Oxidative stress and susceptibility to mitochondrial permeability transition precedes the onset of diabetes in autoimmune non-obese diabetic mice.

Beta cell destruction in type 1 diabetes (TID) is associated with cellular oxidative stress and mitochondrial pathway of cell death. The aim of this study was to determine whether oxidative stress and mitochondrial dysfunction are present in T1D model (non-obese diabetic mouse, NOD) and if they are related to the stages of disease development. NOD mice were studied at three stages: non-diabetic, pre-diabetic, and diabetic and compared with age-matched Balb/c mice. Mitochondria respiration rates measured at phosphorylating and resting states in liver and soleus biopsies and in isolated liver mitochondria were similar in NOD and Balb/c mice at the three disease stages. However, NOD liver mitochondria were more susceptible to calcium-induced mitochondrial permeability transition as determined by cyclosporine-A-sensitive swelling and by decreased calcium retention capacity in all three stages of diabetes development. Mitochondria H2O2 production rate was higher in non-diabetic, but unaltered in pre-diabetic and diabetic NOD mice. The global cell reactive oxygen species (ROS), but not specific mitochondria ROS production, was significantly increased in NOD lymphomononuclear and stem cells in all disease stages. In addition, marked elevated rates of 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation were observed in pancreatic islets from non-diabetic NOD mice. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and lipidomic approach, we identified oxidized lipid markers in NOD liver mitochondria for each disease stage, most of them being derivatives of diacylglycerols and phospholipids. These results suggest that the cellular oxidative stress precedes the establishment of diabetes and may be the cause of mitochondrial dysfunction that is involved in beta cell death.

Avaliação do aprendizado de pacientes com doença pulmonar obstrutiva crônica em um programa de reabilitação pulmonar / Evaluation of learning in patients with chronic obstructive pulmonary disease during a pulmonary rehabilitation program

INTRODUÇÃO: Os pacientes que realizam um programa de reabilitação pulmonar participam, também, de um programa educativo com aulas expositivas abordando assuntos referentes à sua doença e ao tratamento. Esse programa visa levar o conhecimento necessário para que o paciente possa lidar com a doença e suas repercussões. OBJETIVO: Avaliar se o programa educativo aplicado aos pacientes submetidos a reabilitação pulmonar tem resultados efetivos no aprendizado dos mesmos. CASUíSTICA E MÉTODO: Estudo prospectivo, envolvendo 22 pacientes que se submeteram a um programa de reabilitação pulmonar, com idade 63 (DP ± 11,8). Inicialmente foi aplicado um questionário desenvolvido e validado pelo Centro de Reabilitação Pulmonar da UNIFESP/LESF para avaliar o conhecimento da doença pré e pós-intervenção do programa educacional. Os pacientes foram divididos em dois grupos: um grupo com programa educativo e um grupo controle (sem programa educativo). O grupo educativo respondeu ao questionário duas vezes, pré e pós-programa, já o grupo controle respondeu ao questionário apenas uma vez. RESULTADOS: Os pacientes que foram submetidos ao programa educativo apresentaram aumento no percentual de respostas corretas quando comparados o pré e o pós-intervenção, 69 por cento e 84 por cento respectivamente, e uma diminuição na porcentagem de erros quando comparados o pré e o pós-intervenção, 20 por cento e 14 por cento respectivamente. CONCLUSÃO: O programa educativo aplicado aos pacientes do programa de reabilitação pulmonar mostrou-se efetivo, pois aumentou o conhecimento dos pacientes no que se refere à doença, suas conseqüências e seu tratamento.

BACKGROUND: Patients who undergo pulmonary rehabilitation programs also participate in an educational program with classes covering matters related to their disease and treatment. Such programs aim to provide patients with the knowledge needed for them to be able to deal with their disease and its repercussions. OBJECTIVE: To evaluate whether the educational program applied to patients undergoing pulmonary rehabilitation has effective results regarding their learning. METHOD:This was a prospective study involving 22 patients who underwent a pulmonary rehabilitation program. Their mean age was 63 years (SD ± 11.8). Initially, a questionnaire developed and validated by the Pulmonary Rehabilitation Center of UNIFESP/LESF was applied to evaluate the patients' knowledge about the disease before and after the educational intervention. The patients were divided into two groups: one with the educational program and the other serving as a control group (no educational program). The educational program group answered the questionnaire twice (before and after the intervention), and the control group answered only once. RESULTS:The patients who underwent the educational program presented an increase in the percentage of correct answers, from before to after the intervention (69 percent versus 84 percent, respectively), and a decrease in the percentage of mistakes, from before to after the intervention (20 percent versus 14 percent, respectively). CONCLUSION: The educational program applied to patients in the pulmonary rehabilitation program was effective to increase the patients' knowledge about their disease, its consequences and its treatment.

A step test to assess exercise-related oxygen desaturation in interstitial lung disease.

A 6-min step test (6MST) may constitute a practical method for routinely assessing effort tolerance and exercise-related oxyhaemoglobin desaturation (ERD) in the primary care of patients with interstitial lung disease. In total, 31 patients (19 males) with idiopathic pulmonary fibrosis (n = 25) and chronic hypersensitivity pneumonia were submitted, on different days, to two 6MSTs. Physiological responses were compared with those found on maximal and submaximal cycle ergometer tests at the same oxygen uptake (V'(O(2))). Chronic breathlessness was also determined, as measured by the baseline dyspnoea index (BDI). Responses to 6MST were highly reproducible: 1.3+/-2.0 steps x min(-1), +/-5 beats x min(-1) (cardiac frequency), +/-50 mL x min(-1) (V'(O(2))), +/-7 L x min(-1) (minute ventilation) and +/-2% (arterial oxygen saturation measured by pulse oximetry (S(p,O(2)))). The number of steps climbed in 6 min was correlated to peak V'(O(2)) and the BDI. There were significant associations among the tests in relation to presence (change in S(p,O(2)) between rest and exercise > or = 4%) and severity (S(p,O(2)) <88%) of ERD. Four patients, however, presented ERD only in response to 6MST. Resting diffusing capacity of the lung for carbon monoxide and alveolar-arterial oxygen tension difference were the independent predictors of the number of steps climbed. A single-stage, self-paced 6-min step test provided reliable and reproducible estimates of exercise capacity and exercise-related oxyhaemoglobin desaturation in interstitial lung disease patients.

Selective disruption of the E-cadherin-catenin system by an algal toxin.

Yessotoxins (YTXs) are algal toxins that can be accumulated in edible molluscs. YTX treatment of MCF-7 breast cancer cells causes the accumulation of a 100 kDa fragment of E-cadherin, which we have named ECRA(100). A relative decrease in the concentrations of intact E-cadherin did not accompany the accumulation of ECRA(100) in cytosoluble extracts of MCF-7 cells on the first day of YTX treatment, but a collapse of the E-cadherin system was detected after 2-5 days of treatment with the toxin. An analysis of the general structure of ECRA(100) revealed that it consists of an E-cadherin fragment lacking the intracellular domain of the protein. ECRA(100) was not released into culture media of YTX-treated cells. Accumulation of ECRA(100) was observed in other epithelial cells, such as human intestine Caco-2 and MDCK cells after treatment with YTX. In turn, YTX could not induce accumulation of fragments of other members of the cadherin family, such as N-cadherin in the PC12 cell line and K-cadherin in sensitive cells (MCF-7, Caco-2, MDCK). The accumulation of a 100 kDa fragment of E-cadherin devoid of its intracellular domain induced by YTX was accompanied by reduced levels of beta- and gamma-catenins bound to E-cadherin, without a concomitant decrease in the total cytosoluble pools of beta- and gamma-catenins. Taken together, the results we obtained show that YTX causes the selective disruption of the E-cadherin-catenin system in epithelial cells, and raise some concern about the potential that an algal toxin found in seafood might disrupt the tumour suppressive functions of E-cadherin.

The pattern and timing of breathing during incremental exercise: a normative study.

Clinical evaluation of the pattern and timing of breathing during submaximal exercise can be valuable for the identification of the mechanical ventilatory consequences of different disease processes and for assessing the efficacy of certain interventions. Sedentary individuals (60 male/60 female, aged 20-80 yrs) were randomly selected from >8,000 subjects and submitted to ramp incremental cycle ergometry. Tidal volume (VT)/resting inspiratory capacity, respiratory frequency, total respiratory time (Ttot), inspiratory time (TI), expiratory time (TE), duty cycle (TI/Ttot) and mean inspiratory flow (VT/TI) were analysed at selected submaximal ventilatory intensities. Senescence and female sex were associated with a more tachypnoeic breathing pattern during isoventilation. The decline in Ttot was proportional to the TI and TE reductions, i.e. TI/Ttot was remarkably constant across age strata, independent of sex. The pattern, but not timing, of breathing was also influenced by weight and height; a set of demographically and anthropometrically based prediction equations are therefore presented. These data provide a frame of reference for assessing the normality of some clinically useful indices of the pattern and timing of breathing during incremental cycle ergometry in sedentary males and females aged 20-80 yrs.

Giant cell tumor of the rib.

A 27-year-old woman with a giant cell tumor of the rib with a cystic-hemorrhagic appearance underwent surgery consisting of en bloc resection and reconstruction of the thoracic wall with Marlex mesh, reinforced with two titanium plates. When possible this type of tumor requires resection, instead of radiotherapy, since the majority of cases of malignant transformation are linked to prior radiation therapy.

Current trends in chest wall malignant tumor.

Surgery used to treat chest wall sarcomas requires preoperative evaluation in order to document their margins, and to reveal any nodules of recurrence. Because of its considerable sensitivity and specificity ultrasound was associated with routine imaging techniques. The results obtained are useful in defining superficial and lateral tumor margins, but particularly in determining micro-nodules of recurrence which would otherwise go unrecognized. Based on the data obtained using routine methods of diagnosis and ultrasound, we submitted patients to excision that each and every time proved to be wide on histological examination. A particular surgical method was used for reconstruction, involving a polypropylene mesh fixed to one or two mouldable plates. This method exploits the elasticity of polypropylene, which is indispensable for the correct respiratory dynamics of the rib cage, thus avoiding paradoxical or harmful movement. Oncological and functional results were satisfactory.

Chondrosarcoma of the ribs and sternum. Considerations on 20 cases treated.

Over the last 20 years at the Rizzoli Orthopaedic Institute in Bologna 20 cases of chondrosarcoma (CS) of the thoracic wall (14 males, 6 females, mean age 49 years) have been submitted to surgery. Localization was costal in 11 cases, costosternal in 3, sternal in 3, costovertebral in 3. The most frequent histological variety was central with 15 observations. All of the cases were treated surgically. Exeresis was wide in 14 cases, marginal in 6. Reconstruction of the thoracic wall took place either by direct suturing or (14 cases) using prosthetic materials (Marlex mesh, 1 or 2 shapeable metal plates). Of the 20 cases treated, 16 patients are still alive (80%) with a mean survival rate of 33.5 months and a mean reduction in the postoperative ventilative index of function of less than 10%. The results obtained lead us to believe that surgical treatment involving wide exeresis is adequate, and the reconstruction method using Marlex mesh and metal plates is reliable.

Caspase activation and death induced by yessotoxin in HeLa cells.

We have studied the death response induced by yessotoxin (YTX) in cultured HeLa cells, and have compared it to that triggered by okadaic acid (OA) in the same experimental system. Sub-nanomolar concentrations of YTX were found to induce HeLa cell death after a 48-96-h incubation. YTX caused loss of intact poly(ADP-ribose)-polymerase (PARP) in HeLa cells, and detection of the 85kDa fragment, which is indicative of proteolytic attack by caspases. Measurements of caspase activities using extracts prepared from YTX-treated cells and substrates of the caspase-3/7 and caspase-2 isoforms, showed that the relative proteolysis of caspase-3/7 substrate was about eight-fold higher than that of caspase-2, the levels of which were about twice those measured with extracts from control cells. These findings were matched by Western blot analyses of caspase-2, -3 and -7 in HeLa cell extracts, which showed that the levels of pro-caspase-2 were not greatly affected by YTX treatment, whereas pro-caspase-3 and -7 were activated in YTX-treated cells. Taken together, these data complement others previously obtained with OA, and support the notion that caspase isoforms involved in cell death induced by OA and YTX are cell- and toxin-specific.

The toxic responses induced by okadaic acid involve processing of multiple caspase isoforms.

The recognized role of caspases as executioners of apoptosis, led us to investigate their involvement in death responses induced by okadaic acid (OA) in HeLa S(3) and MCF-7 cells. A one-day treatment with OA induced accumulation of the 85kDa poly(ADP-ribose) polymerase (PARP) fragment in cell lysates but the response was prevented if cells were treated with OA in the presence of the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. The HeLa S(3) and MCF-7 cells were found to contain measurable levels of the intact caspase-2, -7, -8 and -9 zymogens, whereas caspase-3 was found only in HeLa cells. After one day of OA treatment, pro-caspase-2, -3, -7 and -9 isoforms were found processed in HeLa cells, whereas only pro-caspase-2 was processed in MCF-7 cells. Pro-caspase-8, in turn, was mostly unprocessed in both cell lines. The possible interference of caspase inhibitors on cell death was also evaluated, and we found that both Z-VAD-FMK and Z-DEVD-FMK could contribute different extents of protection of MCF-7 and HeLa cells from toxic effects caused by OA. We concluded that OA triggers multiple pathways of caspase processing, contributing to death responses triggered by OA in HeLa S(3) and MCF-7 cells.

Computed tomography of pulmonary metastases from osteosarcoma: the less poor technique. A study of 51 patients with histological correlation.

BACKGROUND: The purpose is to evaluate the accuracy of computed tomography (CT) in the pulmonary staging of osteosarcoma. PATIENTS AND METHODS: Fifty-one patients presenting with osteosarcoma and at initial CT considered metastatic to the chest had lung surgery. Two teams of two senior radiologists independently reviewed all CT examinations. Their results were compared to the histological studies. RESULTS: One hundred nineteen CT's were reviewed. The 2 teams found 247 and 268 nodules on the initial. and 143 and 146 nodules on the preoperative CT. Histological studies confirmed metastatic nodules in 29 patients. Two hundred four nodules were excised and studied. One hundred nine were metastases. The 22 patients without metastases had 53 negative nodules removed. In the 29 patients with metastases, 151 nodules were removed, and 42 were non-metastatic. The positive predictive value was 53% with regard to 'nodules', and 57% with regard to 'patients'. Only 4 out of 13 patients with one nodule at surgery were metastatic, but all patients with more than 7 nodules were metastatic. The 46 cases with several available CT's, showed that no change in the number of nodules was more frequent in benign lesions. Other criteria revealed no significant difference. CONCLUSION: CT positive predictive value is limited, but as surgery is the only way to cure metastatic patients, CT will still be used as the reference technique until a more specific approach can be found.

Transient Ca2+-dependent activation of ERK1 and ERK2 in cytotoxic responses induced by maitotoxin in breast cancer cells.

Treatment of MCF-7 breast cancer cells with the marine toxin maitotoxin (MTX) induces cell death. The cytotoxic effects are clearly detectable within 2-4 h after cell treatment with 10(-10)-10(-9) M concentrations of MTX. The response was found to depend on extracellular Ca2+, inasmuch as cell death was prevented when culture dishes received MTX, following addition of EGTA. MTX caused transient phosphorylation of extracellular signal-regulated kinase isoforms 1 and 2 (ERK1 and ERK2) mitogen-activated protein kinase isoforms in MCF-7 cells, which was maximal 15 min after toxin addition to culture vessels. The effect was dependent on influx of extracellular Ca2+, as it was abolished by EGTA, and was induced by ionophores, such as A23187 and ionomycin. Our findings show that signaling pathways involving Ca2+ ions may cause activation of ERK1 and ERK2 in cell death responses.

Different sensitivities of p42 mitogen-activated protein kinase to phorbol ester and okadaic acid tumor promoters among cell types.

The operational equivalence of different types of tumor promoters was studied by comparing immediate, early, and late effects of okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the phosphorylation state of p42 mitogen-activated protein kinase isoform (ERK2) in eight different cell lines. In normal human and mouse fibroblasts, both agents stimulated immediate/early (15-60 min) phosphorylation of ERK2. In mouse 3T3 cells, enhanced phosphorylation of ERK2 was detected only within the first hour of treatment with TPA but not with OA. The early response to both TPA and OA, in turn, was lost in another established cell line, the PNT2 prostate epithelial cells, where we could detect increased levels of phosphorylated ERK2 only after a 24-hr treatment with OA. When the effect of OA was evaluated in different PNT cell strains, we observed that their capacity to respond to this agent, by stabilizing phosphorylated forms of ERK2, was lost in less differentiated strains. In HeLa S3 and HTC tumor cells, however, neither TPA nor OA treatment led to any detectable increase in ERK2 phosphorylation at any time point analyzed. We conclude that the effects of OA and TPA on the phosphorylation states of ERK2 could be related to the cell type, and that the operational equivalence between these two different tumor promoters is maximal in normal cells.

Intraepiphyseal resection of the proximal tibia and its impact on lower limb growth.

From 1989 through 1996, 10 children affected by high grade bone tumors of the proximal tibia underwent an intraepiphyseal intercalary resection. The residual epiphyseal bone segment measured less than 2 cm in thickness in all cases and reconstruction always was performed using the combination of a vascularized fibular autograft and a massive bone allograft. The proximal epiphyseal osteosynthesis was fixed by small fragment screws. The aim of this study was to report the growth pattern of the residual proximal tibial epiphysis and to evaluate any possible lower limb discrepancy and/or deformity after the end of skeletal maturity. At current followup six patients were available for the final evaluation. Radiographic documentation included computed tomography scan of both knees before surgery, a panoramic radiographic view and a computed tomography scan of both lower limbs after the end of skeletal growth. The length of both femurs and tibias, the size of the tibial plateau and of the opposite distal femur, and any possible deformity of femur or tibia were measured and compared with the preoperative data. No patient had a limb length discrepancy greater than 3.5 cm. In all cases the ipsilateral femur had a valgus deformity of the hip develop. In two patients this deformity was associated with an elongation of the femur, partially compensating for the shortening of the tibia. The tibial plateau close to reconstruction grew less than the contralateral one (range 2%-8%) but maintained its normal relationship with the distal femur. None of these patients reported any restriction in recreational activities. They could walk, run, and jump. Their functional result according the International Society of Limb Salvage functional grading system was satisfactory in all cases.

A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system.

We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator. Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine. We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH. We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response. This is demonstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.

Steroid hormones and temperature induce changes of binding parameters of their receptors in intact cells.

When MCF-7 cells were treated with 17 beta-estradiol, dexamethasone, or promegestone at 37 degrees C, the KD of receptors for their cognate ligands was found to decrease as compared to that measured at 2 degrees C. Cell incubation with hormone at 37 degrees C did not affect the Bmax of glucocorticoid and progesterone receptors, but caused a 40% increase of that of estrogen receptor. This increase required the presence of ligand, was insensitive to cycloheximide, and was completed within 10 min of cell incubation at physiological temperature. We conclude that an early step of estrogen action is the recruitment of pre-existing receptor molecules through activation of their binding capacity.

Nanomolar concentrations of untransformed glucocorticoid receptor in nuclei of intact cells.

The subcellular distribution of untransformed glucocorticoid-receptor complex in vivo has been studied by chemical crosslinking of intact cells, and using a procedure adequate for correction of experimental errors due to redistribution of components between cytosolic and nuclear fractions. We found that in HeLa S3 cells 85.4% of total glucocorticoid-receptor complexes are located in nuclei, and 14.6% are cytosolic. If measurements were performed with MCF-7 cells, we determined that the nuclear pool of glucocorticoid-receptor complexes accounts for 75.2% of the total cellular content, whereas the remaining 24.8% are cytosolic. When the subcellular distribution of estrogen-receptor complexes was determined, instead, we found that they are almost exclusively located in nuclei of MCF-7 cells, which contain 88.9% of the total. In order to estimate the molar concentration of receptors in cytosol and nuclei of intact cells, we determined the free water content of the two compartments. The volume of solvent was found to vary in the three cell lines we have studied, and our data showed that these variations are due to the cytosolic fractions, as the free water content of nuclei is essentially the same in those cells. When the free water content and the levels of glucocorticoid-receptor complexes we have measured were used to estimate the molar concentrations of receptors, we found that these range between 0.4 and 18.9 nM in cytosols, and between 3.9 and 6.3 nM in nuclei of the three cell lines we have studied. We then concluded that the relative distribution of untransformed glucocorticoid-receptor complexes between cytosol and nuclei is cell-specific but their molar concentration in the nuclear compartment does not greatly vary among different cells.