Comparative study of carbon nanotubes and C(60) fullerenes as pseudostationary phases in electrokinetic chromatography

The use of carbon nanostructures in different modalities is a topic of growing interest. This article provides a systematic comparison of surfactant-coated single-wall carbon nanotubes, multi-wall carbon nanotubes and C(60) fullerenes as pseudostationary phases (PSPs) in electrokinetic chromatography. The differences on the electrophoretic behaviour as a function of the pseudostationary phase for phenolic compounds, triazines and nitroimidazole derivatives have been explored. Phenolic compounds, triazines and nitroimidazole derivatives has been selected as model compounds of aromatic compounds, pi-exceeding heteroaromatic compounds and pi-deficient heteroaromatic compounds, respectively. Resolution was also evaluated on the basis of the differences in the electrophoretic behaviour and in this sense the best PSP for each group of compounds can be proposed.     

Synthesis of 2,4‐disubstituted 6‐phenyl‐7H‐pyrrolo[3,2‐d]‐pyrimidin‐7‐one 5‐oxides

A relatively short and efficient method for the utilization of 4,6‐dichloro‐2‐methylthio‐5‐nitropyrimidine ( 1 ) in the synthesis of the poly substituted pyrrolo[3,2‐d]pyrimidin‐7‐one 5‐oxides ( 6a ‐g) is reported. Some new 4‐substituted 6‐chloro‐2‐methylthio‐5‐nitropyrimidines ( 2a‐e ) were prepared by reaction of 4,6‐dichloro‐2‐methylthio‐5‐nitropyrimidine ( 1 ) with amines. 4‐Substituted 2‐methylthio‐5‐nitro‐6‐phenylethynylpyrimidines ( 3a‐e ), obtained from 4‐substituted 6‐chloro‐2‐methylthio‐5‐nitropyrimidines ( 2a‐e ) via palladium‐catalyzed Sonagashira coupling reaction with 1‐phenylacetylene, underwent smooth cyclization reaction in boiling 2‐propanol in the presence of catalytic amount of pyridine to give 4‐substituted 2‐methylthio‐6‐phenyl‐7H‐pyrrolo[3,2‐d]pyrimidin‐7‐one 5‐oxides ( 4a‐e ). The methylthio group of the latter compounds can be easily and selectively oxidized by m‐chloroperbenzoic acid and replaced with different amines.     

Amperometric Glucose Biosensors Based on Glassy Carbon and SWCNT‐Modified Glassy Carbon Electrodes

Different carbonaceous materials, such as single‐walled carbon nanotubes (SWCNTs) and glassy carbon submitted to an electrochemical activation at +1.80 V (vs. SCE) for 900 s, have been used with the aim of comparing their performances in the development of enzyme electrodes. Commercial SWCNTs have been pretreated with 2.2 M HNO₃ for 20 h prior to use. The utility of activated GC as promising material for amperometric oxidase‐based biosensors has been confirmed. With glucose oxidase (GOx) as a model enzyme, glucose was efficiently detected up to 1 mM without the use of a mediator. Both electrodes operated in stirred solutions of 0.1 M phosphate buffer (pH 5.5), containing dissolved oxygen, at a potential of ?0.40 V vs. SCE. Although the performances of the two carbonaceous materials were comparable, the biosensors based on activated GC were characterized by a practically unchanged response 40 days after the fabrication, a better signal to noise ratio, and a little worse sensitivity. In addition, the preparation procedure of such biosensors was more simple, rapid and reproducible.     

Demonstration of a protein with enhanced resistance to proteinase K in transmissible cytopathic condition elicited by cell-free lysate of free-living ameba Naegleria gruberi

The cell-free lysate of free-living amebae Naegleria gruberi and Naegleria fowleri were reported to elicit cytopathic effect in various cell lines that could be indefinitely transmitted by the culture media. The causative agent showed sensitivity to treatments detrimental to proteins while resisted exposures damaging to nucleic acids. Here we demonstrate that subsequent to exposure to N. gruberi lysate mild digestion with proteinase K reveals the presence of a protein band in HeLa cells absent from control cell lines. Though the small quantity of this protein with enhanced resistance to proteinase K relative to the total protein content of the sample has proved to date insufficient for its purification, we suppose that it is a human cellular protein that assumed altered conformation in a prion-like fashion. The conformational conversion could have been trigerred by an ameba protein in the lysate. In addition, we showed that HeLa cells treated with N. gruberi lysate display elevated cathepsin B activity which is assumed to be a secondary response to the accumulation of the proteinase K-resistant protein. We propose that a number of degenerative sequelae following previous microbial infections in mammals could have a similar pathomechanism. Moreover, epidemiological data strongly suggest that natural prion disease in sheep, goat and cervids may also have an etiology linked to prior infection/colonization with a microbe, as it had already been proposed by one of us.     

Hydrolytic and photoinduced degradation of tribenuron methyl studied by HPLC-DAD-MS/MS

The paper studies, with the help of HPLC-DAD-MS/MS technique, the hydrolytic and photoinduced degradation processes that take place in aqueous solutions of tribenuron methyl, both when preserved in the dark and when undergoing solar box irradiation under conditions that simulate sun light. The results indicate that the degradation products formed by hydrolysis alone and by photoirradiation are the same, but kinetics of the hydrolysis reaction is much slower. The degradation products are identified as 2-methoxy-4-methylamino-6-methyl-1,3,5-triazine (P1), methyl 2-aminosulfonylbenzoate (P2), and saccharin (P3) and quantified. Ecotoxicological biotests performed on 0.1 microg L(-1) photoirradiated solutions of the herbicide give a border line toxicity situation comparable to that of the precursor and indicate that the herbicide is characterized by low persistence in the environment, as required. Its degradation, however, does not lead to mineralization but to the formation of products of comparable toxicity. To evaluate the matrix effects, the photodegradation of the herbicide is also studied in the presence of rice paddy waters: the process is slower than in ultrapure water but leads to the same products. Experiments performed for comparison by irradiating ultrapure water solutions with UV lamp (254 nm) show that the degradation process is not only faster with respect to sunlight, but gives a different pathway, without in anyway leading to mineralization.     

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides

A modified 2′-deoxycytidine triphosphate derivative ( dC^TOTP ) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dC^TOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dC^TOTP . When dC^TOTP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.     

Validation of the Tessier scheme for speciation of metals in soil using the Bland and Altman test

The Tessier extraction method was used for speciation of Cu, Pb, Zn, As, Fe and Mn in a large concentration range in contaminated soil with various mineralogical compositions. The results were compared by X-ray fluorescence spectrometry (XRF) as a reference method using the Bland and Altman test. A sum of five fractions (exchangeable, bound to carbonates, Fe-Mn oxides, organic matter and residual forms) was compared with the total content determined on solid matrix by the reference method. A good agreement between the methods in the whole concentration range was found for Cu, Zn, As, and Fe. For Mn and Pb, XRF was found suitable to verify the sequential extraction only for concentrations above 250 mg kg⁻¹. This was a consequence of a poorer reproducibility of Pb extraction using the Tessier scheme due to a great difference in the mineralogical composition and the diversity of the Pb species identified in soil. The poorer result of Mn was attributed to the spectral interference of Fe in XRF. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Clustering versus percolation in the assembly of colloids coated with long DNA

We report an experimental study in which we compare the self-assembly of 1 mum colloids bridged through hybridization of complementary single-stranded DNA (ssDNA) strands (12 bp) attached to variable-length double-stranded DNA spacers that are grafted to the colloids. We considered three different spacer lengths: long spacers (48 500 bp), intermediate length spacers (7500 bp), and no spacers (in which case the ssDNA strands were directly grafted to the colloids). In all three cases, the same ssDNA pairs were used. However, confocal microscopy revealed that the aggregation behavior is very different. Upon cooling, the colloids coated with short and intermediate length DNAs undergo a phase transition to a dense amorphous phase that undergoes structural arrest shortly after percolation. In contrast, the colloids coated with the longest DNA systematically form finite-sized clusters. We speculate that the difference is due to the fact that very long DNA can easily be stretched by the amount needed to make only intracluster bonds, and in contrast, colloids coated with shorter DNA always contain free binding sites on the outside of a cluster. The grafting density of the DNA decreases strongly with increasing spacer length. This is reflected in a difference in the temperature dependence of the aggregates: for the two systems coated with long DNA, the resulting aggregates were stable against heating, whereas the colloids coated with ssDNA alone would dissociate upon heating.     

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS): effects of oligomeric dissociation

The present work focuses on the interaction between the zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp). Electronic optical absorption, fluorescence emission and circular dichroism spectroscopy techniques, together with Gel-filtration chromatography, were used in order to evaluate the oligomeric dissociation as well as the autoxidation of HbGp as a function of the interaction with HPS. A peculiar behavior was observed for the HPS–HbGp interaction: a complex ferric species formation equilibrium was promoted, as a consequence of the autoxidation and oligomeric dissociation processes. At pH 7.0, HPS is more effective up to 1 mM while at pH 9.0 the surfactant effect is more intense above 1 mM. Furthermore, the interaction of HPS with HbGp was clearly less intense than the interaction of this hemoglobin with cationic (CTAC) and anionic (SDS) surfactants. Probably, this lower interaction with HPS is due to two factors: (i) the lower electrostatic attraction between the HPS surfactant and the protein surface ionic sites when compared to the electrostatic interaction between HbGp and cationic and anionic surfactants, and (ii) the low cmc of HPS, which probably reduces the interaction of the surfactant in the monomeric form with the protein. The present work emphasizes the importance of the electrostatic contribution in the interaction between ionic surfactants and HbGp. Furthermore, in the whole HPS concentration range used in this study, no folding and autoxidation decrease induced by this surfactant were observed. This is quite different from the literature data on the interaction between surfactants and tetrameric hemoglobins, that supports the occurrence of this behavior for the intracellular hemoglobins at low surfactant concentration range. Spectroscopic data are discussed and compared with the literature in order to improve the understanding of hemoglobin–surfactant interaction as well as the acid isoelectric point (pI) influence of the giant extracellular hemoglobins on their structure–activity relationship.     

Preparation of sigma- and pi-allylcopper(III) intermediates in SN2 and SN2' reactions of organocuprate(I) reagents with allylic substrates

The first pi-allyl complexes of CuIII have been prepared and characterized by using rapid injection nuclear magnetic resonance spectroscopy (RI-NMR). The prototype, (eta3-allyl)dimethylcopper(III), was prepared by injection of allyl chloride into a THF-d8 solution of iodo-Gilman reagent, Me2CuLi.LiI (A), spinning in the probe of an NMR spectrometer at -100 degreesC. A sigma-allyl ate complex, lithium (eta1-allyl)trimethylcuprate(III), was prepared in high yield by including 1 equiv of tributylphosphine in the reaction mixture or by using allyl acetate as the substrate. Cyano ate complex, lithium cis-(eta1-allyl)cyanodimethylcuprate(III) was obtained in high yield by injecting allyl chloride or allyl acetate into the cyano-Gilman reagent, Me2CuLi.LiCN (B), in THF-d8 at -100 degrees C. Reactions of A with allylic substrates show a definite dependence on leaving group (chloride vs acetate), whereas those of B do not. Moreover, these reagents have different regioselectivities, which in the case of A vary with temperature. Finally, the exclusive formation of cis-cyano sigma-allyl CuIII intermediates in both the 1,4-addition of B to alpha-enones and its SN2alpha reaction with allylic substrates now makes sense in terms of pi-allyl intermediates in both cases, thus unifying the mechanisms of these two kinds of conjugate addition.