Fermentative production of acetic acid from various pure and natural cellulosic materials by Clostridium lentocellum SG6

Clostridium lentocellum SG6 fermented various pure crystalline cellulosic materials efficiently with maximum acetic acid yield (gram acetic acid/gram substrate) of 0.67, at low substrate (8 g l⁻¹) concentration. The strain grew poorly on crude biopolymers but fermented them easily after alkali treatment, when grown with 8 g substrate l⁻¹ concentration of alkali-extracted cotton straw (AECS), paddy straw (AEPS) and sorghum stover (AESS) etc. The acetic acid to substrate (A/S) ratios were similar to those obtained with pure cellulosic materials. An increase in substrate concentration led to a decreased A/S ratio and a decreased percentage of substrate degraded. At high substrate concentration of 75 g filter paper l⁻¹, the strain SG6 converted 63.2 g filter paper into 31.28 g acetic acid l⁻¹. At 100 g l⁻¹ concentrations, AECS and AEPS served as the best substrates for acetic acid production when compared with other biopolymers. A maximum amount of 30.98 and 30.86 g acetic acid was produced from 70.6 g AEPS and 70.1 g AESS l⁻¹ of medium by strain SG6, respectively. Acetic acid production of 0.67 g g⁻¹ pure cellulose (Whatman No. 1 filter paper), 0.63 g g⁻¹ of alkali-treated cotton straw (AECS) are the highest among the cellulolytic bacteria reported so far in mono culture fermentations with pure and native cellulosic materials. This revised version was published online in July 2006 with corrections to the Cover Date.     

中脑导水管周围灰质内注射抗阿片肽血清对神经降压素增强电针镇痛的影响

本工作以钾离子透入引起大鼠甩尾反应作为痛反应指标,观察纳洛酮（ＮＸ）和抗阿片肽血清对大鼠中脑导水管周围灰质（ＰＡＧ）内微量注射神经降压素（ＮＴ）增强电针镇痛作用的影响。结果显示：（１）ＰＡＧ内微量注射ＮＴ后,大鼠电针镇痛效应明显提高;（２）ＰＡＧ内注射大剂量ＮＸ,可以显著减弱ＮＴ增强电针镇痛效应;（３）ＰＡＧ内微量注射抗甲脑啡肽血清和抗β内啡肽血清后,ＮＴ增强电针镇痛的作用明显降低;而ＰＡＧ内微量注射抗强啡肽Ａ１１３血清,对于ＮＴ增强电针镇痛的作用并无明显影响。提示：ＰＡＧ内ＮＴ在针刺镇痛过程中发挥重要作用,ＮＴ增强电针镇痛的作用与甲脑啡肽和β内啡肽有较密切的关系。     

中脑导水管周围灰质内注射抗阿片肽血清对神经降压素增强电针镇痛的 …

本工作以钾离子透入引起大鼠甩尾反应作为痛反应指标,观察纳洛酮（ＮＸ）和抗阿片肽血清对大鼠中脑导水管周围灰质（ＰＡＧ）内微量注射神经降压素（ＮＴ）增强电针镇痛作用的影响。     

Effect of cyanocobalamin and p-toluic acid on the fatty acid composition of Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota)

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B₁₂, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14-docosatetraenoic acid (22:4n-9), were detected.     

Analysis of the porcine APOA2 gene expression in liver,polymorphism identification and association with fatty acid composition traits

APOA2 is a protein implicated in triglyceride, fatty acid and glucose metabolism. In pigs, the APOA2 gene is located on pig chromosome 4 (SSC4) in a QTL region affecting fatty acid composition, fatness and growth traits. In this study, we evaluated APOA2 as a candidate gene for meat quality traits in an Iberian × Landrace backcross population. The APOA2:c.131T>A polymorphism, located in exon 3 of APOA2 and determining a missense mutation, was associated with the percentage of hexadecenoic acid [C16:1(n–9)], linoleic acid [C18:2(n–6)], α‐linolenic acid [C18:3(n–3)], dihomo‐gamma‐linolenic acid [C20:3(n–6)] and polyunsaturated fatty acids (PUFAs) in backfat. Furthermore, this SNP was associated with the global mRNA expression levels of APOA2 in liver and was used as a marker to determine allelic expression imbalance by pyrosequencing. We determined an overexpression of the T allele in heterozygous samples with a mean ratio of 2.8 (T/A), observing a high variability in the allelic expression among individuals. This result suggests that complex regulatory mechanisms, beyond a single polymorphism (e.g. epigenetic effects or multiple cis‐acting polymorphisms), may be regulating APOA2 gene expression.     

抗阿片肽血清对催产素增强电针镇痛的影响

本工作观察了大鼠侧脑室注射抗阿片肽血清对催产素增强电针镇痛作用的影响。脑室注射抗Beta-内啡肽血清(AEPS)使电针镇痛效应明显降低,但在注射催产素前预先注射AEPS对催产素增强电针镇痛作用无明显影响。注射抗强啡肽Al-13血清使电针镇痛明显降低,但却使催产素增强电针镇痛作用明显增强。无论抗甲-脑啡肽血清还是抗亮-脑啡肽血清对电针镇痛都无明显影响,对催产素增强电针镇痛的作用也不产生影响。上述结果说明,催产素增强电针镇痛的作用虽被脑内强啡肽Al-13部分对抗,但不受Beta-内啡肽和脑啡肽的影响,表明催产素增强针刺镇痛的作用不依赖于脑内内源性阿片肽系统。     

Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris

Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA.     

Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria

Aims: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4‐vinylphenol [4VP] and 4‐ethylphenol [4EP]) from the metabolism of p‐coumaric acid by lactic acid bacteria (LAB). Methods and Results: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p‐coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p‐coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l^?1) of tannins inhibited the synthesis of 4VP by Lact. plantarum. Conclusions: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p‐coumaric acid. On the other hand, tannins exert an inhibitory effect. Significance and Impact of the Study: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.     

Interaction of Sesamin and Eicosapentaenoic Acid against Δ5 Desaturation and n-6/ n-3 Ratio of Essential Fatty Acids in Rat

Sesamin is a specific inhibitor of Δ5 desaturation, the conversion from dihomo-γ-linolenic acid (20: 3, n-6) to arachidonic acid (AA, 20: 4, n-6). Previously, we reported that sesamin inhibited Δ5 desaturation of n-6 fatty acids in rat hepatocytes but not that of n-3 fatty acids, from 20: 4 (n-3) to eicosapentaenoic acid (EPA, 20: 5, n-3). In this study, we investigated the interaction of sesamin and EPA on Δ5 desaturation of both series and the n-6/n-3 fatty acids ratio by measuring actural fatty acid contents in vivo. Rats were fed three types of dietary oils; 1) linoleic acid (LA, 18: 2, n-6): linolenic acid (LLA, 18: 3, n-3) = 3: 1, n-6/n-3 ratio of 3: 1 (LA group), 2) LA: LLA =1: 3, n-6/n-3 ratio of 1: 3 (LLA group), 3) LA: LLA: EPA =1: 0.5: 3, n-6/n-3 ratio of 1: 3.5 (EPA group) with or without sesamin (0.5% w/w) for 4 weeks. In all groups, sesamin administration increased the content of dihomo-γ-linolenic acid (20: 3, n-6) in the liver and decreased the Δ5 desaturation index of n-6 fatty acid, the ratio of 20: 4/20: 3 (n-6). On the contrary, the Δ5 desaturation index of n-3 fatty acid, the ratio of 20: 5 + 22: 5 + 22: 6/20: 4 (n-3), was increased by the administration of sesamin. These results suggest that sesamin inhibits the A5 desaturation of n-6 fatty acid, but not that of n-3 fatty acid in rat livers. Sesamin administration decreased incorporation of EPA (n-3) and simultaneously increased the AA (n-6) content in the liver. The n-6/n-3 ratio in the liver was increased by administering sesamin under n-3 rich conditions, i.e., the LLA and EPA groups.     

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. VIII. Temperature effects and a perspective on the curious case of Karenia mikimotoi as a producer of the unusual, ‘green algal’ fatty acid hexadecatetraenoic acid [16:4(n-3)]

Previous studies have shown that dinoflagellates with different plastid ancestries have distinct differences in the fatty acid compositions and regiochemistries of their chloroplast-associated galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), thus reflecting plastid origin as a major factor in plastid membrane composition. Specifically, dinoflagellates with aberrant plastids (e.g. Karenia brevis, Kryptoperidinium foliaceum and Lepidodinium chlorophorum) possess certain MGDG- and DGDG-associated fatty acids which are not found in peridinin-containing dinoflagellates (the largest group of photosynthetic dinoflagellates with a red algal plastid ancestry which is thought to be an evolutionary precursor to aberrant plastids), but which are common to other algal groups. For example, hexadecatetraenoic acid (16:4(n-3)) is common to green algae and is found in the MGDG and DGDG of L. chlorophorum, which agrees with its green algal plastid ancestry, while hexadecatrienoic acid (16:3) and hexadecadienoic acid (16:2) are found in the MGDG and DGDG of K. foliaceum, which agrees with its diatom plastid ancestry. Notably, 16:4 has been found by others in the total fatty acids and galactolipids of Karenia mikimotoi, but in no other examined members of the Kareniaceae (all of which have plastids of haptophyte origin). However, these findings lack information as to the regiochemistry of 16:4. We have utilized positive-ion electrospray ionization/mass spectrometry (ESI/MS) and ESI/MS/MS to demonstrate that 16:4, which aside from L. chlorophorum is not found conclusively in the MGDG and DGDG of any other dinoflagellates examined to date irrespective of plastid ancestry, is found in K. mikimotoi as 18:5/16:4 (sn-1/sn-2 regiochemistry) MGDG and DGDG, and that its presence is not modulated (i.e. does not become more saturated) with an increase in growth temperature. Considering an aberrant pigment composition as described by others, we present a perspective where galactolipid-associated 16:4 in K. mikimotoi indicates a plastid ancestry more convoluted than for other members of the Kareniaceae.     

Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii)

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n−6) to di-homo-γ-linolenic acid (20:3 n−6). In addition, the transformed cells converted arachidonic acid (20:4 n−6) and eicosapentaenpic acid (20:5 n−3) to docosatetraenoic acid (22:4 n−6) and docosapentaenoic acid (22:5 n−3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C₁₈Δ6/C₂₀Δ5 but not C₂₂ PUFA substrates.     

Extracellular Formation of O-Butylhomoserine by Anaerobes

Clostridium BB–264, Cl. acetobutyricum 314–48, Cl. kaneboi, Cl. saccharoperbutylacetonicum and other two strains of Cl isolated recently produced an unidentified ninhydrin-positive compound in medium containing 5 % glucose, 1 % ammonium acetate, 0.1 % potassium dihydrogen phosphate, 0.04 % magnesium sulfate, 0.001 % ferrous sulfate, 0.1 % yeast extract, 10 μg/liter of biotin and 1 % calcium carbonate.

This ninhydrin-positive compound was eluted with solvent composed of butanol: acetic acid: water (4: 1: 2) by chromatography on cellulose powder column. It was crystallized from ethanol and then identified as an amino acid, O-butylhomoserine (Abbrev. as O-BHSer). Yield of this amino acid increased by adding homoserine or butanol to the medium. The increase was also recognized with addition of glycine, lysine, serine, threonine or valine. The formation of this amino acid was repressed by adding methionine to the medium.

Gas pressure to the culture is one of the important factors that make the amino acid formation by anaerobes possible.     

Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid and 4-hydroxyoctanoic acid as new constituents of bacterial polyhydroxyalkanoic acids

 A recombinant strain of Pseudomonas putida GPp104 (pHP1014::E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of Thiocapsa pfennigii exhibiting an unusual substrate specificity at a high level was incubated in two-stage batch or fed-batch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second cultivation phase, copolyesters of 3-hydroxybutyric acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic acid (3HHx) plus 5HHx were accumulated as revealed by gas-chromatographic and ¹³C-NMR spectroscopic analysis. When the recombinant P. putida GPp104 was incubated with 4-hydroxyheptanoic acid (4HHp) as carbon source in the second cultivation phase, a copolyester consisting of 3HB, 3-hydroxyvaleric acid and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the second cultivation phase led to the accumulation of a polyester that contained 1–2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3-hydroxyvaleric acid and 3HB. In addition to PHA containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic acid. Eleven strains from five genera have been also analysed for their ability to utilize different carbon sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with unusual constituents. Although most of the carbon sources were utilized by some strains for colony growth, accumulation experiments gave no evidence for the accumulation of new PHA by these wild-type strains. Received: 22 April/Received revision: 23 May 1996/Accepted: 2 June 1996     

2-Methylsorbic acid,an antifungal metabolite of Penicillium vermiculatum

Summary 2-Methylsorbic acid (MA), (2Z,4E)-2-methyl-2,4-hexadienoic acid, is a new metabolite of Penicillium vermiculatum. Antifungal activity of this acid was higher than that of sorbic acid or the bromoderivatives bromomethylsorbic acid and bromosorbic acid. MA suppressed the growth of Talaromyces flavus and germination of its conidia. In P. vermiculatum this acid lowered production of vermiculin and inhibited proteosynthesis in Saccharomyces cerevisiae. Correspondence to: B. Proksa     

High-performance liquid chromatography—thermospray mass spectrometry of epoxy polyunsaturated fatty acids and epoxyhydroxy polyunsaturated fatty acids from an incubation mixture of rat tissue homogenate

A method for the analysis of epoxy polyunsaturated fatty acids (EpPUFAs) and epoxyhydroxy polyunsaturated fatty acids (EpHPUFAs) in rat tissue homogenate, with homo-γ-linolenic acid (20:3, n − 6), arachidonic acid (20:4, n − 6), eicosapentaenoic acid (20:5, n − 3) or docosahexaenoic acid (22:6, n − 3) as a substrate, has been developed. Extraction with dichloromethane at pH 4–5 and concentration in the presence of pyridine were performed. Spectral analysis of chromatograms obtained with high-performance liquid chromatography—thermospray mass spectrometry showed the presence of EpPUFAs, EpHPUFAs and dihydroxy metabolites (DiHPUFAs) of EpPUFAs corresponding to each precursor fatty acid. On a selected-ion monitoring chromatogram, many EpPUFAs, EpHPUFAs and DiHPUFAs in an extract from an incubation mixture of each precursor fatty acid in aged rat tissue homogenate were detected simultaneously within 70 min. EpPUFAs and DiHPUFAs derived from 20:3 (n − 6) or 20:5 (n − 3) were detected in significant amounts. From these results, a highly active cytochrome P450 system or non-enzymic oxidative reactions in aged rat tissue homogenate were suggested.     

Degradation of 3-phenylpropionic acid by Haloferax sp. D1227

Haloferax sp. D1227, isolated from soil contaminated with highly saline oil brine, is the first halophilic archaeon to demonstrate the utilization of aromatic compounds (i.e., benzoic acid, cinnamic acid, and 3-phenylpropionic acid) as sole carbon and energy sources for growth. The degradation of 3-phenylpropionic acid in this strain was studied to examine the strategies utilized by Archaea to metabolize aromatic compounds. Based on our findings of (1) the extracellular accumulation of cinnamic acid, benzoic acid, 3-hydroxybenzoic acid, and gentisic acid in cultures of Haloferax D1227 grown on 3-phenylpropionic acid, (2) the presence of an 3-phenylpropionylCoA dehydrogenase, (3) the ATP, CoA, and NAD-dependent conversion of cinnamic acid to benzoylCoA, and (4) the presence of gentisate 1,2-dioxygenase, we propose that Haloferax D1227 metabolizes 3-phenylpropionic acid by initial 2-carbon shortening of the side chain to benzoylCoA via a mechanism similar to fatty acid β-oxidation, fol-lowed by aromatic degradation using a gentisate pathway. The upper aliphatic pathway from 3-phenylpropionic acid to benzoic acid is regulated separately from the lower gentisate pathway. Received: January 7, 1998 / Accepted: July 22, 1998     

Stereoselective esterification of halogen-containing carboxylic acids by lipase in organic solvent: effects of alcohol chain length

Summary Optical resolution of racemic carboxylic acids containing a halogen atom was attempted with stereoselective esterificatiob by Celite-adsorbed hydrolases in organic solvents. As lipase OF 360 from Candida cylindracea was found to stereoselectively esterify 2-(4-chlorophenoxy)propanoic acid, the (R)-enantiomer (d-isomer) of which is an important herbicide, the effects of alcohol chain length on stereoselectivity as well as reaction rate were investigated. The results revealed that the alcohol chain length markedly affected the stereoselective esterification of 2-(4-chlorophenoxy)propanoic acid: longer-chain alcohols, such as tetradecanol, served as excellent substrates for optical resolution of the acid, although the reaction rate was moderate. Offprint requests to: A. Tanaka     

Simultaneous determination of all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites and all-trans-retinol in human plasma by high-performance liquid chromatography

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.     

Athenolide A,a New Steroidal Lactone from the Leaves of Athenaea martiana (Solanaceae) Determined by Means of HPLC‐HR‐MS‐SPE‐NMR Analysis

Athenaea (Solanaceae) is an endemic genus belonging to the Brazilian Atlantic Rainforest. Recently, botanical investigations suggested the re‐evaluation of the generic status of the genus Athenaea as a synonym of Aureliana. In this study, the first investigation of the Athenaea genus performed on Athenaea martiana by means of HPLC‐HR‐MS‐SPE‐NMR combined with high‐resolution radical scavenging profile led to identification of several phenolic acids as radical scavengers: protocatechuic acid ( 1 ), 4‐hydroxybenzoic acid ( 2 ), caffeic acid ( 3 ), vanillic acid ( 4 ), and ferulic acid ( 6 ). Additional analysis revealed a new steroidal lactone, named athenolide A ( 9 ). Their structures were elucidated by extensive use of NMR spectroscopy as well as HR‐MS. Chemotaxonomic considerations based on these results supported the chemical relationships between the Athenaea and Aureliana genera, in agreement with the recent botanical findings.