Delayed light studies on photosynthetic energy conversion. VII. Effect of the high energy state, coupled to 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow, on millisecond emission from chloroplasts and digitonin subchloroplast particles

The effect of 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow on millisecond delayed light emission from chloroplasts has been compared to the effect on subchloroplast particles. Non-cyclic electron flow of both chloroplasts and subchloroplast particles was blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow increased the millisecond delayed emission by 2–4 times in both chloroplasts and subchloroplast particles. Uncoupling conditions which collapse only the pH gradient component of the proton motive force reduced the 2,3,5,6-tetramethyl p-phenylenediamine stimulation of delayed light in chloroplasts but not in particles. The 2,3,5,6-tetramethyl p-phenylenediamine stimulation of delayed light in particles was sensitive to uncoupling conditions which are presumed to destroy the transmembrane potential. Energy transfer inhibitors were without effect on the 2,3,5,6-tetramethyl p-phenylenediamine stimulation in both chloroplasts and particles.

The 2,3,5,6-tetramethyl p-phenylenediamine stimulation of millisecond delayed emission appears to reflect the particular form of the proton motive force; in chloroplasts it seems to be correlated with the proton concentration gradient, whereas in particles it is more closely correlated with the transmembrane potential.     

4-Substituted-2,3,5,6-tetrafluorobenzenesulfonamides as inhibitors of carbonic anhydrases I,II, VII,XII, and XIII

A series of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides were synthesized and their binding potencies as inhibitors of recombinant human carbonic anhydrase isozymes I, II, VII, XII, and XIII were determined by the thermal shift assay, isothermal titration calorimetry, and stop-flow CO₂ hydration assay. All fluorinated benzenesulfonamides exhibited nanomolar binding potency toward tested CAs and fluorinated benzenesulfonamides posessed higher binding potency than non-fluorinated compounds. The crystal structures of 4-[(4,6-dimethylpyrimidin-2-yl)thio]-2,3,5,6-tetrafluorobenzenesulfonamide in complex with CA II and CA XII, and 2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)sulfonyl]benzenesulfonamide in complex with CA XIII were determined. The observed dissociation constants for several fluorinated compounds reached subnanomolar range for CA I isozyme. The affinity and the selectivity of the compounds towards tested isozymes are presented.     

Application of an N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine-substituted peptide as a heterobifunctional cross-linking agent in a study of protein O-glycosylation in yeast.

In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-N-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-T hr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [14C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [125I]azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the N-(4-azido-2,3,5,6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.     

Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors

A focused library (4 x 14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC50< or =0.1 microM) helping to establish the SAR among these four series of azarene pyrrolidinones.     

Chlorophenol production by anaerobic microorganisms: transformation of a biogenic chlorinated hydroquinone metabolite

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as Desulfitobacterium spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol. 70:385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of Desulfitobacterium sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions. Desulfitobacterium sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.     

Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp. HH35.

The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and (1)H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.     

Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp. HH35

The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and ¹H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.     

Reductive dechlorination of a polychlorinated biphenyl congener and hexachlorobenzene by vitamin B12.

The polychlorinated biphenyl congener 2,3,4,5,6-pentachlorobiphenyl and hexachlorobenzene were reductively dechlorinated in an aqueous biomimetic model system containing vitamin B12. The products of 2,3,4,5,6-pentachlorobiphenyl dechlorination were 2,3,5,6- and 2,3,4,6-tetrachlorobiphenyl. Hexachlorobenzene dechlorinated to pentachlorobenzene and a mixture of 1,2,4,5- and 1,2,3,5-tetrachlorobenzene. The proton from water was shown to be the source of the hydrogen atom used for the replacement of chlorine on the biphenyl ring.     

Reductive dechlorination of a polychlorinated biphenyl congener and hexachlorobenzene by vitamin B12.

The polychlorinated biphenyl congener 2,3,4,5,6-pentachlorobiphenyl and hexachlorobenzene were reductively dechlorinated in an aqueous biomimetic model system containing vitamin B12. The products of 2,3,4,5,6-pentachlorobiphenyl dechlorination were 2,3,5,6- and 2,3,4,6-tetrachlorobiphenyl. Hexachlorobenzene dechlorinated to pentachlorobenzene and a mixture of 1,2,4,5- and 1,2,3,5-tetrachlorobenzene. The proton from water was shown to be the source of the hydrogen atom used for the replacement of chlorine on the biphenyl ring.     

Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl

Anaerobic bacteria reductively dechlorinate polychlorinated biphenyls (PCBs) in aquatic sediments, but these microorganisms remain uncultured and, until now, unidentified. Through denaturing gradient gel electrophoresis (DGGE) of 16S rDNA from a highly enriched ortho -PCB dechlorinating culture, the growth of a single microorganism was shown to be dependent upon the presence and dechlorination of 2,3,5,6-tetrachlorobiphenyl. This is the first identification of a microorganism that catalyses the reductive dechlorination of a PCB. The organism, bacterium o -17, has high sequence similarity with the green non-sulphur bacteria and with a group that includes Dehalococcoides ethenogenes . Bacterium o -17 required acetate for dechlorination and growth. H₂:CO₂ (80:20 at 101 kPa) did not support dechlorination or growth of the dechlorinator. Archaeal 16S rDNA was not detected in actively dechlorinating bromoethanesulphonate-treated non-methanogenic cultures, which indicated that methanogenic Archaea were not required for dechlorination. The consistent association with dechlorinating activity combined with high similarity to other known dechlorinating microorganisms indicates that bacterium o -17 catalyses the reductive ortho -dechlorination of 2,3,5,6-tetrachlorobiphenyl.     

Decomposition of Pentachlorophenol in Paddy Soil

The herbicide, pentachlorophenol decomposes within a few weeks after its application in rice fields. A reductive dechloronation was revealed as one of its decomposition pathways, in which some microorganisms play a dominant role and other soil chemical factors in a reduced condition are relatively of little importance. The stable decomposition products found in the paddy soil were 2,3,4,5-, 2,3,5,6- and 2,3,4,6-tetrachlorophenol, 2,4,5- and 2,3,5-trichlorophenol, 3,4- and 3,5-dichlorophenol, and 3-chlorophenol.     

Mutagenicity of N-nitrosopiperazine derivatives in Saccharomyces cerevisiae

The mutagenic properties of 8 N-nitrosopiperazines were examined in Saccharomyces cerevisiae. Forward mutations to canavanine resistance and reversions of his1-7 were induced by N'-methyl-N-nitrosopiperazine, dinitrosopiperazine, 2-methyldinitrosopiperazine, 2,5-dimethyldinitrosopiperazine, and 2,6-dimethyldinitrosopiperazine, in the presence of rat-liver homogenate. N-nitrosopiperazine, 2,3,5,6-tetramethyldinitrosopiperazine, and 4-benzoyl-3,5-dimethyldinitrosopiperazine were non-mutagenic.     

A binary system of photoreagents for high-efficiency labeling of DNA polymerases

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.     

Molecular modeling studies, synthesis, configurational stability and biological activity of 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPArs) has led to a search for new AMPAr positive modulators. Among them, 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (1) has attracted particular attention, because it is one of the most active benzothiadiazine-derived positive modulators of the AMPA receptor. It possesses two stereogenic centers, C3 and C6, thus it can exist as four stereoisomers. In this work, preliminary in silico studies suggested that 1 interacts stereoselectively with AMPArs. Single stereoisomers of 1 were prepared in order to evaluate their biological activity. However, studies regarding the configurational stability of the investigated compounds suggested a rapid epimerization at C3 in aqueous solvents, and we can expect the same reaction in vivo. Thus, electrophysiological experiments were performed on the two epimeric mixtures, (3?,6R)- and (3?,6S)- 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide, in order to evaluate their activities as positive allosteric modulators of AMPArs. The obtained data suggest that the (3?,6S) epimeric mixture is the most active in positively modulating AMPArs, confirming in silico results.     

白酒酒曲中高产四甲基吡嗪菌株的筛选和鉴定

为探索白酒酿造过程中四甲基吡嗪的合成机制,从白酒高温曲中筛选获得具有高产四甲基吡嗪的菌株。本研究应用Voges．Proskauer（V．P）反应和光度法对获得菌株的发酵液中的四甲基吡嗪含量进行测定从而获得高产菌株,综合菌落生长形态,16SrDNA序列分析及飞行时间质谱分析,对高产四甲基吡嗪菌株进行鉴定。结果表明,从曲中筛选获得的16株产乙偶姻菌株,其中S12、B1、S1001及B3产TTMP含量较高,分别为67．4mg／kg、50．7mg／kg、48．9mg／kg和43．2mg／kg。S12经进一步鉴定为Bacillussp．。     

Multisubstrate monod kinetic model for simultaneous degradation of chlorophenol mixtures

Chlorophenols (CPs) are persistent and highly toxic compounds rated as priority pollutants by the Environmental Protection Agency (EPA). Frequently, these compounds are present as mixtures of CPs in industrial wastewaters. Therefore the study of biodegradation on mixed pollutants is an important aspect of biodegradation and wastewater treatment. In this work, we studied the multisubstrate degradation of CPs by a mixed culture of Pseudomonas aeruginosa and a novel Acromobacter sp. capable of using pentachlorophenol (PCP), 2,4,6 trichlorophenol (2,4,6 TCP) and 2,3,5,6 tetrachlorophenol (2,3,5,6 TeCP) as the sole sources of carbon and energy. The main objective of this work was to evaluate the effect of substrate mixtures on the degradation kinetics of PCP. Batch experiments were conducted with each CP separately and in mixtures of PCP + 2,4,6 TCP, PCP + 2,3,5,6 TeCP, and PCP + 2,4,6 TCP + 2,3,5,6 TeCP. Based upon our results we have concluded that the simultaneous degradation of CPs is a key factor contributing to the improvement of PCP degradation. The kinetic parameters for PCP and 2,4,6 TCP were obtained by fitting the data to a Monod kinetics model. Using such parameters, the model was able to predict simultaneous multisubstrate degradation of PCP with others CPs.     

Chlorophenol Production by Anaerobic Microorganisms: Transformation of a Biogenic Chlorinated Hydroquinone Metabolite

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as Desulfitobacterium spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol. 70:385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of Desulfitobacterium sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions. Desulfitobacterium sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered

Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6-tetrafluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8 nM against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.     

Microbial Reductive Dechlorination of Aroclor 1260 in Anaerobic Slurries of Estuarine Sediments

Reductive dechlorination of Aroclor 1260 was investigated in anaerobic slurries of estuarine sediments from Baltimore Harbor (Baltimore, Md.). The sediment slurries were amended with 800 ppm Aroclor 1260 with and without the addition of 350 μM 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) or 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) and incubated in triplicate at 30°C under methanogenic conditions in an artificial estuarine medium. After 6 months, extensive meta dechlorination and moderate ortho dechlorination of Aroclor 1260 occurred in all incubated cultures except for sterilized controls. Overall, total chlorines per biphenyl decreased by up to 34%. meta chlorines per biphenyl decreased by 65, 55, and 45% and ortho chlorines declined by 18, 12, and 9%, respectively, when 2,3,4,5-CB, 2,3,5,6-CB, or no additional congener was supplied. This is the first confirmed report of microbial ortho dechlorination of a commercial polychlorinated biphenyl mixture. In addition, compared with incubated cultures supplied with Aroclor 1260 alone, the dechlorination of Aroclor 1260 plus 2,3,4,5-CB or 2,3,5,6-CB occurred with shorter lag times (31 to 60 days versus 90 days) and was more extensive, indicating that the addition of a single congener stimulated the dechlorination of Aroclor 1260.