GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Abstract Background: Hypoxia-mediated HIF-1a stabilization and NF-kB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. Methodology/Principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Complexes of Pd(II) and Pt(II) with 9-aminoacridine: reactions with DNA and study of their antiproliferative activity

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and 1H, 13C, and 195Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(μ-Cl)]2 · 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh3 or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl2(DMSO)2]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(μ-Cl)]2 has significant antiproliferative activity, although it is less active than cisplatin.

Complexes of Pd(II) and Pt(II) with 9-aminoacridine: reactions with DNA and study of their antiproliferative activity

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and 1H, 13C, and 195Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(μ-Cl)]2 · 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh3 or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl2(DMSO)2]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(μ-Cl)]2 has significant antiproliferative activity, although it is less active than cisplatin.

Complexes of Pd(II) and Pt(II) with 9-aminoacridine: reactions with DNA and study of their antiproliferative activity

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and 1H, 13C, and 195Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(μ-Cl)]2 · 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh3 or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl2(DMSO)2]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(μ-Cl)]2 has significant antiproliferative activity, although it is less active than cisplatin.

Arvometallien selektiivinen erottaminen kloridiliuoksista

Työssä tutkittiin jalometallien selektiivistä erottamista kloridiliuoksista synteettisten polymeerihartsien avulla. Laboratoriokokeissa keskityttiin tutkimaan kullan erottamista hydrofiilisen polymetakrylaattipohjaisen adsorbentin avulla. Lähtökohtana oli platinarikaste, joka sisälsi kullan lisäksi platinaa, palladiumia, hopeaa, kuparia, rautaa, vismuttia, seleeniä ja telluuria. Mittauksissa tutkittiin eri metallien ja puolimetallien adsorptiota hartsiin tasapaino-, kinetiikka- ja kolonnikokeilla. Työssä käytettiin myös adsorption simulointiin monikomponenttierotuksen dynaamiseen mallintamiseen tarkoitettua tietokoneohjelmaa, johon tarvittavat parametrit estimoitiin kokeellisen datan avulla. Tasapainokokeet yhtä metallia sisältäneistä liuoksista osoittivat, että hartsi adsorboi tehokkaasti kultaa kaikissa tutkituissa suolahappopitoisuuksissa (1-6 M). Kulta muodostaa hartsiin hyvin adsorboituvia tetrakloroauraatti(III)ioneja, [AuCl4]-, jotka ovat erittäin stabiileja pieniin kloridipitoisuuksiin saakka. Suolahappopitoisuudella oli merkitystä ainoastaan raudan adsorptioon, joka kasvoi huomattavasti suolahappopitoisuuden noustessa johtuen raudan taipumuksesta muodostaa hyvin adsorboituvia [FeCl4]--ioneja väkevissä suolahappopitoisuuksissa. Muiden tutkittujen alkuaineiden adsorptiot jäivät alhaisiksi kaikilla suolahappopitoisuuksilla. Rikasteliuoksella tehdyt tasapainokokeet osoittivat, että adsorptiokapasiteetti kullalle riippuu voimakkaasti muista läsnäolevista komponenteista. Kilpaileva adsorptio kuvattiin Langmuir-Freundlich-isotermillä. Kolonnikokeet osoittivat, että hartsi adsorboi kullan lisäksi hieman myös rautaa ja telluuria, jotka saatiin kuitenkin eluoitua hartsista täysin 5 M suolahappopesulla ja sitä seuraavalla 1 M suolahappopesulla. Tehokkaaksi liuokseksi kullan desorboimiseen osoittautui asetonin ja 1 M suolahapon seos. Kolonnierotuksen eri vaiheet pystyttiin tyydyttävästi kuvaamaan simulointimallilla.

An overview of the metabolic differences between Bradyrhizobium japonicum 110 bacteria and differentiated bacteroids from soybean (Glycine max) root nodules: An in vitro 13C-and 31P-nuclear magnetic resonance spectroscopy study

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using 13C- and 31P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B.japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.

Preparation of benzolactams by Pd(II)-catalyzed carbonylation of N-unprotected arylethylamines

An unprecedented NH2-directed Pd(II)-catalytic carbonylation of quaternary aromatic α -amino esters to yield 6-membered 10 benzolactams has been developed. The reaction shows a strong bias to 6-membered lactams over 5-membered ones. The steric hindrance around the amino group seems to be pivotal for the success of the process.

Gas-phase photocatalytic oxidation of volatile organic compounds

Substances emitted into the atmosphere by human activities in urban and industrial areas cause environmental problems such as air quality degradation, respiratory diseases, climate change, global warming, and stratospheric ozone depletion. Volatile organic compounds (VOCs) are major air pollutants, emitted largely by industry, transportation and households. Many VOCs are toxic, and some are considered to be carcinogenic, mutagenic, or teratogenic. A wide spectrum of VOCs is readily oxidized photocatalytically. Photocatalytic oxidation (PCO) over titanium dioxide may present a potential alternative to air treatment strategies currently in use, such as adsorption and thermal treatment, due to its advantageous activity under ambient conditions, although higher but still mild temperatures may also be applied. The objective of the present research was to disclose routes of chemical reactions, estimate the kinetics and the sensitivity of gas-phase PCO to reaction conditions in respect of air pollutants containing heteroatoms in their molecules. Deactivation of the photocatalyst and restoration of its activity was also taken under consideration to assess the practical possibility of the application of PCO to the treatment of air polluted with VOCs. UV-irradiated titanium dioxide was selected as a photocatalyst for its chemical inertness, non-toxic character and low cost. In the present work Degussa P25 TiO2 photocatalyst was mostly used. In transient studies platinized TiO2 was also studied. The experimental research into PCO of following VOCs was undertaken: - methyl tert-butyl ether (MTBE) as the basic oxygenated motor fuel additive and, thus, a major non-biodegradable pollutant of groundwater; - tert-butyl alcohol (TBA) as the primary product of MTBE hydrolysis and PCO; - ethyl mercaptan (ethanethiol) as one of the reduced sulphur pungent air pollutants in the pulp-and-paper industry; - methylamine (MA) and dimethylamine (DMA) as the amino compounds often emitted by various industries. The PCO of VOCs was studied using a continuous-flow mode. The PCO of MTBE and TBA was also studied by transient mode, in which carbon dioxide, water, and acetone were identified as the main gas-phase products. The volatile products of thermal catalytic oxidation (TCO) of MTBE included 2-methyl-1-propene (2-MP), carbon monoxide, carbon dioxide and water; TBA decomposed to 2-MP and water. Continuous PCO of 4 TBA proceeded faster in humid air than dry air. MTBE oxidation, however, was less sensitive to humidity. The TiO2 catalyst was stable during continuous PCO of MTBE and TBA above 373 K, but gradually lost activity below 373 K; the catalyst could be regenerated by UV irradiation in the absence of gas-phase VOCs. Sulphur dioxide, carbon monoxide, carbon dioxide and water were identified as ultimate products of PCO of ethanethiol. Acetic acid was identified as a photocatalytic oxidation by-product. The limits of ethanethiol concentration and temperature, at which the reactor performance was stable for indefinite time, were established. The apparent reaction kinetics appeared to be independent of the reaction temperature within the studied limits, 373 to 453 K. The catalyst was completely and irreversibly deactivated with ethanethiol TCO. Volatile PCO products of MA included ammonia, nitrogen dioxide, nitrous oxide, carbon dioxide and water. Formamide was observed among DMA PCO products together with others similar to the ones of MA. TCO for both substances resulted in the formation of ammonia, hydrogen cyanide, carbon monoxide, carbon dioxide and water. No deactivation of the photocatalyst during the multiple long-run experiments was observed at the concentrations and temperatures used in the study. PCO of MA was also studied in the aqueous phase. Maximum efficiency was achieved in an alkaline media, where MA exhibited high fugitivity. Two mechanisms of aqueous PCO – decomposition to formate and ammonia, and oxidation of organic nitrogen directly to nitrite - lead ultimately to carbon dioxide, water, ammonia and nitrate: formate and nitrite were observed as intermediates. A part of the ammonia formed in the reaction was oxidized to nitrite and nitrate. This finding helped in better understanding of the gasphase PCO pathways. The PCO kinetic data for VOCs fitted well to the monomolecular Langmuir- Hinshelwood (L-H) model, whereas TCO kinetic behaviour matched the first order process for volatile amines and the L-H model for others. It should be noted that both LH and the first order equations were only the data fit, not the real description of the reaction kinetics. The dependence of the kinetic constants on temperature was established in the form of an Arrhenius equation.

Regio- and stereoselective microwave-assisted synthesis of 5-alkyl-4-alkenyl-4-phenyl-1,3-oxazolidin-2-ones

Chiral symmetrical alk-2-yne-1,4-diols have been stereoselectively transformed into 5-alkyl-4-alkenyl-4-phenyl-1,3-oxazolidin- 2-ones, which are precursors of quaternary α-amino β-hydroxy acids. The key step was the cyclization of the bis(tosylcarbamates) of 2- phenylalk-2-yne-1,4-diols, easily obtained from the starting chiral diols. These cyclizations were accomplished with complete regioselectivity and up to 92:8 dr in the presence of catalytic amounts of Ni(0) or Pd (II) derivatives under microwave heating.

Analysis of SKI-1/S1P prodomain provides insight on maturation and activation of SKI-1/S1P zymogen

SKI-l/SlP protease is a member of the proprotein convertase family, with several functions in cellular metabolism and homeostasis. It is responsible for the processing of several cellular substrates, including ATF6, SREBPs, and GlcNAc-1- phosphotranspherase. Furthermore, SKI-1/SlP is also responsible for maturation of arenavirus surface glycoprotein into GP1 and GP2 subunits. This processing is a strict requirement in order to achieve fully mature and fusion-competent virions. Furthermore, SKI-1/SlP itself is synthesized as an inactive zymogen, requiring sequential autocatalytic processing at several sites (B'/B and C) in its prodomain in order to mature and become fully active. Our project focused on the analysis of SKI- 1/S1P prodomain in the biogenesis of the active enzyme. In this context we have additionally developed and characterized a novel cell-based sensor for assessment of cellular activity of the enzyme, with a potential application in screening for novel SKI- 1/S1P inhibitors. In a first aim we have analysed the relevance of cleavage motifs found in the enzyme prodomain. Using molecular and biochemistry tools we have identified and characterized a novel C' maturation site. Furthermore, we found that SKI-1/SlP autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. Contrasting with other proprotein convertases, incompletely matured intermediates of SKI-1/SlP exhibit full catalytic activity toward selected substrates. In a second aim, we turned our attention to the structural basis of SKI-1/SlP N- terminus assisted folding. Studying the folding and activity of prodomain-truncated forms of the enzyme we found that a minimal folding unit is contained in the AB region. Deletion of the BC sequence affected auto-maturation but not folding, and partial activity was retained. However, the BC region seemed required for complete and full activity. Phylogenetic analyses showed that the AB sequence is highly conserved, while the BC fragment is variable in sequence and length. Specifically, replacement of the human prodomain with that of Drosophila, resulted in a fully mature and active chimeric enzyme, suggesting an evolution process of SKI-1/SlP prodomain towards a more complex arrangement and steps of activation. Overall, the additional data we have produced might provide fundamental knowledge crucial for the development of novel SKI-1/SlP inhibitors while also providing new SKI- 1/S1P variants with potential use in crystallization purpose. -- SKI-l/SlP est une protéase membre de la famille des proprotéines convertases (PCs), avec plusieurs fonctions dans le métabolisme cellulaire et de l'homéostasie. Il est responsable pour la maturation de plusieurs substrats cellulaires, y compris ATF6, SREBPs et GlcNAc-1-phosphotranspherase. SKI-l/SlP est également responsable pour la maturation de la glycoprotéine des arénavirus, une exigence stricte pour atteindre des virions infectieuse. Synthétisé comme un zymogène inactif, SKI-l/SlP nécessite d'un traitement autocatalytique séquentiel sur plusieurs sites (B'/B et C) de son prodomaine afin de devenir pleinement active. Notre projet était axé sur l'analyse de SKI-l/SlP prodomaine dans la biogenèse de l'enzyme. Dans ce contexte, nous avons développé un nouveau senseur-cellulaire pour l'évaluation de l'activité de l'enzyme. Ce dernier pourrait avoir une potentielle application dans l'identification de nouveaux inhibiteurs de SKI-l/SlP. Premièrement, nous avons analysé la pertinence des motifs de clivage trouvés dans le prodomaine de l'enzyme. En utilisant des outils moléculaires et biochimiques, nous avons identifié et caractérisé un nouveau site de maturation (C'). Aussi, nous avons constaté que la maturation de SKI-l/SlP a des intermédiaires dont le domaine catalytique reste associé à des fragments du prodomaine de différentes longueurs. Contrastant avec d'autres PCs, les intermédiaires partiellement matures de SKI-1 / SIP présentent une activité catalytique complète envers des substrats spécifiques. Dans un deuxième but nous avons tourné notre attention sur la base structurelle du pliage de SKI-l/SlP assisté par son N-terminus: En étudiant l'activité et pliage des formes tronquées dans le prodomaine de l'enzyme, nous avons constaté qu'une unité de pliage minimale est contenue dans la région de l'AB. La suppression de la séquence d'auto-BC affecte la maturation mais pas le pliage, et l'activité partielle est maintenue. Cependant, la région BC semble nécessaire pour une activité complète. Les analyses phylogénétiques ont montré que la séquence AB est fortement conservée, tandis que le fragment de BC est variable en longueur et en séquence. En particulier, le remplacement du prodomaine humain avec celui de la drosophile, a donné lieu à une enzyme chimérique complètement mature et active. Suggérant un processus d'évolution du prodomaine vers un arrangement et des mesures d'activation plus complexe. Globalement, ces donnees supplémentaires augment les connaissances fondamentales cruciales pour le développement de nouveaux inhibiteurs de SKI-1/ SIP, tout en offrant de nouvelles variantes SKI-1 / SIP dans le but d'obtenir la structure cristallographique de l'enzyme.

Total synthesis of aeruginazole A

The first total synthesis of Aeruginazole A, prepared via a convergent strategy that involved both solid-phase peptide synthesis and solution phase chemistry and that enabled conservation of the stereochemistry of the intermediates, is reported.

Current and future trens in the quality control of pharmaceuticals and biopharmaticals : impacts on Cost of Goods Sold (COGS)

[Summary] 2. Roles of quality control in the pharmaceutical and biopharmaceutical industries. - 2.1. Pharmaceutical industry. - 2.2. Biopharmaceutical industry. - 2.3. Policy and regulatory. - 2.3.1. The US Food and Drug Administration (FDA). - 2.3.2. The European Medicine Agency (EMEA). - 2.3.3. The Japanese Ministry of Work, Labor and Welfare (MHLW). - 2.3.4. The Swiss Agency for Therapeutic Products (Swissmedic). - 2.3.5. The International Conference on Harmonization (ICH). - - 3. Types of testing. - 3.1. Microbiological purity tests. - 3.2. Physiochemical tests. - 3.3. Critical to quality steps. - 3.3.1. API starting materials and excipients. - 3.3.2. Intermediates. - 3.3.3. APIs (drug substances) and final drug product. - 3.3.4. Primary and secondary packaging materials fro drug products. - - 4. Manufacturing cost and quality control. - 4.1.1. Pharmaceutical manufacturing cost breakdown. - 4.1.2. Biopharmaceutical manufacturing cost breakdown. - 4.2. Batch failure / rejection / rework / recalls. - - 5. Future trends in the quality control of pharmaceuticals and biopharmaceuticals. - 5.1. Rapid and real time testing. - 5.1.1. Physio-chemicals testing. - 5.1.2. Rapid microbiology methods

Contribuições recentes da reação de hidroformilação na síntese de produtos farmacêuticos: parte II

The hydroformylation reaction represents one of the most important C1-chemistry area in the chemical industry. This catalytic process, which has been developed up to now mainly to the production of commodities chemicals, has shown a remarkable potential for the preparation of several categories of specialty chemicals and in particular pharmaceutical compounds. Arylpropanoic acids, various amines containing aryl groups, and intermediates for the preparation of vitamins, carbocyclic and heterocyclic compounds and many other classes of organic molecules endowed with pharmacological activity are currently accessible in good-to-high yields through hydroformylation of selected olefinic substrates. The asymmetric hydroformylation is going to reach the stage of maturity and hence to contribute in solving many troublesome synthetic problems connected with the preparation of pharmacologically active compounds with very high enantiomeric purity. The present survey emphasizes the usefulness of synthesis gas as a starting material in fine chemistry, which is expected to be important for industry.

Utilização de α,α-Diclorociclobutanonas em Síntese de Produtos Naturais

The preparation and chemical potentiality of a,a-dichlorocyclobutanones as useful intermediates in the total synthesis of natural products are reviewed. Some aspects related to the recent advances reported in the literature about the mechanism of [2+2] cycloaddition reaction between dichloroketene and olefins are also presented.

Síntese e caracterização estrutural de complexos mono e bimetálicos de paládio e platina com ligantes contendo nitrogênio e enxofre

The synthesis of several mono- and bimetallic platinum and palladium complexes are reported. The monometallic complexes are of the type MCl2(PEt3)2L (L=2-aminothiazol; benzothiazol; 2-imidazolidithione; M=Pt; Pd), and the bimetallic complexes (M2Cl4(PEt3) 2L) are derived from them. Infrared spectroscopic characterization of the monometallic complexes shows that the metal centre are coordinated through the N-endocyclic (L=2-aminothiazol; benzothiazol) and through the sulphur when L=2-imidazolidithione. Coordination of a second metal fragment occurs via the sulphur atom (L=2-aminothiazol; benzothiazol) according to infrared studies. 31P{¹H} NMR spectroscopy suggest that the complexes have a cis- configuration since the ¹J Pt-P are around the value found for this type of compound.