Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct _Cp ) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2-hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1 _Ps6-19 and Pct _Cp was developed and cultured in a chemically defined medium containing 20 g/L of glucose and varying concentrations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6 mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2-ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co-LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.     

Engineering the Monomer Composition of Polyhydroxyalkanoates Synthesized in Saccharomyces cerevisiae

Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical β-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C₆ to C₁₄) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as β-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.     

Poly‐3‐hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies

The substrate specificity of the two polymerases (PhaC1 and PhaC2) involved in the biosynthesis of medium‐chain‐length poly‐hydroxyalkanoates (mcl PHAs) in Pseudomonas putida U has been studied in vivo. For these kind of experiments, two recombinant strains derived from a genetically engineered mutant in which the whole pha locus had been deleted (P. putida U Δpha) were employed. These bacteria, which expresses only phaC1 (P. putida U Δpha pMC‐phaC1) or only phaC2 (P. putida U Δpha pMC‐phaC2), accumulated different PHAs in function of the precursor supplemented to the culture broth. Thus, the P. putida U Δpha pMC‐phaC1 strain was able to synthesize several aliphatic and aromatic PHAs when hexanoic, heptanoic, octanoic decanoic, 5‐phenylvaleric, 6‐phenylhexanoic, 7‐phenylheptanoic, 8‐phenyloctanoic or 9‐phenylnonanoic acid were used as precursors; the highest accumulation of polymers was observed when the precursor used were decanoic acid (aliphatic PHAs) or 6‐phenylhexanoic acid (aromatic PHAs). However, although it synthesizes similar aliphatic PHAs (the highest accumulation was observed when hexanoic acid was the precursor) the other recombinant strain (P. putida U Δpha pMC‐phaC2) only accumulated aromatic PHAs when the monomer to be polymerized was 3‐hydroxy‐5‐phenylvaleryl‐CoA. The possible influence of the putative three‐dimensional structures on the different catalytic behaviour of PhaC1 and PhaC2 is discussed.     

Rapid and simple analysis of poly-β-hydroxybutyrate content by capillary isotachophoresis

Summary We present a rapid method for the direct analysis of poly--hydroxybutyrate (PHB) content in the soil bacteria Alcaligenes eutrophus. PHB from the fresh cells was converted by sulfuric acid to the crotonic acid and measured by capillary isotachophoresis after the neutralization by CaCO₃. The method can be used for rapid and routine monitoring of the fermentation processes in samples containing 0.001 to 20 mg of PHB.     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl PHAs) from cosmetic co-products by Pseudomonas raguenesii sp. nov., isolated from Tetiaroa, French Polynesia

A new bacterium, designated as strain TE9 was isolated from a microbial mat in French Polynesia and was studied for its ability to synthesize medium chain length poly-β-hydroxyalkanoates (mcl PHAs) during cultivation on cosmetics co-products. The composition of PHAs was analysed by coupled gas chromatography mass spectroscopy (GC/MS), nuclear magnetic resonance (NMR) and Fourier Transform InfraRed (FTIR) spectroscopy. PHAs were composed of C6–C14 3-hydroxyacids monomers, with a predominance of 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD). Differential scanning calorimetry (DSC) experiments allowed the characterization of elastomeric materials with a melting point T_m near 50 °C, enthalpy of fusion ΔH_m from 27 to 32 J/g, and glass transition temperature T_g of −43 °C. Molecular weights ranged from 175,000 to 358,000 g/mol. On the basis of the phenotypical features and genotypic investigations, strain TE9 was assigned to the Pseudomonas genus and the name of Pseudomonas raguenesii sp. nov. is proposed.     

Stability of Activated Persulfate in the Presence of Aquifer Solids

Bench-scale experiments were performed to investigate the persistence of activated persulfate using citric acid (CA) chelated ferrous (Fe(II)), peroxide (H₂O₂), or hydroxide (OH^?) activation in the presence of well-characterized aquifer solids. Chelation by citric acid was ineffective in controlling the interaction between persulfate and Fe(II), and oxidation of Fe(II) was observed, causing a rapid initial decrease in persulfate concentration. Subsequent to this loss, first-order persulfate degradation rate coefficients (k_obs) were estimated, which were up to four times higher than the unactivated case due to the interaction with Fe(III), Fe(II), or CA. Total oxidation strength (TOS) was measured for peroxide activation experiments and was observed to decrease rapidly early due to peroxide degradation. This was followed by slow degradation kinetics similar to that of unactivated persulfate, implying that the initial TOS degradation was peroxide-dominated and the long-term kinetics were dominated by persulfate degradation. The k_obs later used to capture TOS degradation were ～1 to 100 times higher than k_obs for unactivated persulfate. For alkaline activation, k_obs were only one to four times higher than unactivated persulfate, and therefore alkaline conditions demonstrated the least overall impact on persulfate stability among the various activation strategies explored.     

Combined metabolic activity within an atrazine-mineralizing community enriched from agrochemical factory soil

The main objective of this work was to characterize an atrazine-mineralizing community originating from agrochemical factory soil, especially to elucidate the catabolic pathway and individual metabolic and genetic potentials of culturable members. A stable four-member bacterial community, characterized by colony morphology and 16S rDNA sequencing, was rapidly able to mineralize atrazine to CO₂ and NH₃. Two primary organisms were identified as Arthrobacter species (ATZ1 and ATZ2) and two secondary organisms (CA1 and CA2) belonged to the genera Ochrobactrum and Pseudomonas, respectively. PCR assessment of atrazine-degrading genetic potential of the community, revealed the presence of trzN, trzD, atzB and atzC genes. Isolates ATZ1 and ATZ2 were capable of dechlorinating atrazine to hydroxyatrazine and contained the trzN gene. ATZ2 further degraded hydroxyatrazine to cyanuric acid and contained atzB and atzC genes whereas ATZ1 contained atzC but not atzB. Isolates CA1 and CA2 grew on cyanuric acid and contained the trzD gene. Complete atrazine degradation was a result of the combined metabolic attack on the atrazine molecule, and complex interactions may exist between the community members sharing carbon and nitrogen from atrazine mineralization.Scientific relevance: Despite numerous reports on atrazine degradation by pure bacterial cultures, the pathways and the atrazine-degrading gene combinations harboured by bacterial communities are only poorly described. In this work, we characterized a four-member atrazine-mineralizing community enriched from an agrochemical factory soil, which was capable of rapidly metabolizing atrazine to CO₂. This study will contribute towards better understanding of the genetic potential and metabolic activities of atrazine-degrading communities, which are generally considered to be responsible for atrazine mineralization in the natural environment.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C₃ to C₅, medium-chain-length (MCL) PHAs consist of monomer units of C₆ to C₁₄, and SCL-MCL PHAs consist of monomers ranging in size from C₄ to C₁₄. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C₄ to C₁₀ and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.     

An efficient method for the application of PHA‐poor solvents to extract polyhydroxybutyrate from Cupriavidus necator

There are many published studies presenting ethanol and acetone as PHAs‐poor solvents, where these two solvents are shown to dissolve <2% (w/v) of PHAs at low temperatures. In this study, the suitability of ethanol and acetone for the recovery of PHB at different temperatures (from room temperature to near boiling point) in Cupriavidus necator was investigated. Experiments were performed using response surface methodology to examine the effects of different temperatures and heating incubation times on recovery percentage using the two solvents. The highest recovery percentage (92.3%) and product purity (up to 99%) were obtained with ethanol‐assisted extraction at 76°C for 32 min of incubation time. Under these conditions the extracted PHB exhibited a molecular mass of 1.2 × 10⁶. The present strategy showed that at temperatures near its boiling point, ethanol, as a nonhalogenated solvent, represents a good alternative to halogenated solvents, like chloroform, when PHB recovery is concerned. DSC analysis showed good thermal properties for ethanol‐ and acetone‐extracted biopolymers. GC and ¹H NMR analysis confirmed the extracted biopolymer to be polyhydroxybutyrate of good purity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1480–1486, 2016     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.     

Absorption characteristics and kinetics of CO2 capture into N-methyldiethanolamine aqueous solution catalyzed by the immobilized carbonic anhydrase

Abstract

Carbonic anhydrase (CA) is the most effective CO₂ hydratase catalyst, but the poor storage stability and repeatability of CA limit its development. Therefore, CA was immobilized on the epoxy magnetic composite microspheres to enhance the CO₂ absorption into N-methyldiethanolamine (MDEA) aqueous solution in this work. In the presence of immobilized CA, the CO₂ absorption rate of MDEA solution (10?wt%) (0.63?mmol·min^?1) was greatly improved by almost 40%, and their reaction equilibrium time was shortened from 150?min to 90?min compared with that into MDEA solution. The results indicated that the absorption of CO₂ into MDEA solution had been significantly enhanced by using CA. After the 7th reuse recycle, the activity of the immobilized CA was still closed to its initial value at 313.15?K. Moreover, enzyme catalytic kinetics of immobilized CA was investigated using the p-nitrophenyl acetate (p-NPA) as substrate. The values of Michaelis–Menten constant (K_m) and the maximum velocity (V_max) of the immobilized CA were calculated to be 27.61?mmol/L and 20.14?×?10^?3?mmol·min^?1·mL^?1, respectively. Besides, the kinetics of CO₂ reaction into MDEA with or without CA were also compared. The results showed that CO₂ absorption into CA/MDEA aqueous solution obeyed the pseudo first order regime and the second order kinetics rate constant (k₂) was calculated to be 929?m³·kmol^?1·s^?1, which was twice higher than that of MDEA aqueous solution without immobilized CA (k₂=414 m³·kmol^?1·s^?1) at 313.15?K.     

Pulsed feeding strategy is more favorable to medium‐chain‐length polyhydroxyalkanoates production from waste rapeseed oil

This article presents the results of production and characterization of medium‐chain‐length polyhydroxyalkanoates (mcl‐PHAs) using Pseudomonas sp. Gl01. Studies have been carried out to find suitable feeding strategies for mcl‐PHAs production and, for the first time, to investigate in‐depth the properties of biopolyesters obtained under controlled conditions with waste rapeseed oil as a substrate. Up to 44% mcl‐PHAs of cell dry weight was produced at 41 h of biofermentor culture by employing pulsed feeding of waste rapeseed oil. GC analysis showed a polymer composition with monomer length of C₆ to C₁₂ with C₈ and C₁₀ as the principal monomers. The monomeric structure of the extracted polyesters did not depend on the cultivation time and the feeding strategy. Molecular weight of the mcl‐PHAs was found to be ranging from 57 to 154 kDa. Thermal analyses showed the obtained mcl‐polyhydroxyalkanaotes to be semi‐crystalline biopolymer with promising thermal stability, having a glass transition temperature of ?38 to ?50°C. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1243–1246, 2014     

Effect of nutritional supplements on bio-plastics (PHB) production utilizing sugar refinery waste with potential application in food packaging

Abstract

Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage reserve material stored by gram-negative bacteria under nutrient limitation. PHAs are best alternative biodegradable plastics (bio-plastics) due to their resemblance to conventional synthetic plastic. The present study investigated the synergistic effect of nutritional supplements (amino acid and vitamin) on the PHA production by Alcaligenes sp. NCIM 5085 utilizing a sugar refinery waste (cane molasses) under submerged fermentation process. Initially, the effect of individual factor on PHA yield was studied by supplementing amino acids (cysteine, isoleucine, and methionine), vitamin (thiamin), and cane molasses at varying concentration in the production medium. Further, the cultivation medium was optimized by varying the levels of cane molasses, methionine and thiamin using response surface methodology to enhance the PHA yield. The maximum PHA yield of 70.89% was obtained under the optimized condition, which was then scaled up on 7.5?L-bioreactor. Batch cultivation in 7.5?L-bioreactor under the optimized condition gave a maximum PHA yield and productivity of 79.26% and 0.312 gL^?1 h^?1, respectively. The PHA produced was subsequently characterized as PHB by FTIR. PHB extracted was of relatively high molecular weight and crystallinity index. DSC analysis gave T_g, T_m, and X_c of 4.2, 179?°C and 66%, respectively. TGA analysis showed thermal stability with maximized degradation occurring at 302?°C, which is above the melting temperature (179?°C) of the purified polymer. The extracted polymer, therefore, possessed desirable material properties to be used in food packaging.     

Domoic acid neurotoxicity in hippocampal slice cultures

Summary.  The neurotoxicity of domoic acid was studied in 2–3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1–100 μM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC₅₀ values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC₅₀ value of 6 μM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 μM NBQX against 3 and 10 μM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 μM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 μM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Comparative metabolism of isoleucine by corpora allata of nonlepidopteran insects versus lepidopteran insects,in relation to juvenile hormone biosynthesis

We studied the metabolism of [U-¹⁴C]isoleucine by intact and homogenized corpora allata (CA) from various insect species to determine how this substrate is converted to precursors of juvenile hormone (JH). CA homogenates of the lepidopterans Manduca sexta, Hyalophora cecropia, and Samia cynthia metabolize [U-¹⁴C]isoleucine to several products including 2-keto-3-methyl-valerate, 2-methylbutyrate, CO₂, propionate, and acetate. Intact CA of male H. cecropia produce particularly high levels of 2-keto-3-methylvalerate, indicating a highly active branched-chain-amino acid transaminase. In contrast, CA homogenates from the nonlepidopterans Periplaneta americana, Schistocerca nitens, Tenebrio molitor, and Diploptera punctata barely metabolize [U-¹⁴C]isoleucine. However, P. americana CA homogenate metabolizes [U-¹⁴C]2-keto-3-methylvalerate, the transamination product of [U-¹⁴C]isoleucine, more rapidly than does a homogenate of M. sexta CA. Furthermore, intact CA from P. americana incubated with [U-¹⁴C]2-keto-3-methylvalerate incorporate low levels of ¹⁴C into JH III, but do not metabolize this substrate to JH II or JH I. Intact CA from female Diploptera punctata produce very high levels of JH III, but are also unable to incorporate radiolabel from [U-¹⁴C]isoleucine into JH III, which substantiates our findings with other nonlepidopteran CA. The results suggest that CA of nonlepidopteran insects lack an active branched-chain amino acid transaminase and, consequently, are unable to utilize these substrates for JH biosynthesis.