Autoreceptor-mediated inhibition of 3H-5-hydroxytryptamine release from rat brain cortex slices by analogues of 5-hydroxytryptamine

Rat brain cortex slices preincubated with ³H-5-hydroxytryptamine (³H-5-HT) were superfused with physiological salt solution containing paroxetine, an inhibitor of 5-hydroxytryptamine (5-HT) uptake. The effects of various indolethylamines on the electrically evoked tritium overflow (containing 66.3% unmetabolized ³H-5-HT) were investigated (the percentage of unmetabolized ³H-5-HT was not altered by the indolethylamines or metitepin). 6,7-Dihydroxytryptamine (6,7-DHT) did not affect the stimulation-evoked tritium overflow, whereas the latter was inhibited by the other tryptamine derivatives investigated; when the compounds were compared to each other on the basis of their inhibitory potencies the following rank order was obtained: unlabelled 5-HT > 5-methoxytryptamine > 4-HT > 6-HT > 5,6-DHT > tryptamine > 7-HT > 5,7-DHT. The inhibitory effects of these compounds were antagonized by metitepin. It is concluded that the indolethylamines inhibit the stimulation-evoked ³H-5-HT release by activating the presynaptic 5-HT autoreceptors on the 5-HT neurones of the rat brain cortex. Similarities may exist between these receptors and the postsynaptic 5-HT_l binding sites of this brain area.     

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The membrane-bound ATPase activity of Bacillus subtilis was inhibited by dicyclohexylcarbodiimide (DCCD). The DCCD-reactive proteolipid of B. subtilis was extracted, from labelled or untreated membranes containing F₁ or depleted of F₁, with neutral or acidic chloroform/methanol. Purification of the [₁₄C]DCCD-binding proteolipid was attempted by column chromatography on methylated Sephadex G-50 and on DEAE-cellulose. The maximal amount of DCCD which could be bound to the purified proteolipid was found to exceed the amount bound by the purified proteolipid extracted from membranes labelled with the lowest [₁₄C]DCCD concentration required for maximal inhibition of the membrane-bound ATPase activity. The radioactive protein peaks eluted by gel filtration and ion-exchange chromatography were analysed by urea-SDS polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was incorporated into two components of M_r 18 000 and 6000 when proteolipid was purified by methylated Sephadex. The 6000 polypeptide was always present, whatever the extraction and purification procedures. However, the 18 000 polypeptide was present in largest quantity only when proteolipid was extracted from membranes containing F₁ and purified by methylated Sephadex. When proteolipid was purified on DEAE-cellulose this [₁₄C]DCCD binding component of M_r 18 000 was absent.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

Phosphorus and osmium as elemental tags for the determination of global DNA methylation—A novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies

The hyphenation of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS) is proposed in this work as a novel approach for the evaluation of DNA methylation, defined as the ratio between methylated cytosine and total cytosine bases in DNA. In the first part, reversed phase separation of 5-methyl-2′-deoxycytidine monophosphate (5mdCMP) and four deoxynucleotides with specific ICP-MS detection on ³¹P had been explored. In further development, selective labeling of 5-methylcytosine in ssDNA was carried out using potassium osmate (K₂OsO₄) in the presence of strong oxidant (K₃Fe(CN)₆) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The sample was then cleaned up and introduced to size exclusion chromatography–ICP-MS for specific detection at ³¹P and ¹⁸⁹Os and for evaluation of the molar ratio between Os and P eluted in DNA molecular mass fraction. The quantification of the two elemental tags was achieved by external calibration with phosphoric acid and Os(VI)-TEMED, respectively. The amount of Os in DNA fraction corresponded to methylated cytosines, while P signal was directly proportional to the total amount of DNA and could be recalculated to the amount of cytosine bases. The two procedures were tested by analyzing salmon testes DNA and a commercial oligonucleotide of known composition. For comparative purposes, these same samples were digested to deoxynucleosides and analyzed by reversed phase HPLC with spectrophotometric detection (DAD) at 280 nm. The results obtained using two procedures based on ICP-MS detection were in good agreement and also in agreement with the results obtained by HPLC–DAD procedure. In conclusion, ICP-MS specific detection at internal or external element tags seems to be an interesting alternative for the evaluation of global DNA in epigenetic studies.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.     

Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-Methylcytosine

A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-([²H₃]-Methyl)cytosine and [methyl-²H₃]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02–1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and ΦX-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.     

Structure characterization of a methylated ester biosurfactant produced by a newly isolated <Emphasis Type="Italic">Dietzia cinnamea</Emphasis> KA1

Biosurfactans are amphiphilic compounds synthesized by a wide group of microorganisms and tend to interact with surfaces of different polarities. In the present study we purified and characterized a biosurfactant produced by Dietzia cinnamea KA-1 when cultured by n-hexadecane as sole carbon source. The crude biosurfactant was extracted with ethyl acetate and purified by freezing at–20°C and then silica Gel column chromatography. The purified biosurfactant applied for more characterization using Elemental analysis (CHNS), Fourier Transform Infrared Spectroscopy (FTIR), Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (¹H and ¹³C-NMR) analysis. CHNS analysis showed the presence of C (74.92%) and H (11.63%) but not N or S. Functional groups of OH, CH₂, CH₃, C=O and aliphatic C?O revealed by FTIR analysis. The presence and position of these groups were confirmed by NMR analysis, and molecular mass of biosurfactant calculated using MS analysis. Finally, the product characterized as a methylated ester compound with molecular formula of C₂₁H₄₂O₄. This is the first report of biosurfactant of species D. cinnamea identified as ester, furthermore the ester was found to be in the methylated form.     

Purification and properties of mannitol dehydrogenase from Agaricus bisporus sporocarps

Mannitol dehydrogenase (mannitol: NADP⁺ 2-oxidoreductase: EC 1.1.1.138) was isolated from Agaricus bisporus by fractionation with protamine sulphate and (NH₄)₂SO₄, followed by chromatography on DEAE-Sephadex, then by affinity and gel chromatography. The products of enzyme reaction were identified by GLC and TLC. K_m, optimum pH, MW and pI of the enzyme as well as the influence of temperature, ions and inhibitors on enzymic activity were determined. In the sugar reducing reaction, the enzyme was specific for fructose but, in the reverse direction, some structurally related polyols could substitute for mannitol. The enzyme was very sensitive to alterations in the NADP⁺/NADPH ratio. The results are discussed in relation to the possible role of mannitol dehydrogenase in fungal metabolism.     

Generation of methylated violapyrones with improved anti-influenza A virus activity by heterologous expression of a type III PKS gene in a marine Streptomyces strain

Heterologous expression of the type III polyketide synthase (PKS) gene vioA in marine-derived Streptomyces youssoufiensis OUC6819 led to production of six violapyrones (VLPs), including four novel compounds VLPs Q–T (1–4) and two known compounds VLPs B and I (5 and 6). The structures of 1–4 were elucidated by a combination of spectroscopic analyses, including HR-ESIMS and 1D and 2D NMR data, demonstrating that 1–4 are novel VLPs which are methylated at 4-OH with their corresponding non-methylated counterparts to be VLP A, 5 and 6 and VLP C, respectively. Anti-influenza A [H1N1 (A/Virginia/ATCC1/2009) and H3N2 (A/Aichi/2/1968)] virus activity of compounds 1–6 as well as VLPs A and C were then evaluated using ribavirin as a positive control (IC₅₀?=?66.7 and 99.6?μM). The results revealed that these VLPs showed considerable anti-H1N1 and anti-H3N2 activities with IC₅₀ values of 30.6–132.4?μM and 45.3–150.0?μM, respectively. Notably, all the methylated VLPs displayed better anti-virus activity than their non-methylated counterparts, among which compound 3 (VLP S) exhibited the best activities. Interestingly, methylation at 4-OH has negative effect on the anti-MRSA (methicillin-resistant Staphylococcus aureus) activity instead, with methylated VLPs displaying decreased (2) or abolished (3 and 4) activities in comparison with each of their non-methylated counterparts.     

Ablation of N-acetylglucosaminyltransferases in Caenorhabditis induces expression of unusual intersected and bisected N-glycans

The modification in the Golgi of N-glycans by N-acetylglucosaminyltransferase I (GlcNAc-TI, MGAT1) can be considered to be a hallmark of multicellular eukaryotes as it is found in all metazoans and plants, but rarely in unicellular organisms. The enzyme is key for the normal processing of N-glycans to either complex or paucimannosidic forms, both of which are found in the model nematode Caenorhabditis elegans. Unusually, this organism has three different GlcNAc-TI genes (gly-12, gly-13 and gly-14); therefore, a complete abolition of GlcNAc-TI activity required the generation of a triple knock-out strain. Previously, the compositions of N-glycans from this mutant were described, but no detailed structures. Using an off-line HPLC-MALDI-TOF-MS approach combined with exoglycosidase digestions and MS/MS, we reveal that the multiple hexose residues of the N-glycans of the gly-12;gly-13;gly-14 triple mutant are not just mannose, but include galactoses in three different positions (β-intersecting, β-bisecting and α-terminal) on isomeric forms of Hex_4-8HexNAc₂ structures; some of these structures are fucosylated and/or methylated. Thus, the N-glycomic repertoire of Caenorhabditis is even wider than expected and exhibits a large degree of plasticity even in the absence of key glycan processing enzymes from the Golgi apparatus.     

Metabolic fate of endogenously synthesized prostaglandin D2 in a human female with mastocytosis

Increased production of prostaglandin D₂ was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D₂ of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D₂. A five percent aliquot of a twenty-four hour urine collection from the patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F₂. This study provides evidence that endogenously synthesized prostaglandin D₂ is converted in substantial part to prostaglandin F₂ metabolites in humans.     

A differential pulse polarographic assay for O-methylation of catechols by catechol-O-methyltransferase

A new method is described for monitoring the enzyme-eatalyzed O-methylation of norepinephrine (or other catechol substrate) by catechol-O-methyltransferase. Norepinephrine and normetanephrine combine with bis-2-(ethylhexyl)hydrogen phosphate at pH 7.4 to form an ion pair which is quantitatively extracted with an immiscible organic solvent by an application of partition chromatography. The mixture of catechol substrate and O-methylated products are then simultaneously determined in 0.5 m H₂SO₄ solution by monitoring their oxidation at a carbon paste electrode by a differential pulse polarographic technique.     

Gas-liquid chromatography-mass spectrometry of methylated and deuteriomethylated per-O-acetyl-aldononitriles from D-mannose

Peracetylated aldononitriles of the tetra-, tri-, and di-methyl ethers of D-mannopyranose were separated by gas-liquid chromatography, and analyzed by mass spectrometry. Through introduction of deuteriomethyl ether groups, various fragmentions constituting the mass spectra were identified and related to the parent methylated sugar structures. Also identified were several characteristic series of fragment ions that are common to two or more methylated D-mannopyranosides. As expected, mass spectra of the D-mannose derivatives were identical to those previously observed for D-glucose methylated in the same positions. Distinctive mass spectra were also recorded for all additional di-O-methyl-D-mannose derivatives. This information permits use of peracetylated aldononitrite derivatives in methylation-fragmentation analysis of aldohexans.     

Hypermethylation and higher stability of a plant (Eleusin coracana) tRNA

Total tRNA isolated from four-day-old ragi (Eleusin coracana) seedlings has been shown to be highly methylated. Each tRNA molecule on average contains two 2′-O-ribose methylated nucleosides. The high molar yields of 1-methyladenosine (1.6%) indicate that nearly a third of all the tRNA molecules contains more than one residue of 1-methyladenosine. Thermal denaturation studies with total tRNA show that the hypermethylated ragi tRNA melts slower that the yeast tRNA which is less methylated but otherwise has similar base composition. Ragi tRNA is also less susceptible to ribonucleases A, T₂ and T₂.     

Two forms of O-methyltransferase in tobacco cell suspension culture

An S-adenosyl-l-methionine: o-dihydric phenol O-methyltransferase was isolated from tobacco cell suspension culture and was partially purified by (NH₄)₂SO₄ precipitation and successive chromatography on DEAE-Sepharose, Sephacryl S-200 and hydroxyapatite columns. It catalysed the O-methylation of 3 cinnamic acids, two coumarins and two flavonoids, but to different extents. Results obtained from polyacrylamide gel electrophoresis, m-/p-methylation ratios and mixed substrate experiments indicated the existence of two forms of the enzyme which were resolved by chromatography on DEAE-cellulose. One form (MW 74000, pI 6.1, opt. pH 7.3) catalysed the meta-methylation of caffeic acid, while the other (MW 70000, pI 6.3, opt. pH 8.3) mediated the para-methylation of quercetin, though each form exhibited some activity against other substrates.     

Dehydrocyclopeptine epoxidase from Penicillium cyclopium

Dehydrocyclopeptine epoxidase (DE) activity was determined in cell free preparations of Penicillium cyclopium. The enzyme transforms dehydrocyclopeptine into cyclopenin by a mixed function oxygenation. It needs molecular oxygen and uses NAD(P)H, ascorbate or d,l-6-methyl-5,6,7,8-tetrahydropteridine as cosubstrates. DE is inhibited by CN^?, SCN^?, 1,10-phenanthroline, EDTA, 2,2′-bipyridine, sodium diethyldithiocarbamate, dicoumarol, p-chloromercuribenzoate and ions of different heavy metals, but not by CO and the lead salt of diethyldithiocarbamate. These properties indicate a specific importance of Fe²⁺-ions, SH-groups and flavins. DE activity is increased by Fe²⁺ and FAD. The enzyme may be therefore a Fe²⁺ activated FAD containing flavoprotein. DE was enriched 268-fold by (NH₄)₂SO₄ precipitation and chromatography on Sephadex G-200. Its MW estimated by Sephadex chromatography, exceeds 480 000.     

Preparation of methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d4

An eight-step synthesis is described which gives an overall yield of ～30% methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d₄. The preparation utilizes easily obtainable starting materials. Tris(triphenylphosphine)chlororhodium (I) catalyst is used for incorporation of the deuterium isotopes. The double bond in the 9 position is created by the Wittig coupling of 1-non-3-enyl-d₄-triphenylphosphonium bromide to methyl 8-formyloctanoate. Various methods for preparation of the intermediate and final products are discussed. Partial argentation resin chromatography was used to remove the ～9% trans/cis, cis/trans, and trans/trans isomers also produced. Analysis of the final product by mass spectrometry (MS) indicated 96%-d₄.     

Cellulase from Fusarium solani: Purification and properties of the C1 component

The C₁ component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H₃PO₄-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O-(carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (C_x enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H₃PO₄-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (C_x, CM-cellulases), indicate that C₁ is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting C_x enzyme when solubilizing cotton fibre.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.