Determination of the absolute stereospecificity of the enzymatic reduction of NAD+ analogs by means of the primary acid cyclization reaction

NADH, (3-acetylpyridine)ADH, (3-thionicotinamide)ADH and (pyridine-3-aldehyde)ADH were labeled with deuterium by the B specific enzyme lipoyl dehydrogenase. The primary acid rearrangement products, α-O^2′-6B cyclotetra-hydropyridine adenine dinucleotides, of the respective reduced coenzyme analogs were synthesized by incubation at pH 2–2.5. Analysis of the spin-spin coupling constants of the 220 MHz proton magnetic resonance spectra establishes that the structure of the ribose-tetrahydropyridine linkage of these analogs is the same as that of the primary acid product of NADH. All four specifically labeled dinucleotides possess the identical absolute configuration at the C4 position with respect to the deuterium label, establishing that the three coenzyme analogs are reduced by lipoyl dehydrogenase with the same absolute stereospecificity as NAD⁺. The primary acid reaction is described as a general method for the determination of the absolute configuration at the C4 position of specifically labeled β-1,3-substituted, 1,4 dihydropyridine nucleotides.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

The effect of a bromide leaving group on the properties of nitro analogs of the duocarmycins as hypoxia-activated prodrugs and phosphate pre-prodrugs for antitumor therapy

Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sulfonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic cytotoxicity ratios (HCRs) of 52-286 in HT29 cells and 41-43 in SiHa cells. These values compare well with a related previously reported chloride analog. The corresponding more water soluble phosphate pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated activity providing statistically significant hypoxic log(10) cell kills of 0.87-2.80 at non-toxic doses, matching or proving superior to those of their chloride analogs.     

Properties of 4-ethenyl and 4-ethynyl analogs of pyridoxal phosphate and their reactions with the apo form of asparatate aminotransferase.

The binding to apoaspartate aminotransferase of analogs of pyridoxal phosphate bearing vinyl, cis- and trans-methylvinyl, and ethynyl groups in place of the fomyl group of the coenzyme has been studied. Details of synthesis of the ethynyl compound are given. The absorption spectra of all of the compounds have been analyzed and pKa values have been determined. The positions of the absorption bands can be related to those of pyridoxine but with bathochromic shifts induced by the ethenyl and ethynyl groups. However, this shift is almost completely lacking for the cis-methylvinyl compound suggesting nonplanarity of the molecule. Binding of the analogs to the apoenzyme is accompanied by a strong bathochromic shift which, from a study of solvent effects on the free analogs, appears to indicate a hydrophobic environment on the enzyme. Nevertheless, the analogs are bound as dipolar ions exclusively. Binding is accompanied by a distinct perturbation of the protein spectrum in the aromatic region. An effect on the spectrum of 1 or more tryptophan residues is indicated. Bands of the bound analogs exhibit positive circular dichroism except for that of the 4-vinyl analog. The 4-ethynyl analog reacts in a more complex way, giving at least two successive products in addition to the initial complex. The final product is reducible by sodium borohydride, is released from the enzyme by boiling, and appears to have the properties of a Schiff base. We postulate that the addition of an amino group of the enzyme to the ethynyl group is followed by tautomeric rearrangement to a Schiff base in which the ring is in a p-quinonoid structure.     

Synthesis and disulfide structure determination of porcine endothelin: an endothelium-derived vasoconstricting peptide

All disulfide analogs (types A, B and C) of porcine or human endothelin, a 21-amino acid peptide having two intramolecular disulfide bonds, were synthesized and their retention times on HPLC were compared with that of natural endothelin. One of the analogs (type A) having disulfide bonds between positions 1 and 15 and between 3 and 11 was found to be identical with natural endothelin. Random oxidation of fully reduced endothelin formed a mixture of type A and B in a ratio of 3:1, with almost none of type C, which has disulfide bonds between positions 1 and 3 and between 11 and 15. Type A endothelin was also synthesized by the segment condensation procedure in solution applying our maximum protection strategy. This product was found to have full vasoconstricting activity in rat pulmonary artery ring preparations; the potency was as high as that of the natural product.     

The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae.

The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae. This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis. Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site. Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally. We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121. Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth. Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability. In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability. In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication. The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication.     

Immobilized artificial membrane chromatography: supports composed of membrane lipids

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.     

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.     

Uptake of biogenic amines by glial cells in culture I. A neuronal-like transport system for serotonin

Rat C6 astrocytoma cells take up serotonin (5HT) via a high affinity carrier mediated system with Km of 1 micromolar, and a second component of lower affinity. This high affinity 5HT transport system is rapid, concentrative, and highly sodium and temperature dependent. Chlorimipramine and Lilly 110140 preferentially block the glial 5HT but not NE uptake. This preferential inhibition has previously been shown for synaptosomes and brain slices. Norepinerphrine (NE) and to a lesser extent dopamine (DA) block the glial 5HT uptake, suggesting a partial overlap between the catecholamine and indoleamine glial carrier systems. 5-Hydroxy but not 6-hydroxy dopamine inhibits the high affinity 5HT transport in glia. A variety of ring hydroxylated indoleamine analogs block this glial 5HT transport; of the compounds tested, 5, 7 dihydroxytryptamine is the least effective inhibitor. Phenylethylamine (PEA) and its 0-methylated derivatives block synaptosomal and glial 5HT transport equally well. These observations suggest that cultured C6 cells used as models of glia possess a 5HT transport system which kinetically and pharmacologically resembles a neuronal 5HT transport system.     

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.     

Cytotoxicity and topoisomerase I/II inhibition of glycosylated 2-phenyl-indoles, 2-phenyl-benzo[b]thiophenes and 2-phenyl-benzo[b]furans

A panel of glycosylated DNA binding agents (1-12) designed as functional anthracycline mimics was screened against three solid-tumor cell lines (MCF-7, HT 29 and HepG2/C3A) and three non-tumor cell lines by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell viability assay. Several compounds showed better in vitro cytotoxicity and selectivity against MCF-7 cells than daunomycin and doxorubicin, two known DNA binding agents that are clinically-used anti-cancer agents. Although the selectivity for HT 29 and HepG2/C3A cells is generally lower, the IC₅₀ values of some analogs against these two cancer cell lines were of the same magnitude as doxorubicin. Because there was no correlation between DNA binding affinity and cytotoxicity, and because topoisomerase (Topo) inhibition is another biological mechanism of action of most anthracycline drugs, Topo I/II inhibition assays with 1-12 were performed. Some of the compounds showed strong inhibition against these enzymes at 100 ??M, but there was no clear correlation between cytotoxicity and Topo I/II inhibition ability. Topo I/II inhibition mode assays were also performed, which verified that these compounds are topoisomerase suppressors, not poisons. Based on these results, we conclude that although DNA binding and/or topoisomerase inhibition may contribute to the observed cytotoxicity of 1-12, other mechanisms of action are also likely to be important.     

Genetic approaches for controlling ratios of related polyketide products in fermentation processes

Simple acyl thioesters are used as precursors for both the initiation and elongation steps in polyketide biosynthetic processes. Several structurally related polyketide products are sometimes made in these processes. These analogs are typically generated by a combination of two factors: availability of structurally similar biosynthetic precursors, and biosynthetic enzymes unable to effectively discriminate between them. Often, only one polyketide product is desired from a fermentation process, requiring a method to control the ratio of these different analogs. Preferential production of one desired analog is accomplished using random mutagenesis and manipulation of fermentation conditions. A genetic enzymatic understanding of polyketide biosynthesis, as well as the pathways that provide the relevant precursors, allows for a rational and more contemporary approach for control of analogs produced in fermentation processes. This approach involves genetic manipulation of either the pathways that provide pools of the acyl CoA thioester precursors, or the function/specificity of the appropriate biosynthetic enzymes. Reviewed herein are three such examples where these approaches have been carried out successfully with polyketide biosynthetic processes. Journal of Industrial Microbiology & Biotechnology (2001) 27, 368–377. Received 01 March 2001/ Accepted in revised form 08 August 2001     

Formation and properties of Langmuir and Gibbs monolayers: a comparative study using hydrogenated and partially fluorinated amphiphilic derivatives of mannitol

The synthesis and surface behavior of a series of nine new hydrogenated nonionic surfactants and their fluorinated analogs, derived from D-mannitol are described. Adsorption monolayers (Gibbs monolayers) were studied by surface pressure (H) measurements as a function of time. For the spread monolayers (Langmuir monolayers), the measurements of surface pressure versus molecular area (A) were performed. For the most hydrophobic amphiphiles at low concentrations, the adsorption at the air/water interface from the bulk solution required extremely long times to attain equilibrium. The A values for two compounds which could be studied in both adsorbed and spread monolayers provided data allowing a direct comparison of the properties of the two types of films formed at the air/water interface. In spite of different mechanisms of formation of Langmuir and Gibbs monolayers, their characteristic parameters were identical, proving the equivalence of these two types of structures.     

Dynamics of the active site architecture in plant-type ferredoxin-NADP reductases catalytic complexes

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP⁺ reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP⁺ coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor–acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.     

Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.     

Specialization and noncompetitive resource partitioning among sponge-eating dorid nudibranchs

Summary Habitat and food resource partitioning ecologically isolate six species of Pacific Northwest dorid nudibranchs. Food resources along a gradient of sponge skeletal structure are partitioned between two exploitive guilds. The guilds are characterized by mutually exclusive predatory and digestive adaptations which allow efficient processing of sponges with poorly- or well-organized skeletons. Habitats along a depth gradient are partitioned within guilds. For a dorid species, feeding rates, extraction efficiencies and food quality are virtually identical for sponges with the appropriate extreme (either poorly- or well-organized) and intermediate skeletal organizations, but growth and reproductive rates supported by the former prey type are twice those supported by the latter prey type. Prey types with the appropriate extreme skeletal organization are thus optimal food and specialization to that food is expected. When two divergent specialist species co-occur, food resource partitioning is demonstrable but arises from self-stabilizing specializations to maximize net energy accumulation and not from competitive interactions. Habitat partitioning is viewed as arising from prey distributions and dorid physiological tolerances. This system represents an example of morphological and behavioral specializations giving rise to resource partitioning as opposed to competition giving rise to resource partitioning and subsequent morphological and behavioral specializations.     

Studies on the mechanism of adenosylcobalamin-dependent ribonucleotide reduction by the use of analogs of the coenzyme.

A series of 17 analogs of 5'-deoxy-5'-adenosylcobalamin(adenosylcobalamin) have been synthesized with modifications in the base or ribose moiety of the nucleoside ligand. These analogs have been examined for their effects on reactions catalyzed by the ribonucleotide reductase of Lactobacillus leichmannii. All the analogs are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme, and hence all are bound to the catalytic site. Only the 3-beta-D-ribofuranosyladenine analog (isoadenosylcobalamin) showed substantial activity as a coenzyme in ATP reduction, giving a rate of 59% of that obtained with the adenosylcobalamin. Lesser rates of reduction were obtained with nebularyl-, 2'-deoxyadenosyl-, tubercidyl-, isopropylideneadenosyl-, L-adenosyl-, and ara-adenosylcobalamin, coenzyme activity decreasing in that order. Other analogs showed no significant coenzyme activity. The rate of hydrogen exchange into water from the 5'-methylene group of the nucleoside ligand appeared to parallel the coenzyme activity in those analogs examined, but only the four cobalamins with highest coenzyme activity (adenosyl, isoadenosyl, nebularyl, 2'-deoxyadenosyl) gave detectable amounts of "active coenzyme B12," THe rapidly formed paramagnetic intermediate of ribonucleotide reduction. The enzyme system produced the slowly formed paramagnetic species characterized by a doublet EPR spectrum only with adenosyl- and isoadenosylcobalamin. By contrast the enzymic degradation of analogs to cob(II)alamin and 5'-deoxynucleoside occurred not only with those analogs active as coenzymes and in the exchange reaction but also with a number of coenzymically inactive analogs, and the rate of degradation was unrelated to the rate of ribonucleotide reduction for those analogs with coenzyme activity.     

Histochemical classification based upon reaction types and its application to carbohydrate histochemistry

Summary A method of classification is presented, which divides histochemical visualization reactions into categories based on general reaction types. This scheme is dependent upon the reaction between two elements, the substrate and the probe. The substrate represents a tissue component(s) with one or more reactive groups that can combine directly with the probe. Alternatively, the substrate reactive groups are chemically modified (activation) with a suitable reagent before reaction with the probe. Probes are of three types: those that yield a coloured product, those that result in a colourless product, and those that produce a coloured product only after a further reaction.Methods used in carbohydrate histochemistry are divided into one, two and three probe reactions. Two probe reactions are further subdivided into sequences involving one or two coloured products (one and two dye sequences); three probe reactions into sequences involving one, two or three coloured products (one, two and three dye sequences). This classification permits the rationalization and organization of methods, and provides a framework for the examination of existing methods and the development of new ones.