Resorufin butyrate as a soluble and monomeric high-throughput substrate for a triglyceride lipase

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z' > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.     

Fluorescent Derivatives of Polysaccharide Dialdehyde as Substrates for Glucanases

A simple and sensitive assay method for glucanase activity was established using fluorescent polysaccharide substrates. Periodate oxidized α-glucans having dialdehyde were covalently attached to the fluorescent reagent via Schiff base formation followed by NaBH₄ reduction. Ethylenediaminonaphthalene (EDAN) was effective to produce a stable and highly fluorescent polysaccharide. EDAN derivatives of glycogen and dextran dialdehyde were useful substrates for Taka-amylase A and endodextranase, respectively. Two cellulases were shown to release water-soluble fluorescent products from the EDAN derivative of cellulose powder. Moreover, the action of the exo type of enzyme such as glucoamylase was readily distinguished from that of the endo type of enzyme because the attached EDAN prevented the attack of exo-enzyme.     

A Comparison of Substrates for Human Umbilical Vein Endothelial Cell Culture

We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time.

Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

Folate Cofactor Biosynthesis by Plasmodium berghei. Comparison of Folate and Dihydrofolate as Substrates*

SYNOPSIS. The ability of Plasmodium berghei to convert folate and dihydrofolate to folinate in vitro was investigated. Neither parasitized mouse erythrocytes nor free parasites synthesized more than trace amounts of folinate from folate under a wide variety of experimental conditions. Since disrupted cell preparations were no more efficient than whole cells, permeability barriers did not seem to be involved. However, dihydrofolate was a good substrate for the synthesis of folinate by uninfected and parasitized erythrocytes, and by free parasites. The reaction by parasitized erythrocytes required NADPH and dihydrofolate, and was inhibited by pyrimethamine, indicating the presence of dihydrofolate reductase in P. bergkei. It was postulated that P. berghei cannot utilize folate directly, since these experiments indicate that the cells lack a folate reductase. This finding is consistent with the hypothesis that plasmodia synthesize folate cofactors de novo, and do not utilize exogenous folates.     

Plant Cell Wall Carbohydrates as Substrates for Azospirillum brasiliense

Carbohydrate components (simple sugars and polysaccharides) of cell walls of pearl millet (Pennisetum americanum L., cv. Gahi) were studied as potential substrates for the root-associated diazotroph Azospirillum brasiliense Sp. 7. Simple sugars were utilized, but no evidence was obtained to support the suggestion that the polysaccharide components tested might serve as substrates for growth following hydrolysis by the associated azospirilla.     

Extracellular Metabolites of Hydrocarbon-Oxidizing Bacteria as Substrates for Sulfate Reduction

The relationship between bacterial oxidation of hydrocarbons and sulfate reduction was studied in an experimental system with liquid paraffin used as a source of organic compounds inoculated with silt taken from a reservoir. Pseudomonads dominated in the hydrocarbon-oxidizing silt bacteriocenosis. However, Rodococcusand Arthrobacteria amounted to no more than 3%. Arthrobacteria dominated the microbial association formed in the paraffin film of the model system. Sulfate-reducing bacteria were represented by genera Desulfomonas, Desulfotomaculum, and Desulfovibrio. The growth of sulfate-reducing bacteria in media containing paraffin, successive products of its oxidation (cetyl alcohol, stearate, and acetate), and extracellular metabolites of hydrocarbon-reducing bacteria was studied. The data showed that sulfate-reducing bacteria did not use paraffin or cetyl alcohol as growth substrates. However, active growth of sulfate-reducing bacteria was observed in the presence of stearate and extracellular water-soluble or lipid metabolites of Arthrobacteria.     

Amyl Acetate as a Clearing Agent for Embryonic Material

Amyl acetate is soluble in 95% alcohol and hot paraffin and produces no hardening in objects exposed to its action for prolonged periods. It may be advantageously employed as a general clearing agent and is especially recommended for refractory material. The following schedule has proven satisfactory for whole frog embryos and young tadpoles: 45 minutes to 1 hour in 95% alcohol, 24 hours in amyl acetate, rinse in toluene, 15 minutes each in three changes of paraffin, imbed. Material so treated may be sectioned at 5 μ with comparative ease.     

Organic Thiols as Organolithotrophic Substrates for Growth of Phototrophic Bacteria

Rhodopseudomonas sp. strain BB1, isolated from a coastal marine sediment, immediately metabolized mercaptomalate when grown on mercaptomalate. Sulfide was detected as an intermediate. Extracts of cells grown on mercaptomalate converted mercaptomalate or 3-mercaptopropionate to equimolar amounts of sulfide and either fumarate or acrylate, respectively. Rhodopseudomonas sp. strain BB1 gave higher growth yields on mercaptomalate than on sulfide or malate, consistent with metabolism of the carbon chain of the thiol and the liberated sulfide; i.e., the organic thiol was an organolithotrophic substrate. In contrast, Thiocapsa roseopersicina, isolated previously from a marine microbial mat, had similar growth yields on sulfide, mercaptomalate, or 3-mercaptopropionate, with fumarate or acrylate accumulation from the thiols. T. roseopersicina did not grow photoorganotrophically on fumarate or acrylate, and the thiols were only a source of sulfide for photolithoautotrophic growth.     

Scaffold-Based Multi-nanoparticle Clusters as Tunable LSPR Substrates for SERS Applications

Tunable local surface plasmon resonance (LSPR) enhancement properties of scaffold-based multi-nanoparitcle clusters were investigated using finite-difference time-domain (FDTD) method with calculated optical spectra, near-field distribution, and average enhancement of hybrid nanostructures as slab/nanoparticls, cylinder/nanoparticles, and sphere/nanoparticles. Focusing on influence factors including surface curvature, coupling effect, and decorated particle number, several models were built for further understanding on the dominate contribution in complicate multi-particle nanostructure and to explore their potential for plasmonic enhancement applications such as surface-enhanced Raman spectroscopy (SERS), solar cells material, LSPR sensor, and nanoantenna.     

Similarities between l-Phenylalanine and m-Fluorophenylalanine as Substrates for l-Phenylalanine Ammonia-Lyase

The ability of l -phenylalanine ammonia-lyase (E.C. 4.3.1.5) to metabolize dl -m-, dl -o- and dl -p-fluorophenylalanine in Avena sativa has been examined. Although all three amino acid analogues served as substrates for this enzyme, there was a marked difference in the behavior of the meta isomer from that of the para and ortho species. The Michaelis constant for the meta analogue was similar to that obtained for the natural substrate, l -phenylalanine, but distinct from the kinetic data for the para and ortho isomers. In addition, in vivo analyses demonstrated that both l -phenylalanine and the meta-fluoro-derivative served to protect against chlorogenic acid loss, whereas previous studies have shown that the para and ortho species depressed levels of this phenolic derivative. Finally, treatment of coleoptile apices with either the meta isomer or l -phenylalanine reversed dl -p-fluorophenylalanine stimulated growth and attendant reduction in chlorogenic acid content. These findings provide further clarification of the effects of fluorophenylalanines upon l -phenylalanine ammonia-lyase mediated biosynthesis of low molecular weight phenols in Avena.     

Choline and N,N-Dimethylethanolamine as Direct Substrates for Methanogens

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.     

Derivatives of L-Adenosine and L-Guanosine as Substrates for Human Deoxycytidine Kinase

Abstract

A series of analogues of L-adenosine and of L-guanosine, including ß-L-dA, ß-L-Ado, ß-L-araA, and ß-L-dG, have been shown to be substrates of human deoxycytidine kinase thus demonstrating the complete lack of enantioselectivity of this enzyme.