Solvolysis of 2'-deoxyribonucleosides and oligo-2'-deoxyribonucleotides in aqueous pyrrolidine or ammonia solutions.

To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the 2'-ß-D-deoxyribosides of 5-bromouracil, 2-aminopurine, uracil, adenine, cytosine, 5-methylcytosine, hypoxanthine, N6-methyladenine, N4-ethylcytosine, and guanine were compared as to their rate of degradation in 0.5 M aqueous pyrrolidine at 110 °C, conditions used earlier in the analysis of oligonucleotides containing only the canonical bases. The reaction mixtures were analyzed by chromatography on Zorbax XDB-CN and UV absorption spectroscopy. The first-order rate constants for the nucleoside degradations decreased in the above order, spanning a wide range of reactivities. Some of these nucleosides were also tested in 0.5 M aqueous ammonia at 110 °C, giving similar first-order rate constants, except for 2'-deoxyguanosine, which is much more reactive with ammonia, due to the lower basicity of this reagent, leaving a larger proportion of the nucleoside in the non-ionized form, susceptible to nucleophilic attack at the base. Short oligothymidylates containing a single 2-aminopurine, adenine, guanine, or cytosine unit in central position were tested in pyrrolidinolysis, to determine the cleavage rates at these sites and the dependence of these cleavage rates on oligonucleotide length. A model decadeoxyribonucleotide containing all four canonical bases was also pyrrolidinolyzed, followed by ion-exchange chromatography, to deduce the nucleotide sequence from the resulting chromatographic profile.

Lubricación in vitro en prótesis articulares / In Vitro Lubrication In Joint Prostheses

Entender el desgaste prematuro en reemplazos articulares debido a una lubricación deficiente, que puede resultar en valores de fricción altos, es un tema amplio e intrincado de abordar. Además, si el lubricante es el fluido sinovial, los mecanismos de lubricación que ocurren son aún más complejos de develar. En este artículo se revisa el estado de conocimiento actual de la lubricación sinovial, así como las características reológicas del fluido lubricante. Asimismo, se mencionan algunas técnicas experimentales y métodos numéricos con los que se ha estudiado el problema de la lubricación. En algunas simulaciones numéricas de la lubricación en reemplazos articulares no se considera el efecto del esfuerzo cortante del líquido sinovial ya que se asume que tiene un comportamiento newtoniano, sin embargo, otras investigaciones han demostrado que al asumir un comportamiento no newtoniano el proceso de lubricación se afecta significativamente. Con todo esto, incorporar todos los factores que pueden afectar la lubricación en reemplazos articulares, en simulaciones numéricas hasta la fecha es un reto. A través de diversas investigaciones se buscan nuevos materiales, diseños y técnicas de análisis que permitan incrementar la vida útil de los implantes para así reducir las cirugías de revisión(AU)

derstanding premature wear in joint replacements due to poor lubrication, which can result in high friction values, is a broad and intricate topic to address. In addition, if the lubricant is the synovial fluid, the lubrication mechanisms that occur are even more complex to unveil. This article reviews the current state of knowledge on synovial lubrication, as well as the rheological characteristics of the lubricating fluid. It is also made a mention of some experimental techniques and numerical methods with which the problem of lubrication has been studied. In some numerical simulations of lubrication in joint replacements the effect of the shear stress of the synovial fluid is not considered since it is assumed to have a Newtonian behavior; however, other research has shown that by assuming a non-Newtonian behavior the lubrication process is significantly affected. With all this, incorporating all the factors that can affect lubrication in joint replacements, in numerical simulations to date is a challenge. Through various investigations, new materials, designs and analysis techniques are sought to increase the useful life of implants in order to reduce revision surgeries(AU)

High-temperature solvolysis of 2'-deoxyribonucleosides in aqueous amine solutions.

Degradation of 2'-deoxyribonucleosides in 0.5 M aqueous pyrrolidine at 110 °C proceeds at different rates, ordered as deoxyuridine > deoxyadenosine > deoxycytidine > deoxyguanosine ≫ deoxythymidine. Deoxyadenosine degradation produces the free base, adenine, while deoxycytidine by deamination produces deoxyuridine, and then uracil. The solvolysis of deoxyadenosine has an activation energy of 23.3 kcal/mol. Ammonolysis is slower than pyrrolidinolysis for deoxyadenosine, but faster for deoxyguanosine. In pyrrolidinolysis of the trinucleotides, d-TGT and d-TAT, the guanine moiety reacts faster than the adenine moiety. These trends are interpreted in terms of the ionization of the guanine moieties under basic conditions, rendering them less susceptible to nucleophilic attack.

Protonation of Nucleobases in Single- and Double-Stranded DNA.

Single-stranded model oligodeoxyribonucleotides, each containing a single protonatable base-cytosine, adenine, guanine, or 5-methylcytosine-centrally located in a background of non-protonatable thymine residues, were acid-titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5-methylcytosine base, were comparatively evaluated in single-stranded and double-stranded form, by UV spectroscopy and high-field NMR. The N3 protonation of the 5-methylcytosine moiety in the double-stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single-stranded case. The central guanine:5-methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O2 of the 5-methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5-methylcytosine sites in double-stranded DNA might be protonated to a significant extent under physiological conditions.

Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures.

Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c7dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide.

Extruded snacks from whole wheat supplemented with textured soy flour: Effect on instrumental and sensory textural characteristics.

The quality of extruded snacks can be affected not only by processing conditions, but also by some factors like the concentration and type of ingredients incorporated in their formulation and the working conditions used. Although the process conditions have been established with measurable textural properties, sensory qualities have not been correlated with these responses in expanded extruded snacks made with added functional ingredients. Therefore, in this study the effect of adding textured soy flour (TSF) and whole wheat flour (WWF) to refined wheat flour in the production of extruded snacks and expanded with hot air was evaluated. A response surface design using two levels with five central points was applied to obtain the best combinations of functional ingredients added, holding the parameters of the extrusion process and moisture of treatments. Some texture characteristics and sensory analysis were used as response variables, such as, hardness, fracturability, toughness, crispness, granularity, and chewiness. Likewise, the rate of expansion was evaluated. The results showed that the level of substitution of WWF, especially levels of 15%, had a significant effect on the hardness perceived by the panelist during sensory evaluation. The TSF at concentrations of ≥15%, favored the fracturability and crispness of the samples. It was found that the best expansion index was with the combination of 5% TSF and 15% WWF. Although a correlation between instrumental and sensory tests carried out on the extruded snacks expanded was not found. PRACTICAL APPLICATIONS: The physical characteristics of the extruded snacks such as expansion, hardness, and density are important parameters in terms of consumer acceptability of the final product as well as their functional properties. In other words, the appearance and texture are two of the most important attributes that can be seen in snack foods. In particular, the texture can be measured by intrinsic tests: objective (instrumental) and subjective (sensory). That is because the instrumental analysis provides parameters such as firmness, brittleness, consistency, chewiness, among others, when subjected to different stress, strain, and strain rates at the snacks. Similarly, sensory analysis, allows us to see features that include mechanical attributes (concerning the reaction to the applied force), geometric attributes (concerning the shape, size, and orientation of the particles within the food), and attributes related to the perception of moisture or fat content.

PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

Determination of pKa values for deprotonable nucleobases in short model oligonucleotides.

The deprotonation of ionizable nucleobases centrally placed in short model oligonucleotides was examined under different physical conditions, using UV absorption spectroscopy. The oligonucleotide sequences were designed so that only the central base would be ionized over the pH range examined. pKa values of 9.90±0.01 and 9.34±0.04 were determined for the guanine group in the oligomer d-ACAGCAC and 2'-deoxyguanosine, respectively, both at 25°C and 0.1M NaCl. Lengthening the oligonucleotide up to the tridecamer stage further increases the pKa of the central guanine moiety. Electrolyte concentration, temperature, and mixed water-ethanol solvents affect the acidity of the central base. Changes in the sequence surrounding the central guanine can also have a significant effect, especially in the case of strongly stacking sequences. The pKa values were also determined for the hepta(2'-O-methyl)ribonucleotide and the heptamer PNA of identical sequence, as well as for oligodeoxyribonucleotides with different deprotonable bases, viz. thymine, uracil, or hypoxanthine, in the central position. The results are interpreted in terms of the electric-field effect exerted on the departing proton by the negative electric charges located on the internucleotide phosphate groups, and calculations show this effect to approximately explain the magnitude of the pKa difference observed between the deoxyriboheptanucleotide and its electroneutral PNA analogue.

Hybridisation of N4-methylcytosine-containing amplicons on DNA microarrays.

5'-Cy5-labelled PCR amplicons containing the analogue base, N(4)-methylcytosine, instead of cytosines were compared in microarray hybridisation experiments with the corresponding amplicons containing the canonical set of bases, with respect to the intensity of the fluorescence signal obtained, and cross hybridisation to non-corresponding probes. In general, higher hybridisation temperatures resulted in reduced signal intensities, particularly in the case of the N(4)-methylcytosine containing amplicons. At the lower hybridisation temperatures tested (40 °C, 30 °C), these modified amplicons gave about equal or stronger fluorescence signal than the corresponding regular amplicons. With the two GC-richest amplicons tested, in one instance the N(4)-methylated target gave a dramatically higher signal intensity than the unmodified amplicon, interpreted as reflecting the reduced formation of hairpin structures in the target sequence, due to the lower thermodynamic stability of the G:N(4)-methylC base pair, making the target more accessible, while in the other case no hybridisation was observed with either version of the amplicon, probably due to interference from a G-tetrad structure. Both for the regular and the N(4)-methylated amplicons, no significant cross hybridisation was seen in these experiments.

Capacity of N4-methyl-2'-deoxycytidine 5'-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases.

The dCTP analog N4-methyl-2'-deoxycytidine 5'-triphosphate (N4medCTP) was evaluated for its performance in the polymerase chain reaction (PCR). Using the HotStart Taq DNA polymerase with a standard thermal protocol, test segments 85 or 200 bp long were amplified equally well using dCTP or N4medCTP:dCTP mixtures ranging in molar ratio from 3:1 to 10:1, while complete replacement of dCTP by N4medCTP gave clearly lower amplicon yields and higher Cq values. Comparable yields with N4medCTP or dCTP were achieved only by using a slowdown protocol. Post-PCR melting analyses showed decreasing Tm values for amplicons obtained with increasing N4medCTP:dCTP input ratios; for the 200-bp amplicon, complete replacement of dCTP by N4medCTP in the reaction reduced the Tm by 11 °C; for the 85-bp amplicon the Tm reduction was 7 °C. In experiments aiming at the 200-bp amplicon, Pfu exo(-) DNA polymerase did not sustain PCR when dCTP was fully replaced by N4medCTP, even with the slowdown protocol, except at elevated N4medCTP concentrations, and, compared to PCR conducted exclusively with dCTP, the use of N4medCTP:dCTP mixtures gave reduced yields and distinctly higher Cq values, regardless of the thermal program employed. PCR experiments with 9°N DNA polymerase using N4medCTP in the conventional thermal protocol failed to produce the 200-bp amplicon.

Comparative studies on Enterococcus, Clostridium perfringens and Staphylococcus aureus as quality indicators in tropical seawater at a Pacific Mexican beach resort.

Three microorganisms were assayed to evaluate the microbiological quality in the seawater at a resort on the Mexican Pacific coast, and to test for possible associations among the titers of the various bacteria, their possible correlations with environmental conditions, and with the location of potential wastewater outflows. Significant microorganism levels were found (at Caletilla beach, Hornos beach, and Papagayo beach, respectively: for Enterococcus 157, 153, and 149, for C. perfringens 35, 89, and 56, for S. aureus 244,137, and 279CFU/100ml), often in excess of the presently set guideline values. In general, bacterial titers were higher during rainy season than in dry season. For S. aureus, in both seasons, highest concentrations were found at 3pm, the time of highest tourist presence at the beaches. Our results argue for the use of these three microorganisms as part of a set of indicators in the routine microbiological evaluation of Mexican beachwaters.

Comparison of different amines for the one-lane sequence determination of 5'-end-labeled oligodeoxyribonucleotides by chemical cleavage.

Hot aqueous solutions of a wide variety of aliphatic amines bring about efficient cleavage of 5'-(32)P-labeled oligodeoxyribonucleotides. Electrophoretic resolution of the product mixtures produces a radioactivity pattern that can be analyzed in terms of relative band intensities and band separations to deduce the base sequence of the original oligonucleotide. Amines differ in their overall reactivity with respect to the oligonucleotide as well as in their preference for reaction with the various heterocyclic bases. Guanine sites, in particular, vary markedly in their cleavage susceptibility when reacted with the different amines, being more vulnerable to scission when reacted with less basic amines. Guanine cleavage propensity can also be affected markedly by the ionic strength of the aminolysis solution. Both effects are interpreted in terms of variable extents of deprotonation of the guanine sites in the basic medium. Some of the amines produce fragment patterns that are marred by the presence of minor extraneous bands; these probably are due to an incomplete extent of the second beta-elimination involved in backbone cleavage. The methodology is applicable to confirmatory sequencing of synthetic oligonucleotides and can also be used to prepare standard ladders for use in footprinting experiments or chemical reactivity studies.

CYP2E1 regulation by benzene and other small organic chemicals in rat liver and peripheral lymphocytes.

The inducibility of CYP2E1 was investigated in liver and peripheral lymphocytes of rats treated with benzene (0-10 mmol/kg body weight (bw), daily for 3 days, i.p., or 0 and 5 mmol/kg bw, daily for 14 days, i.p.) or toluene (0 and 5 mmol/kg bw, daily for 3 days, i.p.) and compared with that of pyridine (5 mmol/kg bw, i.p.) or acetone (5% in drinking water) both daily for 3 days. Acute benzene treatment (5 mmol/kg bw) increased both CYP2E1 apo-protein (2-fold) and p-nitrophenol hydroxylase (p-NPH) activity (1.4-fold) in liver, and CYP2E1 mRNA in both liver (2.2-fold) and peripheral lymphocytes (2.9-fold). The response to toluene was qualitatively similar, although smaller than that to benzene. As expected, acetone and pyridine treatments resulted in a 2- to 3-fold increase of p-NPH activity and CYP2E1 apo-protein content in liver, but not the mRNA levels. In addition, acute benzene and acetone treatments increased the 6-hydroxychlorzoxazone/chlorzoxazone metabolic ratio 1.6- and 3.1-fold, respectively. The subchronic treatment with benzene increased CYP2E1 mRNA and apo-protein from days 2 and 3 to day 14, respectively, whereas the enzyme activity increased transiently on days 3 and 5 only. These results show that acute/subacute benzene and acute toluene treatments induce CYP2E1 expression probably through a similar mechanism which might be different from that of pyridine or acetone, in that the former increase mRNA levels, both in liver and in peripheral lymphocytes, whereas the latter stabilized the apo-protein.