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1.
Proc Natl Acad Sci U S A ; 119(28): e2122122119, 2022 07 12.
Article in English | MEDLINE | ID: mdl-35867750

ABSTRACT

The precise and accurate determination of the radionuclide inventory in radioactive waste streams, including those generated during nuclear decommissioning, is a key aspect in establishing the best-suited nuclear waste management and disposal options. Radiocarbon ([Formula: see text]) is playing a crucial role in this scenario because it is one of the so-called difficult to measure isotopes; currently, [Formula: see text] analysis requires complex systems, such as accelerator mass spectrometry (AMS) or liquid scintillation counting (LSC). AMS has an outstanding limit of detection, but only a few facilities are available worldwide; LSC, which can have similar performance, is more widespread, but sample preparation can be nontrivial. In this paper, we demonstrate that the laser-based saturated-absorption cavity ring-down (SCAR) spectroscopic technique has several distinct advantages and represents a mature and accurate alternative for [Formula: see text] content determination in nuclear waste. As a proof-of-principle experiment, we show consistent results of AMS and SCAR for samples of concrete and graphite originating from nuclear installations. In particular, we determined mole fractions of 1.312(9) F[Formula: see text] and 30.951(7) F[Formula: see text] corresponding to ∼1.5 and 36.2 parts per trillion (ppt), respectively, for two different graphite samples originating from different regions of the Adiabatic Resonance Crossing activator prototype installed on one irradiation line of an MC40 Scanditronix cyclotron. Moreover, we measure a mole fraction of 0.593(8) F[Formula: see text] ([Formula: see text] ppt) from a concrete sample originating from an external wall of the Ispra-1 nuclear research reactor currently in the decommissioning phase.


Subject(s)
Carbon Radioisotopes , Graphite , Radioactive Waste , Waste Management , Carbon Radioisotopes/analysis , Graphite/chemistry , Mass Spectrometry , Radioactive Waste/analysis , Radiometric Dating , Waste Management/methods
2.
Organometallics ; 41(6): 716-732, 2022 Mar 28.
Article in English | MEDLINE | ID: mdl-35368715

ABSTRACT

A cycle of stoichiometric elemental reactions defining the direct arylation promoted by a redox-pair Rh(I)-Rh(III) is reported. Starting from the rhodium(I)-aryl complex RhPh{κ3-P,O,P-[xant(PiPr2)2]} (xant(PiPr2)2 = 9,9-dimethyl-4,5-bis(diisopropylphosphino)xanthene), the reactions include C-Cl oxidative addition of organic chlorides, halide abstraction from the resulting six-coordinate rhodium(III) derivatives, C-C reductive coupling between the initial aryl ligand and the added organic group, oxidative addition of a C-H bond of a new arene, and deprotonation of the generated hydride-rhodium(III)-aryl species to form a new rhodium(I)-aryl derivative. In this context, the kinetics of the oxidative additions of 2-chloropyridine, chlorobenzene, benzyl chloride, and dichloromethane to RhPh{κ3-P,O,P-[xant(PiPr2)2]} and the C-C reductive eliminations of biphenyl and benzylbenzene from [RhPh2{κ3-P,O,P-[xant(PiPr2)2]}]BF4 and [RhPh(CH2Ph){κ3-P,O,P-[xant(PiPr2)2]}]BF4, respectively, have been studied. The oxidative additions generally involve the cis addition of the C-Cl bond of the organic chloride to the rhodium(I) complex, being kinetically controlled by the C-Cl bond dissociation energy; the weakest C-Cl bond is faster added. The C-C reductive elimination is kinetically governed by the dissociation energy of the formed bond. The C(sp3)-C(sp2) coupling to give benzylbenzene is faster than the C(sp2)-C(sp2) bond formation to afford biphenyl. In spite of that a most demanding orientation requirement is needed for the C(sp3)-C(sp2) coupling than for the C(sp2)-C(sp2) bond formation, the energetic effort for the pregeneration of the C(sp3)-C(sp2) bond is lower. As a result, the weakest C-C bond is formed faster.

3.
Organometallics ; 41(16): 2317-2326, 2022 Aug 22.
Article in English | MEDLINE | ID: mdl-36866062

ABSTRACT

The C-H bond activation of methylquinolines, quinoline, 3-methoxyquinoline, and 3-(trifluoromethyl)quinoline promoted by the square-planar rhodium(I) complex RhH{κ3-P,O,P-[xant(PiPr2)2]} [1; xant(PiPr2)2 = 9,9-dimethyl-4,5-bis(diisopropylphosphino)xanthene] has been systematically studied. Results reveal that the activation of the heteroring is preferred over the activation of the carbocycle, and the activated position depends upon the position of the substituent in the substrate. Thus, 3-, 4-, and 5-methylquinoline reacts with 1 to quantitatively form square-planar rhodium(I)-(2-quinolinyl) derivatives, whereas 2-, 6-, and 7-methylquinoline quantitatively leads to rhodium(I)-(4-quinolinyl) species. By contrast, quinoline and 8-methylquinoline afford mixtures of the respective rhodium(I)-(2-quinolinyl) and -(4-quinolinyl) complexes. 3-Methoxyquinoline displays the same behavior as that of 3-methylquinoline, while 3-(trifluoromethyl)quinoline yields a mixture of rhodium(I)-(2-quinolinyl), -(4-quinolinyl), -(6-quinolinyl), and -(7-quinolinyl) isomers.

4.
Molecules ; 25(6)2020 Mar 21.
Article in English | MEDLINE | ID: mdl-32245155

ABSTRACT

To reduce uncertainties in determining the source term and evolving condition of spent nuclear fuel is fundamental to the safety assessment. ß-emitting nuclides pose a challenging task for reliable, quantitative determination because both radiometric and mass spectrometric methodologies require prior chemical purification for the removal of interfering activity and isobars, respectively. A method for the determination of 90Sr at trace levels in nuclear spent fuel leachate samples without sophisticated and time-consuming procedures has been established. The analytical approach uses a commercially available automated pre-concentration device (SeaFAST) coupled to an ICP-DRC-MS. The method shows good performances with regard to reproducibility, precision, and LOD reducing the total time of analysis for each sample to 12.5 min. The comparison between the developed method and the classical radiochemical method shows a good agreement when taking into account the associated uncertainties.


Subject(s)
Mass Spectrometry/methods , Radioactive Waste/analysis , Spectrophotometry, Atomic/methods , Strontium Radioisotopes/analysis , Beta Particles , Linear Models , Mass Spectrometry/instrumentation , Reproducibility of Results , Spectrophotometry, Atomic/instrumentation , Strontium Radioisotopes/chemistry , Strontium Radioisotopes/isolation & purification , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/chemistry
5.
J Med Chem ; 63(2): 591-600, 2020 01 23.
Article in English | MEDLINE | ID: mdl-31850752

ABSTRACT

New drugs that target Plasmodium species, the causative agents of malaria, are needed. The enzyme N-myristoyltransferase (NMT) is an essential protein, which catalyzes the myristoylation of protein substrates, often to mediate membrane targeting. We screened ∼1.8 million small molecules for activity against Plasmodium vivax (P. vivax) NMT. Hits were triaged based on potency and physicochemical properties and further tested against P. vivax and Plasmodium falciparum (P. falciparum) NMTs. We assessed the activity of hits against human NMT1 and NMT2 and discarded compounds with low selectivity indices. We identified 23 chemical classes specific for the inhibition of Plasmodium NMTs over human NMTs, including multiple novel scaffolds. Cocrystallization of P. vivax NMT with one compound revealed peptide binding pocket binding. Other compounds show a range of potential modes of action. Our data provide insight into the activity of a collection of selective inhibitors of Plasmodium NMT and serve as a starting point for subsequent medicinal chemistry efforts.


Subject(s)
Acyltransferases/antagonists & inhibitors , Antimalarials/chemical synthesis , Antimalarials/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Plasmodium/drug effects , Plasmodium/enzymology , Acyltransferases/chemistry , Animals , Binding Sites , Cell Line , Crystallography, X-Ray , Drug Discovery , High-Throughput Screening Assays , Humans , Malaria/drug therapy , Models, Molecular , Plasmodium falciparum/drug effects , Plasmodium vivax/drug effects , Small Molecule Libraries , Structure-Activity Relationship
6.
PLoS One ; 14(7): e0220492, 2019.
Article in English | MEDLINE | ID: mdl-31348804

ABSTRACT

Rhodococcus ruber Chol-4 is a potent steroid degrader that has a great potential as a biotechnological tool. As proof of concept, this work presents testosterone production from 4-androstene-3,17-dione by tailoring innate catabolic enzymes of the steroid catabolism inside the strain. A R. ruber quadruple mutant was constructed in order to avoid the breakage of the steroid nucleus. At the same time, an inducible expression vector for this strain was developed. The 17-ketoreductase gene from the fungus Cochliobolus lunatus was cloned and overexpressed in this vector. The engineered strain was able to produce testosterone from 4-androstene-3,17-dione using glucose for cofactor regeneration with a molar conversion of 61%. It is important to note that 91% of the testosterone was secreted outside the cell after 3 days of cell biotransformation. The results support the idea that Rhodococcus ruber Chol-4 can be metabolically engineered and can be used for the production of steroid intermediates.


Subject(s)
17-Hydroxysteroid Dehydrogenases/metabolism , Bacterial Proteins/metabolism , Metabolic Engineering/methods , Rhodococcus/genetics , Rhodococcus/metabolism , Testosterone/metabolism , 17-Hydroxysteroid Dehydrogenases/genetics , Bacterial Proteins/genetics , Biotransformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/growth & development
7.
J Vis Exp ; (136)2018 06 06.
Article in English | MEDLINE | ID: mdl-29939189

ABSTRACT

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg·g-1 (1.7 Bq/g).


Subject(s)
Strontium Radioisotopes/chemistry , Laboratories
8.
J Radioanal Nucl Chem ; 313(1): 217-227, 2017.
Article in English | MEDLINE | ID: mdl-28680190

ABSTRACT

A LabVIEW®-based software for the control of the fully automated multi-sequential flow injection analysis Lab-on-Valve (MSFIA-LOV) platform AutoRAD performing radiochemical analysis is described. The analytical platform interfaces an Arduino®-based device triggering multiple detectors providing a flexible and fit for purpose choice of detection systems. The different analytical devices are interfaced to the PC running LabVIEW®VI software using USB and RS232 interfaces, both for sending commands and receiving confirmation or error responses. The AUTORAD platform has been successfully applied for the chemical separation and determination of Sr, an important fission product pertinent to nuclear waste.

9.
Nat Commun ; 8: 15160, 2017 05 17.
Article in English | MEDLINE | ID: mdl-28513586

ABSTRACT

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 µM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection.


Subject(s)
Antimalarials/pharmacology , Drug Evaluation, Preclinical , Germ Cells/metabolism , Adenosine Triphosphate/metabolism , Animals , Antimalarials/chemistry , Antimalarials/pharmacokinetics , Antimalarials/therapeutic use , Disease Models, Animal , Female , Flagella/metabolism , Hep G2 Cells , Humans , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Reproducibility of Results
10.
Microb Cell Fact ; 16(1): 42, 2017 Mar 14.
Article in English | MEDLINE | ID: mdl-28288625

ABSTRACT

BACKGROUND: The Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications. RESULTS: The three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters. CONCLUSIONS: At least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated substrates. KstDs differences in their catalytic properties was somehow related to structural differences revealed by a preliminary structural modelling. Transcription of R. ruber kstD genes is driven from specific promoters. The three genes are constitutively transcribed, although an additional induction is observed in kstD1 and kstD3. These enzymes have a wide versatility and allow a fine tuning-up of the KstD cellular activity.


Subject(s)
Isoenzymes/genetics , Isoenzymes/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Rhodococcus/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cholesterol/metabolism , Cloning, Molecular , Open Reading Frames , Oxidoreductases/isolation & purification , Promoter Regions, Genetic , Rhodococcus/genetics , Steroids/metabolism , Substrate Specificity , Transcription Initiation, Genetic
11.
J Med Chem ; 59(11): 5416-31, 2016 06 09.
Article in English | MEDLINE | ID: mdl-27127993

ABSTRACT

Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen.


Subject(s)
Antimalarials/pharmacology , Enzyme Inhibitors/pharmacology , Malaria, Falciparum/drug therapy , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Plasmodium falciparum/drug effects , Pyrimidines/pharmacology , Triazoles/pharmacology , Animals , Antimalarials/chemical synthesis , Antimalarials/chemistry , Dihydroorotate Dehydrogenase , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Mice , Mice, SCID , Molecular Structure , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Parasitic Sensitivity Tests , Plasmodium falciparum/enzymology , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Rats , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistry
12.
ACS Med Chem Lett ; 5(6): 657-61, 2014 Jun 12.
Article in English | MEDLINE | ID: mdl-24944739

ABSTRACT

Antiparasitic oral drugs have been associated to lipophilic molecules due to their intrinsic permeability. However, these kind of molecules are associated to numerous adverse effects, which have been extensively studied. Within the Tres Cantos Antimalarial Set (TCAMS) we have identified two small, soluble and simple hits that even presenting antiplasmodial activities in the range of 0.4-0.5 µM are able to show in vivo activity.

13.
J Steroid Biochem Mol Biol ; 139: 33-44, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24125733

ABSTRACT

The choG ORF of Rhodococcus ruber strain Chol-4 (referred from now as Chol-4) encodes a putative extracellular cholesterol oxidase. In the Chol-4 genome this ORF is located in a gene cluster that includes kstD3 and hsd4B, showing the same genomic context as that found in other Rhodococcus species. The putative ChoG protein is grouped into the class II of cholesterol oxidases, close to the Rhodococcus sp. CECT3014 ChoG homolog. The Chol-4 choG was cloned and expressed in a CECT3014 ΔchoG host strain in order to assess its ability to convert cholesterol into cholestenone. The RT-PCR analysis showed that choG gene was constitutively expressed in all the conditions assayed, but a higher induction could be inferred when cells were growing in the presence of cholesterol. A Chol-4 ΔchoG mutant strain was still able to grow in minimal medium supplemented with cholesterol, although at a slower rate. A comparative study of the removal of both cholesterol and cholestenone from the culture medium of either the wild type Chol-4 or its choG deletion mutant revealed a major role of ChoG in the extracellular production of cholestenone from cholesterol and, therefore, this enzyme may be related with the maintenance of a convenient supply of cholestenone for the succeeding steps of the catabolic pathway.


Subject(s)
Bacterial Proteins/genetics , Cholestenones/metabolism , Cholesterol Oxidase/genetics , Cholesterol/metabolism , Rhodococcus/enzymology , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Biocatalysis , Cholesterol Oxidase/biosynthesis , Cloning, Molecular , Enzyme Induction , Gene Expression , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Oxidation-Reduction , Phylogeny , Promoter Regions, Genetic , Rhodococcus/genetics , Rhodococcus/growth & development , Sequence Deletion
14.
Genome Announc ; 1(3)2013 May 16.
Article in English | MEDLINE | ID: mdl-23682139

ABSTRACT

The whole-genome shotgun sequence of Rhodococcus ruber strain Chol-4 is presented here. This organism was shown to be able to grow using many steroids as the sole carbon and energy sources. These sequence data will help us to further explore the metabolic abilities of this versatile degrader.

15.
J Steroid Biochem Mol Biol ; 132(3-5): 271-81, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22771584

ABSTRACT

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.


Subject(s)
Oxidoreductases/genetics , Oxidoreductases/metabolism , Rhodococcus/enzymology , Androstadienes/metabolism , Androstenedione/analogs & derivatives , Androstenedione/metabolism , Cholesterol/metabolism , Culture Media , Gene Deletion , Genetic Complementation Test , Genome, Bacterial , Isoenzymes/metabolism , Molecular Sequence Data , Rhodococcus/genetics
16.
Microbiol Res ; 166(5): 403-18, 2011 Jul 20.
Article in English | MEDLINE | ID: mdl-20630728

ABSTRACT

Cholesterol catabolism has been reported in different bacteria and particularly in several Rhodococcus species, but the genetic of this complex pathway is not yet very well defined. In this work we report the isolation and sequencing of a 9.8 kb DNA fragment of Rhodococcus sp. strain CECT3014, a bacterial strain that we here identify as a Rhodococcus erythropolis strain. In this DNA fragment we found several ORF that are probably involved in steroid catabolism, and choG, a gene encoding a putative cholesterol oxidase whose functional characterization we here report. ChoG protein is a class II cholesterol oxidase with all the structural features of the enzymes of this group. The disruption of the choG gene does not alter the ability of strain CECT3014 cells to grow on cholesterol, but it abolishes the production of extracellular cholesterol oxidase. This later effect is reverted when the mutant cells are transformed with a plasmid expressing choG. We conclude that choG is the gene responsible for the inducible extracellular cholesterol oxidase activity of strain CECT3014. This activity distributes between the cellular membrane and the culture supernatant in a way that suggests it is produced by the same ChoG protein that occurs in two different locations. RT-PCR transcript analysis showed a dual scheme of choG expression: a low constitutive independent transcription, plus a cholesterol induced transcription of choG into a polycistronic kstD-hsd4B-choG mRNA.


Subject(s)
Cholesterol Oxidase/genetics , Cholesterol Oxidase/metabolism , Rhodococcus/enzymology , Cholesterol/metabolism , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Expression Profiling , Gene Knockout Techniques , Genetic Complementation Test , Molecular Sequence Data , Mutagenesis, Insertional , Open Reading Frames , Phylogeny , Rhodococcus/genetics , Rhodococcus/growth & development , Sequence Analysis, DNA , Sequence Homology
17.
J Med Chem ; 53(16): 6129-52, 2010 Aug 26.
Article in English | MEDLINE | ID: mdl-20672841

ABSTRACT

Falcipain-2 and falcipain-3 are papain-family cysteine proteases of the malaria parasite Plasmodium falciparum that are responsible for host hemoglobin hydrolysis to provide amino acids for parasite protein synthesis. Different heteroarylnitrile derivatives were studied as potential falcipain inhibitors and therefore potential antiparasitic lead compounds, with the 5-substituted-2-cyanopyrimidine chemical class emerging as the most potent and promising lead series. Through a sequential lead optimization process considering the different positions present in the initial scaffold, nanomolar and subnanomolar inhibitors at falcipains 2 and 3 were identified, with activity against cultured parasites in the micromolar range. Introduction of protonable amines within lead molecules led to marked improvements of up to 1000 times in activity against cultured parasites without noteworthy alterations in other SAR tendencies. Optimized compounds presented enzymatic activities in the picomolar to low nanomolar range and antiparasitic activities in the low nanomolar range.


Subject(s)
Antimalarials/chemical synthesis , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Protozoan Proteins/metabolism , Antimalarials/chemistry , Antimalarials/pharmacology , Cysteine Endopeptidases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Protozoan Proteins/chemistry , Recombinant Proteins/chemistry , Structure-Activity Relationship
18.
Curr Microbiol ; 59(5): 548-53, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19688376

ABSTRACT

The aerobic degradation of cholesterol, testosterone, androsterone, progesterone, and further steroid compounds as sole carbon source has been observed in the newly isolated bacterial Gram-positive strain Chol-4. The 16S rRNA gene sequence shares the greatest similarity with members of the genus Rhodococcus, with the closest shared nucleotide identities of 98-99% with Rhodococcus ruber (DSM 43338(T)) and Rhodococcus aetherivorans (DSM 44752(T)). Phylogenetic analysis of Rhodococcus 16S rRNA gene sequences consistently places strain Chol-4 in a clade shared with those both type strains within the Rhodococcus rhodochrous subclade. The results of DNA-DNA hybridization against its two phylogenetically closest neighbors as well as the results of morphological, physiological, and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-4 from Rhodococcus ruber (DSM 43338(T)) on the species level and from the other validly described Rhodococcus species on the genus level. Strain Chol-4 therefore merits recognition as a novel strain of the species Rhodococcus ruber and demonstrates for the first time the capability of this species to utilize a great variety of steroid compounds as growth substrates never shown for other species of this genus so far. The genome of strain Chol-4 harbors at least one gene cluster that may be responsible for the degradation of steroid compounds. This gene cluster was identified in a cloned 5458 bp BamHI-EcoRV DNA fragment and compared to similar genes from other Gram-positive and Gram-negative bacteria described so far.


Subject(s)
Rhodococcus/genetics , Rhodococcus/isolation & purification , Sewage/microbiology , Steroids/metabolism , Bacterial Typing Techniques , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Rhodococcus/classification , Rhodococcus/metabolism
19.
Int J Syst Evol Microbiol ; 59(Pt 5): 1011-5, 2009 May.
Article in English | MEDLINE | ID: mdl-19406784

ABSTRACT

The taxonomic position of the cholesterol-degrading strain Chol-3(T), isolated from a sewage sludge sample, was clarified using a polyphasic taxonomic approach. Phylogenetic analysis of its 16S rRNA gene sequence, whole-cell fatty acid profile and mycolic acid composition revealed that this isolate is a member of the genus Gordonia with the species Gordonia sihwensis, G. hydrophobica and G. shandongensis being the nearest phylogenetic neighbours. The results of DNA-DNA hybridization against its phylogenetically closest neighbours as well as the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-3(T) from the other Gordonia species with validly published names. Strain Chol-3(T) therefore merits recognition as a member of a novel species within the genus Gordonia, for which the name Gordonia cholesterolivorans sp. nov. is proposed. The type strain is Chol-3(T) (=CECT 7408(T) =DSM 45229(T)).


Subject(s)
Cholesterol/metabolism , Gordonia Bacterium/classification , Sewage/microbiology , Bacterial Typing Techniques , Biodegradation, Environmental , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Fatty Acids/analysis , Genes, rRNA , Genotype , Gordonia Bacterium/genetics , Gordonia Bacterium/isolation & purification , Gordonia Bacterium/physiology , Molecular Sequence Data , Mycolic Acids/analysis , Nucleic Acid Hybridization , Phenotype , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Species Specificity
20.
Anal Bioanal Chem ; 384(3): 751-60, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16372182

ABSTRACT

Boron neutron capture therapy (BNCT) is a bimodal radiotherapeutic treatment based on the irradiation of neoplastic tissues with neutrons after the tissues have selectively accumulated molecules loaded with nuclides with large neutron capture cross-sections (such boron-10). Boron-10 carriers have been tested to a limited extent, and clinical trials have been conducted on sulfhydryl borane (10B-BSH) and boronophenylalanine (10B-BPA). However, precise and accurate measurements of boron-10 concentrations (0.1-100 microg/g) in specimens and samples of limited size (microg scale) are needed in order to be able to biologically characterise new compounds in predictive tissue dosimetry, toxicology and pharmacology studies as well as in clinical investigations. A new approach based on fast separation and detection of 10B-BPA performed by coupling capillary electrophoresis to electrospray mass spectrometry is reported. This method allows the quantitative analysis and characterisation of 10B-BPA in a short time with a high separation efficiency. Detection limits of 3 microM for 10B-BPA and 30 ng/mL for 10B were obtained with CE-ESI-MS. A quantification limit of 10 microM for 10B-BPA (100 ng/mL for 10B) was attained. The total boron-10 concentration was determined by high-resolution inductively coupled mass spectrometry in order to validate the method. Boron-10 isotope measurements were carried out by HR-ICP-MS at medium resolution (R=4000) due to the presence of an isobaric interference at mass 10. Good agreement was obtained between the values from CE-ESI-MS and those from HR-ICP-MS. The method has been successfully used to determine the 10B-BPA in two lines of cultured cells.


Subject(s)
Boron Compounds/analysis , Boron Neutron Capture Therapy , Endothelial Cells/chemistry , Glioma/chemistry , Mass Spectrometry/methods , Phenylalanine/analogs & derivatives , Animals , Boron Compounds/pharmacokinetics , Cell Line, Tumor , Electrophoresis, Capillary , Endothelial Cells/metabolism , Glioma/metabolism , Humans , Isotopes , Molecular Structure , Phenylalanine/analysis , Phenylalanine/pharmacokinetics , Rats , Sensitivity and Specificity , Time Factors
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