ANATOMICAL CHARACTERISTICS AND BIOLOGICAL ACTIVITY OF THE STEMS AND LEAVES OF DAPHNE ALBOWIANA WORONOW EX POBED.

The aim of the study was to determine the stable diagnostic traits and the biological activity of the stems and leaves of Daphne albowiana Woronow ex Pobed., a plant native to Georgia. Biological assays of the methanol, chloroform and hexane extracts show the plant to possess cytotoxic and antioxidant activities, but no noteworthy antibacterial or antifungal activities. All extracts show cytotoxic activity on A549 (lung carcinoma) cells. The following stable diagnostic characteristic were identified during the microstructural analysis: leaf surface glabrous, hypostomatic, dorsoventral; epidermal cells chaotic; curved with curved walls on both the upper and lower epidermis; stomata paracytic; well visible spherical crystals of inulin in leaf epidermis; leaf vascular bundles reverse-collateral; vascular system monocyclic, bilateral; wood diffuse-porous; xylem parenchyma is apotracheal, slightly diffuse; vessel walls are predominantly spirally thickened; collenchyma lamellar; radial rays in single rows, heterogeneous. The identified cytotoxic and antioxidant activity showcase this species to be of significant interest to the medicinal field. The identified anatomical peculiarities provide valuable information for the correct identification and standardization of the Daphne albowiana plant material.

Dual ligation hybridization assay for the specific determination of oligonucleotide therapeutics.

BACKGROUND: Oligonucleotide-based therapeutics are quantified with hybridization assays in biological matrices such as plasma and tissues. Current hybridization methods do not entirely discriminate the parent compound from 5´- or 3´-N-X truncated metabolites. RESULTS: A dual ligation-based hybridization assay was developed to circumvent the limitations of current assay formats. Ligation of probes at either end of the analyte is performed via a bi-enzymatic reaction consisting of polynucleotide kinase and DNA ligase. The method was validated with regard to mechanism, specificity, precision and accuracy. CONCLUSION: The dual ligation assay is specific for the parent compound and detects the full-length product with intact 5´- and 3´-ends. The dual ligation assay can also be used to specifically determine individual metabolites in complex mixtures and is currently implemented to quantitative PCR.

Antiplasmodial, anti-inflammatory and cytotoxic activities of various plant extracts from the Mascarene Archipelago.

AIM OF THE STUDY: Antiplasmodial activity, inhibition of nitric oxide (NO) overproduction, and anti-proliferative activity were investigated in vitro to evaluate the bioactive potential of the traditional pharmacopoeia of the Mascarene Archipelago, which is known for its biodiversity and for the richness of its endemic flora. MATERIALS AND METHODS: A total of 45 methanol (MeOH) and dichloromethane (DCM) extracts were prepared from 19 plant species collected on Réunion and Mauritius Islands. Ninety-six-well microplate assays were performed on chloroquine sensitive Plasmodium falciparum 3D7 strain, on LPS-stimulated Raw 264.7 murine macrophages and on A-549, DLD-1 and WS1 human cells. Activity was evaluated through spectrophotometric methods. RESULTS: Activity was attributed to plant extracts expressing IC(50)<50µg/ml for antiplasmodial response, IC(50)<100µg/ml for cytotoxicity, and IC(50)<130µg/ml for anti-inflammatory reaction. The majority of the extracts tested (69%) exhibited potency in at least one of these three types of activity. This is the first report describing promising antiplasmodial activity (IC(50)<15µg/ml) for Psiadia dentata DCM extract and Terminalia bentzoe MeOH bark extract. NO inhibition assay revealed seven interesting plants, described for the first time as anti-inflammatory: Aphloia theiformis, Buddleja salviifolia, Eupatorium riparium, Hiptage benghalensis, Psiadia arguta, Psiadia dentata, and Scutia commersonii. Finally, anti-proliferative activity was observed for two endemic species, Geniostoma borbonicum and Nuxia verticillata. CONCLUSION: Using the criterion of endemism as part of the criteria for traditional medicinal use raises the chances of finding original active principles. In our case, 86% of the endemic plants tested displayed pharmacological interest.

Chemical composition and anticancer activity of leaf essential oil of Myrica gale L.

Myrica gale L. (Myricaceae), a native plant from Canada used in traditional medicine, was extracted by hydrodistillation and the oil was collected after 30 and 60 min. The chemical composition of these two extracts was determined using GC-MS analysis. We identified 53 components and myrcene (23.18-12.14%), limonene (11.20-6.75%), alpha-phellandrene (9.90-6.49%) and beta-caryophyllene (9.31-10.97%) were the major components in the 30- and 60-min fractions, respectively, whereas higher caryophyllene oxide content was detected in the 60-min fraction (9.94%) than in the 30-min fraction (3.47%). The anticancer activities of these extracts were assessed against human lung carcinoma cell line A-549 and human colon adenocarcinoma cell line, DLD-1. The 60-min fraction showed higher anticancer activity against both tumor cell lines with an IC50 value of 88 +/- 1 microg/ml. The 30-min fraction had an IC50 value of 184 +/- 4 microg/ml for A-549 and 160 +/- 3 microg/ml for DLD-1. The higher cell growth inhibition induced by the 60-min fraction, as compared to the 30-min fraction, could be due to sesquiterpene enrichment.

Membrane interactions of a new class of anticancer agents derived from arylchloroethylurea: a FTIR spectroscopic study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and different lipids such as dimyristoylphosphatidylcholine (DMPC) in the absence and presence of 30 mol% of cholesterol, dimyristoylphosphatidylglycerol (DMPG) and a mixture made of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and DMPC by Fourier transform infrared (FTIR) spectroscopy. The results indicate that the drugs incorporate in the bilayer and cause a decrease of the phase transition temperature and an increase of the conformational disorder of the lipid acyl chains. These effects are dependent on the nature (degree of branching, length of the alkyl chain and presence of a sulfur atom), as well as on the position of the R substituent and are related to the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky branched substituent and those for which the disordering effect on the lipid bilayer is the greatest. On the other hand, the disordering effect is small for the long chain CEUs, such as 4-n-hexadecyl CEU, which have been shown to have weak cytotoxic activity.

Antimitotic antitumor agents: synthesis, structure-activity relationships, and biological characterization of N-aryl-N'-(2-chloroethyl)ureas as new selective alkylating agents.

A series of N-aryl-N'-(2-chloroethyl)ureas (CEUs) and derivatives were synthesized and evaluated for antiproliferative activity against a wide panel of tumor cell lines. Systematic structure--activity relationship (SAR) studies indicated that: (i) a branched alkyl chain or a halogen at the 4-position of the phenyl ring or a fluorenyl/indanyl group, (ii) an exocyclic urea function, and (iii) a N'-2-chloroethyl moiety were required to ensure significant cytotoxicity. Biological experiments, such as immunofluorescence microscopy, confirmed that these promising compounds alter the cytoskeleton by inducing microtubule depolymerization via selective alkylation of beta-tubulin. Subsequent evaluations demonstrated that potent CEUs were weak alkylators, were non-DNA-damaging agents, and did not interact with the thiol function of either glutathione or glutathione reductase. Therefore, CEUs are part of a new class of antimitotic agents. Finally, among the series of CEUs evaluated, compounds 12, 15, 16, and 27 were selected for further in vivo trials.

Mitochondrial GPx1 decreases induced but not basal oxidative damage to mtDNA in T47D cells.

The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., +14% 8-oxo-dG versus +54% in Hygro-3 after 1-h exposure to 25 microM menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model.

Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents.

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that betaIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with beta-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of beta-tubulin.

Using data to evaluate hospital inpatient mortality.

This article evaluates the use of hospital inpatient mortality as an indicator of health care outcomes and describes the development of related data. It demonstrates both the strengths and limitations of mortality as a measure of outcomes. It provides guidance concerning the development of raw and severity adjusted mortality data. It also provides information concerning data related to unexpected mortality and complications.

Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.

Preferential localization of DNA damage induced by depurination and bleomycin in a plasmid containing a scaffold-associated region.

Recent evidence suggests that DNA damage of various origins is not randomly distributed in the genome but appears to be clustered in unidentified hypersensitive regions of the chromatin. A model was proposed that stipulates that unpaired DNA stretches, such as those found in scaffold- (or matrix)-associated regions (SARs) under torsional strain, are candidate regions of hypersensitivity to DNA damage in vivo. In this study, we assessed in vitro the relative susceptibility of supercoiled plasmids containing a SAR or chromatin loop DNA segment to DNA damage induced by acid-catalyzed depurination or FeIII-bleomycin. Single-strand specific S1 nuclease was used in combination with 3'-end-labeling to detect single-strand breaks or gaps, after cleavage of abasic sites or removal of 3'-phosphoglycolates by Escherichia coli endonuclease IV. The optimal conditions of DNA cleavage specificity by S1 nuclease were determined. Using these conditions, the DNA cleavage patterns obtained showed (i) a preferential localization of S1 hypersensitive sites in the SAR DNA as compared with plasmid or chromatin loop DNA and (ii) a strikingly similar localization of DNA damage with the two clastogenic treatments.

Prevalence of isolated urethral asymptomatic Chlamydia trachomatis infection in the absence of cervical infection in incarcerated adolescent girls.

BACKGROUND AND OBJECTIVES: Adolescent girls may have isolated asymptomatic urethral Chlamydia trachomatis infection. GOAL OF THIS STUDY: To determine if a single direct fluorescent antibody (DFA) slide can detect as many urethral and cervical infections as the use of separate slides and to determine if isolated urethral infection occurs. STUDY DESIGN: During pelvic examinations upon admission, DFA slides were prepared from the cervix, from the urethra, and with cells from both sites. RESULTS: Of 125 girls, 17 had C. trachomatis infection: 4 in the urethra only (24%), 5 in the cervical sample only (31%), 5 in both sites (31%), 3 had positive results only on the combined slide, and 1 had a positive result on the cervical slide and an inadequate urethral slide. CONCLUSION: Isolated urethral C. trachomatis infection occurs frequently, and sampling both sites as opposed to sampling the cervix alone increases the number of cases found by 24% (P < 0.002). Using a single slide to detect infection in both sites detects as many infections as using two separate slides.

A study of the relationship of community living situation to independence and satisfaction in the lives of mentally retarded adults.

This study examined the relationship between the level of supervision provided at the community residences of 61 adults with mental retardation and their sense of independence and satisfaction in specific aspects of home life, work and community activity. Responses given to interview questions were transformed into scores for independence and satisfaction for each subject. In order to compare the relationship of supervision at home, an individual's intellectual functional ability, and his social support to independence and satisfaction, non-parametric analyses of the data were performed. These revealed statistically significant inverse relationships between the degree of home supervision and independence both at home and within the community, and satisfaction at home. Greater functional ability was found not to be significantly related to any measures of independence or satisfaction. A high level of social support was correlated only with overall measures of community independence, satisfaction at home, and community satisfaction.

Evaluation of an infant car seat program in a low-income community.

This study was designed to assess the influence of an infant car seat loan program on car seat utilization in a low-income community. An adjacent community, with no car seat program, was chosen for comparison. Systematic observations were made in the two neighborhoods, and this information was supplemented by telephone interviews. Greater use of infant seats was observed in the intervention community (41%) than in the control community (27%) for infants younger than 6 months old. The rate of observed utilization of infants between 7 and 18 months of age increased to 50% on average, but no significant differences were noted between the two communities. These findings suggest that a community-based loan program can produce short-term increases in car seat use rates for infants, even in a low-income community. A strategy to facilitate continued accessibility to such restraints is needed, however, to maintain these improvements.

Color preservation of gross specimens for teaching and medical illustration.

We routinely employ cold Jores' fixative to improve color preservation of gross autopsy specimens for conferences and teaching and to facilitate development of a photographic teaching collection of gross pathological specimens. The fixed specimens also permit light microscopic examination. Additionally, we have found that color restoration of previously fixed gross specimens is possible with McCormick's fixative.