

Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocystemine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88 000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km values for S-methyladenosylhomocysteamine was 0.6 microM and about 60 microM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor. Both the reaction products, spermine and 5'-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosylhomocysteamine (Ki value of about 0.3 microM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 mM). Spermine synthase had no requirements for metal or other cofactors.     

Glycerol 1,2-cyclic phosphate in centric diatoms. Observation by 31P NMR in vivo, isolation, and structural determination

We have isolated and identified glycerol 1,2-cyclic phosphate as the compound responsible for a unique prominent resonance at 19.1 ppm in the 31P NMR spectrum in vivo of four species of centric diatoms, where it is responsible for 15 to 30% of the total signals from organophosphates. This appears to be the first observation of this compound as a major metabolite. It was not detectable in nine other species of algae, including four species of pennate diatoms.     

Ciliopathies with skeletal anomalies and renal insufficiency due to mutations in the IFT-A gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.     

Formation of compound I and compound II ferryl species in the reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

The formation of ferryl heme (Fe(IV) = O) species, i.e., compound I and compound II, has been identified as the main intermediates in heme protein peroxidative reactions. We report stopped-flow kinetic measurements which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce ferryl intermediates compound I and compound II. Compound I appears relatively stable displaying an absorption at 648 nm. The rate constant value (k'(2)) for the conversion of compound I to compound II is 3.0 x 10(-2) s(-1), more than 100 times smaller than that reported for myoglobin. The rate constant value for the oxidation of the ferric heme (k'(12) + k'(13)) is 2.0 x 10(2) M(-1) s(-1). These values suggest an alternate route for the formation of compound II (by k'(13)) avoiding the step from compound I to compound II (k'(2)). In HbI from L. pectinata the stabilization of compound I is attribute to the unusual collection of amino acids residues (Q64, F29, F43, F68) in the heme pocket active site of the protein.     

31P NMR Spectra of Alkalophilic Bacillus No. A-59

Some advanced cancer patients suffer from pungent sulfury malodor. To determine the chemical identity of the odorant, we performed gas chromatography-mass spectrometry-olfactometry analysis of volatiles from fungating cancer wounds. We identified the source of the characteristic smell as dimethyl trisulfide, a compound that is known to be emitted from some vegetables and microorganisms. Controlling the production of dimethyl trisulfide should improve quality of life of patients.