Cold Storage Preservation and Warm Ischaemic Injury to Isolated Arterial Segments: Endothelial Cell Injury

Injury to endothelial calls is thought to be important to the development of the vascular lesion of chronic rejection. It was the aim of this study to develop a semiquantitative method to assess endothelial injury in arterial grafts and to document the injury produced by cold storage preservation and additional warm ischaemia. Twelve- and 24-h cold preservation of rat aortic segments, together with an additional 1 h of warm ischaemia, were assessed. Electron micrographs of representative endothelial cells were scored for cytoplasmic, nuclear and mitochondrial injury. The overall injury score was obtained by addition of the individual scores. Storage for up to 24 h in University of Wisconsin (UW) and Terasaki did not produce any injury. Twenty-four hours of storage in Euro-Collins resulted in endothelial cell death. Injury occurred after 12 h of storage in Ross, Collins and normal saline, and the injury increased following 24 h of storage. One hour of warm ischaemia did not increase the injury. Injury to endothelial cells varies with the preservation solution used and the time of cold storage, so that both the type of solution and the storage time should be taken into account in clinical studies looking at the influence of cold ischaemia time and graft outcome.

Distribution kinetics of solutes in the isolated in-situ perfused rat head using the multiple indicator dilution technique and a physiological two-barrier model

The purpose of this study was to determine the pharmacokinetics of [C-14]diclofenac, [C-14]salicylate and [H-3]clonidine using a single pass rat head perfusion preparation. The head was perfused with 3-[N-morpholino] propane-sulfonic acid-buffered Ringer's solution. Tc-99m-red blood cells and a drug were injected in a bolus into the internal carotid artery and collected from the posterior facial vein over 28 min. A two-barrier stochastic organ model was used to estimate the statistical moments of the solutes. Plasma, interstitial and cellular distribution volumes for the solutes ranged from 1.0 mL (diclofenac) to 1.6 mL (salicylate), 2.0 mL (diclofenac) to 4.2 mL (water) and 3.9 mL (salicylate) to 20.9 mL (diclofenac), respectively. A comparison of these volumes to water indicated some exclusion of the drugs from the interstitial space and salicylate from the cellular space. Permeability-surface area (PS) products calculated from plasma to interstitial fluid permeation clearances (CLPI) (range 0.02-0.40 mL s(-1)) and fractions of solute unbound in the perfusate were in the order: diclofenac>salicylate >clonidine>sucrose (from 41.8 to 0.10 mL s(-1)). The slow efflux of diclofenac, compared with clonidine and salicylate, may be related to its low average unbound fraction in the cells. This work accounts for the tail of disposition curves in describing pharmacokinetics in the head.

Angiotensinogen genotype, plasma protein and mRNA concentration in isolated systolic hypertension

Isolated systolic hypertension (ISH) occurs predominantly in the elderly, with a considerable morbidity and mortality. Its etiology is unknown but is likely to involve a significant genetic component. The aim of this study was to examine the angiotensinogen gene in ISH. The M235T and G(- 6)A polymorphisms were genotyped by polymerase chain reaction (PCR) in 86 ISH patients and 120 normotensive controls. Plasma angiotensinogen concentration was determined in 198 subjects by an indirect radioimmunoassay technique. Angiotensinogen mRNA concentration was determined by quantitative competitive reverse transcription (RT)-PCR in subcutaneous adipose tissue from a subset of these patients (n = 8) and controls (n = 6). Both the M235T (p = 0.0015) and G(- 6)A (p = 0.029) polymorphisms were associated with ISH. Plasma angiotensinogen concentration was higher in patients than controls (p < 0.0001), but was not associated with genotype. Angiotensinogen mRNA concentration in adipose tissue from ISH subjects was significantly lower than in adipose tissue from normotensive subjects (p = 0.033). The association of angiotensinogen gene variants with ISH and the elevation of plasma angiotensinogen concentration in these patients suggests a role of the angiotensinogen gene in this form of hypertension. Angiotensinogen gene expression may be altered in ISH, but this requires further examination.

Characterization of E-cadherin endocytosis in isolated MCF-7 and Chinese hamster ovary cells - The initial fate of unbound E-cadherin

The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.

Characterization of Treponema phagedenis-like spirochetes isolated from papillomatous digital dermatitis lesions in dairy cattle

Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar to Treponema phagedenis and 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity among T. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.

Prevalence of antibodies to the BK and JC papovaviruses in isolated populations

A total of 173 sera from isolated Brazilian Indian populations, 39 from the Diauarun area, and 68 from the Alto Xingú area, respectively in the North and the South of the Xingú National Park and 66 Kren-Akorore Indians, were examined for hemagglutination - inhibiting (HI) antibodies against BK and JC viruses. The global percentages of positive sera (> 1:40) were 5.2% for BK virus and 1.7% for JC virus. The distribution of positive sera according to the population groups showed one individual to be positive for BK virus in the Diauarun Indians and none of the sera contained HI antibody to JC virus; in the Alto Xingú Indians, 4 were positive for BK virus and 3 others were positive for JC virus; as regards Kren-Akorore Indians none of the sera contained antibody to JC virus, and only 4 were BK positive. Due to the limited number of observations it was neither possible to determine the time of occurrence of seroconversion nor correlate the positivity rates for both viruses in the different tribes with the respective "contact" with the white population.

Herbicidal activity of volatiles from coriander, winter savory, cotton lavender, and thyme isolated by hydrodistillation and supercritical fluid extraction

The volatiles from Coriandrum sativum L., Satureja montana L., Santolina chamaecyparissus L., and Thymus vulgaris L. were isolated by hydrodistillation (essential oil) and supercritical fluid extraction (volatile oil). Their effect on seed germination and root and shoot growth of the surviving seedlings of four crops (Zea mays L., Triticum durum L., Pisum sativum L., and Lactuca sativa L.) and two weeds (Portulaca oleracea L. and Vicia sativa L.) was investigated and compared with those of two synthetic herbicides, Agrocide and Prowl. The volatile oils of thyme and cotton lavender seemed to be promising alternatives to the synthetic herbicides because they were the least injurious to the crop species. The essential oil of winter savory, on the other hand, affected both crop and weeds and can be appropriate for uncultivated fields.

In vitro transference and molecular characterization of bla TEM genes in bacteria isolated from Portuguese ready-to-eat foods

The principal aim of this study was to investigate the possibility of transference to Escherichia coli of β-lactam resistance genes found in bacteria isolated from ready-to-eat (RTE) Portuguese traditional food. From previous screenings, 128 β-lactam resistant isolates (from different types of cheese and of delicatessen meats), largely from the Enterobacteriaceae family were selected and 31.3% of them proved to transfer resistance determinants in transconjugation assays. Multiplex PCR in donor and transconjugant isolates did not detect bla CTX, bla SHV and bla OXY, but bla TEM was present in 85% of them, while two new TEMs (TEM-179 and TEM-180) were identified in two isolates. The sequencing of these amplicons showed identity between donor and transconjugant genes indicating in vitro plasmid DNA transfer. These results suggest that if there is an exchange of genes in natural conditions, the consumption of RTE foods, particularly with high levels of Enterobacteriaceae, can contribute to the spread of antibiotic resistance.

Purification and structural characterization of compounds isolated from Cystoseira abies-marina

6th Spanish-Portuguese-Japanese Organic Chemistry Symposium, Lisboa, de 18 a 20 de Julho de 2012 (Poster Communication).

Species composition of arbuscular mycorrhizal fungi differ in semi-natural and intensively managed pastures in an isolated oceanic island (Terceira, Azores)

Copyright © Springer Science+Business Media Dordrecht 2014.

Diversity and characterisation of coagulasenegative staphylococci isolated from healthy individuals in Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) has significantly increased in human medicine. CoNS are common commensal colonisers of the human skin, although now also recognised as major nosocomial pathogens. Over the last decades, several studies have been carried out in order to understand the pathogenicity mechanisms of CoNS. The well known determinants in the pathogenesis of CoNS infections are their ability to form biofilms and an exceptional resistance to several antibiotics. Nevertheless, there is a lack of studies regarding the commensal lifestyle of these microorganisms. Additionally, it is now hypothesised that commensal bacteria might be a reservoir of pathogenic determinants. Therefore, the work described throughout this thesis was aimed to perform a phenotypic and genotypic characterisation of different CoNS species isolated from healthy Portuguese individuals. A total of 61 CoNS isolates, comprising 7 different species, were obtained and characterised at the level of biofilm formation and antibiotic susceptibility profiles. According to the results, biofilm formation ability and presence of biofilm-associated genes were commonly found features, highlighting their pivotal role in the colonising lifestyle of CoNS. This study also addressed the correlation between phenotypic and genotypic characteristics of biofilm formation, corroborating and raising questions about the importance of some genes in this process. Moreover, it was observed a great proportion of isolates with decreased susceptibility and multiple resistances to some important antibiotics. A significant association between antibiotic resistance and biofilm formation was also demonstrated, and some hypotheses about the nature of such association were provided. Lastly, the expression patterns of two biofilm-associated genes at two distinct biofilm developmental stages were determined, confirming their importance in the accumulative stage of biofilm formation. Overall, the results presented in this thesis indicate that staphylococcal skin flora might be an important reservoir of potentially pathogenic bacteria and, simultaneously, bring to light new perceptions about the molecular basis of staphylococcal biofilm formation, and the nature of the association between antibiotic resistance and biofilm formation.

Isolated and synergistic effects of PM10 and average temperature on cardiovascular and respiratory mortality

OBJECTIVE To analyze the effect of air pollution and temperature on mortality due to cardiovascular and respiratory diseases. METHODS We evaluated the isolated and synergistic effects of temperature and particulate matter with aerodynamic diameter < 10 µm (PM10) on the mortality of individuals > 40 years old due to cardiovascular disease and that of individuals > 60 years old due to respiratory diseases in Sao Paulo, SP, Southeastern Brazil, between 1998 and 2008. Three methodologies were used to evaluate the isolated association: time-series analysis using Poisson regression model, bidirectional case-crossover analysis matched by period, and case-crossover analysis matched by the confounding factor, i.e., average temperature or pollutant concentration. The graphical representation of the response surface, generated by the interaction term between these factors added to the Poisson regression model, was interpreted to evaluate the synergistic effect of the risk factors. RESULTS No differences were observed between the results of the case-crossover and time-series analyses. The percentage change in the relative risk of cardiovascular and respiratory mortality was 0.85% (0.45;1.25) and 1.60% (0.74;2.46), respectively, due to an increase of 10 μg/m3 in the PM10 concentration. The pattern of correlation of the temperature with cardiovascular mortality was U-shaped and that with respiratory mortality was J-shaped, indicating an increased relative risk at high temperatures. The values for the interaction term indicated a higher relative risk for cardiovascular and respiratory mortalities at low temperatures and high temperatures, respectively, when the pollution levels reached approximately 60 μg/m3. CONCLUSIONS The positive association standardized in the Poisson regression model for pollutant concentration is not confounded by temperature, and the effect of temperature is not confounded by the pollutant levels in the time-series analysis. The simultaneous exposure to different levels of environmental factors can create synergistic effects that are as disturbing as those caused by extreme concentrations.

Periplasmic nitrate reductases: structural and spectroscopic studies

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica, especialidade Bioquímica-Física, pela Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa

Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade de Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

The electric vehicles as a mean to reduce Co2 emissions and energy costs in isolated regions: the São Miguel (Azores) case study

Most of small islands around the world today, are dependent on imported fossil fuels for the majority of their energy needs especially for transport activities and electricity production. The use of locally renewable energy resources and the implementation of energy efficiency measures could make a significant contribution to their economic development by reducing fossil fuel imports. An electrification of vehicles has been suggested as a way to both reduce pollutant emissions and increase security of supply of the transportation sector by reducing the dependence on oil products imports and facilitate the accommodation of renewable electricity generation, such as wind and, in the case of volcanic islands like Sao Miguel (Azores) of the geothermal energy whose penetration has been limited by the valley electricity consumption level. In this research, three scenarios of EV penetration were studied and it was verified that, for a 15% LD fleet replacement by EVs with 90% of all energy needs occurring during the night, the accommodation of 10 MW of new geothermal capacity becomes viable. Under this scenario, reductions of 8% in electricity costs, 14% in energy, 23% in fossil fuels use and CO2 emissions for the transportation and electricity production sectors could be expected.