Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: a model system

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.     

An improved protocol for carrot haploid and doubled haploid plant production using induced parthenogenesis and ovule excision in vitro

In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20–22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.     

Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic cytotoxin-scaffold. Identification of aerobic-antitumoral activity through DNA fragmentation

Phenazine 5,10-dioxides are prodrugs for antitumor therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here we investigate the expanded system benzo[a]phenazine 7,12-dioxides as selective hypoxic cytotoxin-scaffold. The clonogenic survival of V79 cells on aerobic and anaerobic conditions, conduct us to study antiproliferative activity on Caco-2 tumoral cells in normoxia. Electrochemical, DNA-interaction and DNA-damage studies were performed to establish the mode of action. The results demonstrated the potential biological properties of the studied scaffold being derivatives 6–10 structural hits for further chemical-modifications to become into therapeutics for solid tumors. Compounds 6 and 8 with cytotoxicity against V79 cells in both conditions (aerobia and anaerobia) were also cytotoxic against Caco-2 tumoral cells in aerobiosis.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Bioaerosol emission from wastewater treatment plants

In this report we describe the results of astudy conducted to better estimate airbornemicroorganisms present in wastewater treatmentplants (WWTP) environments. Air samples weretaken with the Air Sampler MAS 100 impactor(Merck) at each stage of an activated-sludgeWWTP (pre-treatment, primary clarifiers,aeration basins and sludge processors), inorder to determine levels of contaminationaccording to work stations. Culture methodswere used to investigate heterotrophic platecount, moulds and yeasts, total and fecalcoliforms, Pseudomonas aeruginosa andspecies belonging to Mycobacteriumtuberculosis complex, whereas these lastmicroorganisms were also analysed by PolymeraseChain Reaction (PCR) methods. Statisticalanalysis showed that pretreatment and primaryclarifiers were the stages with the highestemission of bioaerosols, and that bioaerosolconcentration at each stage depended on windspeed and daily inflow at the WWTP. Lowconcentrations of P. aeruginosa werefound isolated at pretreatment and primaryclarifiers; however, species of M.tuberculosis complex were not detected at anystage of the treatment plant.     

Ichthyoplankton distribution as an indicator of hydrodynamic conditions of a lagoon system in the Mexican Caribbean

The salt-water intrusion into a lagoon system of the Sian Ka’anBiosphere Reserve was assessed during 1 year and related tothe marine component of the larval fish assemblage. This systemis environmentally complex because of the penetration of marinewater, and the discharge of surface and subterranean water.Biological samples were collected during four field expeditionsfrom October 1996 to August 1997. Surface circular tows with500-µm-mesh conical plankton nets with a flowmeter weremade during the day. Position, depth, temperature and salinitywere recorded at each sampling station. All fish larvae wereremoved from the samples and identified to the lowest possibletaxon. Ichthyoplankton abundance was normalized to 100 m³ ofwater. The distribution patterns of the carangiid Oligoplitessaurus, the herring Opisthonema oglinum, the dragonet Diplogrammuspauciradiatus and members of the Tetraodontidae were relatedto the wind-driven circulation pattern of the lagoons obtainedusing a numerical model. The analysis of the distribution ofichthyoplankton showed that these species are good indicatorsof the spatial salinity variations controlled by the hydrodynamiccharacteristics of the system. The spatial use of these estuariesas a habitat for fish larvae is therefore strongly influencedby salinity distribution, itself modulated by annual variationof wind stress.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : VI. THE ACTIVITY COEFFICIENTS OF THE IONS IN CERTAIN OXYHEMOGLOBIN SOLUTIONS.

1. The solvent action of a neutral salt upon a protein, oxyhemoglobin, has been found identical to the solvent action of a neutral salt upon a bi-bivalent or uni-quadrivalent compound. 2. The solubility of oxyhemoglobin in phosphate solutions of varying ionic strength has been defined by the equation: log See PDF for Equation in which µ is the ionic strength, and S ₀ is the solubility in the absence of salt. 3. The values of S ₀ have been calculated to be 12.2, 11.2, and 13.1 gm. per liter respectively at pH 6.4, 6.6, and 6.8. 4. The relatively great solubility of oxyhemoglobin in water has been ascribed to the strong affinity constants for acid and base of certain groups in oxyhemoglobin. 5. The small change in the solubility of oxyhemoglobin effected by neutral salts suggests that but few such groups are dissociated in oxyhemoglobin in the state in which it crystallizes near its isoelectric point. 6. Certain of the other properties of oxyhemoglobin, such as its low viscosity, are considered in the light of its molecular weight and its valence type.