Purification and Characterization of an Autolysin from Clostridium acetobutylicum

A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain.     

Structural Studies on the Pseudomonas aeruginosa Sialidase-Like Enzyme PA2794 Suggest Substrate and Mechanistic Variations

Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical β-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site.     

Fertilization acid release in Urechis eggs: I. The nature of the acid and the dependence of acid release and egg activation on external pH

The amount of fertilization acid produced by eggs of Urechis caupo, monitored by automatically back-titrating egg suspensions with base, depends linearly on the pH of the seawater. Above pH 7.0, at which no acid is released (Paul, M., Dev. Biol.43, 299–312, 1975), acid release increased approximately 0.34 pmole/egg/0.1 pH unit. Activation (germinal vesicle breakdown) depended on the amount of acid release in natural seawater; it did not occur if eggs released <1.5 pmole acid/egg. When fertilization acid is released into HCO^?₃-free seawater and the pH permitted to decrease, the supernatant can be tested for the presence of a volatile acid, such as CO₂, by bubbling with N₂ and comparing the increase in pH as volatile acid is driven off with experiments in which HCl or CO₂ is substituted for fertilization acid. An increase in pH of <0.2 pH units occurred on N₂ bubbling when fertilization acid or HCl was used to acidify HCO^?₃-free seawater compared to an increase of >0.5 pH units when CO₂ was used. Therefore, most, if not all, of Urechis fertilization acid is not volatile, and since Paul (1975) showed that it is not a nonvolatile weak acid, it must be H⁺.     

Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methylpenta-cis-2-trans-4-dienoic acid into abscisic acid in plants

(±)-5-(1,2-Epoxy-2,6,6-trimethylcyclohexyl) -3-methyl[2-¹⁴C]penta-cis-2-trans-4-dienoic acid is converted into abscisic acid by tomato fruit in 1.8% yield (or 3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid is derived from the precursor. The 2-trans-isomer is not converted. The amounts of [2-³H]mevalonate incorporated into abscisic acid have shown that the 40-times higher concentration of (+)-abscisic acid in wilted wheat leaves in comparison with unwilted ones reported by Wright & Hiron (1969) arises by synthesis. The conversion of (±)-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methyl-[2-¹⁴C]penta-cis-2-trans-4-dienoic acid into abscisic acid by wheat leaves is also affected in the same way by wilting and it is concluded from this that the epoxide or a closely related compound derived from it is on the biosynthetic pathway leading to abscisic acid. The oxygen of the epoxy group was shown, by ¹⁸O-labelling, to become the oxygen of the tertiary hydroxyl group of abscisic acid.     

Carnosic acid is an efflux pumps modulator by dissipation of the membrane potential in Enterococcus faecalis and Staphylococcus aureus

Bacterial resistance to antibiotics has become a serious problem of public health. Along with the controlled permeability by the cell-wall, active efflux systems can provide resistance by extruding antibiotics. Carnosic acid is capable to potentiate the antimicrobial activity of several antibiotics. However, the underlying molecular mechanism governing this effect remains unclear. The present study aims to investigate the effect of carnosic acid on the transport of ethidium bromide, on the permeability or the membrane potential in Enterococcus faecalis and Staphylococcus aureus. By using fluorimetric assays it was demonstrated that in E. faecalis, carnosic acid is a modulator of the uptake and efflux of ethidium bromide which does not induce cell membrane permeabilization phenomena. Such effect was sensitive to the inhibition caused by both the proton-motive force carbonyl cyanide m-chlorophenylhydrazone and the calcium antagonist verapamil, but not to vanadate, an ATPase inhibitor. In this work it was demonstrated, for the first time, that the activity of carnosic acid on the uptake/efflux of ethidium bromide is correlated with its capacity to change the membrane potential gradient in S. aureus and E. faecalis. In conclusion, carnosic acid is a natural compound, structurally unrelated to known antibiotics, which can function as an efflux pump modulator by dissipation of the membrane potential. Therefore, carnosic acid would be a good candidate to be employed as a novel therapeutic agent to be used in combination therapies against drug-resistant enterococci and S. aureus infections.     

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.     

Interaction between Mitochondrial Cytochromes and Linoleic Acid Hydroperoxide: POSSIBLE CONFUSION WITH LIPOXYGENASE AND ALTERNATIVE PATHWAY

O₂ uptake by tissue extracts in the presence of linoleic acid is generally ascribed to lipoxygenase. Such an O₂ uptake can be observed not only with mitochondria of Solanum tuberosum L. and Arum maculatum L. and pure lipoxygenase but also with cytochrome c. However, the rate of oxidation is highly dependent on the procedure used to prepare the solutions of linoleic acid. Unless special care is taken to prevent contact between linoleic acid and O₂, it appears that linoleic acid hydroperoxide is readily formed. This derivative can be readily oxidized by mitochondria or cytochrome c. On the other hand, the use of a rapid and specific enzymic procedure to estimate the disappearance of linoleic acid demonstrates that linoleic acid itself is not consumed at any appreciable rate by mitochondria or cytochrome c, the true substrate being linoleic acid hydroperoxide. During the reaction, the heme nucleus of added cytochrome c or of mitochondrial cytochromes undergoes deep alterations. Therefore, caution should be exerted when equating an O₂ uptake observed in the presence of linoleic acid to a lipoxygenase activity. The same holds true for the similarity of reaction towards specific inhibitors between lipoxygenase and the cyanide-insensitive pathway oxidase.     

Mutation in Saccharomyces cerevisiae Conferring Streptomycin and Cold Sensitivity by Affecting Ribosome Formation and Function

A cold-sensitive, streptomycin-sensitive mutant of Saccharomyces cerevisiae accumulates a 28S ribonucleoprotein particle when grown at low temperature. This particle contains 17S ribosomal ribonculeic acid which is degraded when exposed to ribonuclease. The particle does not serve as a precursor to 60 and 40S ribosomal subunits nor is it turned over when growth is allowed to resume at the permissive temperature; rather it is only diluted by growth. That streptomycin sensitivity (allelic with cold sensitivity) is ribosomal is evidenced by the inhibition of protein synthesis in vitro by streptomycin and the binding of labeled streptomycin to the mutant but not the parental 40S ribosomal subunit.     

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1

Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O₂ or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O₂. The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.     

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.     

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

A new product obtained by incubation of [2-¹⁴C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent-kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent-kaurene, ent1-kaurenol, ent-kaurenal and ent-kaurenoic acid, but not ent-7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent-kaurenoic acid and proceeds via the dienoic acid.     

A comparison between CLNA and CLA effects on body fat, serum parameters and liver composition

The potential of conjugated linoleic acid (CLA) as an anti-obesity molecule for humans is still a matter for debate. Thus, a great deal of scientific work is focussed on the research of new effective molecules without deleterious effects on health. The aim of the present work was to analyse the effects of jacaranda seed oil, rich in a conjugated linolenic acid (CLNA), jacaric acid (cis-8,trans-10,cis-12), on body fat, serum parameters and liver composition in rats, and to compare these effects with those oftrans-10,cis-12 CLA. Twenty-six male Wistar rats were divided into three groups fed with high-fat diets, supplemented or not (control group) with 0.5%trans-10,cis-12 CLA (CLA group) or 0.5% jacaric acid (CLNA group) for 7 weeks. No statistical differences in food intake or in final body weight were found. Whereas CLA reduced adipose tissue size, CLNA did not. Both CLA and CLNA significantly reduced non-HDL-cholesterol. In spite of a lack of significant changes in glucose and insulin levels, HOMA-IR index was significantly increased, as well as did non-esterified fatty acid levels in CLNA-fed rats. No changes in liver composition were observed. In conclusion, under our experimental conditions, jacaric acid, unlike CLA, does not show a body-fat lowering effect. Even though it leads to a healthy lipoprotein profile, it impairs insulin function. Consequently, it cannot be proposed as an anti-obesity molecule.     

Ancient Evolution and Recent Evolution Converge for the Biodegradation of Cyanuric Acid and Related Triazines

Cyanuric acid was likely present on prebiotic Earth, may have been a component of early genetic materials, and is synthesized industrially today on a scale of more than one hundred million pounds per year in the United States. In light of this, it is not surprising that some bacteria and fungi have a metabolic pathway that sequentially hydrolyzes cyanuric acid and its metabolites to release the nitrogen atoms as ammonia to support growth. The initial reaction that opens the s-triazine ring is catalyzed by the unusual enzyme cyanuric acid hydrolase. This enzyme is in a rare protein family that consists of only cyanuric acid hydrolase (CAH) and barbiturase, with barbiturase participating in pyrimidine catabolism by some actinobacterial species. The X-ray structures of two cyanuric acid hydrolase proteins show that this family has a unique protein fold. Phylogenetic, bioinformatic, enzymological, and genetic studies are consistent with the idea that CAH has an ancient protein fold that was rare in microbial populations but is currently becoming more widespread in microbial populations in the wake of anthropogenic synthesis of cyanuric acid and other s-triazine compounds that are metabolized via a cyanuric acid intermediate. The need for the removal of cyanuric acid from swimming pools and spas, where it is used as a disinfectant stabilizer, can potentially be met using an enzyme filtration system. A stable thermophilic cyanuric acid hydrolase from Moorella thermoacetica is being tested for this purpose.     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

Polygalacturonic acid trans-eliminase of Xanthomonas campestris

Polygalacturonic acid trans-eliminase from the culture fluid of Xanthomonas campestris was purified 66-fold by acetone precipitation, citrate extraction and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. The optimum pH is 9·5 in glycine–sodium hydroxide buffer. Up to 1mm-calcium chloride brings about a remarkable stimulation of the enzyme activity and, at this concentration, no other cations promote or inhibit enzyme action except Ba²⁺ ions, which cause complete inhibition. The enzyme degrades polygalacturonic acid in a random manner; it does not act upon polygalacturonate methyl glycoside, although it can cleave partially (68%) esterified pectin. The end products from polygalacturonic acid at 46% breakdown are unsaturated di- and tri-galacturonic acids, in addition to saturated mono-, di- and tri-galacturonic acids. Pentagalacturonic acid is split preferentially into saturated dimer plus unsaturated trimer, or into saturated trimer plus unsaturated dimer; at a lower rate, it is also split into monomer and unsaturated tetramer. Unsaturated pentamer is split into unsaturated dimer plus unsaturated trimer. Tetragalacturonic acid is split some-what preferentially at the central bond to form dimer and unsaturated dimer, but it is also split into monomer and unsaturated trimer. Unsaturated tetramer is split only at the central bond to yield only unsaturated dimer. Trigalacturonic acid is split into monomer and unsaturated dimer. Unsaturated trimer is cleaved into saturated dimer and probably 4-deoxy-l-5-threo-hexoseulose uronic acid, which has not yet been directly identified. Neither saturated nor unsaturated digalacturonic acid is attacked. The unsaturated digalacturonic acid was isolated and proved to be O-(4-deoxy-β-l-5-threo-hexopyranos-4-enyluronic acid)-(1→4)-d-galacturonic acid.     

Incorporation of cis-parinaric acid,a fluorescent fatty acid,into synaptosomal phospholipids by an acyl-CoA acyltransferase

The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg²⁺ or Ca²⁺ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [³H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.     

The Determinants of Stability and Folding in Evolutionarily Diverged Cytochromes c

Cytochrome c has served as a paradigm for the study of protein stability, folding, and molecular evolution, but it remains unclear how these aspects of the protein are related. For example, while the bovine and equine cytochromes c are known to have different stabilities, and possibly different folding mechanisms, it is not known how these differences arise from just three amino acid substitutions introduced during divergence. Using site-selectively incorporated carbon-deuterium bonds, we show that like the equine protein, bovine cytochrome c is induced to unfold by guanidine hydrochloride via a stepwise mechanism, but it does not populate an intermediate as is observed with the equine protein. The increased stability also results in more similar free energies of unfolding observed at different sites within the protein, giving the appearance of a more concerted mechanism. Furthermore, we show that the differences in stability and folding appear to result from a single amino acid substitution that stabilizes a helix by allowing for increased solvation of its N-terminus.     

Proteolysis of chloroplast thylakoid membranes. II. Evidence for the involvement of the light-harvesting chlorophyll a/b-protein complex in thylakoid stacking and for effects of magnesium ions on photosystem II-light-harvesting complex aggregates in the absence of membrane stacking

Experimental evidence indicates that the major pathway of retinoic acid metabolism in hamster liver microsomes follows the sequence: retinoic acid → 4-hydroxy-retinoic acid → 4-keto-retinoic acid → more polar metabolites. Using all-trans-[10-³H]retinoic acid, it can be shown by reverse-phase high pressure liquid chromatographic analysis that the first and last steps of this sequence require NADPH, whereas the oxidation of 4-hydroxy to 4-keto-retinoic acid is NAD⁺ (or NADP⁺) dependent. Both NADPH-dependent steps, but not the NAD⁺-dependent dehydrogenase reaction, are strongly inhibited by carbon monoxide. The metabolism of retinoic acid but not of 4-hydroxy-retinoic acid is highly dependent on the vitamin A regimen of the animal. Retinoic acid is rapidly metabolized by liver microsomes either from vitamin A-normal hamsters or from vitamin A-deficient hamsters that have been pretreated with retinoic acid, but not by microsomes from vitamin A-deficient animals; in direct contrast, the rate of metabolism of 4-hydroxy-retinoic acid is equivalent in each of these microsomal preparations. Analysis of the kinetics of these reactions yields the following Michaelis constants with respect to the retinoid substrates: retinoic acid, 1 × 10^?6m; 4-hydroxy-retinoic acid, 2 × 10^?5m; and 4-keto-retinoic acid, 1 × 10^?7m. The 4-hydroxy to 4-keto-retinoic acid oxidation has been shown to be experimentally irreversible, to have a K_mNAD⁺of 2 × 10^?5m, to be strongly inhibited by NADH, and to be unaffected by the presence of retinoic acid or its 4-keto-derivative in an equimolar ratio to the 4-hydroxy-substrate.     

Cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and by stimulating insulin secretion in vitro

Although the anti-diabetic activity of cinnamic acid, a pure compound from cinnamon, has been reported but its mechanism(s) is not yet clear. The present study was designed to explore the possible mechanism(s) of anti-diabetic activity of cinnamic acid in in vitro and in vivo non-obese type 2 diabetic rats. Non-obese type 2 diabetes was developed by injecting 90 mg/kg streptozotocin in 2-day-old Wistar pups. Cinnamic acid and cinnamaldehyde were administered orally to diabetic rats for assessing acute blood glucose lowering effect and improvement of glucose tolerance. Additionally, insulin secretory activity of cinnamic acid and cinnamaldehyde was evaluated in isolated mice islets. Cinnamic acid, but not cinnamaldehyde, decreased blood glucose levels in diabetic rats in a time- and dose-dependent manner. Oral administration of cinnamic acid with 5 and 10 mg/kg doses to diabetic rats improved glucose tolerance in a dose-dependent manner. The improvement by 10 mg/kg cinnamic acid was comparable to that of standard drug glibenclamide (5 mg/kg). Further in vitro studies showed that cinnamaldehyde has little or no effect on glucose-stimulated insulin secretion; however, cinnamic acid significantly enhanced glucose-stimulated insulin secretion in isolated islets. In conclusion, it can be said that cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and stimulating insulin secretion in vitro.     

Genetic and Biochemical Characterization of a 2-Pyrone-4,6-Dicarboxylic Acid Hydrolase Involved in the Protocatechuate 4,5-Cleavage Pathway of Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704–2709, 1990), we found the ligI gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the K_m values for PDC and CHM are 74 and 49 μM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557–565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154–1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.