Indole oxygenase from the leaves of Jasminum grandiflorum

An indole oxygenase from the leaves of Jasminum grandiflorum was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 4.8 and at 30°C. Apart from indole, the oxygenase also attacks 5-hydroxy indole and 5-bromoindole. Both sulfhydryl reagents and sulfhydryl compounds inhibited the enzyme activity. Copper specific metal chelators such as salicylaldoxime, diethyl dithiocarbamate and neocuproine, inhibited the enzyme activity drastically. Inhibition caused by atebrine, could be reversed by FAD. Dialysis resulted in complete loss of enzyme activity. Inactive enzyme could be reactivated only by the addition of both FAD and Cu2+, suggesting that indole oxygenase is a cuproflavoprotein.

Development of radiosynthesis methods for 18F-labelled radiopharmaceuticals : General considerations and production of a dopamine transporter radioligand

Positron emission tomography (PET) is a molecular imaging technique that utilises radiopharmaceuticals (radiotracers) labelled with a positron-emitting radionuclide, such as fluorine-18 (18F). Development of a new radiotracer requires an appropriate radiosynthesis method: the most common of which with 18F is nucleophilic substitution with [18F]fluoride ion. The success of the labelling reaction is dependent on various factors such as the reactivity of [18F]fluoride, the structure of the target compound in addition to the chosen solvent. The overall radiosynthesis procedure must be optimised in terms of radiochemical yield and quality of the final product. Therefore, both quantitative and qualitative radioanalytical methods are essential in developing radiosynthesis methods. Furthermore, biological properties of the tracer candidate need to be evaluated by various pre-clinical studies in animal models. In this work, the feasibility of various nucleophilic 18F-fluorination strategies were studied and a labelling method for a novel radiotracer, N-3-[18F]fluoropropyl-2beta-carbomethoxy-3beta-4-fluorophenyl)nortropane ([18F]beta-CFT-FP), was optimised. The effect of solvent was studied by labelling a series of model compounds, 4-(R1-methyl)benzyl R2-benzoates. 18F-Fluorination reactions were carried out both in polar aprotic and protic solvents (tertiary alcohols). Assessment of the 18F-fluorinated products was studied by mass spectrometry (MS) in addition to conventional radiochromatographic methods, using radiosynthesis of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) as a model reaction. Labelling of [18F]beta-CFT-FP was studied using two 18F-fluoroalkylation reagents, [18F]fluoropropyl bromide and [18F]fluoropropyl tosylate, as well as by direct 18F-fluorination of sulfonate ester precursor. Subsequently, the suitability of [18F]beta-CFT-FP for imaging dopamine transporter (DAT) was evaluated by determining its biodistribution in rats. The results showed that protic solvents can be useful co-solvents in aliphatic 18F-fluorinations, especially in the labelling of sulfonate esters. Aromatic 18F-fluorination was not promoted in tert-alcohols. Sensitivity of the ion trap MS was sufficient for the qualitative analysis of the 18F-labelled products; p-[18F]MPPF was identified from the isolated product fraction with a mass-to-charge (m/z) ratio of 435 (i.e. protonated molecule [M+H]+). [18F]beta-CFT-FP was produced most efficiently via [18F]fluoropropyl tosylate, leading to sufficient radiochemical yield and specific radioactivity for PET studies. The ex vivo studies in rats showed fast kinetics as well as the specific uptake of [18F]beta-CFT-FP to the DAT rich brain regions. Thus, it was concluded that [18F]beta-CFT-FP has potential as a radiotracer for imaging DAT by PET.

Synthesis of Reduced Metabolites of Isoflavonoids, and their Enantiomeric Forms

Isoflavonoids are naturally occurring plant derived biochemicals, which act as phytoalexins. Isoflavonoids are of interest due to their estrogenic and other potential physiological properties, particularly in mammals that typically consume isoflavonoid rich nutrients such as soy and red clover. The literature review of this thesis mainly focuses on the reduced metabolites of hydroxy and/or methoxy substituted isoflavones with four groups: isoflavan-4-ols, isoflav-3-enes, isoflavans and α-methyldeoxybenzoins (1,2-diarylpropan-1-ones), which are all reduced metabolites of food derived isoflavones in mammals. Related isoflavan-4-ones are briefly discussed. Results of an extensive survey of the literature concerning the synthesis of polyhydroxy- or methoxysubstituted isoflavonoids and especially asymmetric approaches are discussed. The experimental section describes new synthetic methods to prepare polyphenolic reduced isoflavonoid structures such as isoflav-3-enes, isoflavan-4-ones, cis- and trans-isoflavan-4-ols, 1,2-diarylpropan-1-ones and isoflavans by various hydride reagents and hydrogenations. The specific reactivity differences of various hydride reagents toward isoflavonoids are discussed. The first enantioselective synthesis of natural (S)-(-)-equol and the opposite enantiomer (R)-(+)-equol is also described by the asymmetric iridium PHOX catalysed hydrogenation of isoflav-3-enes. Both of these equol enantiomers are found to possess biological activity in mammals due to estrogen receptor binding activity. The natural enantiomer prefers estrogen receptor β and the R-enantiomer prefers the estrogen receptor α. Also the precursor, isoflav-3-ene, is found to possess positive biological effects on mammals. In connection with the synthetic work, the (S)-(-)-equol was discovered from serum of ewes after isoflavone rich red clover feeding. The chiral HPLC method was developed to identify natural equol enantiomer for the first time in this species. The first synthesis of natural isoflavonoid (R)-(-)-angolensin and its enantiomer (S)-(+)-angolensin is desribed by the use of recyclable chiral auxiliaries (chiral pseudoephedrines). The method offers a general approach also to other natural polyphenolic 1,2-diarylpropan-1-ones and to further study isoflavonoid metabolism in human and other mammals. The absolute configurations of these new chiral isoflavonoid metabolites were determined by X-ray spectroscopy. Also thorough NMR and MS analysis of synthesised structures are presented.

Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid-6-hydroxylase from Micrococcus

3-Hydroxybenzoic acid-6-hydroxylase from Micrococcus sp. was purified to homogeneity in a single step using the substrate-mediated interaction of the enzyme with blue-Sepharose. The enzyme was bound to the affinity matrix in the presence of 3-hydroxybenzoic acid and was eluted in its absence. The molecular weight of the purified enzyme is 70,000 with no subunit structure. The flavoenzyme required the exogenous addition of FAD for its complete activity and had a strict preference for NADH over NADPH. The activity of the enzyme was drastically inhibited by Cu2+ and Hg2+ and the inhibition was reversed by thiol reagents.

Ball wear and its control in the grinding of a lead-zinc sulphide ore

Marked ball grinding tests were carried out in the laboratory with a lead-zinc sulphide ore under different experimental conditions using high carbon low alloy steel (cast and forged) and high chrome cast iron balls. Relative ball wear as a function of grinding period and milling conditions was evaluated for the different types of ball materials. The role of corrosion and abrasion-erosion in the wear of grinding media is brought out. Methods to minimise ball wear through control of mill atmosphere and addition of reagents are discussed.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Purification of 3,5-Dichlorocatechol 1,2-Dioxygenase, a Nonheme Iron Dioxygenase and a Key Enzyme in the Biodegradation of a Herbicide, 2,4-Dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM−1 s−1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s−1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

Standardization of pollen allergens of Parthenium hysterophoms and selection of an in-house reference extract

A standardized in-house reference extract from the pollen of Parthenium hysterophorus, which is responsible for the high incidence of allergic rhinitis in India, was generated and examined by skin test, radio-allergosorbent test inhibition and isoelectric focusing. Parthenium reference allergen discs and positive reference serum were also generated. These reference reagents could not only be used for the quantitation of Parthenium-specific IgE in the sera of rhinitis patients but also for the evaluation of allergenic activity (relative potency and lot-to-lot variation) of different batches of Parthenium pollen.

The use of murine polyclonal anti-idiotypic antibodies as surrogate allergens in the diagnosis of Parthenium hypersensitivity

Background: Anti-idiotypic antibodies (Ab-2), which are the mirror images of idiotypic antibodies (Ab-1), may be useful as diagnostic reagents and for use as immunogen to induce antigen-specific immune responses. Methods and Results: To explore the biologic potential of Ab-2 as diagnostic reagents in allergic diseases, murine mouse (m) Ab-2 were raised by immunizing Balb/c mice with affinity purified rabbit (r) Ab-1 specific for the pollen of Parthenium hysterophorus, an allergenic weed that grows wild on the Indian subcontinent and in Australia, Mexico, and the southern United States. Affinity purified Parthenium-specific human (h)AB-1 could successfully inhibit the binding of mAb-2 to immobilized rAb-1. Further, Balb/c mice immunized with mAb-2 induced Parthenium-specific anti-anti-idiotypic IgE and IgG antibodies. Specificity of the Ab-2 was confirmed by the ability of Parthenium pollen extracts to inhibit the binding of allergen-specific IgE and IgG Ab-1 in the sera of patients with rhinitis to immobilized mAb-2. Parthenium-sensitive patients with rhinitis who had positive results on skin prick tests to Parthenium pollen extracts also responded with a positive skin reaction to mAb-2. Conclusion: Our data demonstrate that Parthenium-specific mAb-2 may be of value as surrogate allergens in allergen standardization and for in vitro diagnosis.

An Approach Toward Homocalystegines and Silyl-homocalystegines. Acid-Mediated Migrations of Acetates in Seven-Membered Ring Systems

A short access to homocalystegine analogues silylated at C7 is described. The synthesis involves the desymmetrization of a (phenyldimethylsilyl)methylcycloheptatriene using osmium-mediated dihydroxylation, followed by the diol protection and a cycloaddition involving the remaining diene moiety and an acylnitroso reagent. Additions of the osmium and acylnitroso reagents were shown, through X-ray diffraction studies of the resulting major isomers, to occur anti and syn, respectively, relative to the SiCH2 substituent. N-O bond cleavage on the resulting cycloadduct then produces the aminopolyol having a silylmethyl substituent. Oxidation of the C-Si bond also afforded an access to unusual amino-heptitols having five contiguous stereogenic centers. In the course of this work, we finally observed a unusual rearrangement taking place on cycloheptanone 18 substituted by two acetyl groups and a neighboring Boc-protected amine. A profound reorganization of the substituents on the seven-membered ring effectively took place under acidic conditions (TFA) leading to the thermodynamically more stable homocalystegine-type compound., DFT calculations of the conformational energy of isomeric silyl homocalystegines indicated that the product observed upon the acid-mediated rearrangement was the most stable of a series of analogues with various distributions of substituents along the seven-membered ring backbone. A tentative mechanism is proposed to rationalize the acetate migrations and inversions of the stereochemistry at various stereocenters.

Application of selective F-1 decoupled H-1 NMR for enantiomer resolution and accurate measurement of enantiomeric excess at low chiral substrate/auxiliary concentration

The higher substrate and chiral auxiliary concentration is a pre-requisite to obtain efficient separation of H-1 NMR signals of enantiomers. The higher concentration of chiral lanthanide shift reagents provides broadened spectral lines resulting in a severe loss of resolution between the enantiomer resonances. In order to circumvent such difficulties, herein we present the application and the usefulness of a selective F-1 decoupled correlation (COSY) experiment which yields proton decoupled proton spectra in the indirect dimension. The potentiality of the experiment is demonstrated on several chiral compounds possessing different functional groups, employing either a lanthanide shift reagent or a solvating reagent as chiral auxiliaries. (C) 2011 Elsevier B.V. All rights reserved.

Synthesis and esterolytic chemistry of some (dialkylamino) pyridine-functionalized micellar aggregates. Evidence of catalytic turnover

Four new (dialkylamino)pyridine-functionalized surfactants have been synthesized. Micelles were generated either from the surfactant alone in aqueous buffer (pH 8.5 or 9.0) or by comicellization in 1 x 10(-3)-1 x 10(-4) M aqueous micellar cetyltrimethylammonium bromide (CTABr) solution at pH 8.5 or 9.0. Such aggregates were used to cleave p-nitrophenyl alkanoates or p-nitrophenyl diphenylphosphate. The nucleophilic reagents and the second-order ''catalytic'' rate constants toward esterolysis of the substrate p-nitrophenyl octanoate (at 25 degrees C, pH 9.0) were [cat.] = 1 x 10(-4) M, [CTABr] = 1 x 10(-3) M, and k(cat.) = 440.13 M(-1) s(-1) for 1b, [cat.] = 5 x 10(-4) M, [CTABr] = 5 x 10(-4) M, and k(cat.) = 30.8 M(-1) s(-1) for 1c, [cat.] = 5 x 10(-4) M, [CTABr] = 5 x 10(-3) M, and k(cat.) = 183.64 M(-1) s(-1) for 2a, and [cat.] = 3 x 10(-4) M and k(cat.) = 54.1 M(-1) s(-1) for 2b. The catalytic systems, especially 1b/CTABr and 2a/CTABr, also conferred significantly greater reactivity toward the esters derived from alkanoic acids of moderate chain length (C-6-C-10) during hydrolytic cleavages relative to their shorter and longer counterparts. Importantly, the catalytic systems comprising the coaggregates of either neutral 1b and CTABr (1:10) or anionic 2a and CTABr (1:10) conformed to the Michaelis-Menten kinetic scheme and demonstrated turnover behavior in the presence of excess substrate.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

Reverse flotation of silica from Kudremukh iron ore .2. Optimization of flotation process

An experimental programme based on statistical analysis was used for optimizing the reverse Rotation of silica from non-magnetic spiral preconcentrate of Kudremukh iron ore. Flotation of silica with amine and starch as the Rotation reagents was studied to estimate the optimum reagent levels at various mesh of grind. The experiments were first carried out using a two level three factor design. Analysis of the results showed that two parameters namely, the concentration level of the amine collector and the mesh of grind, were significant. Experiments based on an orthogonal design of the hexagonal type were then carried out to determine the effects of these two variables, on recovery and grade of the concentrate. Regression equations have been developed as models. Response contours have been plotted using the 'path of steepest ascent', maximum response has been optimized at 0.27 kg/ton of amine collector, 0.5 kg/ton of starch and mesh of grind of 48.7% passing 300 mesh to give a recovery of 83.43% of Fe in the concentrate containing 66.6% Fe and 2.17% SiO2.

Studies on grinding media wear and its effect on flotation of ferrugenous phosphate ore

Marked ball grinding rests were carried out in the laboratory with a low grade phosphate ore under different experimental conditions. Two types of balls were used, namely high carbon low alloy (HCLA) cast steel and high chrome cast iron. Results of marked ball grinding tests indicated that ball wear increased with time and showed a sharp increase for wet grinding over dry grinding. Ball wear under wet grinding conditions was also influenced by the gaseous atmosphere in the mill. The grinding ball materials could be arranged in the following order with respect to their overall wear resistance: High chrome cast iron > HCLA cast steel balls Methods to minimize ball wear through control of mill atmosphere and addition of flotation reagents are discussed. Effect of grinding media and additions of flotation reagents during grinding on phosphate ore flotation are also discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.