Quantitative spin trapping and triplet quenching by radicals for in vivo studies. I. The chemical model

In order to determine quantitatively the free radical content and its changes affected by additives using spin trapping under in vivo conditions, an approach is suggested carrying out experiments in a completely mixed open system (CMOS). Measurements have been carried out for a chemical oxidation process as a model system, and analysis of products and of the spin trap was extended by kinetic ESR spectrometry of the spin adducts. Since in a CMOS differential equations of accumulation of all species can be transformed into algebraic expressions using available rate constants for the formation of the spin adducts, corresponding concentrations of free radicals have been calculated. In addition, it has been established that triplet excited photosensitizers have a double effect: increasing the rate of initiation by decomposing hydroperoxide-type compounds and inhibiting the overall process by interactions with free radicals. Results indicate that by changing the "reaction vessel" the method can be applied for ex vivo and in vivo systems.     

Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis

The genome of Pseudomonas putida KT2440 encodes an unexpected capacity to tolerate heavy metals and metalloids. The availability of the complete chromosomal sequence allowed the categorization of 61 open reading frames likely to be involved in metal tolerance or homeostasis, plus seven more possibly involved in metal resistance mechanisms. Some systems appeared to be duplicated. These might perform redundant functions or be involved in tolerance to different metals. In total, P. putida was found to bear two systems for arsenic (arsRBCH), one for chromate (chrA), four to six systems for divalent cations (two cadA and two to four czc chemiosmotic antiporters), two systems for monovalent cations: pacS, cusCBA (plus one cryptic silP gene containing a frameshift mutation), two operons for Cu chelation (copAB), one metallothionein for metal(loid) binding, one system for Te/Se methylation (tpmT) and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (one nikABCDE, two znuACB and one mobABC). Some of the metal-related clusters are located in gene islands with atypical genome signatures. The predicted capacity of P. putida to endure exposure to heavy metals is discussed from an evolutionary perspective.     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

Obesity is not associated with increased knee joint torque and power during level walking

While it is widely speculated that obesity causes increased loads on the knee leading to joint degeneration, this concept is untested. The purpose of the study was to identify the effects of obesity on lower extremity joint kinetics and energetics during walking. Twenty-one obese adults were tested at self-selected (1.29m/s) and standard speeds (1.50m/s) and 18 lean adults were tested at the standard speed. Motion analysis and force platform data were combined to calculate joint torques and powers during the stance phase of walking. Obese participants were more erect with 12% less knee flexion and 11% more ankle plantarflexion in self-selected compared to standard speeds (both p<0.02). Obese participants were still more erect than lean adults with approximately 6 degrees more extension at all joints (p<0.05, for each joint) at the standard speed. Knee and ankle torques were 17% and 11% higher (p<0.034 and p<0.041) and negative knee work and positive ankle work were 68% and 11% higher (p<0.000 and p<0.048) in obese participants at the standard speed compared to the slower speed. Joint torques and powers were statistically identical at the hip and knee but were 88% and 61% higher (both p<0.000) at the ankle in obese compared to lean participants at the standard speed. Obese participants used altered gait biomechanics and despite their greater weight, they had less knee torque and power at their self-selected walking speed and equal knee torque and power while walking at the same speed as lean individuals. We propose that the ability to reorganize neuromuscular function during gait may enable some obese individuals to maintain skeletal health of the knee joint and this ability may also be a more accurate risk indicator for knee osteoarthritis than body weight.     

Fighting a virus with a virus: a dynamic model for HIV-1 therapy

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.     

The American brine shrimp as an exotic invasive species in the western Mediterranean

The hypersaline environments and salterns present in the western Mediterranean region (including Italy, southern France, the Iberian Peninsula and Morocco) contain autochthonous forms of the brine shrimp Artemia, with parthenogenetic diploid and tetraploid strains coexisting with the bisexual species A. salina. Introduced populations of the American brine shrimp A. franciscana have also been recorded in these Mediterranean environments since the 1980s. Based on brine shrimp cyst samples collected in these countries from 1980 until 2002, we were able to establish the present distribution of autochthonous brine shrimps and of A. franciscana, which is shown to be an expanding invasive species. The results obtained show that A. franciscana is now the dominant Artemia species in Portuguese salterns, along the French Mediterranean coast and in Cadiz bay (Spain). Co-occurrence of autochthonous (parthenogenetic) and American brine shrimp populations was observed in Morocco (Mar Chica) and France (Aigues Mortes), whereas A. franciscana was not found in Italian cyst samples. The results suggest these exotic A. franciscana populations originate as intentional or non-intentional inoculations through aquacultural (hatchery effluents) or pet market activities, and suggest that the native species can be rapidly replaced by the exotic species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Population genetic differentiation in taxa of Lotus (Fabaceae: Loteae) endemic to the Gran Canarian pine forest

A survey of allozyme variation at 17 loci in 14 populations representing four taxonomically problematic Gran Canarian pine forest endemic taxa of Lotus (L. genistoides, L. holosericeus, L. spartioides and some taxonomically uncertain populations collected under the designation Lotus sp.) was conducted to examine their diversification and systematic relationships. All groups exhibited high values of genetic variation, although inbreeding was common within populations. Considerable among-population genetic homogeneity was detected, as inferred from low values of Gst within each of the groups. The high population sizes of these taxa and a lack of evidence for isolation by distance or genetic bottlenecks indicate that diversity has accumulated over a long period of environmental stability. The association of high genetic distances with low linear distances, and the substantial increase in the values of Gst when the taxa considered were merged in different combinations hint at an incipient (yet probably taxonomically insufficient) reproductive isolation. The genetic similarity between L. genistoides, L. holosericeus and L. spartioides, together with the different behaviour of the populations collected under the designation Lotus sp., may have important implications for the restructuring of the taxonomy of this group when the ongoing morphological studies are completed.     

Early embryogenesis study on a dimethoate containing formulation and Cd-sulphate in chicken embryos

The toxic effects of a widely used organophosphate insecticide (BI 58 EC containing 38% dimethoate as active ingredient) applied alone or in combination with cadmium sulphate modelling the heavy metal load of the environment were studied on chicken embryos in the early phase of development. Solutions and emulsions of different concentrations were made from the test materials and injected in 0.1 ml volume into the air space of eggs on the first day (day 0) of incubation. Subsequently, on days 2 and 3 of incubation permanent preparations were made from the embryos in order to study the early developmental stage. Embryos fixed on slides and stained with osmium tetroxide solution were studied under light microscope. Summarising the findings, it can be established that the embryotoxicity increased after the simultaneous administration of Cd-sulphate and 38% dimethoate containing insecticide formulation compared to the control or the individually treated groups.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.