Dual Role of Methionine in the Biosynthesis of Diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii

Biosynthesis of the polar group of diacylglyceryl-O-4′-(N,N,N-trimethyl)homoserine (DGTS) was studied in intact cells of Chlamydomonas reinhardtii Dangeard. Among the three C₄ amino acids tested, only l-methionine could specifically inhibit the photosynthetic incorporation of [¹⁴C]NaHCO₃ into the polar group of DGTS. The radioactivity in l-[¹⁴C]methionine, which was labeled at either the C3 + C4, the C1, or the methyl carbon, was efficiently incorporated into the polar group of DGTS. These results suggest that the C₄ backbone and the S-methyl group of l-methionine are precursors to the C₄ backbone and the N-methyl groups of DGTS, respectively.     

New diacylglyceryltrimethylhomoserines from the marine microalga Nannochloropsis granulata and their nitric oxide inhibitory activity

Chemical investigation of polar lipids from the marine eustigmatophyte microalga Nannochloropsis granulata led to the isolation of six betaine lipid diacylglyceryltrimethylhomoserine (DGTS), namely, (2S)-1,2-bis-O-eicosapentaenoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (1), (2S)-1-O-eicosapentaenoyl-2-O-arachidonoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (2), (2S)-1-O-eicosapentaenoyl-2-O-myristoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (3), (2S)-1-O-eicosapentaenoyl-2-O-palmitoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (4), (2S)-1-O-eicosapentaenoyl-2-O-palmitoleoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (5), and (2S)-1-O-eicosapentaenoyl-2-O-linoleoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (6). Structures of the isolated DGTSs were elucidated based on both spectroscopic technique and degradation methods. This is the first report of isolation of 1 in pure state, and 2–6 are all new compounds. The isolated betaine lipids showed dose-dependent nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells. Further study suggested that these betaine lipids (1–6) inhibit NO production in RAW264.7 macrophage cells through downregulation of inducible nitric oxide synthase expression, indicating the possible use as an anti-inflammatory agent. This is the first report of DGTS with anti-inflammatory activity.     

Anti-AIDS agents 84. Synthesis and anti-human immunodeficiency virus (HIV) activity of 2′-monomethyl-4-methyl- and 1′-thia-4-methyl-(3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogs

In a continuing investigation into the pharmacophores and structure–activity relationship (SAR) of (3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) as a potent anti-HIV agent, 2′-monomethyl substituted 1′-oxa, 1′-thia, 1′-sulfoxide, and 1′-sulfone analogs were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Among them, 2′S-monomethyl-4-methyl DCK (5a)³ and 2′S-monomethyl-1′-thia-4-methyl DCK (7a) exhibited potent anti-HIV activity with EC₅₀ values of 40.2 and 39.1 nM and remarkable therapeutic indexes of 705 and 1000, respectively, which were better than those of the lead compound DCK in the same assay. In contrast, the corresponding isomeric 2′R-monomethyl-4-methyl DCK (6) and 2′R-monomethyl-1′-thia-4-methyl DCK (8) showed much weaker inhibitory activity against HIV-1 replication. Therefore, the bioassay results suggest that the spatial orientation of the 2′-methyl group in DCK analogs can have important effects on anti-HIV activity of this compound class.     

Diazomethane-catalysed rearrangement of α-d-glucopyranosyl esters of N-acylamino acids into 2-O-acylaminoacyl-α-d-glucopyranoses

Both anomers of 1-O-[N-(tert-butoxycarbonyl)-L-α-glutamyl]-d-glucopyranose (2) were converted into the unprotected 1-esters, characterised as the trifluoroacetate salts 3α and 3β. On esterification with diazomethane and acetylation, the N-acetylated derivative of 3β and 2β gave the peracetylated 1-O-[5-methyl N-acetyl- and -tert-butoxycarbonyl-L-glutam-1-oyl]-β-d-glucopyranoses (4β and 6β), respectively. Similar treatment of 2α and 3β led to acyl migration, to yield 1,3,4,6-tetra-O-acetyl-2-O-[5-methyl N-(tert-butoxycarbonyl)-L-glutam-1-oyl]-α-d-glucopyranose (7α,64%) with traces of 7β, and a mixture (≈2:1:0.2) of the N-acetyl analogue of 7α (8α), 4α, and 8β, respectively. Treatment of 1-O-[5-methyl N-(tert-butoxycarbonyl)-L-glutam-1-oyl]-α-d-glucopyranose (10) and the corresponding glutam-5-oyl isomer 12 in N,N-dimethylformamide with diazomethane for 1 h resulted in 1 → 2 O-acyl transfer to give, upon acetylation, 7α and the fully acetylated 2-O-[1-methyl N-(tert-butoxy- carbonyl)-L-glutam-5-oyl]-α-d>-glucopyranose in yields of 70 and 90 %, respectively; in the absence of diazomethane, 10 and 12 remained unchanged. Similar experiments with α-d-glucopyranosyl esters of N-acetylglycine, N-acetylalanine, and N-(tert-butoxycarbonyl)phenylalanine yielded the 2-O-acyl derivatives in high yields and with high retention of anomeric configuration. The structures of the rearrangement products were proved both spectroscopically and chemically. The results imply that diazomethane functions as a base in the migration process.     

Synthesis of a series of fully methylated aldobiouronic acids,and β-elimination reactions of their synthetic precursors

Treatment of methyl 2,3,4-tri-O-acetyl-l-bromo-l-deoxy-α-d-glucopyranuronate severally with 2,4,6-, 2,3,6-, and 2,3,4-tri-O-methyl derivatives of methyl α-d-glucopyranoside and with methyl 4,6-O-benzylidene-3-O-methyl-α-d-glucopyranoside, in the presence of silver carbonate, afforded crystalline aldobiouronic acid derivatives in high yield. Deacetylation followed by methylation gave a series of fully methylated derivatives of laminaribiouronic, cellobiouronic, and gentiobiouronic acids, and the (1 → 2)-linked analogue. Methylation with methyl iodide and silver oxide in N,N-dimethylformamide was invariably accompanied by a small amount ofβ-elimination, with the formation of olefinic disaccharides which were also obtained by β-elimination reactions of the precursor acetates followed by methylation. Methyl 4,5-unsaturated 4-deoxyhexopyranosyluronate derivatives were the main products of the reaction, but these underwent further degradation with cleavage of the interglycosidic linkage and formation of 6-methoxycarbonyl-4-pyrone.     

Synthesis of halogenated 4-quinolones and evaluation of their antiplasmodial activity

Treatment of 4-hydroxyquinolines with (2-methyl)allyl bromide in the presence of K₂CO₃ resulted in the formation of novel N-[(2-methyl)allyl]-4-quinolones through selective N-alkylation. Further reaction of N-(2-methylallyl)-4-quinolones with bromine or N-bromosuccinimide yielded the corresponding 3-bromo-1-(2,3-dibromo-2-methylpropyl)-4-quinolones and 3-bromo-1-(2-methylallyl)-4-quinolones, respectively. Furthermore, a copper-catalyzed C–N coupling of the latter 3-bromo-4-quinolones with (5-chloro)indole afforded novel 3-[(5-chloro)indol-1-yl]-4-quinolone hybrids. Antifungal and antiplasmodial assays of all new 4-quinolones were performed and revealed no antifungal properties but moderate antiplasmodial activities. All 15 compounds displayed micromolar activities against a chloroquine-sensitive strain of Plasmodium falciparum, and the five most potent compounds also showed micromolar activities against a chloroquine-resistant strain of P. falciparum with IC₅₀-values ranging between 4 and 70 μM.     

Ion binding by repeat hexapeptide of elastin

Synthetic elastin hexapeptides, N-formyl-Val-Ala-Pro-Gly-Val-Gly-O-methyl, n-formyl-Ala-Pro-Gly-Val-Gly-Val-O-methyl, and N-formyl-Pro-Gly-Val-Gly-Val-Ala-O-methyl were studied in order to determine their affinity and selectivity for calcium, magnesium, strontium, sodium, and potassium ions. Of the three permutations N-forrnyl-Val-Ala-Pro-Gly-Val-Gly-O-methyl had the highest calcium affinity, k_a ～- 0.5 × 10⁶. All three hexamers displayed a selectivity toward calcium, although strontium did bind well also. The peptides could not compete effectively with water for magnesium ions and had little affinity for sodium and potassium ions. The significance of these results lies in the possible role of this hexameric sequence in calcium binding in natural elastin.     

Syntheses of 2-acetamido-2-deoxy-4-O-α-D-glucopyranosyl-α-d-glucopyranose (n-acetylmaltosamine) and 2-acetamido-2-deoxy-4-O-β-d-glucopyranosyl-α-d-glucopyranose

Condensation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside with 2,3,4,6-tetra-O-benzyl-1-O-(N-methyl)acetimidoyl-β-D-glucopyranose gave benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside which was catalytically hydrogenolysed to crystalline 2-acetamido-2-deoxy-4-O-α-D-glucopyranosyl-α-D-glucopyranose (N-acetylmaltosamine). In an alternative route, the aforementioned imidate was condensed with 2-acetamido-3-O-acetyl-1,6-anhydro-2-deoxy-β-D-glucopyranose, and the resulting disaccharide was catalytically hydrogenolysed, acetylated, and acetolysed to give 2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-α-D-glucopyranose Deacetylation gave N-acetylmaltosamine. The synthesis of 2-acetamido-2-deoxy-4-O-β-D-glucopyranosyl-α-D-glucopyranose involved condensation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide in the presence of mercuric bromide, followed by deacetylation and catalytic hydrogenolysis of the condensation product.     

N-(Phenoxyalkyl)amides as MT1 and MT2 ligands: Antioxidant properties and inhibition of Ca2+/CaM-dependent kinase II

Recently a series of chiral N-(phenoxyalkyl)amides have been reported as potent MT₁ and MT₂ melatonergic ligands. Some of these compounds were selected and tested for their antioxidant properties by measuring their reducing effect against oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) in the DCFH-diacetate (DCFH-DA) assay. Among the tested compounds, N-[2-(3-methoxyphenoxy)propyl]butanamide displayed potent antioxidant activity that was stereoselective, the (R)-enantiomer performing as the eutomer. This compound displayed strong cytoprotective activity against H₂O₂-induced cytotoxicity resulting slightly more active than melatonin, and performed as Ca²⁺/calmodulin-dependent kinase II (CaMKII) inhibitor, too.     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

Determination of the linkages in some methylated,sialic acid-containing,meningococcal polysaccharides by mass spectrometry

The application of gas-liquid chromatography-mass spectrometric (g.l.c.-m.s.) analysis to a number of sialic acid-containing polysaccharides of meningococcal origin has been studied. Methylation of these polysaccharides by the Hakomori conditions resulted in both O- and N-methylation. Methanolysis of the methylated polysaccharides from serogroup C [(2→9)-linked], colominic acid [(2→8)-linked], and serogroups Y and W-135 [both (1→4)-linked], yielded the respective 4,7,8,4,7,9-, and 7,8,9-tri-O-methyl derivatives of methyl N-acetyl-N-methyl-β-D-neuraminate methyl glycoside. As model compounds, methyl N-acetyl-4,7,8,9-tetra-O-methyl-α-D-neuraminate methyl glycoside and its N-methyl derivative were also synthesized. All of the methylated derivatives could be identified on the basis of their typical fragmentation-patterns, indicating that this method is applicable to the determination of the position of linkages to sialic acid residues in biopolymers.     

Copper(II) and zinc(II) complexes with Schiff-base ligands derived from salicylaldehyde and 3-methoxysalicylaldehyde: Synthesis,crystal structures,magnetic and luminescence properties

The following Schiff bases were employed as ligands in synthesizing copper(II) and zinc(II) complexes: N-[(2-pyridyl)-methyl]-salicylimine (Hsalampy), N-[2-(N,N-dimethyl-amino)-ethyl]-salicylimine (Hsaldmen), and N-[(2-pyridyl)-methyl]-3-methoxy-salicylimine (Hvalampy). The first two ligands were obtained by reacting salicylaldehyde with 2-aminomethyl-pyridyne and N,N-dimethylethylene diamine, respectively, while the third one results from the condensation of 3-methoxysalicylaldehyde with 2-aminomethyl-pyridine. Four new coordination compounds were synthesized and structurally characterized: [Cu(salampy)(H₂O)(ClO₄)] 1, [Cu₂(salampy)₂(H₂trim)₂] 2 (H₂trim^? = the monoanion of the trimescic acid), [Cu₄(valampy)₄](ClO₄)₄ · 2CH₃CN 3, and [Zn₃(saldmen)₃(OH)](ClO₄)₂ · 0.25H₂O 4. The crystal structure of 1 consists of supramolecular dimers resulted from hydrogen bond interactions established between mononuclear [Cu(salampy)(H₂O)(ClO₄)] complexes. Compound 2 is a binuclear complex with the copper ions connected by two monoatomic carboxylato bridges arising from two molecules of monodeprotonated trimesic acid. The crystal structure of 3 consists of tetranuclear cations with a heterocubane {Cu₄O₄} core, and perchlorate ions. Compound 4 is a trinuclear complex with a defective heterocubane structure. The magnetic properties of complexes 1–3 have been investigated. Compound 4 exhibits solid-state photoluminescence at room temperature.     

Synthesis and reactions of α- and β-d-glucopyranosyluronic esters of amino acids

The synthesis of the fully benzylated α- and β-d-glucopyranosyluronic esters of 1-benzyl N-benzyloxycarbonyl-l-aspartic and -glutamic acids and N-(tert-butoxycarbonyl)-l-phenylalanine, followed by hydrogenolysis, afforded the respective anomers of the 1-O-acyl-d-glucopyranuronic acids 2, 7, and 12. Esterification of both anomers of the N-acetylated derivatives of 2 and 7 by diazomethane was accompanied by glycosyl-bond cleavage, and, in the case of the α anomers, with concomitant 1→2 acyl migration to give, after O-acetylation, the 2-O-acyl O-acetyl methyl ester derivatives 5 and 10, respectively. Similarly, 12α yielded methyl 1,3,4-tri-O-acetyl-2-O-[N-(tert-butoxycarbonyl)-l-phenylalanyl]-d-glucopyranuronate and an analogue having a furanurono-6,3-lactone structure. Esterification of the C-5 carboxyl group, in 1-O-acyl-α-d-glucopyranuronic acids by methanol in the presence of the BF_3^?-MeOH reagent (1–1.5 equiv.) proceeded without acyl migration. By using this procedure, followed by acetylation, the N-acetylated derivative of 7α afforded methyl 2,3,4-tri-O-acetyl-1-O-(1-methyl N-acetyl-l-glutam-5-oyl)-α-d-glucopyranuronate, and 12α gave methyl 2,3,4-tri-O-acetyl-1-O-(N-acetyl-l-phenylalanyl)-α-d-glucopyranuronate; the formation of the latter involved cleavage of the tert-butoxycarbonyl group by BF₃, followed by N-acetylation in the next step.     

Seven new triterpenoids from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury

Seven new triterpenoids (1–7), together with two known ones (8–9), were isolated from the aerial parts ofIlex cornuta. The leaves of I. cornuta are the major source of “Kudingcha”, a popular herbal tea consumed in China and other countries. The structures of compounds 1–7 were determined as 20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (1), 20-epi-urs-12,18-dien-28-oic acid 2′-O-acetyl-3β-O-α-l-arabinopyranoside (2), 20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (3), 3β,23-dihydroxy-20-epi-urs-12,18-dien-28-oic acid (4), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (5), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronic acid (6), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (7), on the basis of spectroscopic analyses (IR, ESI–MS, HR-ESI–MS, 1D and 2D NMR) and chemical reactions. Protective effects against H₂O₂-induced H9c2 cardiomyocyte injury were tested in vitro for compounds 1–9, and the data showed that compound 4 had significant cell-protective effect. Compounds 1-9 did not show significant DPPH radical scavenging activity.     

Characterization of aminoalkylphosphonyl cerebrosides in muscle tissue of Turbo cornutus

From muscle tissues of the marine snail (Turbo cornutus) aminoalkylphosphonyl cerebrosides, which had been shown to be present in visceral parts, were isolated.Their structure was determined by degradative methods and by characterization of components by gas chromatography-mass spectrometry.The aminoalkylphosphonyl cerebroside fraction consisted of a major portion of 1-O-[6′-O-(N-methylaminoethylphosphonyl)galactosyl] ceramide and a minor portion of a novel lipid, 1-O-[6′-O-(aminoethylphosphonyl)galactosyl] ceramide.The fatty acids of the fraction were mainly palmitic (53.3%) and 2-hydroxy palmitic acid (14.6%). The long chain bases were mainly dihydroxy C_{22 : 2} (36.6%), C_{18 : 1} (14.6%) and C_{18 : 2} (11.3%), and trihydroxy bases were also found as minor components.     

Celecoxib prodrugs possessing a diazen-1-ium-1,2-diolate nitric oxide donor moiety: Synthesis,biological evaluation and nitric oxide release studies

A new class of anti-inflammatory (AI) cupferron prodrugs was synthesized wherein a diazen-1-ium-1,2-diolato ammonium salt, and its O²-methyl and O²-acetoxyethyl derivatives, nitric oxide (NO) donor moieties were attached directly to an aryl carbon on a celecoxib template. The percentage of NO released from the O²-methyl and O²-acetoxyethyl compounds was higher (18.0–37.8% of the theoretical maximal release of one molecule of NO/molecule of the parent compound) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer saline (PBS) at pH 7.4 (3.8–11.6% range). All compounds exhibited weak inhibition of the COX-1 isozyme (IC₅₀ = 5.8–17.0 μM range) in conjunction with weak or modest inhibition of the COX-2 isozyme (IC₅₀ = 1.6–14.4 μM range). The most potent AI agent 5-[4-(O²-ammonium diazen-1-ium-1,2-diolato)phenyl]-1-(4-sulfamoylphenyl)-3-trifluoromethyl-1H-pyrazole exhibited a potency that was about fourfold and twofold greater than that observed for the respective reference drugs aspirin and ibuprofen. These studies indicate that use of a cupferron template constitutes a plausible drug design approach targeted toward the development of AI drugs that do not cause gastric irritation, or elevate blood pressure and induce platelet aggregation that have been associated with the use of some selective COX-2 inhibitors.     

Mass spectrometry and 13C nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins

The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H₂O and D₂O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-β-d-glucopyranosylamine, and 1-N-acetyl-2-acetamido-β-d-glucopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at $\text{m}\text{e}\text{= 269}$, no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the α-O-glycosidic linkage, fully assigned carbon NMR spectra of α-methyl-N-acetylgalactosamine (GalNAc), α-methyl-3-O-methyl GalNAc,and -GlcNAc as well as the disaccharide Glc-β-1 → 3 GalNAc are reported. Because certain anomalies in the chemical shifts and ¹J_CH observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides.     

Effect of adenine nucleotides on the oxidation of N-methyl groups in liver mitochondria

In phosphorylating mitochondria, isolated in 0.25 M sucrose and suspended in a glycylglycine-KC1 medium at pH 7.4, the N-methyl group of sarcosine is oxidized to formaldehyde, formate, and CO₂. The initial rate of O₂ uptake in this system is only about half as great as with phosphate-washed mitochondria, in which the N-methyl carbon is oxidized only to the level of “active formaldehyde” and can be recovered as serine-β-carbon and/or formaldehyde. In the glycylglycine-KC1 medium, the O₂ uptake with sarcosine occurs in a biphasic manner and the initial slower rate can be extended by the addition of Mg²⁺, and ADP, AMP, or ATP. O₂ uptake is similarly restrained by ADP in mitochondria buffered with imidazole or pyrophosphate. The ADP effect is not observed in the presence of dinitrophenol. The patterns of O₂ uptake obtained with ADP in these various media are not altered when the oxidation of the formaldehyde, derived from the N-methyl group, is suppressed by the addition of either semicarbazide or rotenone. With dimethylglycine, another component of the “1-C cycle”, the initial rate of oxidation in glycylglycine or imidazole is enhanced by ADP rather than being decreased. These results together with appropriate coenzyme analyses suggest that reactions of “one carbon compounds” can provide sensitive markers for assessing compartition of cofactors such as the pyridine nucleotides, flavins, and folates in the mitochondrial matrix.     

Mechanism of Sporicidal Activity for the Synergistic Combination of Peracetic Acid and Hydrogen Peroxide

There is still great interest in controlling bacterial endospores. The use of chemical disinfectants and, notably, oxidizing agents to sterilize medical devices is increasing. With this in mind, hydrogen peroxide (H₂O₂) and peracetic acid (PAA) have been used in combination, but until now there has been no explanation for the observed increase in sporicidal activity. This study provides information on the mechanism of synergistic interaction of PAA and H₂O₂ against bacterial spores. We performed investigations of the efficacies of different combinations, including pretreatments with the two oxidizers, against wild-type spores and a range of spore mutants deficient in the spore coat or small acid-soluble spore proteins. The concentrations of the two biocides were also measured in the reaction vessels, enabling the assessment of any shift from H₂O₂ to PAA formation. This study confirmed the synergistic activity of the combination of H₂O₂ and PAA. However, we observed that the sporicidal activity of the combination is largely due to PAA and not H₂O₂. Furthermore, we observed that the synergistic combination was based on H₂O₂ compromising the spore coat, which was the main spore resistance factor, likely allowing better penetration of PAA and resulting in the increased sporicidal activity.