Modelling assimilation and intercellular CO2 from measured conductance: a synthesis of approaches

A spectrum of models that estimate assimilation rate A from intercellular carbon dioxide concentration (C_i) and measured stomatal conductance to CO₂ (g_c) were investigated using leaf‐level gas exchange measurements. The gas exchange measurements were performed in a uniform loblolly pine stand (Pinus taeda L.) using the Free Air CO₂ Enrichment (FACE) facility under ambient and elevated atmospheric CO₂ for 3 years. These measurements were also used to test a newly proposed framework that combines basic properties of the A–C_i curve with a Fickian diffusion transport model to predict the relationship between C_i/C_a and g_c, where C_a is atmospheric carbon dioxide concentration. The widely used Ball–Berry model and five other models as well as the biochemical model proposed by Farquhar et al. (1980) were also reformulated to express variations in C_i/C_a as a function of their corresponding driving mechanisms. To assess the predictive capabilities of these approaches, their respective parameters were estimated from independent measurements of long‐term stable carbon isotope determinations (δ¹³C), meteorological variables, and ensemble A–C_i curves. All eight approaches reproduced the measured A reasonably well, in an ensemble sense, from measured water vapour conductance and modeled C_i/C_a. However, the scatter in the instantaneous A estimates was sufficiently large for both ambient and elevated C_a to suggest that other transient processes were not explicitly resolved by all eight parameterizations. An important finding from our analysis is that added physiological complexity in modeling C_i/C_a (when g_c is known) need not always translate to increased accuracy in predicting A. Finally, the broader utility of these approaches to estimate assimilation and net ecosystem exchange is discussed in relation to elevated atmospheric CO₂.     

Transformation of aldose formazans. Novel synthesis of 2-acetamido-2-deoxypentonolactones and a new pent-2-enose formazan

2-Acetamido-2-deoxypentonolactones were synthesized from per-O-acetylated formazans of d-ribose, d- and l-arabinose, respectively. In dimethyl sulfoxide, a novel spontaneous transformation of the per-O-acetyl-pentose formazans into new 3,4,5-tri-O-acetyl-pent-2-enose formazans has been recognized. Additional examples for the occurrence of the isomerism between pseudo-aromatic chelate and open phenylazo-phenylhydrazone system were demonstrated by ¹H NMR spectroscopy in both the unprotected pentose formazans and 3,4,5-tri-O-acetyl-pent-2-enose formazans. Computational calculations supported higher stability of the ring form.     

Facile synthesis of (R)N-2-hydroxyacyl-L-cysteine derivatives: (R)N-2-hydroxyacyl transfer from enzymatically-synthesized (R)S-2-hydroxyacylglutathione derivatives to L-cysteine

Summary N-(R)-2-Hydroxyacyl-L-cysteine derivatives were conveniently synthesized by the reaction of the corresponding S-(R)-2-hydroxyacyl-glutathione with cysteine. The (R)2-hydroxyacyl group was transferred from the S-glutathionyl moiety to S-cysteinyl, forming the corresponding (R)S-2-hydroxyacylcysteine; this rearranged to the (R)N-hydroxyacylcysteine. These compounds have anti-proliferative activity associated with the inhibition ofde novo pyrimidine synthesis.Abbreviations TRIS tris(hydroxymethyl) aminomethane - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

β-N-Acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides

A large panel of fungal β-N-acetylhexosaminidases was tested for the regioselectivity of the β-GlcNAc transfer onto galacto-type acceptors ( -galactose, lactose, 2-acetamido-2-deoxy- -galactopyranose). A unique, non-reducing disaccharide β- -GlcpNAc-(1→1)-β- -Galp and trisaccharides β- -GlcpNAc-(1→4)-β- -GlcpNAc-(1→1)-β- -Galp, β- -Galp-(1→4)-β- -Glcp-(1→1)-β- -GlcpNAc and β- -Galp-(1→4)-α- -Glcp-(1→1)-β- -GlcpNAc were synthesised under the catalysis of the β-N-acetylhexosaminidase from the Aspergillus flavofurcatis CCF 3061 with -galactose and lactose as acceptors. The use of 2-acetamido-2-deoxy- -galactopyranose as an acceptor with the β-N-acetylhexosaminidases from A. flavofurcatis CCF 3061, A. oryzae CCF 1066 and A. tamarii CCF 1665 afforded only β- -GlcpNAc-(1→6)- -GalpNAc.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Enzymatic synthesis of 2-keto- -gluconate and 2-keto- -galactonate from -glucose and -galactose with cell culture of Pseudomonas fluorescens and 2-keto-galactonate from -galactono 1,4-lactone with partially purified 2-ketogalactonate reductase

2-Keto- -gluconate and 2-keto- -galactonate were prepared from -glucose (with a yield of 40%) and -galactose (with a yield of 25%), respectively, with cell culture of Pseudomonas fluorescens. However, 2-keto- -mannoate was not prepared in this method. The time courses of the reactions showed that 2-keto- -gluconic acid and 2-keto- -galactonic acid were produced from -glucose and -galactose through -gluconate and -galactonate, respectively. When using -galactono 1,4-lactone as a starting material, 2-keto- -galactonate was produced with partially purified NADP-dependent 2-ketogalactonate reductase from P. fluorescens. Some fundamental properties of the 2-ketogalactonate reductase were compared with those of 2-ketogluconate reductase from Acetobacter and Gluconobacter.     

Design,synthesis and evaluation of 2-phenylisothiazolidin-3-one-1,1-dioxides as a new class of human protein kinase CK2 inhibitors

Abstract

The synthesis and in vitro evaluation of 40 new 2-phenylisothiazolidin-3-one-1,1-dioxide derivatives are described. The optimization based on biological screening data and molecular modeling resulted in a 10-fold increase in inhibitory activity compared with previously reported inhibitors of this class and led to the identification of 3-{[2-chloro-4-(1,1-dioxido-3-oxoisothiazolidin-2-yl)benzoyl]amino}benzoic acid, a potent inhibitor of human protein kinase CK2 (?C₅₀?=?1.5?μM).     

Solvent effect on enzyme-catalyzed synthesis of β- -glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β- -glucopyranosides

Almond β- -glucosidase was used to catalyze alkyl-β- -glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β- -glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.     

Design,synthesis, characterization and in vitro antimicrobial evaluation of 4,6-diaryl-4,5-dihydro-2-phenyl-2H-indazol-3-ols

New 4,6-diaryl-4,5-dihydro-2-phenyl-2H-indazol-3-ols 25-32 were designed, synthesized and in vitro microbially evaluated using clinically isolated bacterial strains viz Staphylococcus aureus, β-Heamolytic streptococcus, Vibreo cholerae, Salmonella typhii, Shigella felxneri and fungal strains viz Aspergillus flavus, Mucor, Rhizopus and Microsporum gypsuem. Results of this study showed that the nature of the substituents on the phenyl rings viz., methyl, methoxy, chloro, nitro as well as the bromo functions at the meta and para positions of the aryl moieties determined the nature and extent of the activity of the fused indazolonol compounds 25-32.     

Design,synthesis, spectral analysis and in vitro microbiological evaluation of 2-phenyl-3-(4,6-diarylpyrimidin-2-yl)thiazolidin-4-ones

A series of novel 2-phenyl-3-(4,6-diarylpyrimidin-2-yl)thiazolidin-4-ones 23-33 were synthesized, and studied for their in vitro antibacterial and antifungal activities against clinically isolated strains. Generally compounds possessing electron donating groups showed good antibacterial activity. Compound 31, which contain both electron withdrawing chloro and electron donating methyl groups showed potent activity against all the tested Gram positive and Gram negative bacterial strains whereas compounds 32 and 33 which contain electron donating methoxy functional group at the para position of the phenyl ring attached to pyrimidine ring showed promising activity against S.aureus, S.typhii and E.coli. Compounds 32 and 33, both containing electron withdrawing groups (-Cl, -F) showed excellent activities against all the tested A. flavus, Mucor, Rhizopus and M.gypsuem fungal strains. while against Mucor, compound 27 which contains an electron donating methyl group at the para position of the phenyl ring attached to pyrimidine ring showed promising activity. Also compound 31, which contains both electron withdrawing chloro and electron donating methyl groups showed potent activity against A. flavus and Rhizopus.     

Design,synthesis and application of chiral tetraoxacalix[2]arene[2]triazine‐based organocatalysts in asymmetric Michael addition reactions

A novel type of oxacalix[2]arene[2]triazine‐based organocatalysts for asymmetric Michael reactions are reported for the first time. Chiral subunits were attached to the heteroatom‐bridged calixaromatic platform by a reaction of (R)‐ and (S)‐1‐aminotetraline with tetraoxacalix[2]arene[2]triazine in both enantiomeric forms. To evaluate the catalytic efficiency of the novel organocatalysts, isobutyraldehyde reacted with various substituted and unsubstituted aromatic trans‐β‐nitrostyrenes in tetrahydrofuran (THF), leading to Michael adducts in excellent yields and enantioselectivites (up to 97% yield and 99% ee).     

Tobacco TTG2 and ARF8 function concomitantly to control flower colouring by regulating anthocyanin synthesis genes

• Recently we elucidated that tobacco TTG2 cooperates with ARF8 to regulate the vegetative growth and seed production.
• Here we show that TTG2 and ARF8 control flower colouring by regulating expression of ANS and DFR genes, which function in anthocyanin biosynthesis.
• Genetic modifications that substantially altered expression levels of the TTG2 gene and production quantities of TTG2 protein were correlated with flower development and colouring. Degrees of flower colour were increased by TTG2 overexpression but decreased through TTG2 silencing, in coincidence with high and low concentrations of anthocyanins in flowers. Of five genes involved in the anthocyanin biosynthesis pathway, only ANS and DFR were TTG2‐regulated and displayed enhancement and diminution of expression with TTG2 overexpression and silencing, respectively. The floral expression of ANS and DFR also needed a functional ARF8 gene, as ANS and DFR expression were attenuated by ARF8 silencing, which concomitantly diminished the role of TTG2 in anthocyanin production. While ARF8 required TTG2 to be expressed by itself and to regulate ANS and DFR expression, the concurrent presence of normally functional TTG2 and ARF8 was critical for floral production of anthocyanins and also for flower colouration.
• Our data suggest that TTG2 functions concomitantly with ARF8 to control degrees of flower colour by regulating expression of ANS and DFR, which are involved in the anthocyanin biosynthesis pathway. ARF8 depends on TTG2 to regulate floral expression of ANS and DFR with positive effects on anthocyanin production and flower colour.

     

Stereoselective total synthesis of a potent natural antifungal compound (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one

A practical stereoselective synthesis of (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one (1), a potent natural antifungal compound, is described. The sequence involves diastereoselective iodine-induced electrophilic cyclization, epoxide ring opening with a vinyl Grignard reagent and ring closing metathesis (RCM) as the key steps.     

Lipase-catalyzed synthesis of medium- and long-chain diesters of 2-oxoglutaric acid

Abstract

The influence of various reaction parameters, such as alcohol-to-substrate ratio, enzyme-to-substrate ratio, solvent and temperature, on the enzymatic preparation of a series of novel medium- and long-chain esters of 2-oxoglutaric acid has been evaluated. Among the tested lipases, those from Candida antarctica and Carica papaya appeared to be the best catalysts. Mild reaction conditions and low environmental impact make the biocatalytic procedure a convenient way to prepare the reported products, which are potential fat substitutes in the food industry.     

A novel S-enantioselective amidase acting on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide from Arthrobacter sp. S-2

A novel S-enantioselective amidase acting on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide was purified from Arthrobacter sp. S-2. The enzyme acted S-enantioselectively on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to yield (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid. Based on the N-terminal amino acid sequence of this amidase, the gene coding S-amidase was cloned from the genomic DNA of Arthrobacter sp. S-2 and expressed in an Escherichia coli host. The recombinant S-amidase was purified and characterized. Furthermore, the purified recombinant S-amidase with the C-His₆-tag, which was expressed in E. coli as the C-His₆-tag fusion protein, was used in the kinetic resolution of (±)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to obtain (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid and (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide.     

VEGFR-2 inhibitors and apoptosis inducers: synthesis and molecular design of new benzo[g]quinazolin bearing benzenesulfonamide moiety

Two series of novel 4-(2-(2-(2-(substituted) hydrazinyl)-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 5–17 and 4-(2-(2-(substituted-1H-pyrazol-1-yl)-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 18–24 were synthesised from the starting material 4-(2-(2-hydrazinyl-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 5, to be evaluated for their inhibitory activity towards VEGFR-2. The target compounds 5–24, were screened for their cytotoxic activity against MCF-7 breast cancer cell line and the percentage inhibition against VEGFR-2. Compounds 9, 20, 22 and 23, showed excellent VEGFR-2 inhibitory activity with IC₅₀ ranging from 0.64 to 1.04?µm. Being the most potent, compound 9 was evaluated for its apoptotic inducer effect by studying the effect on caspase-3, it was found to increase its level. Compound 9 boosted the level of Bax and reduced the level of BCl2, compared to the control. Cell cycle analysis was conducted, compound 9 showed cell cycle arrest at G2/M phase. Moreover, mild cytotoxic effect (IC₅₀?=?29.41?µm, respectively) in normal breast cells MCF-12?A, was observed when treated with the same compound. Finally, a molecular docking study was performed to investigate the possible binding interaction inside the active site of the VEGFR-2 enzyme.     

Design,synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A2A receptor

The development of adenosine A_2A receptor antagonists has received much interest in recent years for the treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles has been identified as potential A_2AR antagonists. Structure-affinity relationship was investigated in position 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards the A_2A receptor. Compound F1, with an affinity of 1?μm, presented good absorption, distribution, metabolism and excretion properties with an excellent aqueous solubility (184?μm) without being cytotoxic at 100?μm. This compound, along with low-molecular weight compound D1 (K_i?=?10?μm), can be easily modulated and thus considered as relevant starting points for further hit-to-lead optimisation.     

Calcium-independent phospholipases A2 in murine osteoblastic cells and their inhibition by bromoenol lactone: impact on arachidonate dynamics and prostaglandin synthesis

Context: Bromoenol lactone (BEL) is an inhibitor of group VI phospholipases (iPLA₂s), but has been shown to have severe side effects. Objective: iPLA₂ characterization in osteoblasts and effect of BEL on prostaglandin (PG) E₂ formation. Methods: iPLA₂ expression: RT-PCR, Western Blotting. PGE₂ formation: GC–MS after stimulation, treatment with inhibitors or gene silencing. Arachidonate (AA) reacylation into phospholipids, inhibitor reaction products, PGHS-1 modification proteomic analysis: HR-LC–MS/MS. AA accumulation: ¹⁴C-AA. Results: iPLA₂ß and iPLA₂γ were expressed and functionally active. BEL inhibition up to 20 μM caused AA accumulation and enhanced PGE₂ formation, followed by a decrease at higher concentrations. BEL reacted with intracellular cysteine and GSH leading to GSH depletion and oxidative stress.

Discussion: Initial PGE₂ enhancement after BEL inhibition is due to iPLA₂-independent accumulation of AA. GSH depletion caused by high BEL concentrations is responsible for the decrease in PGE₂ production. Conclusion: BEL must be used with caution in a cellular environment due to conditions of extreme oxidative stress.     

Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration

In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O₂ tension (pO₂) in the medium. The pO₂ values that gave rise to half-maximal synthesis of the products (pO_0.5) were 0.2–0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate. The pO_0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar. Thus, the pO₂ for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO_0.5≤ 5 mbar), which was determined earlier. An essential role for quinol oxidase bd in microaerobic growth was demonstrated. A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O₂), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain. In the presence of nitrate, the amount of lactate was largely decreased. Therefore, under microoxic conditions, the pO₂ appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo. Received: 7 February 1997 / Accepted: 2 May 1997