C-phycocyanin to overcome the multidrug resistance phenotype in human erythroleukemias with or without interaction with ABC transporters.

The phenotype of multidrug resistance (MDR) is one of the main causes of chemotherapy failure. Our study investigated the effect of C-phycocyanin (C-PC) in three human erythroleukemia cell lines with or without the MDR phenotype: K562 (non-MDR; no overexpression of drug efflux proteins), K562-Lucena (MDR; overexpression of ATP-binding cassette, sub-family B/ABCB1), and FEPS (MDR; overexpression of ABCB1 and ATP-binding cassette, sub-family C/ABCC1). Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we showed that 20 and 200 µg/mL C-PC decreased K562 viable cells after 24 h and 200 µg/mL C-PC decreased K562-Lucena cell proliferation after 48 h. C-PC did not decrease viable cells of FEPS cells. On the other hand, the MTT assay showed that exposure of 2, 20, and 200 µg/mL C-PC for 24 or 48 h was not cytotoxic to peritoneal macrophages. At 72 h, the trypan blue exclusion assay showed that 20 µg/mL C-PC decreased K562 and K562-Lucena cell proliferation and in FEPS cells, only 200 µg/mL C-PC decreased proliferation. In addition, protein-protein docking showed differences in energy and binding sites of ABCB1 and ABCC1 for C-PC, and these results were confirmed by the efflux protein activity assay. Only ABCC1 activity was altered in the presence of C-PC and FEPS cells showed lower C-PC accumulation, suggesting C-PC extrusion by ABCC1, conferring C-PC resistance. In combination with chemotherapy (vincristine [VCR] and daunorubicin [DNR]), the sensitivity of K562-Lucena cells for C-PC + VCR did not increase, whereas FEPS cell sensitivity for C-PC + DNR was increased. In molecular docking experiments, the estimated free energies of binding for C-PC associated with chemotherapy were similar (VCR: -6.9 kcal/mol and DNR: -7.2 kcal/mol) and these drugs were located within the C-PC cavity. However, C-PC exhibited specificity for tumor cells and K562 cells were more sensitive than K562-Lucena cells, followed by FEPS cells. Thus, C-PC is a possible chemotherapeutic agent for cells with the MDR phenotype, both alone in K562-Lucena cells (resistance due to ABCB1), or in combination with other drugs for cells similar to FEPS (resistance due to ABCC1). Moreover, C-PC did not damage healthy cells (peritoneal macrophages of Mus musculus).

Serratia liquefaciens Infection of a Previously Excluded Popliteal Artery Aneurysm.

INTRODUCTION: Popliteal artery aneurysms (PAAs) are rare in the general population, but they account for nearly 70% of peripheral arterial aneurysms. There are several possible surgical approaches including exclusion of the aneurysm and bypass grafting, or endoaneurysmorrhaphy and interposition of a prosthetic conduit. The outcomes following the first approach are favorable, but persistent blood flow in the aneurysm sac has been documented in up to one third of patients in the early post-operative setting. Complications from incompletely excluded aneurysms include aneurysm enlargement, local compression symptoms, and sac rupture. Notably infection of a previously excluded and bypassed PAA is rare. This is the third reported case of PAA infection after exclusion and bypass grafting and the first due to Serratia liquefaciens. METHODS: Relevant medical data were collected from the hospital database. RESULTS: This case report describes a 54 year old male patient, diagnosed with acute limb ischaemia due to a thrombosed PAA, submitted to emergency surgery with exclusion and venous bypass. A below the knee amputation was necessary 3 months later. Patient follow-up was lost until 7 years following surgical repair, when he was diagnosed with aneurysm sac infection with skin fistulisation. He had recently been diagnosed with alcoholic hepatic cirrhosis Child-Pugh Class B. The patient was successfully treated by aneurysm resection, soft tissue debridement and systemic antibiotics. CONCLUSION: PAA infection is a rare complication after exclusion and bypass procedures but should be considered in any patient with evidence of local or systemic infection. When a PAA infection is diagnosed, aneurysmectomy, local debridement, and intravenous antibiotic therapy are recommended. The "gold standard" method of PAA repair remains controversial. PAA excision or endoaneurysmorrhaphy avoids complications from incompletely excluded aneurysms, but is associated with a high risk of neurological damage.

Coronary Stent Embolisation to an Iatrogenic Arteriovenous Fistula: An Unexpected Complication.

INTRODUCTION: Systemic embolisation of stents represents a rare complication of coronary interventions. REPORT: The case of a patient with peripheral embolisation of a coronary stent while undergoing a coronary procedure for an ischaemic event is reported. The stent lodged at a previously created iatrogenic fistula joining the profunda femoral artery and the common femoral vein. An endovascular attempt was ineffective in treating this complication. The stent was removed surgically and the fistula repaired. DISCUSSION: This unusual clinical setting of stent embolisation concomitant to an iatrogenic arteriovenous femoral fistula is reported.

Microbiota of high-pressure-processed Serrano ham investigated by culture-dependent and culture-independent methods.

The microbiota of Serrano dry-cured ham of different chemical composition, subjected or not to high-pressure processing (HPP), was investigated using culture-dependent and culture-independent methods. Microbial counts were submitted to analysis of variance with physicochemical parameters (aw, NaCl concentration, salt-in-lean ratio and intramuscular fat content) or HPP as main effects. In untreated hams, physicochemical parameters significantly affected counts of aerobic mesophiles, psychrotrophs, and moulds and yeasts. NaCl concentration and fat content influenced the levels of four and three of the five studied microbial groups, respectively, whereas no influence of aw was stated. The HPP treatment had a significant effect on counts of all investigated microbial groups. Culture-independent methods showed the presence of bacteria such as Staphylococcus equorum, Staphylococcus succinus, Bacillus subtilis and Cellulosimicrobium sp., moulds like Penicillium commune, Aspergillus fumigatus, Sclerotinia sclerotiorum, Eurotium athecium and Moniliella mellis, and yeasts like Debaryomyces hansenii and Candida glucosophila. Absence of B. subtilis bands and weaker bands of E. athecium were recorded for HPP-treated hams. The higher microbial levels found in lean ham might result in a quicker deterioration. HPP treatment confirmed its suitability as a procedure to control spoilage microorganisms. DGGE did not seem to be sensitive enough to highlight changes caused by HPP treatment in the microbiota of ham, but contributed to the detection of microbial species not previously found in ham.

Influence of physicochemical parameters and high pressure processing on the volatile compounds of Serrano dry-cured ham after prolonged refrigerated storage.

One hundred and three volatile compounds were detected by solid-phase microextraction followed by gas chromatography-mass spectrometry in 30 ripened Serrano dry-cured hams, submitted or not to high pressure processing (HPP) and afterwards held for 5months at 4°C. The effect of ham physicochemical parameters and HPP (600MPa for 6min) on volatile compounds was assessed. Physicochemical parameters primarily affected the levels of acids, alcohols, alkanes, esters, benzene compounds, sulfur compounds and some miscellaneous compounds. Intramuscular fat content was the physicochemical parameter with the most pronounced effect on the volatile fraction of untreated Serrano ham after refrigerated storage, influencing the levels of 38 volatile compounds while aw, salt content and salt-in-lean ratio respectively influenced the levels of 4, 4 and 5 volatile compounds. HPP treatment affected 21 volatile compounds, resulting in higher levels of alkanes and ketones and lower levels of esters and secondary alcohols, what might affect Serrano ham odor and aroma after 5months of refrigerated storage.

Brazilian Pampa Biome Honey Protects Against Mortality, Locomotor Deficits and Oxidative Stress Induced by Hypoxia/Reperfusion in Adult Drosophila melanogaster.

We aimed to investigate the potential beneficial effects of the Brazilian Pampa biome honey in a Drosophila-based hypoxia model. Adult flies were reared in standard medium in the presence or absence of honey (at a final concentration of 10 % in medium). Then, control flies (4 % sucrose in medium) and honey-treated flies were submitted to hypoxia. Subsequently, flies were analyzed for mortality, neurolocomotor behavior (negative geotaxis), mitochondrial/oxidative stress parameters and expression of hypoxia/stress related genes by RT-qPCR. The HPLC analysis revealed the presence of phenolics and flavonoids in the studied honey. Caffeic acid was the major compound followed by p-coumaric acid and kaempferol. The presence of such compounds was correlated with a substantial antioxidant activity in vitro. Flies subjected to hypoxia presented marked mortality, locomotor deficits and changes in oxidative stress and mitochondrial activity parameters. Honey treatment was able to completely block mortality and locomotor phenotypes. In addition, honey was able to reverse ROS production and hypoxia-induced changes in mitochondrial complex I and II activity. Hypoxia also induced an up-regulation in mRNA expression of Sima (HIF-1), NFκß, NRF2, HOX, AKT-1, InR, dILP2, dILP5 and HSP27. Honey treatment was not able to modulate changes in the tested genes, indicating that its protective effects involve additional mechanisms other than transcriptional activity of hypoxia-driven adaptive responses in flies. Our results demonstrated, for the first time, the beneficial effects of honey against the deleterious effects of hypoxia/reperfusion processes in a complex organism.

Endogenous vascular endothelial growth factor and angiopoietin-2 expression in critical limb ischemia.

AIM: The aim of this paper was to contribute to a better understanding of the angiogenesis in peripheral arterial disease (PAD); we evaluated the expression of vascular endothelial growth factor (vegf) and angiopoietin-2 (Ang-2) in critical limb ischemia (CLI). METHODS: Skin and muscle biopsies were collected from 12 patients submitted to major amputation for CLI, proximal samples from amputation level and distal ones from the more ischemic region. Three controls were obtained from orthopedic patients. Capillary density was determined in random selected high-power fields. Expression pattern of VEGF and Ang-2 was studied by immunohistochemistry and quantification was performed by enzyme-linked immunosorbent assay. RESULTS: In skin, capillary density and levels of VEGF and Ang-2 were higher in distal samples when compared to proximal (capillary density, P=0.003, VEGF, P=0.008, Ang-2, P=0.041). Distal muscle had also elevated capillary number (P=0.005) and Ang-2 concentration (P=0.023). VEGF concentration in distal muscle was found to be similar to proximal muscle (P=1). Immunohistochemical expression of VEGF was clearly more evident in distal samples and was predominantly present in epidermis and skeletal myocytes. Ang-2 was essentially detected distally and only observed in endothelial cells. CONCLUSION: The capillary density is enhanced in distal samples, suggesting an effective angiogenic drive in CLI. In addition, the observed increase of VEGF expression in ischemic skin and Ang-2 in ischemic skin and muscle may contribute to clarify the potential role of VEGF and Ang-2 supplementation for therapeutic angiogenesis in CLI.

Proteolysis, lipolysis, volatile compounds, texture, and flavor of Hispánico cheese made using frozen ewe milk curds pressed for different times.

Hispánico cheese is manufactured in Spain from a mixture of cow and ewe milk. Production of ewe milk varies throughout the year, with a peak in spring and a valley in summer and autumn. To overcome this seasonal shortage, curd from spring ewe milk may be frozen and used for cheese manufacture some months later. In the present work, ewe milk curds pressed for 15, 60, or 120 min were held at -24 degrees C for 4 mo, thawed, cut to 1-mm pieces, and mixed with fresh cow milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized cow and ewe milk in the same (80:20) proportion. Cheeses, made in duplicate experiments, were analyzed throughout a 60-d ripening period. No significant differences between cheeses were found for lactic acid bacteria counts, dry matter content, hydrophilic peptides, 47 out of 68 vol.tile compounds, texture, and flavor characteristics. On the other hand, differences of minor practical significance between experimental and control cheeses, unrelated to the use of frozen ewe milk curd or the pressing time of ewe milk curd, were found for pH value, aminopeptidase activity, proteolysis, hydrophobic peptides, free amino acids, free fatty acids, and the remaining 21 vol.tile compounds. It may be concluded that the use of frozen ewe milk curd in the manufacture of Hispánico cheese does not alter its main characteristics.

Thrombosed popliteal aneurysm - first manifestation of bilateral popliteal entrapment syndrome.

Popliteal artery entrapment syndrome is a rare and underdiagnosed cause of claudication in the young adult. One of its rare and potentially limb threatening complications is aneurysmal degeneration. We present a case of a fifty years old man with a bilateral popliteal entrapment syndrome admitted with an acutely thrombosed aneurysm at the right inferior limb. An urgent bypass was required. The contralateral limb presented with a type III entrapment and an elective section of a musculotendinous structure was performed. This case suggests that an early diagnosis is mandatory to avoid a dramatic clinical outcome and to limit the surgical treatment to a myotomy.

Association of the HLA-G 14 bp polymorphism with systemic lupus erythematosus.

Human leukocyte antigen-G (HLA-G) is a nonclassical class I major histocompatibility complex molecule which is induced at the course of inflammatory pathologies, and its expression has been suggested as a possible mechanism of tissue protection against autoimmune inflammatory responses, therefore acting as a mechanism of immune surveillance. We investigated the influence of the 14 bp polymorphism of the HLA-G gene on systemic lupus erythematosus (SLE) by analyzing 293 patients with SLE and 460 healthy controls. The patient's group was not in Hardy-Weinberg equilibrium, presenting an excess of heterozygotes (P = 0.014). The heterozygote group exhibited lower systemic lupus erythematosus disease activity indexes than the homozygous deletion group and the homozygous insertion group (mean value = 2.29 against 2.97 and 3.4, respectively, P = 0.035). Photosensitive patients showed a higher frequency of heterozygotes and an equivalent lower frequency of homozygotes for deletion; on the other hand, patients without arthritis presented a higher frequency of heterozygotes than the arthritis group and also a lower frequency of the del/del genotype. Overall, our results support the idea of a role of the HLA-G insertion/deletion polymorphism and therefore a role for the HLA-G molecule, on the pathology of SLE.

The chemokine receptor CCR5 genetic polymorphism and expression in rheumatoid arthritis patients.

OBJECTIVES: To identify the genetic polymorphism of the chemokine receptor CCR5 (the Delta32 allelic variant) in patients with rheumatoid arthritis (RA) and compare the findings with healthy controls. To compare the CCR5 phenotypic expression in T cells and monocytes isolated from the peripheral blood and synovial fluid in a subgroup of RA patients. METHODS: CCR5 genes of 92 RA patients and 160 healthy controls were genotyped using specific primers flanking the region of deletion. The ethnic distribution was similar between the groups. Flow cytometric analysis was used for immunophenotyping the T cells and monocytes isolated from the peripheral blood and synovial fluid of eight RA patients. The isolated cells were triple stained with CD4 or CD8, CD25 and CCR5 monoclonal antibodies. RESULTS: There was no difference in the CCR5Delta32 genotypic frequency between the RA patients and the control group (0.055 and 0.063, respectively, p = 0.989). No homozygote for the CCR5Delta32 allele was seen in either group. Five heterozygotes were identified in the RA patient group, whose disease was shown to be aggressive. A significant enrichment of activated CCR5+ monocytes was seen in the synovial fluid of the RA patients subjected to arthrocentesis, who were all homozygotes for the CCR5 wild-type genotype. CONCLUSION: A protective role for the CCR5 allelic variant in RA development was not observed. Disease severity in the heterozygotes suggests that other proinflammatory mechanisms might overcome this mutation in vivo. The activated CCR5+ monocyte enrichment in the rheumatoid synovial fluid might indicate that this cell population has an important role in the pathogenesis of the disease.

GABA(B) receptor-mediated modulation of cutaneous input at the cuneate nucleus in anesthetized cats.

This study examined the modulatory influence exerted by GABA(B) receptors on the transmission of cutaneous afferent input to cuneate nucleus neurons in anesthetized cats. Electrical stimulation at the center of a receptive field activated cuneate nucleus cells at latencies of < or = 7 ms whereas stimulation at neighboring sites (receptive field edge) increased the response latency. Extracellular recording combined with microiontophoresis demonstrated that GABA(B) receptors are tonically active. Blockade of GABA(B) receptors prolonged sensory-evoked response durations and decreased times of occurrence of successive bursts whereas the agonist baclofen suppressed both these effects. Ejection of baclofen delayed the evoked response from the receptive field edge with respect to the receptive field center response and inhibited responses from the receptive field edge more effectively than responses from the receptive field center. From these results it is concluded that activation of GABA(B) receptors precludes cuneate cells from reaching firing threshold when afferent inputs are weak, spatially modulate cuneate nucleus excitability, play a major role in temporal pattern of discharges, and shape cutaneous receptive fields.

Induction of fetal haemoglobin expression in erythroid cells--a model based on iron availability signalling.

The K562 cell line has erythroid origin and is used for the study of fetal haemoglobin (HbF) production after treatment with several drugs, such as hydroxyurea, cisplatin and cytosine arabinoside (Ara C). It represents an important tool for the study of cancer differentiation therapy and treatment of thalassaemia and sickle cell disease. Although subject to intense research, the mechanisms involved in the induction of HbF are not fully established, and the regulation of several genes and signalling pathways has been proposed. Using the methodology of differential display, we investigated the changes in gene expression in K562 cells treated with doxorubicin and aclarubicin, which induce HbF expression and cell cycle arrest. Several genes were shown to present differential expression patterns, many of them related to the iron signalling pathway. Particular attention was given to Ndrg1, expressed as early as 24 h after treatment, which can be regulated by iron and is involved with blocking of the cell cycle. A review of the literature shows that, similar to doxorubicin and aclarubicin, most of the drugs used to induce HbF present some kind of effect on the iron signalling pathway, activating in the cells the machinery necessary for the incorporation of extracellular iron. Considering these results, as well as the fact that in erythroid cells the synthesis of haemoglobin is of vital importance, we propose that the production of fetal haemoglobin in erythroid cells is highly dependent on the iron signalling pathway.

Nonviral in vivo gene transfer in the mucopolysaccharidosis I murine model.

Mucopolysaccharidosis I (MPS I) is a lysosomal disorder characterized by a deficiency of the enzyme alpha-L: -iduronidase (IDUA), which is responsible for the degradation of glycosaminoglycans (GAGs). This deficiency leads to the accumulation of dermatan and heparan sulphate in lysosomes. Presently available treatments include bone marrow transplantation and enzyme replacement therapies, both of which are limited in their effects. In this work, knockout (KO) MPS I mice were treated with a nonviral vector containing the human IDUA cDNA. KO mice were transfected by hydrodynamic injection of pRIDUA in the caudal vein (i.v., n = 3) or by intraperitoneal injection of pRIDUA/Superfect complexes (i.p., n = 3). GAG concentration and IDUA activity were analysed in the kidneys, spleen, lungs, brain and liver. The expression of IDUA in the organs of i.v.- and i.p.-treated mice was also analysed by real-time reverse-transcription (RT) PCR and compared by relative quantification. The concentration of GAGs in the organs differed between KO and wild-type mice. In the spleen and liver, GAG levels were lower in i.v.- and i.p.-treated KO mice than in control nontreated animals. Real-time RT-PCR showed that the transgene is expressed in all the analysed organs of i.p.- and i.v.-treated KO mice. Enzyme activity was similarly observed in all the organs analysed. Our data suggest that this kind of transfection may be a useful tool for studies of nonviral protocols for gene therapy of MPS.

B-F DNA sequence variability in Brazilian (blue-egg Caipira) chickens.

A total of 100 chickens from the Brazilian (blue-egg Caipira) native breed were studied in relation to exon 2 of the B-F genes of the major histocompatibility complex (MHC) region. After a first screening on 100 birds, 22 animals were selected for amplification, cloning and sequencing experiments of exons 2-4 (a total of 1048 bp) of their DNA. Twenty-three sequences were obtained, of which at least 10 appear novel. Inferred protein sequences were compared with those previously described, totalling 41 different sequences with amino acid changes in 33 of the 88 sites in alpha1, and 34 of the 91 sites in alpha2 domains. Allele expression was investigated in these animals through cloning experiments. The blue-egg Caipira chickens may provide a source of novel B-F alleles for genetic improvement programmes.

[Functional heterogeneity of the piramidal system: corticobulbar and corticospinal tracts]. / Heterogeneidad funcional del sistema piramidal: tractos corticobulbar y corticoespinal.

INTRODUCTION AND OBJECTIVE: The pyramidal system is constituted by the cortical fibers reaching the bulbar pyramids at the level of the inferior Olive. Close to 75% of these fibers decussate terminating at bulbar level about half of them while the other half form the crossed corticospinal tract. The majority of the uncrossed fibers descend to the cord forming the direct corticospinal tract. This revision presents evidence demonstrating the functional heterogeneity of the pyramidal system. DEVELOPMENT: The corticobulbar fibers are slower conducting and branch more than the corticospinal fibers. The conduction speed of the corticospinal fibers increases as they reach more caudal segments, thus assuring that the different signals modulating the inter segmental activity reach their targets simultaneously. Those from the somatosensory cortex end in the dorsal horn while those from the primary motor cortex end primarily on interneurons of the reflex pathways to distal limb muscles although in rats, primates and humans about 10% of them terminate directly on motoneurons. CONCLUSIONS: The corticobulbar component of the pyramidal system selects the adequate motor synergies and modulates other descending systems to coordinate the activity of the distal musculature and the associated postural adjustments. The corticospinal component is functionally related to motor activities requiring accuracy, and to motoneuronal recruitment to adjust the contractile force. Both pyramidal subsystems modulate the processing and integration of the ascending somatosensory information generated by the movement itself.

Heterogeneidad funcional del sistema piramidal: tractos corticobulbar y corticoespinal / Functional heterogeneity of the piramidal system: corticobulbar and corticospinal tracts

Introducción y objetivo. El sistema piramidal está formado por los axones corticales que alcanzan las pirámides bulbares cerca de la oliva inferior. Cerca del 75 por ciento de dichas fibras se decusan, de modo que terminan en el bulbo aproximadamente la mitad, mientras que la otra mitad forma el tracto corticoespinal cruzado. La mayoría de las fibras que no se decusan forman el tracto corticoespinal directo. En esta revisión se presentan evidencias que demuestran la heterogeneidad funcional del sistema piramidal. Desarrollo. Las fibras corticobulbares son más lentas y presentan un mayor grado de colateralización que las corticoespinales. La velocidad de conducción de estas últimas es tanto mayor cuanto más caudal es su lugar de terminación en la médula espinal. Así, las diferentes señales que modulan la actividad intersegmentaria alcanzan simultáneamente sus destinos. Las procedentes de la corteza somatosensitiva terminan en el asta dorsal y las de la corteza motora primaria lo hacen mayoritariamente sobre las interneuronas de las vías reflejas que controlan la musculatura distal de los miembros, aunque alrededor del 10 por ciento lo hace directamente sobre las motoneuronas en ratas, primates y humanos. Conclusiones. El componente corticobulbar del sistema piramidal selecciona las sinergias adecuadas para cada acto motriz y modula la actividad de otros sistemas descendentes para coordinar la actividad de la musculatura distal y los ajustes posturales asociados. El componente corticoespinal está relacionado con las actividades motrices que requieren precisión y con el reclutamiento de motoneuronas para ajustar la fuerza contráctil. Ambos subsistemas modulan el procesamiento y la integración de la información somatosensorial ascendente producida por el movimiento en curso (AU)

Introduction and objective. The pyramidal system is constituted by the cortical fibers reaching the bulbar pyramids at the level of the inferior Olive. Close to 75% of these fibers decussate terminating at bulbar level about half of them while the other half form the crossed corticospinal tract. The majority of the uncrossed fibers descend to the cord forming the direct corticospinal tract. This revision presents evidence demonstrating the functional heterogeneity of the pyramidal system. Development. The corticobulbar fibers are slower conducting and branch more than the corticospinal fibers. The conduction speed of the corticospinal fibers increases as they reach more caudal segments, thus assuring that the different signals modulating the inter-segmental activity reach their targets simultaneously. Those from the somatosensory cortex end in the dorsal horn while those from the primary motor cortex end primarily on interneurons of the reflex pathways to distal limb muscles although in rats, primates and humans about 10% of them terminate directly on motoneurons. Conclusions. The corticobulbar component of the pyramidal system selects the adequate motor synergies and modulates other descending systems to coordinate the activity of the distal musculature and the associated postural adjustments. The corticospinal component is functionally related to motor activities requiring accuracy, and to motoneuronal recruitment to adjust the contractile force. Both pyramidal subsystems modulate the processing and integration of the ascending somatosensory information generated by the movement itself (AU)