Multiplexed, Tethered Particle Microscopy for Studies of DNA-Enzyme Dynamics.

DNA is the carrier of genetic information and, as such, is at the center of most essential cellular processes. To regulate its physiological function, specific proteins and motor enzymes constantly change conformational states with well-controlled dynamics. Twenty-five years ago, Schafer, Gelles, Sheetz, and Landick employed the tethered particle motion (TPM) technique for the first time to study transcription by RNA polymerase at the single-molecule level. TPM has since then remained one of the simplest, most affordable, and yet incisive single-molecule techniques available. It is an in vitro technique which allows investigation of DNA-protein interactions that change the effective length of a DNA tether. In this chapter, we will describe a recent strategy to multiplex TPM which substantially increases the throughput of TPM experiments, as well as a simulation to estimate the time resolution of experiments, such as transcriptional elongation assays, in which lengthy time averaging of the signal is impossible due to continual change of the DNA tether length. These improvements allow efficient study of several DNA-protein systems, including transcriptionally active DNA-RNA polymerase I complexes and DNA-gyrase complexes.

A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties.

The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.

Five-year results of a randomized trial comparing de novo sirolimus and cyclosporine in renal transplantation: the SPIESSER study.

Calcineurin inhibitors improve acute rejection rates and short-term graft survival in renal transplantation, but their continuous use may be deleterious. We evaluated the 5-year outcomes of sirolimus (SRL) versus cyclosporine (CsA) immunosuppressive treatment. This observational study was an extension of the SPIESSER study where deceased donor kidney transplant recipients were randomized before transplantation to a SRL- or CsA-based regimen and followed up 1 year. Data from 131 (63 SRL, 68 CsA) out of 133 patients living with a functional graft at 1 year were collected retrospectively at 5 years posttransplant. Seventy percent of CsA patients versus 54% of SRL patients were still on the allocated treatment at 5 years (p = 0.091), most discontinuations in each group being due to safety issues. In intent-to-treat, mean MDRD eGFR was higher with SRL: 54.2 versus 45.3 mL/min with CsA (p = 0.019); SRL advantage was greater in on-treatment analyses. There were no differences for patient survival (p = 0.873), graft survival (p = 0.121) and acute rejection (p = 0.284). Adverse events were more frequent with SRL (80% vs. 60%, p = 0.015). Results confirmed the high SRL discontinuation rate due to adverse events. Nevertheless, a benefit was evidenced on renal function in patients (more than 50%) still on treatment at 5 years.

Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins.

Proteins in bile may have important physiological functions and serve as disease biomarkers. Here, the protein composition of human gallbladder bile was analyzed using a recently described chromatography-like technology capable to enhance the signal of low-abundance species. First, proteins present in bile fluid were treated with immobilized peptide ligand libraries to concentrate dilute and very dilute species while concomitantly diluting the high-abundance proteins. The analysis of resulting protein mixture was then performed using LC-MS/MS after having classically separated proteins by a mini preparative gel electrophoresis. Overall 222 gene products were found; 143 of them were not reported before in proteomics studies. Ligand libraries by themselves contributed to find 81 new gene products distributed throughout different categories. The described chromatographic approach provides a significant contribution to the bile protein repertoire and opens new perspectives for the discovery of markers for specific biliary tract diseases.

Atomic force microscopy study of DNA deposited on poly L-ornithine-coated mica.

Analyses of individual biomolecules, like DNA, or DNA-protein complexes, via atomic force microscopy, require 'gentle' methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly-L-ornithine-coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B-form helical rise (0.34 nm bp(-1)). The conformations of DNA that result are reminiscent of three-dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.

[Oesophageal cancer in patients with head and neck cancers: therapeutic implications]. / Association cancers de l'oesophage et des voies aérodigestives supérieures : implications thérapeutiques.

AIM OF THE STUDY: To determine therapeutic and prognostic implications of an associated head and neck primary cancer in patients undergoing oesophagectomy for squamous cell carcinoma of the oesophagus. PATIENTS AND METHODS: Between 1982 and 2000, 868 patients with oesophageal cancer were operated in our institution, including 78 (9%) who underwent oesophagectomy for associated oesophageal and head and neck cancers; the latter was synchronous (n = 52) or anterior metachronous (n = 26). Influence of head and neck cancer on the treatment of oesophageal carcinoma was analysed retrospectively in terms of surgical therapeutic strategy and survival. RESULTS: Oesophageal resection consisted of oeso-pharyngolaryngectomy (n = 14, 17.9%), subtotal oesophagectomy (n = 62, 79.5%) and cervical oesophagectomy (n = 2, 2.6%). Radical resection (R0) was obtained in 85% of cases. Postoperative mortality rate was 5 % (4/78). Main complications were pulmonary (18% = 14/78) and anastomotic leaks (14% = 11/78), all of them cervical. Follow-up (mean = 25 +/- 27 months) was complete for all 78 patients. Five-year survival after R0 resection was 25%. Survival pronostic factors were denutrition, complete resection, and pT status of oesophageal tumor. CONCLUSION: In patients with associated carcinomas of oesophagus and head and neck, agressive treatment -including an oesophagectomy- allowed a 5-year survival rate more than 25% without increased mortality or morbidity rates, compared with patients operated on for isolated oesophageal carcinoma.

Labeling phosphorylated LHCII with microspheres for tracking studies and force measurements.

We report a method to selectively label phosphorylated, membrane proteins with microscopic particles. This technology is particularly useful in single particle studies. In such studies, the particles may serve to visualize protein diffusion and/or as 'handles' to study the force of interaction between the labeled protein and the membrane matrix. In the latter kind of experiments, forces can be applied and measured by calibrated optical tweezers. Optical tweezers were used in this work to test the strength of the particle labeling. Labeling a single protein with a particle produces a long-lived, distinct tag and is particularly useful for proteins in photosynthetic membranes, which contain endogenous fluorophores that would render single fluorescent proteins difficult to detect.

Three-dimensional magneto-optic trap for micro-object manipulation.

A magneto-optic trap for micro-objects is described. Magnetic beads were trapped by optical tweezers while being rotated by a new integrated magnetic manipulator. Rotation was achieved with eight electromagnets with tip-pole geometry. The time orbital potential technique was used to achieve rotation of magnetic beads. Trapping in three dimensions and rotation of magnetic beads on three axes are demonstrated with forces up to 230 pN and force momenta of up to 10(-16)N m . A position-detection apparatus based on an interferometric scheme provides nanometer sensitivities in a few milliseconds.

Superior vena cava syndrome of malignant origin. Which surgical procedure for which diagnosis?

OBJECTIVE: Since some malignancies causing superior vena cava syndrome (SVCS) are only sensitive to a specific treatment regimen, it is crucial to diagnose the underlying pathology in such cases. The aim of the present study was to review the surgical procedures used to establish the aetiology of SVCS of a malignant origin. METHODS: This retrospective study was based on a series of 88 patients referred to surgeons for SVCS, for whom biological and/or endoscopic procedures had failed to establish the diagnosis. On the basis of the results of clinical examination, biological tests and CT-scan presentation, we performed 99 sampling procedures to obtain a diagnosis for all 88 patients. These procedures were the following: biopsy of peripheral adenopathy (n=11), CT-guided biopsy (CTGB; n=23), axial mediastinoscopy (MDS; n=23), anterior mediastinotomy (n=26), anterior mediastinoscopy (n=6), biopsy of the suprascapular mass (n=3), pericardioscopy (n=3), thoracoscopy (n=1), thoracotomy (n=2) and sternotomy (n=1). RESULTS: Per-operative morbidity consisted of one case of massive venous bleeding during MDS requiring a salvage sternotomy to achieve hemostasis. The diagnoses finally established for the 88 patients were non-Hodgkin's lymphoma (NHL) for 36, small cell lung cancer for 25, non-small cell lung cancer for 17, Hodgkin's disease for five, thymoma for three, germ cell tumour for one and sarcoma for one. For the diagnosis of lung cancer, the sensitivities of CTGB and MDS were 85 and 100%, respectively. For the diagnosis of NHL, the sensitivity of anterior mediastinotomy was 95%. CONCLUSION: The surgical diagnostic procedure, chosen on the basis of the clinical presentation and CT-scan, can be performed safely in the case of SVCS, with the same accuracy as in the absence of this syndrome. Among the patients referred to surgeons, NHL is the most frequent aetiology of SVCS, together with small cell lung cancer.

Pericardoscopy for primary management of pericardial effusion in cancer patients.

OBJECTIVE: To assess the usefulness of pericardoscopy via the subxyphoid route for the diagnosis and treatment of pericardial effusion in patients with a history of cancer. METHODS: All patients with a recent or remote history of cancer and a pericardial effusion of unknown origin requiring drainage for diagnostic and therapeutic purposes were included in the study. They underwent complete exploration and cleansing of the pericardial cavity. Abnormal structures or deposits were biopsied under direct visual control, with a 24 cm long rigid pericardoscope. RESULTS: Between 1985 and 1998, pericardoscopy was completed in 112 of the 114 patients included (feasibility 98%), resulting in the immediate relief of symptoms in all the cases. Peri-operative mortality was 3.5%, and post-operative morbidity, 6.1%. After pericardioscopy pericardial effusions were considered malignant in 43 cases. One more case (2.3%) due to a false negative result of pericardioscopy was diagnosed during follow-up. Overall, 44 of the 114 patients (38.6%) had a malignant effusion, and 70 (61.4%), a non-malignant effusion according the follow up. In 10 of the 44 patients with a malignant pericardial effusion (22.7%), pericardoscopy corrected the results of cytological pericardial fluid studies and pericardial window biopsy, both false negatives. The sensitivities of cytological studies of the pericardial fluid, pathological examinations of pericardial window biopsy and pericardioscopy were 75, 65 and 97%, respectively. One patient with a malignant effusion had a non-symptomatic recurrence 1 month after pericardioscopy (2.3%). CONCLUSION: We recommend pericardioscopy to ascertain the malignant nature of the effusion and to diminish the recurrence rate, this avoiding repeat procedures in patients with a short life expectancy.

Thermal sensitivity of the red absorption tail of the photosystem II reaction center complex.

The red tail of the absorption spectrum of the D1-D2-cytb559 complex, defined as the absorption signal not described by the two Gaussian sub-bands associated with the intense electronic transitions at 680 and 683 nm, exhibits anomalous temperature behavior. This tail was analyzed in the temperature interval between 80 and 300 K in terms of the mean square deviation (sigma2) of the total Qy absorption band and by Gaussian sub-band decomposition. The value of the average optical reorganization energy (Snum) obtained from the temperature dependence of sigma2 for the whole absorption band was 32 cm(-1), and changed to 16-20 cm(-1) after subtraction of the sub-bands describing the red tail. This latter value is in agreement with the hole burning literature data for chlorophyll bound to proteins, and indicates that the rather high value for the apparent optical reorganization energy obtained by analysis of the total Qy band of the D1-D2-cytb559 complex is determined by the temperature sensitivity of the red tail. This suggests that the long wavelength absorption tail might be due to vibrational transitions associated with vibrational modes in the range of 80-150 cm(-1) which are thermally accessible and give rise to an absorption signal on the low-energy side of the (0,0) transition. On the basis of this assumption, the electron-phonon coupling strength (S) for these modes is estimated to be in the range 0.028-0.18. This interpretation furthermore supports the idea that the electronic transition near 683 nm is that of a monomer chlorophyll.

[Clinical course of patients treated with partial left ventriculectomy]. / Evolução clínica de pacientes submetidos à ventriculectomia esquerda parcial.

OBJECTIVE: To assess the efficacy of partial left ventriculectomy as a treatment for patients with end-stage heart failure. METHODS: From February to June 1995, 7 patients with end-stage heart failure underwent partial left ventriculectomy. Subsequently, patients underwent clinical evaluation every 2 months, and 2-dimensional echocardiography at the 6th and 12th months after cardiac surgery. All patients were given digitalis and diuretics at conventional doses, and captopril or enalapril at maximal tolerated doses. RESULTS: Two (28%) patients died; 1 from cardiac arrhythmia associated with gastrointestinal hemorrhage, and the other suddenly. One (14%) patient developed an embolic cerebrovascular accident. Four (57%) patients were hospitalized for congestive heart failure; all of them had either decreased the daily dose of captopril or enalapril or discontinued the drugs by themselves. Twelve months after ventriculectomy, left ventricular ejection fraction values were greater and left ventricular diastolic dimension and functional class values lower than those found before cardiac operation. CONCLUSION: Beneficial effects of partial left ventriculectomy are observed one year after the surgical procedure. This technique, therefore, can be useful for the treatment of patients with end-stage heart failure.

[Atrial septal defect percutaneous transvenous occlusion with the buttoned device]. / Oclusão percutânea transvenosa de comunicação interatrial mediante utilização do buttoned device.

PURPOSE: To analyse the experience with the use of Sideris' device for percutaneous transvenous occlusion of atrial septal defect (ASD). METHODS: Thirty six procedures of ASD occlusion were performed in 32 patients; 27 (84.4%) female and five (15.6%) male, mean age of 16 years (4-61). All patients had secundum atrial septal defects with clinical and hemodynamic compromise. The ASD diameter measured < 31 mm and occupied less than 50% of the septal length in all cases. The basic device was used in 28 (87.5%) patients and the self centered model in four (12.5%). The direct implant technique was employed in 15 (41.7%) procedures while in the other 21 (58.3%) an over a wire technique was used. The size of the device was selected according with the Sideris' normogram. RESULTS: Occlusion of the defect was achieved in 29 patients (90.63%). In two (6.25%) the position of the device was not adequate and in one (3.12%) the device embolized to the pulmonary artery. In these three cases the device was removed surgically during the defect closure. There was neither morbidity nor mortality in this series. Trivial or small residual shunt was detected with color flow mapping in three (9.4%) patients immediately after the procedure. In 22 patients with 12 months follow-up, trivial residual shunt was present in only one (4.5%). Structural modification of the device was detected in one patient, however with complete ASD occlusion and neither clinical nor hemodynamic disturbance. CONCLUSION: The Sideris' device is safe and efficient for ASD occlusion in selected patients. Although there is a relatively high incidence of residual shunt immediately after the procedure, the shunt itself tends to become smaller or disappear during the follow-up and it does not preclude clinical and hemodynamic improvement.

[Initial experience with partial left ventriculectomy as a treatment for end-stage heart failure]. / Experiência inicial com a ventriculectomia parcial esquerda no tratamento da insuficiência cardíaca terminal.

PURPOSE: To evaluate the efficacy of left ventriculectomy, on a short term basis, as a treatment for patients with end-stage heart failure. METHODS: From February to June 1995, 7 patients with end-stage heart failure underwent partial left ventriculectomy. Before the surgical procedure, 7 (100%) patients were in functional class IV. Three (42%) patients needed inotropic support for hemodynamic stability. The mean daily dose of furosemide was l48.67 +/- 128.27 mg, of captopril 87.50 +/- 95.20 mg and of digoxin 0.23 +/- 0.04. Mean left ventricular diastolic dimension determined by 2-D echocardiography was 78.29 +/- 12.63 mm, mean left ventricular ejection fraction, determined by radionuclide ventriculography, was 0.15 +/- 0.05 whereas mean transpulmonary gradient and pulmonary vascular resistance in Wood units, determined by right heart catheterization, were 16.80 +/- 8.80 and 6.57 +/- 3.22, respectively. RESULTS: Sixty days after the surgery, the mean functional class was 1.71 +/- 0.48 (p = 0.009), the mean left ventricular diastolic dimension 64.67 +/- 11.41 mm (p = 0.02) and the mean left ventricular ejection fraction 0.22 +/- 0.04 (p = 0.02). CONCLUSION: The left ventriculectomy is a promising treatment for patients with end-stage heart failure.

[Complete heart block after percutaneous mitral valvotomy with Inoue balloon]. / Bloqueio atrioventricular completo após valvoplastia mitral percutânea com balão de Inoue.

We report a permanent complete heart block, in a 27 year-old female patient, developed during mitral percutaneous valvotomy using a single inflation of the Inoue balloon catheter. After one month, even using corticoids, a Mobitz II second-degree atrioventricular block persisted, the patient became symptomatic to moderate efforts and a cardiac pacemaker was implanted.

Bloqueio atrioventricular completo após valvoplastia mitral percutânea com baläo de Inoue / Complete Hear Block after Percutaneous Inoue Balloon Mitral Valvotomy

A valvoplastia mitral percutânea (VMP) tem sido utilizada como tratamento näo cirúrgico para pacientes selecionados portadores de estenose mitral, sendo uma laternativa à comissurotomia cirúrgica. Complicaçöes maiorese da VMP incluem: morte durante o procedimento, perfuraçäo cardíaca, embolismo sistêmico (causando acidente vascular cerebral, infarto do miocárdio ou êmbolo periférico), e lesäo valvar grave resultando em regurgitaçäo mitrla. Complicaçöes menores incluem: criaçäo de comunicaçäo interaial, reflexos vaso-vagais, lesäo vascular ou snagramento nos locais de inserçào dos cateteres, taquiarrtimais atriais e ventriculares, e distúrbios de conduçäo temporários. Descreve-se o caso de uma paciente que desenvolveu bloqueio atrioventricular total (BAVT) permanente após valvoplastia mitral percutânea utilizando-se o cateter-baläo de Inoue. Os disturbios da conduçäo cardíaca neste procedimento näo säo infrequentes, sendo em geral transitórios. O levantamento da bibliografia médica, täo extenso quno possível, revelou relatos de apenas dois casos de BAVT permanente como complicaçäo da VMP. Um destes pacientes foi tratado com a técnica do duplo baläo, o outro tratado com o baläo de Inoue, e ambos necessitaram de implante de marcapasso cardíaco.

Slow exciton trapping in Photosystem II: A possible physiological role.

Photosystem II, which has a primary photochemical charge separation time of about 300 ps, is the slowest trapping of all photosystems. On the basis of an analysis of data from the literature this is shown to be due to a number of partly independent factors: a shallow energy funnel in the antenna, an energetically shallow trap, exciton dynamics which are partly 'trap limited' and a large antenna. It is argued that the first three of these properties of Photosystem II can be understood in terms of protective mechanisms against photoinhibition. These protective mechanisms, based on the generation of non photochemical quenching states mostly in the peripheral antenna, are able to decrease pheophytin reduction under conditions in which the primary quinone, QA, is already reduced, due to the slow trapping properties. The shallow antenna funnel is important in allowing quenching state-protective mechanisms in the peripheral antenna.

A thermal broadening analysis of absorption spectra of the D1/D2/cytochrome b-559 complex in terms of Gaussian decomposition sub-bands.

Absorption spectra of the isolated D1/D2/cytochrome b-559 complex have been measured in the temperature range 80-300 K. All spectra were analyzed in terms of a linear combination of Gaussian bands and the thermal broadening data interpreted in terms of a model in which the spectrum of each pigment site is broadened by (a) a homogeneous component due to linear electron-phonon coupling to a low-frequency protein vibration and (b) an inhomogeneous component associated with stochastic fluctuations at each pigment site. In order to obtain a numerically adequate description of the absorption spectra, a minimum number of five sub-bands is required. Further refinement of this sub-band description was achieved by taking into account published data from hole burning and absorption difference spectroscopy. In this way, both a six sub-band description and a seven sub-band description were generated. In arriving at the seven sub-band description, the original five sub-band wavelength positions were essentially unchanged. Thermal broadening analysis of the seven sub-band description yielded data which displayed the closest correspondence with the literature observations. The wavelength positions of the sub-bands were near 661, 667, 670, and 675 nm, with two bands near 680 and 684 nm. The two almost isoenergetic sub-bands near 680 nm, identified as P680 and pheophytin, have optical reorganization energies around 40 and 16 cm-1, respectively. All other sub-bands, identified as accessory pigments, have optical reorganization energies close to 16 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)