JOURNAL OF PHYCOLOGY, Issue 2000
K.M. McGinnis
The role of the plastid in fatty acid biosynthesis in the non-photosynthetic diatom Nitzschia alba was studied and compared to that in the photosynthetic, closely related Nitzschia laevis. Transmission electron microscopy was used to analyze structural features of the plastid that may be relevant to biochemical function. Typical of a photosynthetic diatom, N. laevis had a chloroplast envelope composed of four membranes, and had abundant chloroplast ribosomes. The leucoplast of N. alba also had a multi-membrane envelope, chloroplast ribosomes, and a genome that encodes plastid specific proteins. This suggested that the plastid of N. alba may still possess the biochemical functions of the chloroplast, aside from photosynthesis. To determine whether plastidial fatty acid biosynthesis occurred in N. alba, the response of the two diatoms to the compound thiolactomycin was compared. Thiolactomycin has been shown to effect keto-acyl-ACP-synthases, and specifically inhibits the plastidial fatty acid biosynthetic pathway. While growth of N. alba was not impacted by thiolactomycin as in N. laevis, neutral lipid accumulation and fatty acid composition was impacted by thiolactomycin in both organisms. These findings suggest that the plastidial fatty acid biosynthetic pathway does exist in the leucoplast of N. alba, although it lacks photosynthetic capacity. [source]

LIGHT REGULATION OF PHYCOBILISOME BIOSYNTHESIS AND CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTOR

JOURNAL OF PHYCOLOGY, Issue 2000
K. Terauchi
Ambient light quality changes dramatically affect the composition of light harvesting structures, the phycobilisomes, in many cyanobacterial species. In the cyanobacterium Fremyella diplosiphon, shifts in the ratio of red to green light lead to transcriptional changes and altered synthesis of several phycobilisome components. This process is called complementary chromatic adaptation (CCA). These two colors have opposite effects: red light activates an operon encoding the biliprotein phycocyanin (PC) and inactivates the operon encoding phycoerythrin (PE), whereas green light activates PE synthesis and shuts down PC synthesis. The effects of red and green light on CCA are photoreversible. Thus, CCA is similar to transcriptional processes that are controlled by phytochromes, a family of eukaryotic red/far red photoreversible photoreceptors. We are using molecular genetics to determine the mechanisms by which F. diplosiphon senses changes in the color of light of its environment. Initial mutant generation and complementation lead to the discovery of three CCA regulatory components that are part of a complex two component system. The most interesting of these is RcaE (regulator of chromatic adaptation), a histidine kinase-class protein containing a region in its amino-terminal half with similarity to the chromophore binding domains of phytochromes. Within this region, RcaE contains a cysteine residue in a similar location as that used for covalent attachment of the open-chain tetrapyrrole chromophore in phytochromes. We will present recent data characterizing RcaE, including in vivo analysis of the chromophore that is attached to RcaE, as well as results from our recent isolation of a new CCA regulatory component. [source]

The expression of cytosolic phospholipase A2 and biosynthesis of leukotriene B4 in acute myeloid leukemia cells

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2007
Gudmundur Runarsson
Abstract Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B4 (LTB4) in acute myeloid leukemia (AML) cells (16 clones) and G-CSF mobilized peripheral blood CD34+ cells. CD34+ cells from patients with non-myeloid malignancies expressed cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase but not 5-lipoxygenase (5-LO). The enzyme cPLA2 was abundantly expressed in AML cells and the activity of the enzyme was high in certain AML clones. The expression of 5-LO, FLAP, and LTA4 hydrolase in AML clones was in general lower than in healthy donor polymorphonuclear leukocytes (PMNL). The calcium ionophore A23187-induced release of [14C] arachidonic acid (AA) in AML cells was low, compared with PMNL, and did not correlate with the expression of cPLA2 protein. Biosynthesis of LTB4, upon calcium ionophore A23187 activation, was only observed in five of the investigated AML clones and only three of the most differentiated clones produced similar amounts of LTB4 as PMNL. The capacity of various cell clones to produce LTs could neither be explained by the difference in [1 , 14C] AA release nor 5-LO expression. Taken together, these results indicate that LT synthesis is under development during early myelopoiesis and the capacity to produce LTs is gained upon maturation. High expression of cPLA2 in AML suggests a putative role of this enzyme in the pathophysiology of this disease. [source]

Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class,Ia and HLA-E expression: impact on target susceptibility to NK cell subsets

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2003
Manuel Llano
Abstract We compared in an inducible expression system the individual effect of US2, US6 and US11 human cytomegalovirus (HCMV) proteins on HLA-E and HLA class,Ia surface expression, assessing in parallel their influence on target susceptibility to NK cell clones. To this end, the RPMI,8866 B,lymphoma cell line (HLA-A2, HLA-A3, HLA-B7, HLA-Cw7, HLA-ER, HLA-EG) was stably cotransfected with the ecdysone receptor, together with the US sequences under the control of an ecdysone-inducible promoter. Biosynthesis of viral proteins was turned on by incubating transfectants with Ponasterone,A. US6 down-regulated expression of all class,I molecules, hampering target resistance to NK cell clones controlled by the CD94/NKG2A, KIR2DL2 and/or CD85j (ILT2 or LIR-1) inhibitory receptors. By contrast, US11 reduced the surface levels of class,Ia molecules but preserved HLA-E; this rendered US11+ cells sensitive to NK clones under the control of KIR2DL2 and/or CD85j, while their resistance to CD94/NKG2A+KIR2DL2, effector cells was maintained. US2 preserved as well HLA-E expression but selectively targeted class,Ia molecules; in fact, HLA-A and HLA-C allotypes were down-modulated whereas HLA-B7 remained unaltered. US2+ targets became sensitive to KIR2DL2+ cells but remained resistant to CD94/NKG2A+CD85j+ NK clones. The differential effects of US proteins on HLA class,Ia and HLA-E likely reflect the evolutionary adaptation of HCMV to counteract NK-mediated surveillance. [source]

Total Synthesis of Silyl-Protected Early Intermediates of Polyketide Biosynthesis,

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 16 2010
Karsten Krohn
Abstract The ketal- or dithioketal-protected isocoumarins 15,18 gave the corresponding 1-naphthols 21,26 in their reactions with the acetoacetate (10) or pentane-2,4-dione (19) dianions and the acetone monoanion. Subjection of the dithioketal-protected ester 28 to Baker,Venkataraman reaction conditions led to the 8-deoxy tautomeric, protected forms 29/30 of the early decaketide antibiotic intermediate 2b. However, the dithioketal protecting groups could not be removed without destruction of the molecule. Consequently the silyl-protected unstable early tri- and tetracyclic decaketide biosynthesis intermediates 37a, 37b, and 38a (precursors of angucycline and anthracycline antitumor antibiotics) were prepared through silylation of 33a and 33b, to afford 34a and 34b, and subsequent treatment with acetylacetone dianion. The ultimate synthetic goal, the silyl-protected 2,3-dialkylated naphthol derivative 41, was achieved by selective elongation of the bottom chain of the bis-silyl-protected methyl ester 36 with acetylacetone dianion. [source]

Studies on the Biosynthesis of Bovilactone-4,4 and Related Fungal Meroterpenoids,

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 20 2008
Martin Lang
Abstract The initial step in the biosynthesis of suillin (1), boviquinone-4 (2) and bovilactone-4,4 (3) in Suillus species is the geranylgeranylation of 3,4-dihydroxybenzoic acid at the 2-position. Feeding experiments with advanced precursors have identified boviquinone-4 and deacetylsuillin (9) as building blocks for the dilactone and catechol moieties, respectively, of bovilactone-4,4 (3). In order to explain the failure of boviquinone-4 (2) to incorporate side-chain-labelled deacetylsuillin (9#), an alternative sequence for the formation of 2 is proposed. During these experiments an interesting change in metabolism was noticed: after administration of larger quantities of aromatic carboxylic acids, the boviquinone-4 present in the fruit bodies disappeared and de novo synthesis of bovilactone-4,4 occurred. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source]

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferase 1 (GalNAc-T1)

FEBS JOURNAL, Issue 17 2002
Mari Tenno
Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p- chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc. [source]

Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin)

FEBS JOURNAL, Issue 17 2000
Mechanism of the cyanophycin synthetase reaction studied with synthetic primers
Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi- l -arginyl-poly- l -aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25,30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate. [source]

Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361

FEMS MICROBIOLOGY LETTERS, Issue 2 2009
Hyungjae Lee
Abstract Thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361 isolated from honey, strongly inhibited the growth of Bacillus cereus F4552. The bacteriocin was purified by 65% ammonium sulfate precipitation of the culture supernatant, followed by octyl-sepharose CL-4B and reverse-phase HPLC. The molecular mass of the bacteriocin was determined to be 3139.51 Da and the 14 amino acids of the bacteriocin at the N-terminus were identified. The complete amino acid sequence of mature thurincin H was deduced from three structural genes, thnA1, thnA2, and thnA3 found in tandem repeats on the chromosome, all of which encode for the same bacteriocin, thurincin H. The genetic determinants for thurincin H biosynthesis consist of 10 ORFs, including three thurincin H structural genes. Northern hybridization elucidated that the transcription of all three bacteriocin structural genes was regulated by a putative promoter located upstream of thnA1. [source]

The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides

FEMS MICROBIOLOGY LETTERS, Issue 2 2000
Michelle C Moffitt
Abstract Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems. [source]

A member of the YER057c/yjgf/Uk114 family links isoleucine biosynthesis and intact mitochondria maintenance in Saccharomyces cerevisiae

GENES TO CELLS, Issue 6 2001
Jong-Myong Kim
Background Two paralogs, YIL051c and YER057c, in the Saccharomyces cerevisiae genome are members of the YER057c/Yigf/Uk114 family, which is highly conserved among Eubacteria, Archaea and Eukarya. Although the molecular function of this protein family is not clear, previous studies suggest that it plays a role in the regulation of metabolic pathways and cell differentiation. Results Yil051cp is 70% identical in amino acid sequence to Yer057cp, and differs in that the former is longer by 16 amino acids containing, in part, the mitochondrial targeting signal at the N-terminus of the protein. An HA-tagged protein of Yil051cp is localized strictly in mitochondria, while that of Yer057cp is found in both cytoplasm and nucleus. Disruption of YIL051c (yil051c,) resulted in severe growth retardation in glucose medium due to isoleucine auxotroph, and no growth in glycerol medium due to the loss of mitochondria. An extract prepared from yil051c, cells showed no transaminase activity for isoleucine, while that for valine or leucine was intact. Haploid yil051c, cells newly isolated from the YIL051c/yil051c, hetero-diploids gradually lost mitochondrial DNA within 24 h in the absence of, but not in the presence of, an isoleucine. Mutants either requiring leucine (leu2,112) or isoleucine-valine (bat1,, bat2,) in a YIL051c background showed no changes in mitochondrial DNA maintenance in the absence of requirements. Conclusions Based on these results, we named Yil051c as Ibm1 (Isoleucine Biosynthesis and Mitochondria maintenance1) and concluded that: (i) Ibm1p determines the specificity of isoleucine biosynthesis, probably at the transamination step, (ii) Ibm1p is required for the maintenance of mitochondrial DNA when isoleucine is deficient, and (iii) Isoleucine compensates for the lack of Ibm1p. Taken together, Ibm1p may act as a sensor for isoleucine deficiency as well as a regulator determining the specificity for branched amino acid transaminase. [source]

Structure-Based Design and Synthesis of the First Weak Non-Phosphate Inhibitors for IspF, an Enzyme in the Non-Mevalonate Pathway of Isoprenoid Biosynthesis

HELVETICA CHIMICA ACTA, Issue 6 2007
Corinne Baumgartner
Abstract In this paper, we describe the structure-based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C5 precursors to isoprenoids, isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2; Scheme,1). The non-mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non-mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs.,2 and 3), new cytosine derivatives and analogues (Fig.,1) were selected as potential drug-like inhibitors of IspF protein, and synthesized (Schemes,2,5). Determination of the enzyme activity by 13C-NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine-2,5-diamine derivatives in the upper micromolar range (IC50 values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56,, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub-pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to ,Pocket III') and rings that occupy the flexible hydrophobic region of ,Pocket II'. The proposed binding mode remains to be further validated by X-ray crystallography. [source]

Mutations in human monoamine-related neurotransmitter pathway genes,

HUMAN MUTATION, Issue 7 2008
Jan Haavik
Abstract Biosynthesis and metabolism of serotonin and catecholamines involve at least eight individual enzymes that are mainly expressed in tissues derived from the neuroectoderm, e.g., the central nervous system (CNS), pineal gland, adrenal medulla, enterochromaffin tissue, sympathetic nerves, and ganglia. Some of the enzymes appear to have additional biological functions and are also expressed in the heart and various other internal organs. The biosynthetic enzymes are tyrosine hydroxylase (TH), tryptophan hydroxylases type 1 and 2 (TPH1, TPH2), aromatic amino acid decarboxylase (AADC), dopamine beta-hydroxylase (D,H), and phenylethanolamine N -methyltransferase (PNMT), and the specific catabolic enzymes are monoamine oxidase A (MAO-A) and catechol O -methyltransferase (COMT). For the TH, DDC, DBH, and MAOA genes, many single nucleotide polymorphisms (SNPs) with unknown function, and small but increasing numbers of cases with autosomal recessive mutations have been recognized. For the remaining genes (TPH1, TPH2, PNMT, and COMT) several different genetic markers have been suggested to be associated with regulation of mood, pain perception, and aggression, as well as psychiatric disturbances such as schizophrenia, depression, suicidality, and attention deficit/hyperactivity disorder. The genetic markers may either have a functional role of their own, or be closely linked to other unknown functional variants. In the future, molecular testing may become important for the diagnosis of such conditions. Here we present an overview on mutations and polymorphisms in the group of genes encoding monoamine neurotransmitter metabolizing enzymes. At the same time we propose a unified nomenclature for the nucleic acid aberrations in these genes. New variations or details on mutations will be updated in the Pediatric Neurotransmitter Disorder Data Base (PNDDB) database (www.bioPKU.org). Hum Mutat 29(7), 891,902, 2008. © 2008 Wiley-Liss, Inc. [source]

Microcapsules Containing a Biomolecular Motor for ATP Biosynthesis,

ADVANCED MATERIALS, Issue 15 2008
Qiang He
Abstract Over the past two decades, advances in modern biology and nanotechnology have enabled a rapid development in the design and building of biomimetic functional materials. ATP synthase is one of the most extensively studied molecular machines because it can be used as a rotary motor in the design of novel nanodevices and it can also continuously synthesize ATP in an artificial environment. A lot of research efforts have focused on assembling ATP synthase in biomimetic systems so that a complex cellular process can be constructed in a controllable manner. As we summarize here, layer-by-layer assembled microcapsules have proved to be a suitable cellular mimetic structure, which can be applied for engineering active biomimetic systems with a cellular process. An added benefit is that these assembled microcapsules can be used as bioenergy containers and thus ATP supply on demand. [source]

Dispatches from the Last Frontier of Molecular and Cell Biology: Biosynthesis of Polysaccharides and Proteoglycans of the Cell Surface and Extracellular Matrix

IUBMB LIFE, Issue 4 2002
Bruce Stone
No abstract is available for this article. [source]

Heparin and Heparan Sulfate Biosynthesis

IUBMB LIFE, Issue 4 2002
Kazuyuki Sugahara
Abstract Heparan sulfate is one of the most informationally rich biopolymers in Nature. Its simple sugar backbone is variously modified to different degrees depending on the cellular conditions. Thus, it matures to have an enormously complicated structure, which most likely exhibits a considerable number of unique overlapping sequences with peculiar sulfation profiles. Such sequences are recognized by specific complementary proteins, which form a huge group of "heparin-binding proteins," and the sugar sequences in turn support unique functions of the respective proteins through specific interactions. The heparan sulfate sequences are not directly encoded by genes, but are created by elaborate biosynthetic mechanisms, which ensure the generation of these indispensable sequences. In heparan sulfate biosynthesis, the tetrasaccharide sequence (GlcA-Gal-Gal-Xyl-), designated the protein linkage region, is first assembled on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparan sulfate chain is then polymerized on this fragment by alternate additions of GlcNAc and GlcA through the actions of glycosyltransferases with overlapping specificities encoded by the tumor suppressor EXT family genes. Then follow various modifications by N -deacetylation and N -sulfation of glucosamine, C5-epimerization of GlcA and multiple O -sulfations of the component sugars. Recent studies have achieved purification of several, and molecular cloning of most, of the enzymes responsible for these reactions. Some of these enzymes are bifunctional. The availability of cDNA probes has facilitated elucidation of the crystal structures for two of the biosynthetic enzymes, demonstration of their intracellular location, and their occurrence in complexes to achieve rapid and efficient synthesis of complex sugar sequences. Genomic structure and transcript analysis have shown the existence of multiple isoforms for most of the sulfotransferases. Many aspects of the heparan sulfate biosynthetic scheme are shared by the structural analog heparin, which is synthesized in mast cells and some other mammalian cells and is several-fold higher degree of polymerization and more extensive modification than heparan sulfate. [source]

Partial Recovery of Light-Independent Chlorophyll Biosynthesis in the chlL -Deletion Mutant of Synechocystis sp.

IUBMB LIFE, Issue 5 2001
PCC 680
Abstract A chlL -deletion mutant of Synechocystis sp. PCC 6803 designated as chlL - was unable to make significant amounts of chlorophyll in darkness. However, an apparent pseudorevertant has been generated spontaneously that can synthesize an increased amount of chlorophyll under light-activated heterotrophic growth conditions. Under these conditions, the chlorophyll content in this pseudorevertant was about 20% of that in the wild-type strain and about 4 times more than that in the original and in the recently recreated chlL -deletion mutant. This is paralleled by increased performance of dark-grown cells in terms of chlorophyll fluorescence induction and oxygen evolution rates in the pseudorevertant versus in the original mutant. PCR analysis confirmed that the chlL - pseudorevertant mutant still lacked the chlL gene. These results imply that the light-independent chlorophyll biosynthesis pathway was partly recovered. [source]

Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003

JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008
L.P. Thapa
Abstract Aims:, To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003. Methods and Results:, The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA, speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine. Conclusions:, The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis. Significance and Impact of the Study:, This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides. [source]

Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers

JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2007
P.K. Anil Kumar
Abstract Aims:, The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. Methods and Results:, In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l,1 sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) , P(HB-co-HV) , of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l,1) in a 3·0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2·7 g l,1, respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2·5 g l,1. The polymer was characterized by proton NMR. Conclusions:, Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. Significance and Impact of the Study:, Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production. [source]

Improvements in the production of bacterial synthesized biocellulose nanofibres using different culture methods

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2010
Amir Sani
Abstract This review summarizes previous work that was done to improve the production of bacterial cellulose nanofibres. Production of biocellulose nanofibres is a subject of interest owing to the wide range of unique properties that makes this product an attractive material for many applications. Bacterial cellulose is a natural nanomaterial that has a native dimension of less than 50 nm in diameter. It is produced in the form of nanofibres, yielding a very pure cellulose product with unique physical properties that distinguish it from plant-derived cellulose. Its high surface-to-volume ratio combined with its unique properties such as poly-functionality, hydrophilicity and biocompatibility makes it a potential material for applications in the biomedical field. The purpose of this review is to summarize the methods that might help in delivering microbial cellulose to the market at a competitive cost. Different feedstocks in addition to different bioreactor systems that have been previously used are reviewed. The main challenge that exists is the low yield of the cellulosic nanofibres, which can be produced in static and agitated cultures. The static culture method has been used for many years. However, the production cost of this nanomaterial in bioreactor systems is less expensive than the static culture method. Biosynthesis in bioreactors will also be less labour intensive when scaled up. This would improve developing intermediate fermentation scale-up so that the conversion to an efficient large-scale fermentation technology will be an easy task. Copyright © 2009 Society of Chemical Industry [source]

Chemistry of some fluorescamine,amine derivatives with relevance to the biosynthesis of benzylpenicillin by fermentation

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 12 2002
L Baker
Abstract The reaction of fluorescamine with 6-aminopenicillanic acid (6-APA) at pH 4 was investigated for its potential use in the biosynthesis of benzylpenicillin. A number of amino acids and penicillin amine derivatives, that reacted with fluorescamine at pH 7,9, were unlikely to do so to a significant extent at pH 4 and hence were unlikely to interfere in results that led to the biosynthesis of benzylpenicillin. Biosynthesis was followed using Penicillium chrysogenum Wis F3-64, growing in a corn steep liquor medium in a shake flask fed with phenylacetic acid daily. Analysis of benzylpenicillin formation was effected enzymically and fluorimetrically. A sample of the fermentation broth was treated with buffer at pH 7.8 and an active penicillin acylase solution for 1,h at 37,°C. The pH was then lowered to 4 by swamping with acetate buffer and the solution was treated with fluorescamine. The resulting fluorescence was compared with that of a standard 6-aminopenicillianic acid solution treated in the same manner. In this case the biosynthesis of benzylpenicillin was found to increase over 6 days. The results were compared with those for a control broth where the penicillin acylase was not added. © 2002 Society of Chemical Industry [source]

Biosynthesis of ascorbic acid by extant actinopterygians

JOURNAL OF FISH BIOLOGY, Issue 3 2000
R. Moreau
Polypterus senegalus, the longnose gar Lepisosteus osseus and the bowfin Amia calva had gulonolactone oxidase activity in the kidney and thus can synthesize ascorbic acid de novo. The enzyme activity was associated with the microsomal fraction. The common carp Cyprinus carpio and the goldfish Carassius auratus had no gulonolactone oxidase activity. Antibodies directed against white sturgeon gulonolactone oxidase showed cross-reactivity with lake sturgeon, bowfin and longnose gar kidney enzymes, but not with enzymes from Polypterus, sea lamprey, and tadpole kidney or pig liver. Given cross-reactivity, gulonolactone oxidase relatedness matched actinopterygian phylogeny, and suggested homology of the character throughout fishes. Modern teleosts may have lost the ability to synthesize ascorbic acid since the late Triassic as a result of a single reversal in the founding population. Wild bowfin and longnose gar exhibited high ascorbate concentrations in liver and spleen when compared with the teleosts rainbow trout Oncorhynchus mykiss and common carp fed vitamin C-supplemented diets. [source]

Study of the Biosynthesis of 1-Octen-3-ol Using a Crude Homogenate of Agaricus bisporus in a Bioreactor

JOURNAL OF FOOD SCIENCE, Issue 3 2008
R.O. Morawicki
ABSTRACT:, 1-Octen-3-ol and 10-oxo- trans -8-decenoic acid are metabolites of the breakdown of linoleic acid (LA) by mushroom enzymes. These compounds can be produced in a bioreactor using a crude mushroom homogenate and the exogenous addition of LA and oxygen. The factors' duration of blending, mushroom,buffer ratio, effect of a surfactant, whole against partially clarified reaction broths, purity of LA, and utilization of stumps instead of whole mushrooms were studied for their effect on reaction yield using a 1-L bioreactor. The results showed the feasibility of using the more inexpensive 60%-pure LA instead of the 99%-pure LA even when a yield loss was involved. Waste stumps could be used instead of whole mushrooms with a yield decline of 26%. [source]

Current Opinions on the Functions of Tocopherol Based on the Genetic Manipulation of Tocopherol Biosynthesis in Plants

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2008
Yin Li
Abstract As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp. PCC 6803. At the basis of these foundations, genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol. In addition, genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems. The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants. The roles of tocopherol in the network of reactive oxygen species, antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed. [source]

Terpenoid Indole Alkaloids Biosynthesis and Metabolic Engineering in Catharanthus roseus

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 7 2007
Dong-Hui Liu
Abstract Catharanthus roseus L. (Madagascar periwinkle) biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids (vinblastine and vincristine) and antihypertensive alkaloids (ajmalicine and serpentine). The multi-step terpenoid indole alkaloids (TIAs) biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed. [source]

An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thaliana

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 1 2006
Jun Liu
Abstract Auxin and jasmonic acid (JA) are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions. Although extensive investigations have been undertaken to study the biological functions of auxin and JA, little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall down regulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings. (Managing editor: Ya-Qin Han) [source]

Inhibiting Biosynthesis and/or Metabolism of Progestins in the Ventral Tegmental Area Attenuates Lordosis of Rats in Behavioural Oestrus

JOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2005
S. M. Petralia
Abstract In the ventral tegmental area (VTA), lordosis of rats is facilitated by 5,-pregnan-3,-ol-20-one (3,,5,-THP). Central 3,,5,-THP results from metabolism of peripheral progesterone, from the ovaries and/or adrenals, by sequential enzymatic activity of 5,-reductase and 3,-hydroxysteroid oxidoreductase (3,-HSOR). In addition, in glial cells, cholesterol is converted into pregnenolone by the P450 side-chain cleavage enzyme (P450scc), which is then metabolized to progesterone by 3,-hydroxysteroid dehydrogenase, and subsequently reduced to 3,,5,-THP. We hypothesize that, in the VTA, formation of 3,,5,-THP by both metabolism and biosynthesis is necessary for facilitation of lordosis of female rats. In Experiment 1, naturally-receptive rats received bilateral VTA infusions of a P450scc inhibitor, digitoxin (1 µg/side); a 5,-reductase inhibitor, finasteride (10 µg/side); digitoxin (1 µg/side) + finasteride (10 µg/side); or vehicle and were tested 3 h later for lordosis. In Experiment 2, the effects of VTA infusions of digitoxin, finasteride, digitoxin + finasteride, or vehicle on lordosis and midbrain and plasma 3,,5,-THP levels were examined. In Experiment 3, we investigated whether infusions of 3,,5,-THP to the VTA reinstated lordosis and midbrain 3,,5,-THP levels following administration of inhibitors. VTA infusions of digitoxin, finasteride, or digitoxin + finasteride, significantly and similarly reduced lordosis and midbrain, but not plasma 3,,5,-THP levels, compared to vehicle. Following receipt of inhibitor infusions, 3,,5,-THP to the VTA restored lordosis and midbrain 3,,5,-THP levels. These data suggest that, in the VTA, both central biosynthesis of progesterone and metabolism of progesterone (from central and/or peripheral sources) to 3,,5,-THP are important for mediating lordosis of rats. [source]

Processing of Frameshifted Vasopressin Precursors

JOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2000
Evans
Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones of the hypothalamo-neurohypophysial system comprises endoplasmic reticulum (ER) transit, sorting into the regulated secretory pathway and subsequent processing in the individual proteins VP, neurophysin and a glycoprotein. These processes are severely disrupted in the homozygous diabetes insipidus (di/di) Brattleboro rat, which expresses a mutant VP precursor due to a single nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional frameshift mutations in VP transcripts, in solitary magnocellular neurones of the di/di rat due to a GA dinucleotide deletion resulting in two different mutant VP precursors with partly restored reading frame. Frameshifted VP precursors are also expressed in several magnocellular neurones in wild-type rats. In this study, we determined if the +1 frameshifted precursors from di/di and wild-type rats can lead to biosynthesis of the hormone VP. Therefore, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were analysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells expressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-immunoreactive products were detected. In contrast, in AtT20 cells, frameshifted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Comparison between the two cell lines showed a very slow ER transit of the wild-type prohormone combined with inefficient processing in Neuro2A cells. The results show that mutant precursors can reach the regulated secretory pathway if ER transport is sufficiently rapid as in the case of AtT20 cells. This suggests that the di/di rat may regain the capacity to biosynthesize authentic VP through these +1 frameshifted precursors in magnocellular neurones. [source]

Biosynthesis of peptide fragments of eukaryotic GPCRs in Escherichia coli by directing expression into inclusion bodies

JOURNAL OF PEPTIDE SCIENCE, Issue 5 2010
Leah S. Cohen
Abstract Biosynthesis of peptides in heterologous systems is often a prerequisite to biophysical analyses. Large amounts of peptides, in particular portions of membrane proteins, are needed to optimize conditions for secondary and tertiary structure analysis. Chemical synthesis of these peptides is limited by their high hydrophobicity and also due to the need to incorporate isotopic labels for high resolution NMR analysis. In this protocol, we describe a method for the heterologous expression and purification of unlabeled and isotopically labeled peptide fragments of Ste2p, an integral membrane G protein-coupled receptor. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source]

Ethanol Modulation of TNF-alpha Biosynthesis and Signaling in Endothelial Cells: Synergistic Augmentation of TNF-alpha Mediated Endothelial Cell Dysfunctions by Chronic Ethanol

ALCOHOLISM, Issue 6 2005
Corinne Luedemann
Despite reported cardio-protective effects of low alcohol intake, chronic alcoholism remains a risk factor in the pathogenesis of coronary artery disease. Dose related bimodal effects of alcohol on cardiovascular system might reflect contrasting influences of light versus heavy alcohol consumption on the vascular endothelium. Chronic ethanol induced damage to various organs has been linked to the increased release of TNF-alpha (TNF). We have previously shown that TNF, expressed at the sites of arterial injury, suppresses re-endothelialization of denuded arteries and inhibits endothelial cell (EC) proliferation in vitro. Here we report that in vitro chronic ethanol exposure enhances agonist-induced TNF mRNA and protein expression in EC. Ethanol-mediated increment in TNF expression involves increased de novo transcription without affecting mRNA stability. DNA binding assays revealed that ethanol-induced TNF up regulation was AP1 dependent. Functionally, TNF induced EC dysfunction, including reduced proliferation, migration and cyclin A expression, were all markedly enhanced in the presence of ethanol. Additionally, expression of cyclin D1 was significantly attenuated in cells co-treated with TNF and ethanol while each treatment alone had little effect on cyclin D1 expression. Furthermore, exposure to ethanol potentiated and prolonged agonist-induced activation of JNK. Inhibition of JNK by over-expression of dominant negative JNK1 substantially reversed ethanol/TNF-mediated inhibition of cyclin A expression and EC proliferation, suggesting modulation of JNK1 signaling as the mechanism for ethanol/TNF-induced EC dysfunctions. Taken together, these data indicate that chronic ethanol consumption may negatively influence post angioplasty re-endothelialization thereby contributing to the development of restenosis. [source]