Primer-independent initiation of RNA synthesis by SeMV recombinant RNA-dependent RNA polymerase

Sesbania mosaic virus (SeMV),a single-strand positive-sense RNA plant virus, belongs to the genus Sobemoviruses. Mechanism of replication in Sobemoviruses is poorly understood. In the present study, SeMV RNA-dependent RNA polymerase (RdRp) was overexpressed and purified as a thioredoxin-tagged protein. The recombinant SeMV RdRp could synthesize RNA from genomic or subgenomic RNA templates, even in the absence ofthe protein primer, VPg. Analysis of the product indicated that it was double-stranded and that the mode of initiation was de novo. Mutational analysis of the 3' UTR of subgenomic RNA revealed that a stem-loop structure at the 3' end was important. Further, analysis of this stem-loop showed that the SeMV RdRp was capable of recognizing stem-loop structures of various lengths and forms. These results demonstrate that the SeMV RdRp is capable of primer-independent RNAsynthesis in vitro. (C) 2010 Elsevier Inc. All rights reserved.

Characterization of two M17 family members in Escherichia coli, Peptidase A and Peptidase B

Escherichia coil encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in Delta pepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of Delta pepN during nutritional downshift and hightemperature stress. Purified PepA and PepB display broad substratespecificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in Delta pepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms. (C) 2010 Elsevier Inc. All rights reserved.

A study of the purification and properties of tryptophan synthetase of Bengal gram (Cicer arietinum)

Active preparations of tryptophan synthetase were obtained from Bengal gram (Cicer arietinum) by the following procedure: (1) precipitation of inactive materials by manganous sulfate, (2) Adsorption of impurities on Alumina Cγ, (3) Adsorption of tryptophan synthetase on tricalcium phosphate gel, removal of inert protein from the gel by treatment with phosphate buffer (pH 7.2), and selective elution of the enzyme by 0.15 M phosphate buffer pH 7.2 containing 10% ammonium sulfate and 10−3 M serine. A 220-fold purification of the enzyme with 44% recovery of the activity was achieved. The pH optimum, effect of temperature, and substrate concentration and other properties of the purified enzyme have been studied in detail. Only the Image -isomer of serine takes part in the reaction. The Km values for indole, Image -serine, and Image -serine were calculated to be 0.66, 4.1, and 8.6 × 10−4 M, respectively. A kinetic study of the inhibition of tryptophan synthetase by indole-propionic acid has shown that it is of a competitive type. It has been demonstrated for the first time that 4-nitro-salicylaldehyde can replace pyridoxal phosphate as a coenzyme for the tryptophan synthetase reaction.

Studies of the enzymes involved in nicotinamide adenine dinucleotide metabolism in Aspergillus niger

The enzyme nicotinamide amidase (nicotinamide amidohydrolase) was purified 57-fold from Aspergillus niger. The purified preparation was specific towards its substrate nicotinamide and did not deamidate NADP, NAD, NMN, N′-methyl nicotinamide, asparagine, glutamine, benzamide, α-naphthaleneamide and indoleacetamide. The asparagine, glutamine, benzamide, α-naphthaleneamide and indoleacetamide.vThe optimum pH was found to be 7.5. Temperature optimum was 40°. It had a Km value of 6.504 · 10−4 M towards nicotinamide. The enzyme exhibited Mg2+ ion requirement for its optimum activity. NAD-glycohydrolase (EC 3.2.2.5) was purified 109-fold from the mold. A. niger. The enzyme preparation was active only towards NAD and NADP and did not attack NMN, N′-methylnicotinamide and NADH. The Km value for NAD was found to be 7.693 · 10−6 M. The enzyme did not require any metal ion for its activity. It is suggested that A. niger will serve a better source for a large scale preparation of NAD-glycohydrolase than the Neurospora mold. The biological role of both NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD level has been discussed. It is, further, observed that NAD did not exert its feedback control on nicotinamide amidase at least in A. niger.

Biosynthesis of valine and isoleucine in plants I. Formation of α-acetolactate in Phaseolus radiatus

1. 1. The presence of an enzyme system in plants catalyzing the formation of α-acetolactate from pyruvate has been demonstrated; the system in green gram (Phaseolus radiatus) has been partially purified and its characteristics have been studied.2. Free acetaldehyde is formed as a product of the reaction and so the reaction is mainly diverted towards the formation of acetoin. 3. The system requires thiamine pyrophosphate and a divalent metal ion (Mn2+ or Mg2+) for maximum activity. The optimum pH is around 6.0 and the optimum temperature is 60°. 4. The system is very labile in absence of pyruvate, Mn2+ and DPT. 5. The Km values for pyruvate, Mn2+, Mg2+ and DPT are 3·10−2 M. 5·10−5 M, 2·10−5 M, and e·10−6 M respectively. The activation energy is 3540 cal/mole. 6. The enzyme is strongly inhibited by p-chloromercuribenzoate and the inhibition can be reversed partially by 2-mercaptoethanol, BAL or cysteine. Heavy metals, such as Hg2+ and Ag+, are inhibitory but l-valine does not inhibit the reaction.

Biosynthesis of isoleucine and valine in mycobacterium tuberculosis H37Rv. II. Purification and properties of acetohydroxy acid isomeras

Acetohydroxy acid isomerase (AHA isomerase) was purified about 110-fold and separated from reductase and acetohydroxy acid isomeroreductase. The AHA isomerase was found to be homogeneous by agar and polyacrylamide gel electrophoreses at different pHs. The properties of AHA isomerase have been studied. The purified enzyme showed requirement for l-ascorbic acid and sulfate ions for its activity. Synthetic ascorbic acid sulfate could replace l-ascorbic acid and sulfate. α-Methyllactate and α-ketoisovalerate were found to inhibit AHA isomerase activity competitively whereas l-valine and l-isoleucine had no significant inhibitory effect. p-Hydroxymercuribenzoate inhibited AHA isomerase activity and the inhibition was reversed by β-mercaptoethanol.

Biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid, the lathyrus sativus neurotoxin

The biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (ODAP) the Lathyrus sativus neurotoxin has been found to follow the scheme depicted below: {A figure is presented}. The first reaction is catalysed by oxalyl-CoA synthetase which has properties similar to that of the enzyme in peas. The second reaction is catalysed by another enzyme which is specific to L. sativus and is designated as oxalyl-CoA-α,β-diaminopropionic acid oxalyl transferase. The enzymes have been purified by about 60-fold and their properties studied. A partial resolution of the two enzyme activities has been achieved using CM-sephadex columns.

Oxidation of vitamin A1 and vitamin A2 aldehydes to the corresponding acids by enzymes from pig and rat livers

VITAMIN A is stored in rat liver largely as its ester with small amounts of the alcohol, but is transported in the normal circulating blood in the latter form1. Although it was generally believed that the alcohol form is the more physiological state of the vitamin, since the work of Dowling and Wald2, it is being recognized that vitamin A acid and not the alcohol may be nearer to the 'active vitamin A'. If this were to be so, it would be important to demonstrate that a mechanism exists in the rat for the production of vitamin A acid from vitamin A alcohol through the intermediate, the aldehyde. Regarding the formation of the aldehyde, it has been well established that the alcohol dehydrogenase can bring about the conversion of vitamin A alcohol to retinene3. The presence of an enzyme in rat and pig liver catalysing the oxidation of retinene1 and retinene2 to the corresponding acids has been demonstrated in the present work and the partially purified enzyme preparation shown to be completely devoid of alcohol dehydrogenase activity.

Indoleacetaldoxime hydro-lyase (4.2.1.29). III. Further studies on the nature and mode of action of the enzyme

Further purification of indoleacetaldoxime (IAOX) hydro-lyase from Gibberella fujikuroi by DEAE-cellulose chromatography is described. The purified enzyme was activated by dehydroascorbic acid (DHA), ascorbic acid (AA), and pyridoxal phosphate (PALP) and was inhibited by thiol compounds and thiol reagents including phenylthiocyanate. Ferrous ions but not ferric ions activated the purified enzyme. The enzyme was activated by dihydrofolic acid but inhibited by tetrahydrofolic acid. Phenylacetaldoxime, a competitive inhibitor, afforded partial protection of the enzyme from the action of N-ethylmaleimide suggesting the involvement of a thiol function at the active site or substrate-binding site. The inhibition of the enzyme by 2,3-dimercaptopropanol was reversed by DHA, PALP, or frozen storage. KCN inhibition of the enzyme was reversed by PALP. NaBH4 reduction of the purified enzyme in the presence of PALP gave an active enzyme which was further activated by PALP or DHA but not by ferrous ions. These results suggested a "structural" role for PALP in the activity of IAOX hydro-lyase. Dilute solutions of the purified enzyme, obtained during DEAE-cellulose chromatography and concentrated using sucrose, showed enhanced activity upon frozen storage and thawing. The increase in activity of the enzyme during certain culture conditions, the activation and inhibition of the enzyme by several unrelated compounds, and the effect of freezing indicate that IAOX hydro-lyase is probably a metabolically regulated enzyme with a structure composed of subunits.

Metabolism of glycerol by an Arthrobacter species

An Arthrobacter species (tentatively identified as A. citreus), isolated by the enrichment culture method with glycerol as the sole source of carbon, was studied with a view to elucidate its pathway of glycerol breakdown. Evidence has been obtained against the functioning of the phosphorylative pathway by the study of (1) oxygen uptake with phosphorylated intermediates, (2) uptake of inorganic phosphorus by intact resting cells, (3) action of inhibitors like sodium fluoride, sodium azide, sodium arsenite, sodium iodoacetate, and parachloromercurybenzoate on oxygen uptake with resting cell suspensions and cell-free extracts in some cases. Evidence presented for the functioning of a non-phosphorylative pathway includes studies on the oxidation of glycerol, D-glyceraldehyde, glycerate, glycolic aldehyde, glycolic acid, glyoxylic acid, and formic acid to carbon dioxide and water. Further, the possibility of glyoxylate metabolism through the tricarboxylic acid cycle by its formation of malate was shown. The significance of the above pathway is that it has pointed to an alternative route of carbohydrate metabolism and entry into the tricaboxylic acid cycle without the intervention of pyruvate or the condensing enzyme.

Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Endo-polygalacturonate lyase of Cytophaga johnsonii

An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca++ ions for its maximum activity. The apparent Kmfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.

Italian ja Veneton läänin kolmas sektori. Maaraportti maaseutualueiden palvelutuotannon haasteista ja hyvistä käytännöistä.

Tiivistelmä Maaraportin tavoitteena on luoda yleiskatsaus Italian kolmanteen sektoriin ja tutkimus kohdistuu erityisesti Pohjois-Italiassa sijaitsevan Veneton läänin kolmannen sektorin palvelutuotantoon. Raportissa tarkastellaan erityisesti kolmannen sektorin ja julkisen palvelutuotantosektorin sekä julkishallinnon toiminnallisia suhteita. Kolmannen sektorin mahdollisuuksia vastata maaseutualueiden palvelujen tarjonnan haasteisiin on pyritty analysoimaan. Italia on julkishallinnon ja kolmannen sektorin palvelutuotannon kannalta tarkasteltuna nykytilanteessa mielenkiintoinen testilaboratorio koko Euroopan mittakaavassa johtuen maan historiallisista, kulttuuri-sista, kieli- ja maantieteellisistä sekä itse väestöpohjan tarjoamista haasteista. Italiasta ei voida puhua yhden homogeenisen valtiokäsitteen alla, koska maa toimii kulttuurisista perinteistään johtuen kolmella eri nopeudella. Itse valtio käsitettäkin tulkitaan Italiassa monin eri tavoin pohjautuen ”valtiona” nähdyn organisaation käytännön funktioon sitoutuneena alueellisiin ja kulttuurisiin tekijöihin. Teollinen, dynaaminen yritysten varakas Pohjois-Italia henkilöityneenä Milanoon, hallinnon ja kulttuurin leimaama Keski-Italia kiteytyneenä ikuiseen kaupunkiin Roomaan ja Napolin alapuolinen Etelä-Italia, joka painii ”järjestelmässä sisällä olevan järjestelmänsä” kanssa eivät ole koskaan muodos-taneet oikeasti yhtenäistä Italian valtiota. Italiassa erillisinä itsehallinnollisina alueina toimivat lääneiksi luettavat Sisilia, Trentino Alto-Adige, Val di Aosta, Sardegna ja Friuli-Venezia Giulia, mikä lisää hallinnollista kirjavuutta. Viimeisten vuosien aikana Italian julkishallinto on ollut jatkuvassa transitiotilassa yrittäessään kohdata uudistumisen vaateita globalisoituvassa maailmassa. Italiassa on meneillään hidas siirtyminen liittovaltiomaiseen järjestelmään ja jopa syvempään paikallisuuteen. Massiivinen ja raskas julkishallintokoneisto neljällä eri tasolla (valtio - lääninhallinnolliset alueet - vahvat maakunnat - kunnat) on erittäin hankalasti modernisoitavissa, mutta poliittinen tahtotila on vaihtumassa klassista vahvaa keskushallintoa kannattavasta ajattelutavasta hyväksymään federalistisia, alueellisia hallintoratkaisuja. Italiassa on herätty havaitsemaan, että ongelmien ratkaisu täytyy viedä itse alueille ja niiden ihmisille, koska unilateraalinen keskushallinnointitapa ei enää vastaa monikerroksisiin, moniulotteisiin alueellisiin haasteisiin. Jokaisella alueella on erilaiset lähtökohdat ongelmien ratkaisuihin myös globalisoitumisen asettamissa yhteisissä haasteissa. Kolmannen sektorin asema arvostettuna italialaisten kansalaispalvelujen täydentäjänä ja tuottajana alkaa olla nykypäivää. Kolmannen sektorin elintärkeä osuus uusien palvelutarpeiden tunnistamisessa on jo yleisesti tunnustettu tosiasia Italiassa. Vapaaehtoisjärjestöt toimivat eturivissä kansalaisten arkipäivässä ja pystyvät näin kanavoimaan ensimmäisinä sosiaalista kehityskulkua ja kohottamaan esiin erilaisia marginaalistenkin ryhmien tarpeita sekä vastaamaan niihin. Kolmas sektori laajentaa, tuottaa ja paikkaa paikallista julkista palvelutuotantoa. Ratkaiseva askel lainsäädännölliseltä kannalta on ollut rahoitusvirtojen ohjaaminen kolmannen sektorin ns. ONLUS-organisaatioille ja ONLUS-statuksen perustaminen vuonna 1997. Italiassa on 60 231.214 asukasta (31.7.2009) ja se on hallinnollisesti jakaantuneena 8 100 kuntaan. Suomessa vastaavat luvut ovat 5 350 712 asukasta ja 348 kuntaa (11/2009). Maantieteelliseltä kooltaan Italia ei eroa 301 338 km2:n pinta-alallaan paljonkaan Suomesta (338 424 km2). Väestömäärän erojen vuoksi Italiassa on keskimäärin 199,9 asukasta neliökilometrillä ja Suomessa 17,1. Maiden välinen suora vertailu ei ollut mittakaavaerojen vuoksi järkevää. Kuntien hallinnollinen rooli on Italiassa painotukseltaan erilainen kuin Suomessa. Suomessa kuntien vastuulla oleva sosiaali-, terveydenhuolto- ja koulutoimi eivät ole samassa laajuudessa italialaisten kuntien vastuulla vaan näiden toimialojen vastuulliset tahot ovat läänit terveydenhoitopiireineen sekä maakuntahallinto koulupiirien osalta. Italialaisten kuntien vastuualueet voivat myös vaihdella mittavasti perustuslaissa määriteltyjen perustoimien lisäksi. Tutkimuksen kohteeksi on rajattu Koillis-Italiassa sijaitseva Veneton läänin alue, koska se on väkimäärältään lähes yhtä suuri kuin Suomi, 4 893 309 asukasta (31.3.2009). Kuntien lukumäärä on huimaava 581 kpl. Veneto on maantieteelliseltä alueeltaan pieni ja harvaan asuttua maaseutua on pinta-alasta melko niukasti. Pohjoisen osan vuoristoalueet karuine olosuhteineen muodostavat palvelutuotannollisesti haas-teellisen ympäristön, joka on verrattavissa suomalaisten maaseutualueiden tilanteeseen. Venetossa on omaksuttu Italian mittakaavassa innovatiivisia ratkaisuja julkishallinnon palvelutuotannon ja kolmannen sektorin toiminnan osalta. Veneton alue on luokiteltu maailman pienyritysintensiivisimmäksi alueeksi, mikä heijastuu myös alueen palvelutuotantoratkaisuissa. Raportti esittelee ensin Italian hallintoa, keskushallinnon, paikallishallinnon ja tutkimuksen asettelulle olennaisten kolmannen sektorin toimijoiden osalta. Tämä on välttämätöntä, sillä kolmannen sektorin toiminta on Italiassa hyvin pitkälle lainsäädännöllä ohjattua ja rajattua. Kolmannen sektorin käsitteistöä, toimijoita, järjestäytymistä ja sen toimintaa säätelevää lainsäädäntöä esitellään tarkemmin. Veneton läänin osalta kolmanteen sektoriin perehdytään sekä hallinnon että toimijakentän näkökulmia tulkiten. Raportin lopussa esitellään kolmannen sektorin palvelutuotantoon ja hallinnointiin liittyviä case-esimerkkitapauksia.

Studies on nucleotide pyrophosphatase I. Partial purification and properties of a sheep liver enzyme that catalyzes the hydrolysis of dinucleotides

A partially purified sheep liver enzyme that hydrolyzed dinucleotides at the pyrophosphate bond was obtained by solubilizing the 18,000g sediment with n-butanol and fractionating the solubilized enzyme with acetone. The enzyme activity when measured using FAD as substrate, (FAD → FMN + AMP), was optimal at pH 9.7 and temperatures between 30 °–36 ° and at 60 °. The rate of release of FMN with time occurred with an initial lag of 30 sec, a linear increase for 1 min, and a subsequent irregular rate. In the presence of orthophosphate (Pi; 10 μImage ), FMN was released at an uniformly continuous and enhanced rate. 32Pi was not incorporated into the substrate or products. Sodium arsenate counteracted the effects of Pi. The apparent Km and Vmax were 0.133 mImage and 100 units; and 0.133 mImage and 200 units, in the absence and presence of Pi, respectively. The temperature optimum was 42 ° in the presence of Pi.Negative cooperative interactions observed at low concentrations of FAD were abolished by the addition of Pi. The inhibition by AMP was sigmoid and Pi abolished this sigmoidal response. The enzyme hydrolyzed in addition to FAD, NAD+ and NADP+. Nucleoside triphosphates were potent inhibitors of the enzyme activity. The partial inhibition of the enzyme by o-phenanthroline and by p-hydroxymercuribenzoate could be reversed by Fe2+ ions and by reduced glutathione, respectively.