Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].     

Microbial oxidases of acidic -amino acids

Two microbial oxidases of acidic -amino acids have been purified to homogeneity. One is a -aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with -aspartate and is far more active toward -aspartate than -glutamate. The other is a -glutamate oxidase of Candida boidinii 2201 that preferred -glutamate to -aspartate as a substrate in terms of k_cat/K_m, but was not induced very effectively by -glutamate. The most potent competitive inhibitor of the C. humicolus -aspartate oxidase was malonate, and that of the C. boidinii -glutamate oxidase was -malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus -aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus -aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K_d for the apoprotein-FAD complex was very low (8.2×10⁻¹² M) consistent with the observed tight binding. The C. humicolus -aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral -amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.     

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free -alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both -to- and -to- alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K_m and V_max values for -alanine were 37.3 mM and 520 nmol/min/mg, respectively; for -alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various -amino acids was investigated. The enzyme catalyzes the racemization of -serine (relative reaction rate, 144% of rate for -alanine), -alanine (100%), -homoserine (17.1%), -2-aminobutyrate (5.6%), -glutamate (4.5%), and -asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl- -cysteine and o-phthaldialdehyde

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl- -cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free -serine (0.22 μmol/g of tissue; + RATIO = 0.25) in the brain whereas -aspartate and -alanine were established to be at trace levels. These results further support the presence of -serine in adult brain tissues as demonstrated by recent work using gas chromatography.     

Die Wirkung von d-Aminosäuren auf die Struktur und Biosynthese des Peptidoglycans

Zusammenfassung In einem Konzentrationsbereich von 0,02–0,2 M hemmt d-Serin das Wachstum aller untersuchten Bakterien. Gleichzeitig traten morphologische Veränderungen der Bakterienzellen auf. In den nucleotidaktivierten Vorstufen von gehemmten Zellen wurden die d-Alaninreste des Peptidanteils ganz oder teilweise durch d-Serin ersetzt. Auch das Peptidoglycan enthielt d-Serin anstelle von d-Alanin, jedoch weiniger als in den Vorstufen. Zusätzlich war das modifizierte Peptidoglycan zu einem geringeren Anteil quervernetzt als das normale. Vier weitere d-Aminosäuren (Threonin, Valin, Leucin, Methionin) verursachten bei einer Konzentration von 0.2 M ähnliche Wirkungen wie d-Serin. Die Wirkungsweise von d-Aminosäuren auf die Peptidoglycansynthese kann daher allgemein wie folgt beschreiben werden: In Gegenwart von wachstumshemmenden Konzentrationen an d-Aminosäuren werden modifizierte nucleotidaktivierte Peptidoglycanvorstufen synthetisiert, die zu einem geringeren Ausmaß in das Peptidoglycan eingebaut und im Peptidoglycan schlechter quervernetzt werden. Der Ersatz von d-Alanin in Position 4 der Peptiduntereinheit ist dabei in der Regel am wirkungsvollsten. Nur bei Corynebacterium insidiosum und Staphylococcus aureus erwies sich der Ersatz in Position 5 als stärker hemmend. Diese Wirkungsweise entspricht weitgehend derjenigen von Glycin. Im Unterschied zur Wirkung von Glycin kann l-Alanin in Position 1 der Peptiduntereinheit nicht durch d-Aminosäuren ersetzt werden.

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Mode of action of d-amino acids on the biosynthesis of peptidoglycan

The mechanism of growth inhibition by d-amino acids was studied. d-Serine at concentrations from 0.02–0.2 M was sufficient to cause partial growth inhibition in seven species of bacteria representing the four most common types of peptidoglycan. The inhibited cells displayed morphological alterations. In the nucleotide-activated peptidoglycan precursors of these cells, d-alanine residues in position 4 and/or 5 of the peptide moiety were partially or even completely replaced by d-serine. The peptidoglycan also contained d-serine instead of d-alanine, but the percentual content of d-serine was significantly lower than that in the precursors. In addition, the modified peptidoglycan was less cross-linked than the normal one. Four other d-amino acids (d-threonine, d-valine, d-leucine, d-methionine) at concentrations of about 0.2 M caused similar effects as did d-serine when applied to Corynebacterium callunae and Bacillus subtilis. Thus the mode of action of d-amino acids on peptidoglycan synthesis can be generally described as follows: in their presence, at growth inhibiting concentrations modified nucleotide-activated peptidoglycan precursors are formed in which d-alanine residues are replaced by the d-amino acids. They are less efficiently incorporated into peptidoglycan. A high percentage of the modified muropeptides remains non-cross-linked, since they are poor substrates for the transpeptidation reaction. In the majority of the organisms, cross-linking was decreased when d-alanine in position 4 of the peptide subunit was replaced, in two organisms (Corynebacterium insidiosum and Staphylococcus aureus) replacement in position 5 was most effective, however. The low extent of crosslinkage is consistent with the morphological aberrations of inhibited cells. In previous studies with glycine, results were described that were in close analogy to those obtained with d-amino acids. However, glycine can replace not only d-alanine residues in position 4 and 5 but also l-alanine in position 1 of the peptide subunit.

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Verwendete Abkürzungen Dab Diaminobuttersäure - m-Dmp meso-Diaminopimelinsäure - GlcNAc oder G N-Acetylglucosamin - MurNAc oder M N-Acetylmuraminsäure     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Screening of microorganisms producing α-methylserine hydroxymethyltransferase, purification of the enzyme, gene cloning, and application to the enzymatic synthesis of α-methyl-l-serine

Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85 kDa and contains 1 mol of pyridoxal-5′-phosphate per mol of the subunit. The K_m for α-methyl-l-serine and tetrahydrofolate was 0.54 mM and 73 μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278 bp, encoding a polypeptide with a calculated molecular mass of 46.0 kDa. Using E. coli cells as whole-cell catalysts, 9.7 mmol of α-methyl-l-serine was stereoselectively obtained from 15 mmol of d-alanine and 13.2 mmol of formaldehyde.     

Biodegradation of lindane by a native bacterial consortium isolated from contaminated river sediment

The aerobic biodegradation of lindane (γ-hexachlorocyclohexane) by a consortium of acclimated bacteria from sediment at a polluted site on the Suquia River, Cordoba, Argentina, is reported. The bacteria were acclimated for 30 days under aerobic conditions, using a minimal culture medium containing lindane (0.034 mM) as sole carbon source. Growth of the bacterial consortium decreased at a lindane concentration of 1.03 mM and was totally inhibited at 2.41 mM. The consortium showed initial lindane degradation rates of 4.92×10⁻³, 11.0×10⁻³ and 34.8×10⁻³ mM h⁻¹ when exposed to lindane concentrations of 0.069, 0.137 and 0.412 mM, respectively. Chloride concentration increased during aerobic biodegradation, indicating lindane mineralization. A metabolite identified as γ-2,3,4,5,6-pentachlorocyclohexene appeared during the first 24 h of biodegradation. Four different bacteria, identified as Sphingobacterium spiritivorum, Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis, were isolated. Pure strains of B. thiooxidans and S. paucimobilis degraded lindane after 3 days of aerobic incubation. This is the first report of lindane biodegradation by B. thiooxidans.     

Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension

Cell–cell signaling regulated by retinoic acid (RA), Wnt/β-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/β-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2^−/− mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2^−/− mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/β-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/β-catenin signaling as the body axis extends.     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a γ-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-β- -arabinofuranosyladenine-5′-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2′,3′-dideoxythymidine-5′-triphosphate (IC₅₀>400 μM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase γ. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC₅₀>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing -amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize -amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various -amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from -methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm⁻² h⁻¹ were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in -amino acid oxidase [ -amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria – Micrococcus luteus and Bacillus silvestris.Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and α-keto/α-hydroxy acids, but did not produce catecholates.This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

d-Alanine metabolism in the lucinid clam Lucinoma aequizonata

The chemoautotrophic symbiont-bearing clam Lucinoma aequizonata contains very high levels of free d-alanine in all tissues. The possible sources for this amino acid and its involvement in the clams' metabolism were investigated. Very low levels of d-alanine (generally below 1 mol·l^-1) were measured in the sediment porewaters from the habitat of the clams. Experiments with ¹⁴C-labeled tracers demonstrate an active metabolism of d-alanine in the clams rather than a role as inert waste product. d-alanine is metabolized at about 0.12 mol·g fw^-1·h^-1. Label from aspartate, but not glucose and CO₂, is incorporated into d-alanine. Incubation with labeled d-alanine did not result in formation of radioactive l-alanine. Tests for alanine racemase (EC 5.1.1.1) and d-amino acid oxidase (EC 1.4.3.3.) did not show activity in either gill, i.e. symbiont and host, or foot tissue. d-Alanine amino transferase (EC 2.6.1.b.) was demonstrated in gill and foot tissues. Two sources for d-alanine are proposed: a degradation of cell walls of symbiotic bacteria and production by the host using a d-specific alanine transaminase.Abbreviations aa amino acid(s) - fw fresh weight - HPLC high-performance liquid chromatography - MBH methyl benzethonium hydroxyde - NAC N-acetyl-l-cysteine - OPA ortho-phthaldialdehyde - TCA tricarbonic acid     

A new ecdysteroid and other constituents from two Dioscorea species

Phytochemical investigation of the rhizome of Dioscorea dumetorum has led to the isolation by several chromatographic steps on normal and reversed phase silica gel of a new ecdysteroid, (20R)-5β,11α,20-trihydroxyecdysone (1), and two known ecdysteroids, ajugasterone C (2) and herkesterone (3). Their structures were determined by spectroscopic methods including 1D- and 2D-NMR (COSY, TOCSY, HSQC and HMBC). This is the first report on the occurrence of phytoecdysteroids in the Dioscoreaceae family. These compounds were devoid of antifungal activity against three Candida species (Candida albicans, Candida glabrata and Candida tropicalis, MIC > 200 μg/ml).     

Sesquiterpenoids from Teucrium ramosissimum

An antiplasmodial bioguided investigation of the EtOAc extract of the aerial parts of Teucrium ramosissimum led to isolation and identification of three sesquiterpenoids, teucmosin, 4α-hydroxy-homalomenol C, 1β,4β,7α-trihydroxy-8,9-eudesmene and two trinorsesquiterpenoids, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione and 1β,4β-dihydroxy-11,12,13-trinor-8,9-eudesmen-7-one together with five known sesquiterpenoids, oplopanone, homalomenol C, oxo-T-cadinol, 1β,4β,6β-trihydroxyeudesmane, 1β,4β,7α-trihydroxyeudesmane and four flavonoids, 5-hydroxy-7,4′-dimethoxyflavone, salvigenin, genkwanin and cirsimaritin. The structures and the relative stereochemistry were elucidated by extensive spectroscopic studies including 1D and 2D NMR and mass spectrometry (MS). Homalomenol C, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione, oxo-T-cadinol and 1β,4β,6β-trihydroxyeudesmane displayed a significant in vitro antiplasmodial activity against Plasmodium falciparum with IC₅₀ values ranging from 1.2 to 5.0 μg/ml. Furthermore, no cytotoxicity was observed upon the human diploid lung cell line MRC-5 for these compounds.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

The complete amino acid sequence of the antenna polypeptide B806-866-β from the cytoplasmic membrane of the green bacterium Chloroflexus auranliacus

The bacteriochlorophyll a-binding polypeptide B806–866-β was extracted from membranes of the green thermophilic bacterium Chloroflexus aurantiacus with chloroform/methanol/ammonium acetate. Purification of the antenna polypeptide (6.3 kDa) was achieved by chromatography on Sephadex LH-60, Whatman DE-32 and by FPLC. The complete amino acid sequence (53 amino acid residues) was determined. The B806–866-β polypeptide is sequence homologous to the antenna β-polypeptides of purple bacteria (27–40%) and exhibits the characteristic three domain structure of the B870, B800–850 and B800–820 antenna complexes. The two typical His residues, conserved in all antenna β-polypeptides of purple bacteria, were found: His-24 lies within the N-terminal hydrophilic domain and His-42 within the central hydrophobic domain. This polypeptide together with the previously described α-polypeptide form the basic structural unit of the B806–866 antenna complex from C. aurantiacus.