Nuovi coniugati fra acido azelaico ed azaeterocicli: sintesi e valutazione dell'attività biologica

The well-known antiproliferative properties of the 9-hydroxystearic acid (9-HSA) on human colon cancer cells (HT-29 cell line) have inspired this thesis work in order to obtain new derivatives maintaining the C1-C8 chain of the HSA linked to an heterocyclic moiety at the C-9 carbon atom and to investigate their biological activity. First, thiazoles, thiadiazoles and benzothiazoles, that are compounds of interest in many fields for their biological activities, have been introduced through an amide bond starting from their 2-amino precursors. The products have been obtained by treatment with methyl 9-chloro-9-oxononanoate according to a Schotten-Baumann type reaction. The acylation reaction occurred at the endocyclic nitrogen atom of the heterocycle, as ascertained through NOESY-1D experiment. After, methyl 9-chloro-9-oxononanoate was reacted with indole, N-methylindole, and triptamine giving a serie of new indole derivatives. Finally, the biological activity of some compounds has been tested through assays on HT-29 cancer cells and bacterial and fungal microorganisms; docking calculations have also been performed to evaluate the possible interactions with the active site of histone deacetylase, which are molecular targets of the 9-HSA.

Dearomatizzazione organocatalitica regio- ed enantioselettiva di sali di piridinio attivati

In this work, we present the first regio- and enantioselective organocatalytic nucleophilic dearomatization of activated N-alkyl pyridinium salts. In particular, N-benzyl pyridinium bromides bearing electron-withdrawing substituents at the C3 position of the pyridine ring were chosen as substrates. These compounds were easily obtained through an alkylation reaction between benzyl bromides and the corresponding 3-substituted pyridines. Then, a wide range of nucleophiles and organocatalysts was tested, providing the best results when indole, a thiourea derived from quinidine and 1-benzyl-3-nitropyridinum bromide were employed as the nucleophile, the catalyst and the pyridinium salt, respectively. Subsequently, the reaction conditions were optimised evaluating different bases, solvents, N-benzylic protecting groups, molar concentrations and temperatures. With the optimized condition in hand, the scope of the reaction with different substituted indoles was explored, affording the corresponding 1,4-dihydropyridines in good yields, regio- and enantio-selectivities. In addition, several experiments were carried out in order to understand the mechanism of the reaction, showing an unusual pathway involving a covalently bound intermediate formed by addition of the catalyst to the pyridine unit.

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

Xenobiotic metabolism: a view through the metabolometer

The combination of advanced ultraperformance liquid chromatography coupled with mass spectrometry, chemometrics, and genetically modified mice provide an attractive raft of technologies with which to examine the metabolism of xenobiotics. Here, a reexamination of the metabolism of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), the suspect carcinogen areca alkaloids (arecoline, arecaidine, and arecoline 1-oxide), the hormone supplement melatonin, and the metabolism of the experimental cancer therapeutic agent aminoflavone is presented. In all cases, the metabolic maps of the xenobiotics were considerably enlarged, providing new insights into their toxicology. The inclusion of transgenic mice permitted unequivocal attribution of individual and often novel metabolic pathways to particular enzymes. Last, a future perspective for xenobiotic metabolomics is discussed and its impact on the metabolome is described. The studies reviewed here are not specific to the mouse and can be adapted to study xenobiotic metabolism in any animal species, including humans. The view through the metabolometer is unique and visualizes a metabolic space that contains both established and unknown metabolites of a xenobiotic, thereby enhancing knowledge of their modes of toxic action.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Classification of organisms previously reported as the SP and Stewart-Letscher groups, with descriptions of Necropsobacter gen. nov. and of Necropsobacter rosorum sp. nov. for organisms of the SP group

To allow classification of bacteria previously reported as the SP group and the Stewart-Letscher group, 35 isolates from rodents (21), rabbits (eight), a dog and humans (five) were phenotypically and genotypically characterized. Comparison of partial rpoB sequences showed that 34 of the isolates were closely related, demonstrating at least 97.4 % similarity. 16S rRNA gene sequence comparison of 20 selected isolates confirmed the monophyly of the SP group and revealed 98.5 %-100 % similarity between isolates. A blast search using the 16S rRNA gene sequences showed that the highest similarity outside the SP group was 95.5 % to an unclassified rat isolate. The single strain, P625, representing the Stewart-Letscher group showed the highest 16S rRNA gene similarity (94.9-95.5 %) to members of the SP group. recN gene sequence analysis of 11 representative strains resulted in similarities of 97-100 % among the SP group strains, which showed 80 % sequence similarity to the Stewart-Letscher group strain. Sequence similarity values based on the recN gene, indicative for whole genome similarity, showed the SP group being clearly separated from established genera, whereas the Stewart-Letscher group strain was associated with the SP group. A new genus, Necropsobacter gen. nov., with only one species, Necropsobacter rosorum sp. nov., is proposed to include all members of the SP group. The new genus can be separated from existing genera of the family Pasteurellaceae by at least three phenotypic characters. The most characteristic properties of the new genus are that haemolysis is not observed on bovine blood agar, positive reactions are observed in the porphyrin test, acid is produced from (+)-L-arabinose, (+)-D-xylose, dulcitol, (+)-D-galactose, (+)-D-mannose, maltose and melibiose, and negative reactions are observed for symbiotic growth, urease, ornithine decarboxylase and indole. Previous publications have documented that both ubiquinones and demethylmenaquinone were produced by the proposed type strain of the new genus, Michel A/76(T), and that the major polyamine of representative strains (type strain not included) of the genus is 1,3-diaminopropane, spermidine is present in moderate amounts and putrescine and spermine are detectable only in minor amounts. The major fatty acids of strain Michel A/76(T) are C(14 : 0), C(16 : 0), C(16:1)omega7c and summed feature C(14 : 0) 3-OH/iso-C(16 : 1) I. This fatty acid profile is typical for members of the family Pasteurellaceae. The G+C content of DNA of strain Michel A/76(T) was estimated to be 52.5 mol% in a previous investigation. The type strain is P709(T) ( = Michel A/76(T) = CCUG 28028(T) = CIP 110147(T) = CCM 7802(T)).

Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen

Gram-negative, coccoid, non-motile bacteria that are catalase-, urease- and indole-negative, facultatively anaerobic and oxidase-positive were isolated from the bovine rumen using an improved selective medium for members of the Pasteurellaceae. All strains produced significant amounts of succinic acid under anaerobic conditions with glucose as substrate. Phenotypic characterization and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, infB and recN genes were performed on seven independent isolates. All four genes showed high sequence similarity to their counterparts in the genome sequence of the patent strain MBEL55E, but less than 95 % 16S rRNA gene sequence similarity to any other species of the Pasteurellaceae. Genetically these strains form a very homogeneous group in individual as well as combined phylogenetic trees, clearly separated from other genera of the family from which they can also be separated based on phenotypic markers. Genome relatedness as deduced from the recN gene showed high interspecies similarities, but again low similarity to any of the established genera of the family. No toxicity towards bovine, human or fish cells was observed and no RTX toxin genes were detected in members of the new taxon. Based on phylogenetic clustering in the MLSA analysis, the low genetic similarity to other genera and the phenotypic distinction, we suggest to classify these bovine rumen isolates as Basfia succiniciproducens gen. nov., sp. nov. The type strain is JF4016(T) (=DSM 22022(T) =CCUG 57335(T)).

The inhibition of bone resorption in rats treated with (-)-menthol is due to its metabolites

(-)-Menthol, a monoterpene from Mentha species (Lamiaceae), has been shown to inhibit bone resorption in vivo by an unknown mechanism. In the present study, plasma and urine profiling in rats determined by GC/MS demonstrate that (-)-menthol is extensively metabolized, mainly by hydroxylation and carboxylation, and excreted in the urine, in part as glucuronides. In plasma, very low concentrations of (-)-menthol metabolites were detected after a single dose of (-)-menthol, whereas after repeated treatment, several times higher concentrations and long residence times were measured. In contrast, the elimination of unchanged (-)-menthol was increased by repeated treatment. (-)-Menthol, at concentrations found in plasma, did not inhibit bone resorption in cultured mouse calvaria (skull). However, the neutral metabolites of (-)-menthol, extracted from urine of rats fed with (-)-menthol, inhibited bone resorption in vitro, the concentrations being at plasma level or higher. These results suggest that not (-)-menthol itself, but one or several of its neutral metabolites inhibit the bone resorbing cells in vivo.

Herbal medicine in the treatment of liver diseases

Herbal drugs have become increasingly popular and their use is widespread. Licensing regulations and pharmacovigilance regarding herbal products are still incomplete and clearcut proof of their efficacy in liver diseases is sparse. Nevertheless, a number of herbals show promising activity including silymarin for antifibrotic treatment, phyllantus amarus in chronic hepatitis B, glycyrrhizin to treat chronic viral hepatitis, and a number of herbal combinations from China and Japan that deserve testing in appropriate studies. Apart from therapeutic properties, reports are accumulating about liver injury after the intake of herbals, including those advertised for liver diseases. Acute and/or chronic liver damage occurred after ingestion of some Chinese herbs, herbals that contain pyrrolizidine alkaloids, germander, greater celandine, kava, atractylis gummifera, callilepsis laureola, senna alkaloids, chaparral and many others. Since the evidence supporting the use of botanicals to treat chronic liver diseases is insufficient and only few of them are well standardised and free of potential serious side effects, most of these medications are not recommended outside clinical trials. Particularly with regard to the latter, adequately powered randomised-controlled clinical trials with well-selected end points are needed to assess the role of herbal therapy for liver diseases.

Structural characterization of water-soluble atmospheric organic matter by ultrahigh-resolution mass spectrometry

Isolated water-soluble analytes extracted from fog water collected during a radiation fog event near Fresno, CA were analyzed using collision induced dissociation and ultrahigh-resolution mass spectrometry. Tandem mass analysis was performed on scan ranges between 100-400 u to characterize the structures of nitrogen and/or sulfur containing species. CHNO, CHOS, and CHNOS compounds were targeted specifically because of the high number of oxygen atoms contained in their molecular formulas. The presence of 22 neutral losses corresponding to fragment ions was evaluated for each of the 1308 precursors. Priority neutral losses represent specific polar functional groups (H2O, CO2, CH3OH, HNO3, SO3, etc., and several combinations of these). Additional neutral losses represent non-specific functional groups (CO, CH2O, C3H8, etc.) Five distinct monoterpene derived organonitrates, organosulfates, and nitroxy-organosulfates were observed in this study, including C10H16O7S, C10H17NO7S, C10H17 NO8S, C10H17NO9S, and C10H17NO10S. Nitrophenols and linear alkyl benzene sulfonates were present in high abundance. Liquid chromatography/mass spectrometery methodology was developed to isolate and quantify nitrophenols based on their fragmentation behavior.

Sulfoxides as an intramolecular sulfenylating agent for indoles and diverse applications of the sulfide-sulfoxide redox cycle in organic chemistry

This dissertation involves study of various aspects of sulfoxide chemistry. Specifically designed t-butyl and propanenitrile sulfoxides tethered to indole-2-carboxamide were used as a source of intramolecular sulfenylating agents to synthesize novel indolo[3,2-b]-1-5-benzothiazepinones which are structurally analogous to the other biologically active benzothiazepinones. This study reveals that the intramolecular cyclization of sulfoxide follows an electrophilic sulfenylation (Sulfoxide Electrophilic Sulfenylation, SES) reaction pathway. Evidence of the absence of sulfenic acid as a transient reactive intermediate in such intramolecular cyclization is also provided. In another study, sulfoxide was used as a “protecting group” of thioether to synthesize 8-membered, indole substituted, thiazocine-2-acetic acid derivative via Ring Closing Metathesis (RCM). Protection (oxidation) of inert (to RCM) sulfide to sulfoxide followed by RCM produced cyclized product in good yields. Deprotection (reduction) of sulfoxide was achieved using Lawessons Reagent (L.R.). Application of the sulfide-sulfoxide redox cycle to solve the existing difficulties in using RCM methodology to thioethers is illustrated. A new design of a “molecular brake”, based on the sulfide-sulfoxide redox cycle is described. N-Ar rotation in simple isoindolines is controlled by the oxidation state of the proximate sulfur atom. Sulfide [S(II)] shows “free” [brake OFF] N-Ar rotation whereas sulfoxide displayed hindered [brake ON] N-Ar rotation. The semi-empirical molecular orbital (PM3) calculations revealed concerted pyramidalization of amidic nitrogen with N-Ar rotation.

Isolation of plant growth promoting bacteria from torch lake sediments and their role in phytoremediation of metal contaminated soil

In this study, we isolated eight copper-resistant bacteria from Torch Lake sediment contaminated by copper mine tailings (stamp sand). Sequence analysis of gyrB and rpoD genes revealed that these organisms are closer to various Pseudomonas species. These eight bacterial isolates were also resistant to zinc, cesium, lead, arsenate and mercury. Further characterization showed that all the strains produced plant growth promoting indole-3-acetic acid (IAA), iron chelating siderophore and solubilized mineral phosphate and metals. The effect of bacterial inoculation on plant growth and copper uptake by maize (Zea mays) and sunflower (Helianthus annuus) was investigated using one of the isolates (Pseudomonas sp. TLC 6-6.5-4) with higher IAA production and phosphate and metal soubilization, which resulted in a significant increase in copper accumulation in maize and sunflower, and an increase in the total biomass of maize. Genes involved in copper resistance of Pseudomonas sp. TLC 6-6.5-4 was analyzed by transposon mutational analysis. Two copper sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trp A gene (tryptophan synthase alpha subunit); CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analysis were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. The effect of different bacterial inoculation methods on plant growth, copper uptake and soil enzyme activities was investigated. Four different delivery methods were used including soil inoculation (before or after plant emergence), seed coating and root dipping. Soil inoculation before sowing seeds and coating seeds with PGPB led to better growth of maize, higher copper uptake and an increase in soil invertase and dehydrogenase activities. Proteomic and metabolomic analyses were performed to investigate the effect of bacterial inoculation on maize grown in normal soil and stamp sand. Our results revealed that bacterial inoculation led to environment-dependent effects on maize proteome and metabolome.

DESIGN AND SYNTHESIS OF NOVEL SYNTHETIC ANTIOXIDANTS FOR THE TREATMENT OF OXIDATIVE STRESS RELATED DISEASES

Free radicals play an important role in many physiological processes that occur in the human body such as cellular defense responses to infectious agents and a variety of cellular signaling pathways. While at low concentrations free radicals are involved in many significant metabolic reactions, high levels of free radicals can have deleterious effects on biomolecules like proteins, lipids, and DNA. Many physiological disorders such as diabetes, ageing, neurodegenerative diseases, and ischemia-reperfusion (I/R) injury are associated with oxidative stress.1 In particular, the deleterious effects caused by I/R injury developed during organ transplantation, cardiac infarct, and stroke have become the main cause of death in the United States and Europe.1,2 In this context, we synthesized and characterized a series of novel indole-amino acid conjugates as potential antioxidants for I/R injury. The synthesis of indole-phenol conjugate compounds is also discussed. Phenolic derivatives such as caffeic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), resveratrol, and its analogues are known for their significant antioxidative properties. A series of resveratrol analogues have been designed and synthesized as potential antioxidants. The radical scavenging mechanisms for potential antioxidants and assays for the in vitro evaluation of antioxidant activities are also discussed.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.

Proposal of Histophilus somni gen. nov., sp. nov. for the three species incertae sedis 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis'

Earlier investigations have shown that 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis' represent the same species. In the present investigation, the taxonomic position of this species is explored further by sequencing the 16S rRNA and rpoB genes of strains that were investigated previously by DNA-DNA hybridization. These results clearly support the allocation of this species to a novel genus within the family PASTEURELLACEAE: The phenotypic separation of Histophilus somni gen. nov., sp. nov. from other members of the family can, for most strains, be based on capnophilia, yellowish pigmentation and indole production. However, due to phenotypic variation, the use of a species-specific PCR test based on the 16S rRNA gene is included in the species description. This is justified by the high sequence similarity of the 16S rRNA gene within the species and the fact that the highest sequence similarity to any other taxon within the family is 93.4 %. The type strain, 8025(T)=ATCC 43625(T)=CCUG 36157(T), was isolated in the USA from a bovine brain with lesions of thromboembolic meningoencephalitis.