Pharmacological study of Stachytarpheta cayennensis Vahl in rodents

Freeze-dried aqueous extracts (AEs, 0.1–1 g/kg body wt., p.o.) obtained from entire or selected parts of Stachytarpheta cayennensis were tested for their effects on gastric secretion, gastric motility, inflammation and pain in rodents, with the purpose of validating the plant's ethnomedical uses. The AE-Total, AE-Flowers and AE-Leaves but not AE-Stems inhibited the gastric acid secretion in pylorus-ligated rats with varying potency. Purification of AEs yielded the semipurifed fractions EtFs rich in iridoids. All the EtFs with exception of EtF-Stems inhibited gastric acid secretion of pylorus ligated mice. While AE-Total stimulated the intestinal transit of mice by 43%, AE-Leaves delayed it by 38%. These effects on intestinal transit were not observed when the EtFs were tested. Only AE-Leaves and AE-Flowers altered the gastric emptying of semisolids, increasing it by 45% and 69%, respectively. These results indicate that the compounds related to inhibition of gastric acid secretion and gastrointestinal motility are different. The AE-Total reduced abdominal writhing induced by acetic acid potently (ED₅₀ VALUE=700 mg/kg, p.o.) without altering the writhes induced by acetylcholine. Attempts to identify the mechanism of analgesia were unsuccessful since the AE-Total did not show analgesic effects when tested in different models of pain such as formalin and capsaicin or the tail-flick test. Pretreatment of animals with AE-Total did not show antiinflammatory activity in any of the acute (paw edema induced by carrageenin, dextran or histamine, pleurisy induced by carrageenin and capsaicin-induced mouse ear edema) or chronic (air pouch) models used. No toxic signs were observed after administration of the different extracts up to 2 g/kg body wt., p.o.

Collectively, the results confirmed folk information indicating presence of analgesic, mild laxative and potent inhibition of gastric secretion activities in the aqueous extracts of S. cayennensis. The results do not, however confirm the folk use of the plant as an antiinflammatory medicine.     

Constitutive expression of IL-2Rbeta chain and its effects on IL-2-induced vascular leak syndrome

IL-2-induced vascular leak syndrome (VLS) is an important mechanism explaining the toxic effects of this cytokine and limiting its therapeutic use. We previously characterized a mouse model of IL-2-induced pulmonary VLS used to demonstrate that NK lymphocytes are involved in early/acute phase VLS (after one IL-2 injection). We also showed that NK cells and polymorphonuclear neutrophils (PMN) are involved in the late/chronic phase of the syndrome (after four daily IL-2 injections). In this study we use our mouse model to evaluate the role played by the IL-2 receptor (IL-2R) in VLS induction. Mouse and human IL-2R are different since the mouse IL-2Rbeta chain does not recognize IL-2. Here, we compare the acute and late VLS responses in human IL-2Rbeta transgenic and C57BL/6 wild type mice. Parameters linked to early phase VLS (bronchoconstriction and PMN mobilization) are enhanced in human IL-2Rbeta transgenic mice. By contrast, parameters used to measure late events (protein leakage and edema) are similar in human IL-2Rbeta transgenic mice and C57BL/6 wild type animals. However, after four IL-2 injections, the cellular content of the bronchoalveolar lavage fluids was different between the two types of animals. This study also characterizes a humanized animal model that could be further used to study human IL-2 activity and side effects in vivo.     

Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.     

New Synthetic Route to CY5-Labeled 2'-Deoxycytidine- 5'-Triphosphates Using Sonogashira Reaction

Herein, we present a new synthetic route to fluorescently-labeled nucleoside triphosphates via Sonogashira cross-coupling of iodinated deoxycytidine monophosphate and cyanine dye with a terminal alkyne group.     

Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo–) and Deep Vent (exo–) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chromophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.     

IFNL4 and IFNL3 Associated Polymorphisms Strongly Influence the Spontaneous IFN-Alpha Receptor-1 Expression in HCV-Infected Patients

Single-nucleotide polymorphism in IFNL3 gene (rs12979860) predicts spontaneous and therapy-induced HCV clearance. In a previous study from our group PBMC from patients with favourable rs12979860 genotype showed higher levels of IFNAR-1 mRNA. Recently, a dinucleotide polymorphism, ss469415590 (TT or ΔG), has been discovered in the region upstream IFNL3 gene, which is in high linkage disequilibrium with rs12979860. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designed IFNL4, encoding the interferon-lambda 4 protein (IFNL4). The aim of the present study was to extend the analysis of IFNAR-1 mRNA levels to the ss469415590 variants. Our results highlight that the difference of IFNAR-1 mRNA levels between favourable and unfavourable genotype combinations, at both rs12979860 and ss469415590 loci, is stronger than that observed for single polymorphisms at each locus. These findings suggest may represent the biological basis for the observed association between IFNL3 CC and IFNL4 TT/TT genotypes and favourable outcome of either natural HCV infection (clearance vs chronic evolution) or IFN-based therapy.     

Chromosome number of <Emphasis Type="Italic">Orthophytum</Emphasis> species (Bromeliaceae)

The genus Orthophytum Beer comprises 53 species, all narrow endemics to south-eastern and north-eastern Brazil. In this study we present meiotic and mitotic chromosome numbers of 12 species of this important genus in Bromeliaceae. For six of these taxa we are reporting the first cytogenetic study. Orthophytum albopictum, O. amoenum and O. burle-marxii presented 2n = 100 chromosomes and O. hatschbachii, O. mucugense, O. vagans, O. supthutii, O. zanonii and O. ophiuroides showed 2n = 50 chromosomes. These results are consistent with the proposed basic number of x = 25 for Bromeliaceae family. In the genus Orthophytum, polyploidy seems to play an important role in chromosome evolution associated with habitat differentiation among diploid and polyploid species.     

An evaluation of p16INK4a expression in cervical intraepithelial neoplasia specimens, including women with HIV-1

Although several studies have evaluated the role of p16INK4a as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16INK4a in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16INK4a as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16INK4a was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16INK4a expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16INK4a in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16INK4a under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16INK4a expression in CIN 2-3.     

Anti-inflammatory activity of Urera baccifera (Urticaceae) in Sprague-Dawley rats

On a preliminary test, anti-inflammatory and analgesic dose-related activities on rats were observed for the aqueous fraction of Urera baccifera; this extract was bioassay-guided fractionated and the final aqueous fraction was used according the ethnobotanical use. Carrageenan-induced edema (n = 6), was used as an assay in the fractionating process. The anti-inflammatory and antinociceptive properties of the final aqueous fraction were studied using in vivo models. For the anti-inflammatory activity rat paw edema (n = 6), pleurisy induced by carrageenan (n = 6) and ear edema induced by topical croton oil (n = 6) models were used, and tail-flick test (n = 6), abdominal constrictions induced by acetic acid (n = 6), and formalin test (n = 6), were used for the antinociceptive activity. The tests performed showed an inhibition effect on leukocyte migration, and a reduction on pleural exudate, as well as dose-dependant peripheral analgesic activity, at a range of 25-100 mg/kg i.p. The final aqueous fraction contains most of the anti-inflammatory activity of the plant U. baccifera. A possible mechanism of action is discussed and based on the results we conclude that this plant has a potential for both anti-inflammatory and analgesic activity at the clinical level.