Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Synthesis and characterization of block copolymers from D,L-lactide and poly(tetramethylene ether glycol)

The copolymer from D ,L -lactide and poly(tetramethyene ether glycol) (PTMG) was prepared in bulk with an isotributyl aluminum–water–phosphoric acid complex catalyst as the initiator and characterized by H-NMR, GPC, and DSC. The effects of the temperature and the amount of PTMG on the polymerization rate and the molecular weight of copolymers were studied. The behavior of the degradation and delivery rate of Levonorgestrel microspheres in vitro was observed. The results show that the degradation and the delivery rate can be controlled by adjusting the molar rate of hydrophilic and hydrophobic segments of the copolymer © 1995 John Wiley & Sons, Inc.     

Vascular manifestations in systemic sclerosis (scleroderma)

Progressive systemic sclerosis (PSS), a disease still of unknown origin, is a generalized autoimmune disorder characterized by immunological abnormalities, microvascular dysfunction, and tissue fibrosis. The mechanism leading to selective microvascular injury in PSS is not completely known, however it is now clear that neuropeptides, vascular endothelium, and disturbances in the regulation of fibroblast function are the three major contributors to the increased fibrosis of skin and internal organs. Thus, endothelial cell and fibroblast dysfunction may be linked through the paracrine activity of soluble endothelial cell products: the cytokine cascade (IL-1, TGF-beta-1, PDGF, TNF, etc.). In systemic sclerosis, the exaggerated generalized vasospastic tendency is clinically represented by Raynaud's phenomenon as shown by an early digital arterial closure after cold stimulation, and by an inadequate vasodilatory response to heat. In this review we summarize recently established data that center around the role of adhesion molecules, immune reactions, and aberrant fibroblast biology and metabolism in effecting vascular and connective tissue alterations in this disease. Only a better knowledge of the pathophysiological process involved in scleroderma might lead to the development of new therapeutic approaches.     

Ion microbeam radiation system

An ion microbeam radiation test system has been built for studying radiation-induced charge collection and single event upsets in advanced semiconductor circuits. With this system, it is possible to direct an ion beam of a diameter as small as 1 μm onto a circuit or test structure with a placement accuracy of 1 μm. The components of the system and its operation are described. Applications are described which demonstrate the capabilities of the system     

Spin coating over topography

A model for predicting film thickness profiles around topographical features during spin coating is presented. This model is applicable to features of arbitrary geometry in the two lateral dimensions. This generally permits study of the planarization of real device structures, including both isolated and neighboring features, with any orientation with respect to the wafer center. Predictions from this model agree qualitatively with measured thin-film profiles from interferograms taken during spinning. Phenomena such as pile-up and wakes result from interactions between surface tension and other driving forces in the flow