Tetramethylpyrazine protects Schwann cells from ischemia-like injury and increases cell survival in cold ischemic rat nerves

Tetramethylpyrazine (TMP), a major active ingredient of Ligusticum wallichi Franchat extract (a Chinese herb), exhibits neuroprotective properties in ischemia. In this study, we assessed its protective effects on Schwann cells (SCs) by culturing them in the presence of oxygen glucose deprivation (OGD) conditions and measuring cell survival in cold ischemic rat nerves. In the OGD-induced ischemic injury model of SCs, we demonstrated that TMP treatment not only reduced OGD-induced cell viability losses, cell death, and apoptosis of SCs in a dose-dependent manner, and inhibited LDH release, but also suppressed OGD-induced downregulation of Bcl-2 and upregulation of Bax and caspase-3, as well as inhibited the consequent activation of caspase-3. In the cold ischemic nerve model, we found that prolonged cold ischemic exposure for four weeks was markedly associated with the absence of SCs, a decrease in cell viability, and apoptosis in preserved nerve segments incubated in University of Wisconsin solution (UWS) alone. However, TMP attenuated nerve segment damage by preserving SCs and antagonizing the decrease in nerve fiber viability and increase in TUNEL-positive cells in a dose-dependent manner. Collectively, our results indicate that TMP not only provides protective effects in an ischemia-like injury model of cultured rat SCs by regulating Bcl-2, Bax, and caspase-3, but also increases cell survival and suppresses apoptosis in the cold ischemic nerve model after prolonged ischemic exposure for four weeks. Therefore, TMP may be a novel and effective therapeutic strategy for preventing peripheral nervous system ischemic diseases and improving peripheral nerve storage.

Tetrametilpirazina (TMP), o principal componente do extrato de Ligusticum wallichi Franchat (erva chinesa), apresenta propriedades neuroprotetoras na isquemia. Nesse estudo, avaliamos seus efeitos protetores nas células de Schwann (SC), cultivando-as na presença de condições de depleção de oxigênio da glicose (OGD) e medindo a sobrevivência dos nervos de ratos isquêmicos pelo resfriamento. No modelo de lesão isquêmica em SC induzida por OGD, demonstramos que o tratamento com TMP não somente reduziu as perdas de viabilidade celular induzida por OGD, a morte celular, a apoptose de SC dose-dependente e inibiu a liberação de LDH, mas, também, suprimiu a infra-regulação do Vcl-2 e a supra-regulação de Bax e caspase-3, e inibiu a consequente ativação da caspase-3. No modelo de nervo isquêmico por resfriamento, observamos que a exposição prolongada ao resfriamento por quatro semanas estava, marcadamente, associada com a ausência de SC, com o decréscimo da viabilidade celular e a apoptose em segmentos de nervo incubados na solução da Universidade de Wisconsin apenas. Entretanto, a TMP atenuou o dano no segmento do nervo preservando SC e antagonizando a diminuição da viabilidade da fibra nervosa e o aumento das células TUNEL-positiva de modo dose-dependente. De forma conjunta, nossos resultados indicam que o TMP não só fornece efeitos protetores em um modelo de dano semelhante à isquemia de SC de ratos cultivados pela regulação de BCl-2, Bax e caspase 3, mas, também, aumenta a sobrevivência celular e suprime a apoptose no modelo de isquemia por resfriamento por exposição prolongada por quatro semanas. Então, TMP pode ser uma estratégia terapêutica eficaz para prevenir doenças isquêmicas do sistema nervoso periférico e melhora a armazenagem do nervo periférico.

A highly sensitive capillary electrophoresis method using p-nitrophenyl 5'-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities.

A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length×75.5µm (id)), electrokinetic injection for 60s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80µA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K(m) values for NPP1 and NPP3, K(i) values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.

A liquid chromatographic method for routine analysis of 5'-mononucleotides in pediatric formulas.

An RP-HPLC method for the routine determination of supplemented 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in pediatric formulas and milk products is described. Following sample dissolution, potential interferences were removed by anion-exchange SPE. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92-101% and repeatability RSDs of 1.0-2.3%. The method was optimized for the rapid, routine analysis of nucleotide-supplemented bovine milk-based infant and follow-on formulas.

[Quantum-mechanical conformational analysis of the 5'-thymidilic acid molecule].

The conformational analysis of the DNA structural unit--the nucleotide with thymine base and electroneutral phosphate group at 5'-position-has been carried out with the applied quantum mechanics methods at the MP2/6-311++G(d,p) // B3LYP/6-31G(d,p) theory level. As many as 660 conformations with relative Gibbs energies under standard conditions from 0 to 11.1 kcal/mole have been found. Among them, six conformations are similar to the structure of the nucleotide of AI-DNA, one--to AII- and seven--to the DNA in BI-form. The lowest Gibbs energy among the DNA-like conformations (deltaG = 2.7 kcal/mole) belongs to BI-DNA-like structure. It is shown that the glycoside chemical bond is the most labile one. The role of intramolecular CH...O hydrogen bonds in formation of the 5'-thymidilic acid molecule structure is demonstrated.

Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS.

To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.

Influence of antidepressant drugs on Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) from salivary glands of rats.

Xerostomia is commonly caused by antidepressant drugs and ATP can influence the saliva production. Adenosine is the product of extracellular hydrolysis of adenine nucleotides in submandibular gland cells, which occurs by the action of ectonucleotidases. In this study, we have evaluated the effect of three different antidepressants in ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP1-3) activities in cultured cells from salivary glands. Rats received imipramine (10mg/ml), fluoxetine (20mg/ml) or moclobemide (30mg/ml) by oral gavage. The drugs were administered once a day for 14 days. Our results have shown that the hydrolysis of p-nitrophenyl-5'-thymidine monophosphate increased in all treatments. These effects were not consequence of transcriptional control of E-NPP1-3 genes. The results reported here can highlight the importance of ectonucleotidases in the most common side effect caused by antidepressant therapy.

Simultaneous determination of thymidylate and thymidine diphosphate by capillary electrophoresis as a rapid monitoring tool for thymidine kinase and thymidylate kinase activities.

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of thymidylate (TMP) and thymidine 5'-diphosphate (TDP) in enzyme assays without using radioactive-labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. The separation of micromolar TMP and TDP from millimolar adenosine 5'-triphosphate (ATP) was performed at 25 degrees C using sodium tetraborate as the background electrolyte. Under the optimal condition, a good separation with high efficiency was achieved in 6 min. Several parameters affecting the separation were studied, including the pH of electrolyte, the applied voltage, and acetonitrile-salt sample stacking. The fronting of the ATP peak resulting from the interference of magnesium ion in the enzyme assay buffer was suppressed by the addition of sodium ethylenediaminetetraacetate to the sample solution. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of TMP and TDP were 2.6 and 3.8 microM, respectively. Application of the proposed method for simultaneous determination of TMP and TDP in enzyme assays was demonstrated by the activity assays of thymidine kinase and thymidylate kinase from white spot syndrome virus. This is a sensitive, nonradioactive method for thymidine kinase and thymidylate kinase assays.

Analysis of melphalan adducts of 2'-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography-electrospray tandem mass spectrometry.

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.

A new method for quantitative determination of tritium-labeled nucleoside kinase products adsorbed on DEAE-cellulose.

The counts of tritiated compounds--adsorbed to paper disks, paper chromatograms, electrophoretograms or TLC plates--can be strongly affected by extended self-absorption of tritium beta-particles on the matrix, due to their low energy. Therefore fully quantitative results can be obtained only by elution of the substances or decomposition of the matrix and subsequent counting in homogeneous solution. In this study we describe a new method for fast and proper decomposition of cellulose matrices by cellulase digestion prior to scintillation counting. This new approach yields up to 98% recovery. For method validation recombinant herpes simplex type 1 thymidine kinase was characterised kinetically. The Km of 0.2 microM remained the same as expected but Vmax was considerably higher yielding 1050 pmol/microgram/min.

Specific base recognition of oligodeoxynucleotides by capillary affinity gel electrophoresis using polyacrylamide-poly(9-vinyladenine) conjugated gel.

Poly(9-vinyladenine) was synthesized and utilized as an affinity macroligand entrapped within the gel matrix. Base-specific separation of oligodeoxynucleotides was achieved with high resolution and high speed by electrophoresis, using capillaries filled with conjugated polyacrylamide-poly(9-vinyladenine) gel. Oligothymidylic acids were selectively separated from the mixture of oligothymidylic and oligodeoxyadenylic acids by utilizing a specific hydrogen bonding between poly(9-vinyladenine) and oligothymidylic acids. Migration time and resolution of oligodeoxynucleotides were influenced by several parameters, such as the size of poly(9-vinyladenine), capillary temperature, and concentrations of poly(9-vinyladenine) and urea. Some guidelines are presented, based on the theoretical formulation of the effect of these parameters, in order to find optimum electrophoretic conditions. Analytical capillary affinity gel electrophoresis was developed for the selective and sensitive base recognition of oligodeoxynucleotides with efficiencies as high as several 10(6) plates/m by using a urea-gel capillary with poly(9-vinyladenine) and temperature-programming.

Determination of deoxymononucleotides and deoxynucleosides as a tool for toxicological examination--a trial for conversion of absorbance unit into molar amount in deoxydinucleotide.

Conversion of absorbance unit into molar amount was tried as to deoxydinucleotide. In order to achieve this trial, a reversed-phase high-performance liquid chromatographic method with a linear gradient mode was established for separation and quantification of deoxymononucleotides and deoxynucleosides. Selecting 2'-deoxycytidylyl-(3'-5')-thymidylic (5') acid as an example of deoxydinucleotides, this compound dissolved in water was estimated in absorbance unit at 260 nm, and it was treated by alkaline phosphatase and furthermore by snake venom phosphodiesterase. Based on quantification of the cleaved nucleic acid constituents including 2'-deoxycytidine 5'-monophosphoric acid and thymidine by the above method, the molar amount per 1.000 absorbance unit at 260 nm in the deoxydinucleotide was determined. Thus, the conversion of absorbance unit into molar amount is considered to be applicable to other deoxyoligonucleotides.

CpG and TpA frequencies in the plant system.

Higher plant nuclear sequences reveal avoidance of CpG and TpA doublets. Chloroplast sequences avoid the TpA doublet in all codon positions. The chloroplast genome is not methylated but codon positions II-III and untranslated regions avoid CpG. The mitochondrial genome, also unmethylated, avoids CpG in all codon positions. We therefore deduce that methylation is not sufficient to explain CpG avoidance in the higher plant systems. Other factors must be taken into account such as amino acid composition, codon choices and perhaps stability of the DNA helix.

[Complementarily addressed alkylation and cleavage of T7 phage DNA adjacent to the oligothymidylic sequences]. / Komplementarno adresovannoe alkilirovanie i fragmentatsiia DNK fara T17 vblizi oligotimidilovykh posledovatel'nostei.

Effect of temperature and reagent excess on the alkylation of the T7 phage DNA by an alkylating derivative of hexaadenylate that containes a modifying group on the 3'-terminus has been studied. Under condition of saturation at 20 degrees the reagent covalently binds adjacent to the 133 5'-PuN2--4Tn greater than or equal to 4 sequences in the T7 DNA; at 40 degrees the reagent alkylates 24 5'-PuN2Tn greater than or equal to 6 sequences. DNA containing specific apurinic sites has been prepared due to elimination of the alkylated purines. This DNA has been cleaved at the apurinic sites and specific DNA fragments have been obtained. Three types of DNA fragments are formed due to alkylation at 20 degrees: one fragment with 26,500 nucleotides in length, seven fragments with 4500 nucleotides in length and 103--111 fragments with an average length of 190 nucleotides. Alkylation at 40 degrees with following apurinization and cleavage yields 21--23 fragments with the lengths: 1--31,500; 1--19,000; 3--9500 and 17--19 with 1100 nucleotides in average.

[Specific cleavage of glycosylyl denatured DNA of T2 and T4 phages adjacent to the oligothymidilic or oligoadenylic sequences]. / Fragmentatsiia gliukozilirovannykh odnotiazhevykh DNK fagov T2 i T4 vblizi oligotimidilovykh i oligoadenilovykh posledovatel'nostei.

A method of complementarily directed alkylation with following elimination of the alkylated bases and with DNA cleavage at apurinic sites was used for specific fragmentation of glucosylyl DNA of T2 and T4 phages. It was shown that denatured glucosylyl T2 and T4 DNA's are modified by alkylating derivatives of hexaadenylate and heptauridylilate. The extent of alkylation reached the maximum and then stopped. The extent of elimination and chain cleavage corresponded to that of alkylation. Treatment of DNA after alkylation with (Ap)5ARCl under condition of saturation at 20 degrees gives 572 +/- 28 fragments from T4 DNA with 200--25,000 nucleotides long and 578 +/- 33 fragments from T2 DNA. Alkylation under condition of DNA saturation with (Ap)5ARCl at 40 degrees leads to 138 +/- 15 fragments from T4 DNA and 170 +/- 16 fragments from T2 DNA. Characteristics of the fragments obtained are given.

Interactions between exogenous deoxyribonucleic acid and membrane vesicles isolated from competent and noncompetent Bacillus subtilis.

Competent cultures of Bacillus subtilis 168 have been fractionated into a high-competent and a low-competent fraction by a large-scale separation technique. Membrane vesicles isolated from both cell fractions are equally active in the transport of L-glutamate. Both membrane vesicle preparations seem to have similar endo- and exonuclease activities. Also, both preparations are capable of binding deoxyribonucleic acid. However, especially at low deoxyribonucleic acid concentrations (1 mug or less per ml), vesicles obtained from competent cells bind significantly more deoxyribonucleic acid (up to sixfold) than vesicles from noncompetent cells.