Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (I) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.     

Autonomic dysfunction as the presenting feature of Guillain-Barré syndrome

Autonomic dysfunction has been demonstrated in various conditions associated with peripheral neuropathy such as acute intermittent porphyria, amyloidosis, and Guillain-Barré syndrome (GBS). In the latter, hypertension is an associated complication that typically occurs after neurological signs are already present. We report a case of a patient with autonomic dysfunction as the presenting feature who was admitted to the coronary unit with chest pain and hypertension. Subsequently, he developed progressive symmetric muscle, weakness, sensory changes, and areflexia. GBS was then diagnosed based on the clinical picture, albuminocytologic dissociation in the cerebrospinal fluid, and electrodiagnostic abnormalities suggestive of demyelinative polyneuropathy with conduction block. Few cases in the literature have reported autonomic dysfunction as the presenting feature of GBS, such as in this case. In a previously asymptomatic patient, acute onset of autonomic dysfunction should alert the physician to the possibility of an acute polyneuropathy, such as GBS.     

A five year follow up into changes in caries experience amongst a sample of 12 year old children from Athens

The airway functions in pregnancy have been widely studied but reports obtained from Western and Indian population show divergence. While the Indian populations show significant changes in total and timed vital capacity (FVC and FEV1), the Western counterparts dismiss such changes as insignificant. Our results show insignificant alteration in airway function and support the results reported for Western population.     

Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.     

Avian adenovirus induces ion channels in model bilayer lipid membranes

The action of duck egg drop syndrome 1976 (EDS-76) adenovirus on model bilayer lipid membranes (BLM) has been investigated on planar egg phosphatidylcholine membranes and small unilamellar vesicles. It was found that the adenovirus formed channels in planar BLM in a pH-dependent manner. The addition of EDS-76 to planar BLM at pH 5 induced voltage-independent channel activity of about 60 pS conductivity after a lag phase. At pH 3, EDS-76 induced irregular spikes of current across the planar BLM which disappeared after several minutes. The adenovirus also was able to induce pH-dependent leakage of calcein-loaded liposomes. EDS-76 did not induce channel activity in planar BLM or liposome leakage at neutral pH.