The isolation of Nepsilon-formyl-L-lysine from the reaction between formaldehyde and L-lysine and its identification by OPLC and NMR spectroscopy

From the reaction between l-lysine and formaldehyde, Nepsilon-formyl-L-lysine was isolated by means of ion-exchange column chromatography. The identification of Nepsilon-formyl-L-lysine was carried out by ion-exchange overpressured-layer chromatography (OPLC) and 1H NMR and 13C NMR spectroscopies. The m.p. and mixed m.p. values, the retention characteristics and the chemical shifts of the isolated product were identical with those of an authentic sample of Nepsilon-formyl-L-lysine.     

A nitro-arginine derivative of trimebutine (NO2-Arg-Trim) attenuates pain induced by colorectal distension in conscious rats

Irritable bowel syndrome (IBS) is characterized by dysfunction of the afferent pathways that may lead to visceral hypersensitivity. Trimebutine is a weak μ opioid receptor agonist used in the treatment of IBS. We report on the effects of a novel derivative in which trimebutine has been salified with nitro-arginine (NO₂-Arg-Trim), in modulating nociception to colorectal distension (CRD) in intact and post-colitis rats, an animal model that mimics some features of IBS. Colorectal sensitivity and pain were assessed by measuring the abdominal withdrawal score (AWR) during CRD. Healthy rats were treated with vehicle, trimebutine (10 mg/kg i.p.) or NO₂-Arg-Trim (4, 8 and 16 mg/kg i.p.). Post-colitis, allodynic rats were investigated 4 weeks after colitis induction. Treating healthy rats with NO₂-Arg-Trim resulted in a dose-dependent attenuation of CRD-induced nociception and in an inhibition of CRD-induced overexpression of spinal cFOS mRNA. NO₂-Arg-Trim-induced antinociception was reversed by the opioid receptor antagonist naloxone and by the NO synthase–cGMP pathway inhibitor methylene blue, while L-NAME had no effect. The antinociceptive effect of NO₂-Arg-Trim was maintained in a rodent model of post-inflammatory allodynia. In this setting, NO₂-Arg-Trim but not trimebutine, significantly down-regulated the spinal cFOS mRNA expression and increased blood concentrations of NO₂ + NO₃. Moreover, the expression of several genes involved in inflammation and pain, as IL-1β, TNFα, COX2 and iNOS, was up-regulated in colonic tissue from post-colitis rats and NO₂-Arg-Trim, but not trimebutine, effectively reversed this effect. In summary, these data suggest that NO₂-Arg-Trim inhibits nociception induced by CRD in both healthy and post-colitis, allodynic rats. The NO₂–arginine moiety interacts with the opioid agonist trimebutine to potentiate its analgesic activity. This study provides evidence that NO₂–arginine derivative of trimebutine might have beneficial effect in the treatment of painful intestinal disorders.     

Strength of the human mixed lymphocyte reaction (MLR) and its suppression by azathioprine or 6-mercaptopurine.

Azathioprine (36 microM) had a significantly (P less than 0.00003) greater inhibitory effect on human MLR responses below 20,000 counts/min than on responses above 20,000 counts/min. In contrast, 6-mercaptopurine (100 microM) had a similar suppressive effect on MLR responses below and above 20,000 counts/min suggesting it has a different mode of action to azathioprine.     

Conformational analysis of the dopamine-receptor agonist 5-hydroxy-2-(dipropylamino)tetralin and its C(2)-methyl-substituted derivative

The conformational preferences of the dopamine (DA) receptor agonist 5-hydroxy-2-(di-n-propylamino)tetralin (1) and the DA-inactive 5-hydroxy-2-methyl-2-(di-n-propylamino)tetralin (2) have been studied by use of molecular mechanics (MMP2) calculations and NMR spectroscopy. A good agreement is demonstrated between the experimentally determined (by NMR) and the calculated (by MMP2) conformational distribution of 1 and 2. In addition, there is a good agreement between bond distances and bond angles in the X-ray structure of the hydrobromide of 1 and those in the corresponding MMP2 conformation. Results obtained demonstrate that the energetically preferred conformations of 1 and 2 are different: Compound 1 preferentially adopts half-chair conformations with a pseudoequatorial nitrogen substituent whereas the low-energy conformations of compound 2 have a pseudoaxial nitrogen substituent. However, the results also indicate that the difference in conformational preferences is too small to account for the dopaminergic inactivity of 2. Therefore it is suggested that the steric bulk of the C(2)-methyl group per se prevents a proper alignment of (2S)-2 with DA receptors.     

Formation of spiroiminodihydantoin nucleoside by reaction of 8-oxo-7,8-dihydro-2'-deoxyguanosine with hypochlorous acid or a myeloperoxidase-H(2)O(2)-Cl(-) system

Hypochlorous acid (HOCl), generated by myeloperoxidase from H(2)O(2) and Cl(-), is a strong chlorinating and oxidizing agent, playing an important role in host defense and inflammatory tissue injury. As several recent studies have shown that various oxidizing agents including peroxynitrite and singlet oxygen react readily with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) to yield further oxidized products, we have studied the reaction of 8-oxodGuo with reagent HOCl and with a myeloperoxidase-H(2)O(2)-Cl(-) system. When 1 mM 8-oxodGuo was reacted with 0.5 mM HOCl at pH 7.4 and 37 degrees C, two major products were formed. They were identified as the diastereomers of spiroiminodihydantoin deoxyribonucleoside (dSph) on the basis of their identical ESI-MS and UV spectra and HPLC retention times with those of the major reaction products which were reported to be formed in other oxidation systems including potassium monopersulfate plus cobalt (II) chloride, peroxynitrite plus thiol, and type II photosensitization. Under the above reaction conditions, the yield of the diastereomers of dSph was 0.38 mM, with 0.57 mM 8-oxodGuo remaining unreacted. Since the presence of 50% D(2)O, 10 mM sodium azide, or 2% ethanol did not affect the yield of the products, involvement of singlet oxygen and hydroxyl radical in the formation of dSph from 8-oxodGuo with HOCl was ruled out. A 1000-fold excess of dGuo did not inhibit the reaction of 8-oxodGuo with HOCl, indicating that 8-oxodGuo reacts more readily than dGuo with HOCl. dSph was also formed by reaction of 8-oxodGuo with myeloperoxidase in the presence of H(2)O(2) and Cl(-). Our results suggest that formation of dSph from 8-oxodGuo is mediated, possibly via an addition of Cl(+) to, or two-electron oxidation of 8-oxodGuo, with HOCl or the myeloperoxidase-H(2)O(2)-Cl(-) system.     

2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxyadenosine adducts resulting from the reaction of tetrahydrofuran with DNA bases

A recent study showed that tetrahydrofuran (THF), a widely used solvent, is carcinogenic in experimental animals. Despite its carcinogenic activity, there is a paucity of information regarding cellular toxicity, biomolecular damage, and genotoxicity induced by THF. We describe here the structural characterization of adducts produced by the reaction of oxidized THF with 2'-deoxyguanosine (dGuo-THF 1 and dGuo-THF 2), 2'-deoxyadenosine (dAdo-THF), and 2'-deoxycytidine (dCyd-THF). Adducts were isolated from in vitro reactions by reverse-phase HPLC and fully characterized on the basis of spectroscopic measurements. The stable derivatives obtained by the reduction of adducts with NaBH(4) (the case of dGuo-THF 1, dCyd-THF, and dAdo-THF) and the stable adduct dGuo-THF 2 were used as standards for optimization of chromatographic separations for adduct detection in DNA through HPLC/ESI/MS-MS. Using this methodology, we successfully detected the four adducts in calf thymus DNA reacted with oxidized THF. The present study also provides evidence that rat liver microsomal monooxigenases oxidize THF to the reactive electrophilic compounds that are able to damage the DNA molecule, as indicated by a significant increase in adduct dGuo-THF 1 level when NADPH was added to the THF/microsomes/dGuo incubation mixtures. Our data point to DNA-THF adducts as possible contributing factors to the toxicological effects of THF exposure.     

Nitric oxide (NO) modulation of PAF-induced cardiopulmonary action: interaction between NO synthase and cyclo-oxygenase-2 pathways

1. To further investigate into the mechanisms of PAF-induced cardiopulmonary actions, we examined the effects of the nitric oxide synthase (NOS) inhibitor L-N(omega)-nitro-L-arginine (L-NNA), of the specific cyclooxygenase-2 (COX-2) inhibitor NS 398, and of the combined presence of both COX and NOS inhibitors on the PAF responses in the heart lung preparation of guinea-pig (HLP). 2. In HLPs perfused with homologous blood, dose-response curves for the haemodynamic and bronchial effects of PAF (1 - 32 ng) were carried out in the absence or presence of L-NNA (200 microM). L-NNA caused an increase in the resting pulmonary arterial pressure (PAP) without affecting the other basal values, and strongly potentiated the bronchoconstriction and pulmonary hypertension elicited by PAF. An enhancement of the PAF-induced actions on right atrial pressure (RAP) and cardiac output (CO) was also observed. All the effects of L-NNA were antagonized by L-arginine (2 mM). 3. The presence of L-NNA in the perfusing blood of HLPs failed to affect the pulmonary hypertensive and bronchoconstrictor responses induced by the thromboxane A(2) mimetic U46619 (0.05 - 1.6 microg), 5-hydroxytryptamine (0.1 - 1.6 microg), and histamine (0.1 - 1.6 microg), thus suggesting that these PAF secondary mediators are not responsible for the hyper-responsiveness to PAF induced by L-NNA. 4. Blocking COX-2 pathway with NS 398 (15 - 30 microM) did not alter the cardiopulmonary resting variables. However, a reduction of the PAF-mediated pulmonary hypertension, but not of bronchoconstriction, was observed. When L-NNA was added to the perfusing medium of HLPs pre-treated with NS 398 or with indomethacin (15 microM), the basal PAP values were enhanced. However, in the combined presence of COX and NOS inhibitors, only a slight increase in the hypertensive responses to the highest doses of PAF was observed, whereas the PAF mediated actions at bronchial and cardiac level were unaffected. 5. This study indicates that (i) the cardiopulmonary actions induced by PAF are specifically modulated by endogenous NO through the NOS pathway, and (ii) COX-2 isoform is involved in the pulmonary hypertensive, but not bronchoconstrictor, effects of PAF. Furthermore, an interaction between PAF stimulated COX, particularly COX-2, and NOS pathways appears to take a functional role at both bronchial and cardiovascular level.     

5-AZA-2'-deoxycytidine (5-AZA-CdR): a demethylating agent affecting development and reproductive capacity

The objective was to evaluate the effects of 5-AZA-2'-deoxycytidine (5-AZA-CdR) on postnatal development and reproductive capacity. Pregnant mice were administered 1 mg kg-1 5-AZA-CdR at gestation day 10. The body weights of F1 control and treated (in uterine-exposed) pups were recorded. To evaluate the reproductive capacity, 5-AZA-CdR F1 males and females were mated with control mice. The presence of plugs and the number of pregnancies were recorded. The 5-AZA-CdR F1 male mice were killed. Total body, testes and epididymis weights were recorded. Spermatid head counting, histological analyses and serum testosterone levels were performed. Body weights of 5-AZA-CdR F1 mice were statistically lower than controls (P < 0.01), with the females more strongly affected (P < 0.05). Male mating capacity appeared to be more adversely affected. Mating of 5-AZA-CdR F1 males with control females resulted in a lower pregnancy rate compared with control mating groups (P < 0.01). Gross testicular and epididymis weights were lower in 5-AZA-CdR F1 mice (P < 0.01). However, testicular and epididymis weights in these mice were higher than controls when correlated to body weight (P < 0.01). In 5-AZA-CdR F1 male mice, all measured reproductive parameters, including total number of spermatid heads per testis, are significantly lower (P < 0.01) than the controls except for the number of spermatid heads per milligram of testis.     

Racemization of L-lysine for pharmaceutical synthesis and its chiral separation by GC-MS spectroscopy

In order to improve physico-chemical properties and to enhance stability of drugs, amino acid salt has been widely adopted in pharmaceutical synthesis. Acetylsalicylic acid lysinate is one of the widely used analgesics and it is a good example of this synthesis. In the case of acetylsalicylic acid lysinate synthesis, racemization of naturally occurred lysine is essential because the racemic lysine salt of the drug shows better yield, crystallinity and dryness than that of the L-lysine salt. To establish a simple, practical and economical process for L-lysine racemization, L-lysine treatments with phosphoric acid and with acetic acid were compared and the optimum conditions for its process and derivatization were investigated by chiral separation methods using GC-MS spectroscopy.     

寡核苷酸聚赖氨酸修饰物的脂质体制备及其对HepG2细胞的细胞毒活性研究

目的研究反义寡核苷酸的聚赖氨酸修饰物对脂质体包封率的影响及对HepG2细胞活性的初步测定。方法利用反义药物的聚赖氨酸修饰物,采用薄膜分散法制备反义寡核苷酸及其聚赖氨酸修饰物的脂质体;紫外分光光度法测定包封率的差异并考察对HepG2细胞细胞毒活性的影响。结果高密度正电荷的聚赖氨酸与带负电荷的反义寡核苷酸偶联后,脂质体的载药量大大增加,并且细胞的摄入量增加,对HepG2细胞生长具有抑制作用。结论聚赖氨酸修饰物增加了反义寡核苷酸的脂质体包封率,有效诱导人肝癌细胞的凋亡作用。     

Synthesis of 8-(hydroxymethyl)-3,N(4)-etheno-2'-deoxycytidine, a potential carcinogenic glycidaldehyde adduct, and its site-specific incorporation into DNA oligonucleotides

The previously unreported glycidaldehyde adduct, 8-(hydroxymethyl)-3, N(4)-etheno-2'-deoxycytidine (8-HM-epsilondC), has been synthesized for the first time by reaction of 2'-deoxycytidine with bromoacetaldehyde at pH 4.5, followed by reduction with sodium borohydride. The adduct was characterized by UV, MS, and NMR. The compound was stable to neutral and acidic conditions but not in alkaline solution. The corresponding phosphoramidite was synthesized in good yield from the intermediate, 3, N(4)-ethenocarbaldehyde-2'-deoxycytidine, using the standard methodology and site-specifically incorporated in both 15- and 25-mer oligonucleotides, for studies on biochemical and biophysical properties. The resulting oligonucleotides were purified using HPLC, and the base composition was verified by HPLC after enzymatic digestion.     

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 2.5 mM) was allowed to react with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC and HPLC-MS technique. Although the predominant reaction observed was chlorambucil hydrolysis, 1 reacted with various heteroatoms of the nucleosides. The principal site of alkylation with all pyrimidine nucleosides was N3, as judged by 1H NMR and HPLC-MS analyses. Also, several other adducts were detected, which could be tentatively characterized by means of HPLC-MS and MS/MS. As expected, thymidine was the least reactive pyrimidine nucleoside studied, and in addition of the N3 derivative, it reacted only at the carbohydrate moiety. Overall reactivity of cytosine nucleosides with 1 was considerably higher. The N3 adducts of dCyd and 5-Me-dCyd partially deaminated under the reaction conditions employed, but the reaction was not catalyzed by the participation of the omega-hydroxy function of the alkyl substituent but presumably by the nitrogen atom of the chlorambucil moiety. In the case of cytosine nucleosides, the O2 derivatives were the second most abundant species. 5-Me-dCyd reacted more readily at O2 than dCyd. These O2 adducts were labile under acidic, neutral, and basic conditions. No N4 derivatives or cross-links were detected, but dCyd reacted also at C5, although the yield of this derivative was very low. The role of chlorambucil-pyrimidine 2'-deoxyribonucleoside adducts on the cytotoxicity and mutagenity of 1 is also discussed.     

Synthesis and antimicrobial activity of 3-hydroxyimino-5-methyl-2-hexanone(HIMH) and its dioxime derivative

A new oxime, 3-hydroxyimino-5-methyl-2-hexanone (HIMH) has been synthesized by the reaction of 1-pentyl nitrite with 5-methyl-2-hexanone under acidic conditions. The subsequent treatment of HIMH with NH2OH x HCl gives 5-methyl-2,3-hexanedione dioxime (H2MHDDO). The structures of these compounds have been confirmed by physicochemical and spectral data. A preliminary screening of these compounds for biological activity against several microorganisms has indicated that they are selective growth inhibitors of m-tuberculosis, in particular.     

Critical review of the human data on short-term nitrogen dioxide (NO2) exposures: Evidence for NO2 no-effect levels

Nitrogen dioxide (NO₂) is a ubiquitous atmospheric pollutant due to the widespread prevalence of both natural and anthropogenic sources, and it can be a respiratory irritant when inhaled at elevated concentrations. Evidence for health effects of ambient NO₂ derives from three types of studies: observational epidemiology, human clinical exposures, and animal toxicology. Our review focuses on the human clinical studies of adverse health effects of short-term NO₂ exposures, given the substantial uncertainties and limitations in interpretation of the other lines of evidence. We examined more than 50 experimental studies of humans inhaling NO₂, finding notably that the reporting of statistically significant changes in lung function and bronchial sensitivity did not show a consistent trend with increasing NO₂ concentrations. Functional changes were generally mild and transient, the reported effects were not uniformly adverse, and they were not usually accompanied by NO₂-dependent increases in symptoms. The available human clinical results do not establish a mechanistic pathway leading to adverse health impacts for short-term NO₂ exposures at levels typical of maximum 1-h concentrations in the present-day ambient environment (i.e., below 0.2 ppm). Our review of these data indicates that a health-protective, short-term NO₂ guideline level for susceptible (and healthy) populations would reflect a policy choice between 0.2 and 0.6 ppm.

Extended abstract

Nitrogen dioxide (NO₂) is a ubiquitous atmospheric pollutant due to the widespread prevalence of both natural and anthropogenic sources, and it can be a respiratory irritant when inhaled at elevated concentrations. Natural NO₂ sources include volcanic action, forest fires, lightning, and the stratosphere; man-made NO₂ emissions derive from fossil fuel combustion and incineration.

The current National Ambient Air Quality Standard (NAAQS) for NO₂, initially established in 1971, is 0.053 ppm (annual average). Ambient concentrations monitored in urban areas in the United States are ~0.015 ppm, as an annual mean, i.e., below the current NAAQS. Short-term (1-h peak) NO₂ concentrations outdoors are not likely to exceed 0.2 ppm, and even 1-h periods exceeding 0.1 ppm are infrequent. Inside homes, 1-h NO₂ peaks, typically arising from gas cooking, can range between 0.4 and 1.5 ppm.

The health effects evidence of relevance to ambient NO₂ derives from three lines of investigation: epidemiology studies, human clinical studies, and animal toxicology studies. The NO₂ epidemiology remains inconsistent and uncertain due to the potential for exposure misclassification, residual confounding, and co-pollutant effects, whereas animal toxicology findings using high levels of NO₂ exposure require extrapolation to humans exposed at low ambient NO₂ levels. Given the limitations and uncertainties in the other lines of health effects evidence, our review thus focused on clinical studies where human volunteers (including asthmatics, children, and elderly) inhaled NO₂ at levels from 0.1 to 3.5 ppm during short-term (½–6-h) exposures, often combined with exercise, and occasionally combined with co-pollutants. We examined the reported biological effects and classified them into (a) lung immune responses and inflammation, (b) lung function changes and airway hyperresponsiveness (AHR), and (c) health effects outside the lungs (extrapulmonary).

We examined more than 50 experimental studies of humans inhaling NO₂, finding that such clinical data on short-term exposure allowed discrimination of NO₂ no-effect levels versus lowest-adverse-effects levels. Our conclusions are summarized by these six points: For lung immune responses and inflammation: (1) healthy subjects exposed to NO₂ below 1 ppm do not show pulmonary inflammation; (2) at 2 ppm for 4?h, neutrophils and cytokines in lung-lavage fluid can increase, but these changes do not necessarily correlate with significant or sustained changes in lung function; (3) there is no consistent evidence that NO₂ concentrations below 2 ppm increase susceptibility to viral infection; (4) for asthmatics and individuals having chronic obstructive pulmonary disease (COPD), NO₂-induced lung inflammation is not expected below 0.6 ppm, although one research group reported enhancement of proinflammatory processes at 0.26 ppm. With regard to NO₂-induced AHR: (5) studies of responses to specific or nonspecific airway challenges (e.g., ragweed, methacholine) suggest that asthmatic individuals were not affected by NO₂ up to about 0.6 ppm, although some sensitive subsets may respond to levels as low as 0.2 ppm. And finally, for extra-pulmonary effects: (6) such effects (e.g., changes in blood chemistry) generally required NO₂ concentrations above 1–2 ppm.

Overall, our review of data from experiments with humans indicates that a health-protective, short-term-average NO₂ guideline level for susceptible populations (and healthy populations) would reflect a policy choice between 0.2 and 0.6 ppm. The available human clinical results do not establish a mechanistic pathway leading to adverse health impacts for short-term NO₂ exposures at levels typical of maximum 1-h concentrations in the present-day ambient environment (i.e., below 0.2 ppm).