Expression of hyoscyamine 6 beta-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L.

Hyoscyamine 60-hydroxylase (H6H: EC 1.14.11.11), a key enzyme at the terminal step of tropane alkaloid biosynthesis, converts hyoscyamine to scopolamine. The accumulation of scopolamine in different organs, in particular the aerial parts for storage, is subject to the expression of hyoscyamine 6-phydroxylase as well as its transport from the site of synthesis. To understand the molecular basis of this regulation, we have analyzed, in parallel, the relative levels of hyoscyamine and scopolamine, and the accumulation of H6H (both protein and transcript) in leaves, stems and roots of D. metel. The root, stem and leaf tissues all contain about 0.51-0.65 mg g(-1) dry weight of scopolamine. Hyoscyamine content was extremely low in leaf and stem tissues and was about 0.28 mg g(-1) dry weight in the root tissue. H6H protein and its transcript were found only in roots but not in the aerial parts viz. stems and leaves. The immunolocalization studies performed on leaf, stem, root as well as hairy root tissues showed that H6H was present only in the pericycle cells of young lateral and hairy roots. These studies suggest that the conversion of hyoscyamine to scopolamine takes place in the root pericycle cells, and the alkaloid biosynthesized in the roots gets translocated to the aerial parts in D. metel. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis

Osteoporosis is a disease of low bone mass most often caused by an increase in bone resorption that is not sufficiently compensated for by a corresponding increase in bone formation(1). As gut-derived serotonin (GDS) inhibits bone formation(2), we asked whether hampering its biosynthesis could treat osteoporosis through an anabolic mechanism (that is, by increasing bone formation). We synthesized and used LP533401, a small molecule inhibitor of tryptophan hydroxylase-1 (Tph-1), the initial enzyme in GDS biosynthesis. Oral administration of this small molecule once daily for up to six weeks acts prophylactically or therapeutically, in a dose-dependent manner, to treat osteoporosis in ovariectomized rodents because of an isolated increase in bone formation. These results provide a proof of principle that inhibiting GDS biosynthesis could become a new anabolic treatment for osteoporosis.

The quest for golden bananas: Investigating carotenoid regulation in a Fe’i group Musa cultivar

The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fe’i group Musa cultivar, “Asupina”, has been examined and compared to that of a low-carotenoid-accumulating cultivar, “Cavendish”, to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in “Cavendish” and the conversion of amyloplasts to chromoplasts during fruit ripening in “Asupina”. Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in “Cavendish” fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.

Modulation of growth by aromatase inhibitor treatment in boys: Efficacy and safety

Without estrogen action, the fusion of the growth plates is postponed and statural growth continues for an exceptionally long time. Aromatase inhibitors, blockers of estrogen biosynthesis, have therefore emerged as a new potential option for the treatment of children with short stature. We investigated the efficacy of the aromatase inhibitor letrozole in the treatment of boys with idiopathic short stature (ISS) using a randomised, placebo-controlled, double-blind research setting. A total of 30 boys completed the two-year treatment. By decreasing estrogen-mediated central negative feedback, letrozole increased gonadotrophin and testosterone secretion in pubertal boys, whereas the pubertal increase in IGF-I was inhibited. Treatment with letrozole effectively delayed bone maturation and increased predicted adult height by 5.9 cm (P0.001), while placebo had no effect on either parameter. The effect of letrozole treatment on near-final height was studied in another population, in boys with constitutional delay of puberty, who received letrozole (n=9) or placebo (n=8) for one year, in combination with low-dose testosterone for six months during adolescence. The mean near-final height of boys randomised to receive testosterone and letrozole was significantly greater than that of boys who received testosterone and placebo (175.8 vs. 169.1 cm, P=0.04). As regards safety, treatment effects on bone health, lipid metabolism, insulin sensitivity, and body composition were monitored in boys with ISS. During treatment, no differences in bone mass accrual were evident between the treatment groups, as evaluated by dual-energy x-ray absorptiometry measurements of the lumbar spine and femoral neck. Bone turnover and cortical bone growth, however, were affected by letrozole treatment. As indicated by differences in markers of bone resorption (U-INTP) and formation (S-PINP and S-ALP), the long-term rate of bone turnover was lower in letrozole-treated boys, despite their more rapid advancement in puberty. Letrozole stimulated cortical bone growth in those who progressed in puberty: the metacarpal index (MCI), a measure of cortical bone thickness, increased more in letrozole-treated pubertal boys than in placebo-treated pubertal boys (25% vs. 9%, P=0.007). The change in MCI correlated positively with the mean testosterone-to-estradiol ratio. In post-treatment radiographic evaluation of the spine, a high rate of vertebral deformities - mild anterior wedging and mild compression deformities - were found in both placebo and letrozole groups. In pubertal boys with ISS treated with letrozole, stimulated testosterone secretion was associated with a decrease in the percentage of fat mass and in HDL-cholesterol, while LDL-cholesterol and triglycerides remained unchanged. Insulin sensitivity, as evaluated by HOMA-IR, was not significantly affected by the treatment. In summary, treatment with the aromatase inhibitor letrozole effectively delayed bone maturation and increased predicted adult height in boys with ISS. Long-term follow-up data of boys with constitutional delay of puberty, treated with letrozole for one year during adolescence, suggest that the achieved gain in predicted adult height also results in increased adult height. However, until the safety of aromatase inhibitor treatment in children and adolescents is confirmed, such treatment should be considered experimental.

X-ray and neutron scattering studies on some nanoscale structures in molecular biology

Scattering of X-rays and neutrons has been applied to the study of nanostructures with interesting biological functions. The systems studied were the protein calmodulin and its complexes, bacterial virus bacteriophage phi6, and the photosynthetic antenna complex from green sulfur bacteria, chlorosome. Information gathered using various structure determination methods has been combined to the low resolution information obtained from solution scattering. Conformational changes in calmodulin-ligand complex were studied by combining the directional information obtained from residual dipole couplings in nuclear magnetic resonance to the size information obtained from small-angle X-ray scattering from solution. The locations of non-structural protein components in a model of bacteriophage phi6, based mainly on electron microscopy, were determined by neutron scattering, deuterium labeling and contrast variation. New data are presented on the structure of the photosynthetic antenna complex of green sulfur bacteria and filamentous anoxygenic phototrophs, also known as the chlorosome. The X-ray scattering and electron cryomicroscopy results from this system are interpreted in the context of a new structural model detailed in the third paper of this dissertation. The model is found to be consistent with the results obtained from various chlorosome containing bacteria. The effect of carotenoid synthesis on the chlorosome structure and self-assembly are studied by carotenoid extraction, biosynthesis inhibition and genetic manipulation of the enzymes involved in carotenoid biosynthesis. Carotenoid composition and content are found to have a marked effect on the structural parameters and morphology of chlorosomes.

Metabolic enzymes as potential drug targets in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria that is fatal in many cases. Emergence of drug resistant strains of P. falciparum requires that new drug targets be-identified. This review considers in detail enzymes of the glycolytic pathway, purine salvage pathway, pyrimidine biosynthesis and proteases involved in catabolism of haemoglobin. Structural features of P. falciparum triosephosphate isomerase which could be exploited for parasite specific drug development have been highlighted. Utility of P. falciparum hypoxanthine-guanine-phosphoribosyltransferase, adenylosuccinate synthase, dihydroorotate dehydrogenase, thymidylate synthase-dihydrofolate reductase, cysteine and aspartic proteases have been elaborated in detail. The review also briefly touches upon other potential targets in P. falciparum

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of similar to 3.5 angstrom in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C-alpha and C-beta of Ser-37 in tetradecanoyl-ACP. C-13, N-15-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C-8)- and dodecanoyl (C-12)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C-14)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK(a) value for the carboxylate, similar to 1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diamino-pelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1 s(-1). The K-m values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 mu M and 308.28 mu M, respectively whereas the V-max and k(cat) values for the enzyme are 0.02074 mu moles/min/ml and 0.003 s(-1), respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.

Solution structures of conformationally equilibrium forms of holo-acyl carrier protein (PfACP) from Plasmodium falciparum provides insight into the mechanism of activation of ACPs

Acyl Carrier Protein (ACP) from the malaria parasite, Plasmodium falciparum (PfACP) in its holo form is found to exist in two conformational states in solution. Unique 3D solution structures of holo-PfACP have been determined for both equilibrium conformations, using high-resolution NMR methods. Twenty high-resolution solution structures for each of the two forms of holo-PfACP have been determined on the basis of 1226 and 1218 unambiguously assigned NOEs (including NOEs between 4 '-phosphopantetheine prosthetic group (4 '-PP) and protein), 55 backbone dihedral angles and 26 hydrogen bonds. The atomic rmsd values of the determined structures of two equilibrium forms, about the mean coordinates of the backbone and heavy atoms, are 0.48 +/- 0.09 and 0.92 +/- 0.10 and 0.49 +/- 0.08 and 0.97 +/- 0.11 angstrom, respectively. The interaction of 4 '-PP with the polypeptide backbone is reported here for the first time for any of the ACPs. The structures of holo-PfACP consist of three well-defined helices that are tightly packed. The structured regions of the molecule are stabilized by extensive hydrophobic interactions. The difference between the two forms arises from a reorientation of the 4 '-PP group. The enthalpy difference between the two forms, although small, implies that a conformational switch is essential for the activation of holo-ACP. Sequence and structures of holo-PfACP have been compared with those of the ACPs from type I and type II fatty acid biosynthesis pathways (FAS), in particular with the ACP from rat and the butyryl-ACP from E. coli. The PfACP structure, thus determined has several novel features hitherto not seen in other ACPs.

Specificity for the Exchange of Phospholipids Through Polymyxin B Mediated Intermembrane Molecular Contacts

Structural specificity for the direct vesicle−vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996) Biochemistry 35, 299−308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle−vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange in covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that stable PxB-mediated vesicle−vesicle contacts may be structurally and functionally distinct from “stalks”, the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis and trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundations of interfacial equilibria and catalysis.

Studies on the metabolism of o-aminophenol purification and properties of isophenoxazine synthase from Bauhenia monandra

An enzyme which catalyzes the oxidative conversion of o-aminophenol to 2-amino-3-H-isophenoxazin-3-one has been purified 396-fold by using standard fractionation procedures. The enzyme is specific for o-aminophenol and has pH and temperature optima at 6.2 and 40 °, respectively. It is insensitive to metal chelating agents but is inhibited by several reducing substances. There is no cofactor or metal ion requirement for the reaction. A competitive type of inhibition was observed with structural analogs such as anthranilic acid and 3-hydroxyanthranilic acid. There are no free sulfhydryl groups in the enzyme, but preincubation of the enzyme with substrate or substrate analogs resulted in the liberation of titratable free sulfhydryl groups. The mechanism of biosynthesis of isophenoxazine ring is discussed.

Valittujen makrolidien ominaisuudet ja niiden vaikutukset fermentoinnin jälkeiseen jatkokäsittelyyn

Työn kirjallisessa osuudessa tarkasteltiin makrolideja yleisellä tasolla keskittyen kahden makrolidin ominaisuuksiin molekyylitasolla. Näiden tautomerisoitumista käsitellään tuoden esiin sekä yhdisteiden rakenteelliset yhteneväisyydet ja eroavaisuudet että niiden vaikutukset yhdisteiden vaikutusmekanismiin ja metaboliaan. Kirjallisessa osuudessa perehdyttiin myös makrolidien biosynteesiin ja tuotantoprosessiin keskittyen downstream-prosessointiin ja erityisesti biosynteesistä peräisin oleviin epäpuhtauksiin. Lisäksi kirjallisessa osuudessa käsiteltiin argentaatiokromatografian perusteita. Kokeellisessa osuudessa yhdelle makrolidille kehitettiin argentaatiokromatografiaan perustuva puhdistusmenetelmä. Perinteisillä kromatografisilla menetelmillä yhdistettä ei voida puhdistaa kaikista sen epäpuhtauksista. Makrolidin puhtaus argentaatiokromatografian jälkeen oli 98,6 %. Lisäksi kehitettiin uusi kiteytysmenetlmä, jossa yhdiste kiteytettiin anhydridina tavanomaisen monohydraattimuodon sijasta. Makrolidin analysointiin kehitettiin HPLC-menetelmä, joka validoitiin ICH:n ohjeiden mukaisesti. Yhditeen tautomeerimuodot ja siinä esiintyvät epäpuhtaudet tutkittiin käyttäen LC/MS-analyysia. Yhden epäpuhtauden rakenne varmistettiin eristämisen jälkeen NMR:n avulla. Saatavilla olevien tietojen mukaan yhdisteen tulkittuja NMRspektrejä ei ole julkaistu. Lisäksi aiemmin tuntematon epäpuhtaus identifioitiin perustuen retentioaikaan ja MS-analyysiin.

MECHANISMS AND EFFECTS OF MITOCHONDRIAL DNA INSTABILITY AND COPY NUMBER MANIPULATION

Defects in mitochondrial DNA (mtDNA) maintenance cause a range of human diseases, including autosomal dominant progressive external ophthalmoplegia (adPEO). This study aimed to clarify the molecular background of adPEO. We discovered that deoxynucleoside triphosphate (dNTP) metabolism plays a crucial in mtDNA maintenance and were thus prompted to search for therapeutic strategies based on the modulation of cellular dNTP pools or mtDNA copy number. Human mtDNA is a 16.6 kb circular molecule present in hundreds to thousands of copies per cell. mtDNA is compacted into nucleoprotein clusters called nucleoids. mtDNA maintenance diseases result from defects in nuclear encoded proteins that maintain the mtDNA. These syndromes typically afflict highly differentiated, post-mitotic tissues such as muscle and nerve, but virtually any organ can be affected. adPEO is a disease where mtDNA molecules with large-scale deletions accumulate in patients tissues, particularly in skeletal muscle. Mutations in five nuclear genes, encoding the proteins ANT1, Twinkle, POLG, POLG2 and OPA1, have previously been shown to cause adPEO. Here, we studied a large North American pedigree with adPEO, and identified a novel heterozygous mutation in the gene RRM2B, which encodes the p53R2 subunit of the enzyme ribonucleotide reductase (RNR). RNR is the rate-limiting enzyme in dNTP biosynthesis, and is required both for nuclear and mitochondrial DNA replication. The mutation results in the expression of a truncated form of p53R2, which is likely to compete with the wild-type allele. A change in enzyme function leads to defective mtDNA replication due to altered dNTP pools. Therefore, RRM2B is a novel adPEO disease gene. The importance of adequate dNTP pools and RNR function for mtDNA maintenance has been established in many organisms. In yeast, induction of RNR has previously been shown to increase mtDNA copy number, and to rescue the phenotype caused by mutations in the yeast mtDNA polymerase. To further study the role of RNR in mammalian mtDNA maintenance, we used mice that broadly overexpress the RNR subunits Rrm1, Rrm2 or p53R2. Active RNR is a heterotetramer consisting of two large subunits (Rrm1) and two small subunits (either Rrm2 or p53R2). We also created bitransgenic mice that overexpress Rrm1 together with either Rrm2 or p53R2. In contrast to the previous findings in yeast, bitransgenic RNR overexpression led to mtDNA depletion in mouse skeletal muscle, without mtDNA deletions or point mutations. The mtDNA depletion was associated with imbalanced dNTP pools. Furthermore, the mRNA expression levels of Rrm1 and p53R2 were found to correlate with mtDNA copy number in two independent mouse models, suggesting nuclear-mitochondrial cross talk with regard to mtDNA copy number. We conclude that tight regulation of RNR is needed to prevent harmful alterations in the dNTP pool balance, which can lead to disordered mtDNA maintenance. Increasing the copy number of wild-type mtDNA has been suggested as a strategy for treating PEO and other mitochondrial diseases. Only two proteins are known to cause a robust increase in mtDNA copy number when overexpressed in mice; the mitochondrial transcription factor A (TFAM), and the mitochondrial replicative helicase Twinkle. We studied the mechanisms by which Twinkle and TFAM elevate mtDNA levels, and showed that Twinkle specifically implements mtDNA synthesis. Furthermore, both Twinkle and TFAM were found to increase mtDNA content per nucleoid. Increased mtDNA content in mouse tissues correlated with an age-related accumulation of mtDNA deletions, depletion of mitochondrial transcripts, and progressive respiratory dysfunction. Simultaneous overexpression of Twinkle and TFAM led to a further increase in the mtDNA content of nucleoids, and aggravated the respiratory deficiency. These results suggested that high mtDNA levels have detrimental long-term effects in mice. These data have to be considered when developing and evaluating treatment strategies for elevating mtDNA copy number.

Vitamin C status of vitamin A-deficient rats

IT was initially suggested that vitamin A-deficiency leads to an interference in the biosynthesis of ascorbic acid, because depletion of vitamin A was found to cause a fall in the tissue-levels of ascorbate and diminished urinary ascorbic acid excretion in animals1-3. Mapson4, however, concluded that lowered ascorbic acid-levels in vitamin A-deficient rats is due to inanition only, because he was able to show that following chloretone treatment vitamin A-deficient and pair-fed vitamin A normal rats excrete comparable amounts of ascorbic acid in their urine and that restriction of food intake reduces the urinary ascorbate even in the chloretone-treated normal rats. Results of our preliminary experiments reported here clearly indicate that the synthesis of ascorbic acid in rats is markedly reduced during vitamin A-deficiency.