Tandem mass spectrometry: a convenient approach in the dosage of steviol glycosides in Stevia sweetened commercial food beverages.

The use of sweeteners extracted from leaves of the plant species Stevia rebaudiana is increasing worldwide. They are recognized as generally recognized as safe by the US-FDA and approved by EU-European Food Safety Authority, with some recommendation on the daily dosage that should not interfere with glucose metabolism. The results presented here introduce an easy analytical approach for the identification and assay of Stevia sweeteners in commercially available soft drink, based on liquid chromatography coupled to tandem mass spectrometry, using a natural statin-like molecule, Brutieridin, as internal standard. Copyright © 2017 John Wiley & Sons, Ltd.

[Repositioning accuracy of cerebral fractionated stereotactic radiotherapy using CT scanning]. / Evaluation par tomodensitométrie du repositionnement en radiothérapie stéréotaxique fractionnée cérébrale.

PURPOSE: To evaluate the accuracy of patient repositioning in fractionated cerebral stereotactic radiotherapy using a Brain Lab stereotactic cranial mask in conjunction with standard dental fixation. PATIENTS AND METHODS: Fifty planning and checking CT scans were performed in 25 patients. The check CT scan was performed before or after one of the three sessions of treatment. Coregistration to the planning CT scan was used to assess alignment of the isocentre to the reference markers. The relative position of the PTV with regard to isocentre allowed us to determine its total displacement (3D vector). RESULTS: Mean isocentre translations (+/-SD) taking into account direction were -0.01+/-0.7, -0.2+/-1.3 and 0.07+/-0.5mm in mediolateral, craniocaudal and anteroposterior directions respectively. Mean rotations (+/- SD) were -0.02+/-0.6, -0.08+/-0.3 and -0.1+/-0.3 degree in mediolateral, craniocaudal and anteroposterior axes respectively. Mean overall PTV displacement was 1.8+/-1.5mm. PTV displacement was smaller than 2 and 3mm in 19/25 and 23/25 patients respectively. CONCLUSION: The accuracy of patient positioning using a stereotactic cranial mask system is similar to those reported in the literature and shows a satisfactory reproducibility with a standard dental fixation.

Direct costs of asthma in Brazil: a comparison between controlled and uncontrolled asthmatic patients.

Asthma is a common chronic illness that imposes a heavy burden on all aspects of the patient's life, including personal and health care cost expenditures. To analyze the direct cost associated to uncontrolled asthma patients, a cross-sectional study was conducted to determine costs related to patients with uncontrolled and controlled asthma. Uncontrolled patient was defined by daytime symptoms more than twice a week or nocturnal symptoms during two consecutive nights or any limitations of activities, or need for relief rescue medication more than twice a week, and an ACQ score less than 2 points. A questionnaire about direct cost stratification in health services, including emergency room visits, hospitalization, ambulatory visits, and asthma medications prescribed, was applied. Ninety asthma patients were enrolled (45 uncontrolled/45 controlled). Uncontrolled asthmatics accounted for higher health care expenditures than controlled patients, US$125.45 and US$15.58, respectively [emergency room visits (US$39.15 vs US$2.70) and hospitalization (US$86.30 vs US$12.88)], per patient over 6 months. The costs with medications in the last month for patients with mild, moderate and severe asthma were US$1.60, 9.60, and 25.00 in the uncontrolled patients, respectively, and US$6.50, 19.00 and 49.00 in the controlled patients. In view of the small proportion of uncontrolled subjects receiving regular maintenance medication (22.2%) and their lack of resources, providing free medication for uncontrolled patients might be a cost-effective strategy for the public health system.

Direct costs of asthma in Brazil: a comparison between controlled and uncontrolled asthmatic patients

Asthma is a common chronic illness that imposes a heavy burden on all aspects of the patient's life, including personal and health care cost expenditures. To analyze the direct cost associated to uncontrolled asthma patients, a cross-sectional study was conducted to determine costs related to patients with uncontrolled and controlled asthma. Uncontrolled patient was defined by daytime symptoms more than twice a week or nocturnal symptoms during two consecutive nights or any limitations of activities, or need for relief rescue medication more than twice a week, and an ACQ score less than 2 points. A questionnaire about direct cost stratification in health services, including emergency room visits, hospitalization, ambulatory visits, and asthma medications prescribed, was applied. Ninety asthma patients were enrolled (45 uncontrolled/45 controlled). Uncontrolled asthmatics accounted for higher health care expenditures than controlled patients, US$125.45 and US$15.58, respectively [emergency room visits (US$39.15 vs US$2.70) and hospitalization (US$86.30 vs US$12.88)], per patient over 6 months. The costs with medications in the last month for patients with mild, moderate and severe asthma were US$1.60, 9.60, and 25.00 in the uncontrolled patients, respectively, and US$6.50, 19.00 and 49.00 in the controlled patients. In view of the small proportion of uncontrolled subjects receiving regular maintenance medication (22.2 percent) and their lack of resources, providing free medication for uncontrolled patients might be a cost-effective strategy for the public health system.

Cancer predisposition caused by elevated mitotic recombination in Bloom mice.

Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.

Novel protein isoforms of the APC tumor suppressor in neural tissue.

The conventional protein isoform of the APC tumor suppressor is 310 kD and is encoded by exons 1 - 15 of the APC gene. Other RNAs are expressed from the APC gene and include one form that contains an exon upstream of exon 1, designated BS, but this transcript does not include exon 1. This transcript recently has been shown to be enriched in non-dividing, terminally-differentiated cells (Santoro and Groden, 1997). To determine if the BS-containing transcript encoded an alternate APC protein isoform, we generated and affinity-purified a polyclonal antibody directed to protein sequence predicted by exon BS. The BS antibody labeled a band of approximately 300 kD on immunoblots of cerebral and cerebellar tissue from adult human, baboon, rat and mouse. These same tissue lysates also contained prominent BS-reactive proteins of 290 kD, 200 kD and 150 kD. Lysates from mitotically active cells did not contain these APC isoforms. To verify that BS-reactive proteins were APC isoforms, BS-immunoprecipitates were blotted and labeled with commercially available APC antibodies. All four high molecular weight BS-antibody-precipitated proteins were recognized by antibodies directed against epitopes encoded by APC exons 2 and 15. BS isoforms were not, however, labeled with antibodies to an epitope encoded by APC exon 1, consistent with the prediction that BS - APC isoforms lack the domain encoded by these sequences. Like conventional APC, at least one of the four BS-APC protein isoforms also interacts with beta-catenin. BS-APC isoforms that lack exon 1-encoded sequences are incapable of dimerization with the conventional form of APC, yet retain the ability to bind beta-catenin. Such isoforms are likely to be functionally distinct from the conventional APC protein.

Calcitonina monomérica plasmática e hipercalcemia em pacientes portadores de neoplasia pulmonar / Monomeric plasmatic calcitonin and hypercalcemia in lung cancer patients

A calcitonina (CT) é um hormonio peptídico relacionado ao metabolismo de calcio produzido pelas células C da tiróide. Encontra-se com níveis plasmaticos bastante elevados no carcinoma medular de tiróide e mostra-se como excelente marcador dessa doença. No entanto, existem relatos na literatura que demonstraram níveis elevados deste peptídio em pacientes portadores de outras neoplasias, principalmente no carcinoma de pulmao. Objetivo. Avaliar a validade da dosagem da CT sérica como possível marcador tumoral em pacientes portadores de tumor de pulmao de diferentes tipos histológicos. Métodos. Foram dosados CT plasmatica e calcio ionizado sanguíneo em 56 pacientes portadores de tumores malignos de pulmao. Para as dosagens de CT os autores utilizaram um método de radioimunoensaio específico, realizado após extraçao prévia do soro em coluna de sílica. Resultados. Observou-se prevalência de hipercalcemia de 21,4 por cento; apenas três (5,4 por cento) dos 56 pacientes investigados apresentaram níveis pouco elevados de calcitonina, e o restante manteve níveis normais ou identectaveis do peptídio. Conclusao. Os resultados demonstram que, com a utilizaçao de um método bastante específico para dosagem da calcitonina em sua forma monomérica, nao se encontram níveis elevados deste hormonio em pacientes portadores de neoplasia pulmonar, desestimulando sua utilizaçao como marcador tumoral nesta patologia.

[Monomeric plasmatic calcitonin and hypercalcemia in lung cancer patients]. / Calcitonina monomérica plasmática e hipercalcemia em pacientes portadores de neoplasia pulmonar.

BACKGROUND: Calcitonin (CT) is a peptidic hormone produced by the thyroid C cells and related to calcium metabolism. High plasmatic levels of this hormone are found in patients with medullary thyroid carcinoma, what makes it an excellent tumor marker for this disease. However, there are reports that showed an increase of plasmatic CT levels in patients with other tumors, mainly in lung cancer. PURPOSE: These data prompt us to investigate the validity of the CT level determinations as a potential tumor marker in different histologic lung cancer, and its correlation with hypercalcemia, a very common complication in these tumors. METHOD: Blood were sampled from 56 patients with malignant lung disease for the CT and ionized calcium determinations. Calcitonin was measured using a specific radioimmunoassay for the monomeric form of the molecule, in a previous silica extracted serum probe. RESULTS: We did not find elevated levels of monomeric CT in lung cancer. Only 3 patients had mild elevated levels, while in the others CT was normal or undetectable. Hypercalcemia was found in 21.4% of these patients, but only one with supranormal CT levels. CONCLUSION: Monomeric CT serum levels are normal in lung cancer, what makes the latter use an unreliable tumor marker.

Alternative splicing of the APC gene and its association with terminal differentiation.

The human tumor suppressor gene, APC, is composed of at least 21 exons, 7 of which are alternatively expressed. Sixteen APC transcripts that differ in their 5'-most regions and arise by the alternative inclusion of 6 of these exons have been identified by reverse transcription-PCR analysis of RNA prepared from human, mouse, and rat cell lines and tissues. Tissue-specific differences were observed in the expression of APC transcripts without exon 1, a coding region for a heptad repeat that supports APC homodimerization. Transcripts without exon 1 were observed at high levels in postmitotic, differentiated tissues and in two cell lines following the induction of differentiation. Sequence analysis of these novel open reading frames predicts APC proteins with different amino-terminal domains and therefore potentially different abilities to associate with other proteins. Our findings suggest that the alternative splicing of APC leads to alternative forms of APC proteins with potentially unique functions in growth control and differentiation.

Parameters that influence the extent of site occupancy by a candidate telomere end-binding protein.

The MF3 protein specifically recognizes telomeric and non-telomeric DNA probes that can form G.G base-paired structures (Gualberto, A., Patrick, R. M., and Walsh, K. (1992) Genes & Dev. 6, 815-824). Here we further characterize the nucleic acid recognition properties of MF3 and present a mathematical analysis that evaluates the potential extent of telomere site occupancy by this factor. The substitution of dI at dG positions in telomeric DNA probes revealed that a single dG at any position within the internal repeat was sufficient for high affinity binding to MF3. The RNA analogs of high affinity DNA sites were not bound specifically by MF3, but the substitution of dU for dT in a DNA probe had little or no effect on binding. These data demonstrate that ribose ring structure is a critical feature of nucleoprotein complex formation, and this ribose specificity may enable MF3 to occupy sites of unusual DNA structure while minimizing interactions with cellular RNAs. Collectively, the nucleic acid binding properties of MF3 suggest that it may occupy a significant fraction of sites at telomere ends or other G-rich regions of altered DNA structure in vivo.

Radiofrequency catheter ablation of idiopathic left ventricular tachycardia guided by a Purkinje potential.

BACKGROUND: Verapamil-sensitive, idiopathic left ventricular tachycardia (ILVT) with right bundle branch block configuration and left-axis deviation has been suggested to originate from the left posterior fascicle. The purpose of this study was to determine how frequently potentials generated by the Purkinje fiber network (P potential) can be recorded preceding ventricular activation, and the role of the P potential in guiding radiofrequency catheter ablation. METHODS AND RESULTS: Eight patients (mean age, 26 +/- 10 years) with ILVT (cycle length, 346 +/- 59 milliseconds) were studied. Right and left ventricular endocardial mapping during tachycardia identified earliest ventricular activation at the posteroapical left ventricular septum. In all patients, earliest ventricular activation during tachycardia was preceded by a distinct potential. This potential also preceded ventricular activation during sinus rhythm, consistent with activation of a segment of the left posterior fascicle (P potential). The earliest recorded P potential preceded the QRS during tachycardia by 15 to 42 milliseconds (mean, 27 +/- 9 milliseconds). The application of radiofrequency current at 1 to 4 sites (median, 1) eliminated ILVT in all eight patients. In the seven patients with P potentials recorded at multiple sites within the posteroapical septum, ablation was successful at the site of the earliest P potential and unrelated to the timing of ventricular activation. In the remaining patient, ablation was successful at a site recording a late P potential fusing with earliest ventricular activation. During follow-up (1 to 67 months; median, 10.5) ILVT recurred only in the latter patient. Pace mapping during tachycardia at the successful ablation site in four patients produced a similar QRS with stimulus-QRS interval equal to P-QRS interval during tachycardia. However, a similar QRS was obtained by pacing at nearby sites that recorded a later P potential. CONCLUSIONS: These findings support the hypothesis that ILVT originates from the Purkinje network of the left posterior fascicle. A P potential can be recorded at the posteroapical left ventricular septum during ILVT, and ablation is successful at the site recording the earliest P potential. Pace mapping with similar QRS is not specific due to capture of the Purkinje fiber network at a site remote from the origin of the tachycardia.

Radiofrequency ablation of a concealed accessory pathway as treatment for incessant supraventricular tachycardia in a dog.

A new technique for the treatment of certain types of cardiac arrhythmias was used in a 3-year-old dog that was evaluated for incessant supraventricular tachycardia (220 to 280 beats/min), which had been refractory to several treatment regimens. The mechanism of supraventricular tachycardia was atrioventricular (AV) reentry, using a dorsoseptal accessory pathway (AP) for retrograde ventriculoatrial conduction (concealed AP). With the dog under general anesthesia and with fluoroscopic monitoring, electrode catheters were introduced into the heart via peripheral vessels. Electrical recordings allowed localization of the accessory AV pathway. Programmed electrical stimulation was used to verify the function of the abnormal AV connection. At the atrial insertion site of the AP, 2 applications of radiofrequency current (45 V, 21.6 W) were delivered to the dorsoseptal right atrium (near the coronary sinus ostium), which eliminated AP conduction and AV reentrant tachycardia. The dog has remained free of tachycardia and has not required medication during more than 1 year of follow-up.

Prevalência de hipercalcemia em pacientes portadores de câncer de pulmäo / Prevalence of hypercalcemia in patients carriers of lung neoplasm

A hipercalcemia é uma das maiores freqüentes manifestaçöes paraneoplásicas, principalmente dentre as neoplasias malignas de pulmäo, mas ainda pouco valorizada em nosso meio. Neste trabalho estudamos a prevalência de hipercalcemia em uma populaçäo de pacientes portadores de tumor de pulmäo que freqüentaram o ambulatório específico da Escola Paulista de Medicina no Hospital Säo Paulo. Foram investigadso 90 pacientes assim distribuídos: 35 carcinomas espinocelulares (CEC), 30 adenocarcinomas (AdenoCa), 11 carcinomas indiferenciados de pequenas células (CIPC), 2 tumores de grandes células (RGC), 1 carcinóide, 1 mesotelioma, 2 indiferenciados, 1 adenoescamoso, 1 carcinoma in situ e 3 tumores metastáticos de origens diversas. Em todos estes pacientes foi dosado Ca ionizado (Ca-i) em amostras de sangue total. Nos pacientes em que se constataram níveis de Ca-i acima do limite de normalidade )>1,29 mmol/L), foram realizadas dosagens de paratormônio (PTH) sérico e AMP cíclico urinário para se afastar um possível hiperparatiroidismo como causa da hipercalcemia. Encontramos níveis de Ca-i elevados (variando de 1,3 a 2,0 mmol/L em 18 destes pacientes (20//), sendo: 12 CEC (66,7//), 3 AdenoCa (16,7//), 2 CIPC (11,1//) e 1 TGC (5,6//). O PTH estava baixo ou suprimido em todos os pacientes hipercalcêmicos, afastando, desta forma, um hiperparatiroidismo. A dosagem de AMP cíclico urinário näo se mostrou útil no diagnóstico diferencial, estando elevada em 6 dos 12 pacientes avaliados. Concluímos ser hipercalcemia um achado bastante comum em pacientes portadores de tumores de pulmäo, principalmente dentre os carcinomas espinocelulares, sendo seu diagnóstico sindrômico e etiológico de grande interesse para melhor tratamento desses pacientes

[Prevalence of hypercalcemia in patients with lung cancer]. / Prevalência de hipercalcemia em pacientes portadores de câncer de pulmão.

Although hypercalcemia is a serious and frequent complication of lung cancer, it is not commonly investigated in our country. We studied the prevalence of hypercalcemia in 90 random lung cancer patients from the Department of Pneumology, Escola Paulista de Medicina. The following histological types were found: 35 Squamous cells carcinomas (CEC), 30 Adenocarcinomas (AdenoCa), 11 Small cells carcinomas (CIPC), 2 Large cells carcinomas (TGC), 1 Carcinoid, 1 Mesothelioma, 2 Undifferentiated carcinomas, 1 Adenosquamous, 1 in situ carcinoma and 4 metastatic tumors. Ionized Ca (Ca-i) was measured in blood samples of all patients. Hyperparathyroidism was excluded by PTH and cAMP determinations in the hypercalcemic patients (Ca-i > 1.29 mmol/L). We found elevated levels of Ca-i (range = 1.30 to 2.0 mmol/L) in 18 patients (20%), being: 12 CEC (66.7%), 3 AdenoCa (16.7%), 2 CIPC (11.1%) and 1 TGC (5.6%). The PTH levels were low or suppressed in all 18 patients, but cAMP determinations were elevated in 6 out of 12 patients. Hypercalcemia is then a very frequent complication of lung cancer (20%), and PTH measurement was able to exclude a hyperparathyroidism in all cases studied.

Natural and synthetic DNA elements with the CArG motif differ in expression and protein-binding properties.

DNA elements with the CC(A/T)6GG, or CArG, motif occur in promoters that are under different regulatory controls. CArG elements from the skeletal actin, c-fos, and myogenin genes were tested for their abilities to confer tissue-specific expression on reporter genes when the individual elements were situated immediately upstream from a TATA element. The c-fos CArG element, also referred to as the serum response element (SRE), conferred basal, constitutive expression on the test promoter. The CArG motif from the myogenin gene was inactive. The skeletal actin CArG motif functioned as a muscle regulatory element (MRE) in that basal expression was detected only in muscle cultures. Muscle-specific expression from the 28-bp MRE and the 2.3-kb skeletal actin promoter was trans repressed by the Fos and Jun proteins. The expression and factor-binding properties of a series of synthetic CArG elements were analyzed. Muscle-specific expression was conferred by perfect 28-bp palindromes on the left and right halves of the skeletal actin MRE. Chimeric elements of the skeletal actin MRE and the c-fos SRE differed in their expression properties. Muscle-specific expression was observed when the left half of the MRE was fused to the right half of the SRE. Constitutive expression was conferred by a chimera with the right half of the MRE fused to the left half of the SRE and by chimeras which exchanged the central CC(A/T)6GG sequences. At least three distinct proteins specifically bound to these CArG elements. The natural and synthetic CArG elements differed in their affinities for these proteins; however, muscle-specific expression could not be attributed to differences in the binding of a single protein. Furthermore, the MRE did not bind MyoD or the myogenin-E12 heterodimer, indicating that muscle-specific expression from this element does not involve a direct interaction with these helix-loop-helix proteins. These data demonstrate that the conserved CArG motifs form the core of a family of functionally different DNA regulatory elements that may contribute to the tissue-specific expression properties of their cognate promoters.

Pulsus paradoxus: a definition revisited.

Pulsus paradoxus is associated with many clinical conditions and is defined as a greater than 10 mm Hg end-inspiratory decrease in systolic blood pressure. Kussmaul's original definition of pulsus paradoxus is presented, along with an explanation of his choice of the term "pulsus paradoxus." A case of pulsus paradoxus is graphically described using simultaneous two-dimensional targeted M-mode, Doppler echocardiographic, and high-fidelity pressure recordings.

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements.

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.