The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H₂O₂ 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941?mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8?μM, respectively. The kinetics of the reaction between DBBF and H₂O₂ was also studied.     

Antineoplastic effects of clove buds (Syzygium aromaticum L.) in the model of breast carcinoma

It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF‐7 cells was carried out. Dietary administered CLO caused the dose‐dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl‐2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase‐3 and ALDH1 expression increase after high‐dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro‐apoptotic effects of CLO extract in MCF‐7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase‐7, Bcl‐2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.     

Delivery as "physiological stress" and its influence on liver enzymatic systems in asphyxial newborns

A new aspect on delivery as "physiological stress" provides the possibility for assessing its influence in asphyxial newborns. Asphyxia is characterized by hypoxic ischemic damage of organs with subsequent damage due to reoxygenation and reperfusion. The aim of the study was to compare the activity of liver enzymatic systems of healthy newborns with asphyxial newborns. Reference values of the AST on the 1st day of life 0.80 +/- 0.12 microkat/l and on the 5th day of life 0.78 +/- 0.11 microkat/l, of the ALT on the 1st day of life 0.29 +/- 0.04 microkat/l and on the 5th day of life 0.54 +/- 0.21 microkat/l and of the quotient of DeRitis on the 1(st) day of life 3.35 +/- 0.6 and on the 5th day of life 2.14 +/- 0.22 were established. The quotient of DeRitis expresses better the influence of stress on healthy term newborns since isolated evaluation of liver enzymes and their dynamics, which is decreased on the 5th day of life shows stabilization of the baby and improvement of liver cell function in comparison with the 1st day of life. The authors found that delivery is considered to be a certain form of physiological stress.     

The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae)

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8× depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373 bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,657 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperzia chloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophyte chloroplast genome data also enable a better reconstruction of the basal tracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, inferred amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.     

The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA.     

Development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new P genotype

Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.     

Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O(6)-methylguanine DNA-methyltransferase in other organisms

Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.     

Juxtaglomerular apparatus tumor: a rare, surgically correctable cause of hypertension

Although uncommon, presentation of juxtaglomerular cell tumor is distinct and should allow a correct preoperative diagnosis in most patients. Typical clinical presentations include headaches, polyuria, or isolated, asymptomatic, severe hypertension. The diagnosis of a juxtaglomerular apparatus (JGA) tumor typically results from identification of plasma renin levels two- to sevenfold greater than the normal value. Although JGA tumors are considered benign, with no reports of metastases or recurrence, they are potentially lethal if left untreated. Surgical excision is curative.     

The anther-culture response of triticale line x tester progenies

Seven triticale cultivars (Ampiac, Aubrac, Trinidad, Ticino, Lamberto, Pronto and Prado) and their F1 hybrids obtained after crossing in a line x tester scheme were examined with respect to their androgenetic effectiveness. The embryo induction rate (number of embryos per 100 anthers), green plant regeneration rate (number of green plantlets per 100 embryos), plant yield (number of green and albino plantlets per 100 anthers) and green plant yield (number of green plantlets per 100 anthers) were assessed. The multivariate and univariate effects of general (GCA) and specific (SCA) combining abilities for the studied traits were estimated and tested. Significant differences between the genotypes were found for individual traits as well as for all the traits treated jointly. Hybrids generally showed a better response in anther culture than their parental genotypes. Heterosis effects were observed in some hybrids for embryo induction rate and green plant yield. GCA and SCA variances were significant and a dominance of the GCA over the SCA variation was found. Among the examined cultivars, Ticino and Pronto were characterised by positive and significant GCA for embryo induction and green plant yield, and these cultivars may be recommended for the improvement of anther culture responsiveness in triticale.