Therapeutic Applications of Cannabinoids in Cardiomyopathy and Heart Failure.

A large number of cannabinoids have been discovered that could play a role in mitigating cardiac affections. However, none of them has been as widely studied as cannabidiol (CBD), most likely because, individually, the others offer only partial effects or can activate potential harmful pathways. In this regard, CBD has proven to be of great value as a cardioprotective agent since it is a potent antioxidant and anti-inflammatory molecule. Thus, we conducted a review to condensate the currently available knowledge on CBD as a therapy for different experimental models of cardiomyopathies and heart failure to detect the molecular pathways involved in cardiac protection. CBD therapy can greatly limit the production of oxygen/nitrogen reactive species, thereby limiting cellular damage, protecting mitochondria, avoiding caspase activation, and regulating ionic homeostasis. Hence, it can affect myocardial contraction by restricting the activation of inflammatory pathways and cytokine secretion, lowering tissular infiltration by immune cells, and reducing the area of infarct and fibrosis formation. These effects are mediated by the activation or inhibition of different receptors and target molecules of the endocannabinoid system. In the final part of this review, we explore the current state of CBD in clinical trials as a treatment for cardiovascular diseases and provide evidence of its potential benefits in humans.

Impact of shear rate pattern on post-occlusive near-infrared spectroscopy microvascular reactivity.

The primary aim of the present study was to determine the impact of acute changes in shear rate patterns, in particular retrograde shear rate, on microvascular function in 15 healthy, young men and women as determined via the post-occlusive near-infrared spectroscopy (NIRS) microvascular reactivity response. Microvascular reactivity, via NIRS-derived measurements of post-occlusion tissue saturation index (TSI%) and total microvascular hemoglobin+myoglobin concentration ([Hb]total), were assessed in each participant before and immediately after exposure to a 30min retrograde shear treatment. Retrograde shear was achieved via a blood pressure cuff placed below the knee inflated to 75mmHg. One leg was exposed to the retrograde shear (Treatment leg) and the contralateral leg served as a non-treatment control. In the Treatment leg, significant increases in retrograde shear rate occurred during the retrograde intervention. Following the intervention, the area under the TSI% post-occlusion response curve, which represents the total microvascular reactivity response, and the absolute peak TSI% response were significantly increased compared to pre-intervention in the Treatment leg, but not the Control leg. The absolute peak [Hb]total response was significantly increased post-intervention in both legs. These results are in contrast to our hypothesis that 75mmHg cuff inflation, designed to increase retrograde shear rate in the femoral artery would negatively affect post-occlusive microvascular reactivity. These data suggest that the current method of increasing retrograde shear rate in the intact human does not adversely impact NIRS derived measurements of microvascular reactivity.

Eficacia de la fisioterapia en la insuficiencia venosa crónica en evolución / Efficacy of physical therapy in chronic venous insufficiency in evolution

Objetivos: Estudiar la eficacia de la fisioterapia en la insuficiencia venosa y la úlcera como complicación de ésta. Método: Búsqueda realizada en PEDro (puntuación mayor de 6), Cochrane, MEDLINE y CEBP. Resultados: El edema venoso se redujo hasta 2,2ml mediante movilizaciones activas y derivación circulatoria. En la úlcera venosa se comprobó que el ultrasonido no fue eficaz tras la revisión de siete ensayos; en cuatro se comparó con el ultrasonido simulado y en los tres restantes se cotejó con un tratamiento estándar. Otros estudios con ultrasonidos pulsátil de 0,5W/cm2 a 1MHz durante 12 semanas tampoco evidenciaron mejoría. El láser no mostró significación para la úlcera si bien evidenció sus efectos terapéuticos en aplicación combinada con luz infrarroja. Conclusiones: La cinesiterapia de la bomba venomuscular periférica es eficaz en la insuficiencia venosa. Los tratamientos mediante láser y ultrasonidos en forma aislada no modifican la evolución de la úlcera flebostática(AU)

Objectives: To study the effectiveness of physical therapy in venous insufficiency and ulcer as a complication of it. Method: Search conducted in PEDro (score greater than 6), Cochrane, MEDLINE and CEBP. Results: Venous edema was reduced to 2.2 cc by active mobilizations and circulatory shunt. It was found that ultrasound was not effective on the venous ulcer after 7 trials were reviewed. Sham ultrasound was compared in 4 of them and the remaining 3 were collated with the standard treatment. Other studies with ultrasound pulses of 0.5W/cm2 to 1MHz for 12 weeks also showed no improvement. Laser showed no significance for the ulcer although its therapeutic effects were verified when it was combined with infrared light. Conclusions: Pump kinesiotherapy is effective in venomuscular peripheral venous insufficiency. Therapeutic laser and ultrasound when used separately do not alter the development of the phlebostatic ulcer(AU)

Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E.

Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with (14)C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.

Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate.

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.

A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.

Responses of Gram-negative bacteria to certain environmental stressors.

Bacteria in nature are exposed to variations in temperature, and are affected by the availability of nutrients and water and the presence of toxic molecules. Their reactions to these changes require a series of rapid adaptive responses. Although transcriptional regulation is of primary importance in these responses, translational regulation and even activation of 'silenced' enzymes are critical for survival in changing environments. Bacteria have developed a series of mechanisms at the membrane structure level to cope with high concentrations of solvents. In addition, solvent-tolerant strains express highly effective efflux pumps to remove solvents from the cytoplasm. Desiccation tolerance is based on the synthesis and accumulation of osmoprotectants together with changes in fatty acid composition to preserve membrane structure. Both cold shock and heat shock responses are mainly regulated at a post-transcriptional level, translation efficiency in the case of cold shock and mRNA half-life and sigma32 stability in the case of heat shock.

Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow with toluene as the sole C-source. Tn5 mutagenesis was carried out and a mutant unable to use toluene as the sole C-source was isolated. DNA was sequenced upstream and downstream of the site where the Tn5 was inserted. Analysis of the DNA revealed 13 open reading frames (ORFs) homologous to the tod genes for the toluene dioxygenase pathway of P. putida F1, which are organized in two operons: todXFC1C2BADEGIH and todST. The Tn5 was inserted at the todH gene. The role of the todXFC1C2BADEGIH operon in toluene metabolism was further confirmed in a todC1 mutant (generated by insertional inactivation), which was unable to use toluene as the sole C-source. Primer extension analysis identified a single promoter upstream from the todX gene. The -10 and -35 regions of this promoter showed no significant homology to known promoters. Expression from the todX promoter occurred in response to toluene, ethylbenzene, styrene, xylenes and other aromatic hydrocarbons. Expression from the todS gene was constitutive. Sensitivity to toluene of the todH and todC1 mutants was similar to that of the wild-type strain. This suggests that toluene metabolism is not involved in toluene tolerance.

Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440.

A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strain E. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter, bolAp1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of the nlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele of rpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultant rpoS mutant of P.putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein of P. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell cell envelope.

Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/'phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low-osmolarity medium. The P. putida DNA surrounding the mini-Tn5/'phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro.

Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid.

The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient. The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P. putida strains, a process that appears to involve a single conjugational event. The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient). The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions. No interspecific mobilization of the chromosomal marker in matings involving P. putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed.

Fate of Pseudomonas putida after release into lake water mesocosms: Different survival mechanisms in response to environmental conditions.

To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number of P. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming cells in mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.

Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440.

Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms. We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids. Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P. putida bacteria. Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot. A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D. Detection of the cell antigen depended on the bacterial growth phase and culture medium. The O antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium. MAb 7.3B was used successfully to track P. putida (pWW0) released in unsterile lake mesocosms.

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.

Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid.

Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.