Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.

A Ku-band active transmit-array module with a horn or patch array as a signal launching/receiving device

This paper is concerned with the design of a Ku-band active transmit-array module of transistor amplifiers excited by either a pyramidal horn or a patch array Optimal distances between the active transmit array and the signal-launching:receiving device, which is either a passive corporate-fed array or a horn, are determined to maximise the power gain at a design frequency: Having established these conditions, the complete structure is investigated in terms of operational bandwidth and near-field and far-field distributions measured at the output side of the transmit array, The experimental results show that the use of a corporate-fed array as an illuminating/receiving device gives higher gain and significantly larger operational bandwidth, An explanation for this behavior is sought.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Clinical trials with glycoprotein IIB/IIIA antagonists - No benefit without bleeding?

As the glycoprotein GPIIb/IIIa receptor is the final common pathway in platelet aggregation, antagonists of this receptor cause a profound inhibition of aggregation induced by any agonist. The short-term efficacy and safety of GPIIb/IIIa antagonists in patients undergoing coronary angioplasty was demonstrated with murine 7E3 Fab, but this antibody was immunogenic. Abciximab is a chimeric human-mouse monoclonal antibody that is less immunogenic. The first major trial with a GPIIb/IIIa antagonist was the EPIC trial with abciximab, which showed that abciximab reduced the ischemic complications of coronary balloon angioplasty and atherectomy in high-risk patients, but increased the risk of bleeding. Subsequent studies showed that using less concurrent heparin reduced bleeding. Abciximab also reduced the rate of revascularization. Further studies have shown that the benefits of abciximab extended to all patients undergoing angioplasty (EPILOG), including patients with unstable angina (CAPTURE) and acute myocardial infarction (RAPPORT). Clinical trials with eptifibatide and tirofiban have failed to demonstrate benefit, at the doses used, in angioplasty. Abciximab and eptifibatide, but not oral xemilofiban, improve the safety of the coronary stenting procedure. Shortterm intravenous treatment with lamifiban, eptifibatide or tirofiban is beneficial in acute coronary syndromes (unstable angina, non-Q wave myocardial infarction). Orally active GPIIb/IIIa antagonists are being developed for use in acute coronary syndromes and myocardial infarction. However, no benefit has been shown with lefradafiban in acute coronary syndromes and sibrafiban and orbofiban are harmful. Eptifibatide, lamifiban and abciximab improve coronary patency in myocardial infarction, and long-term trials of GPIIb/IIIa antagonists are being conducted in acute myocardial infarction. Abciximab can cause thrombocytopenia, and all the GPIIb/IIIa antagonists increase the incidence of bleeding, but there is no excess of intracranial hemorrhage. (C) 2001 Prous Science. All rights reserved.

Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design. (C) 2002 Elsevier Science (USA). All rights reserved.

Deep and superficial fibers of the lumbar multifidus muscle are differentially active during voluntary arm movements

AB Study Design. A cross-sectional study was conducted. Objective. To determine the activity of the deep and superficial fibers of the lumbar multifidus during voluntary movement of the arm. Summary of Background Data. The multifidus contributes to stability of the lumbar spine. Because the deep and superficial parts of the multifidus are near the center of lumbar joint rotation, the superficial fibers are well suited to control spine orientation, and the deep fibers to control intervertebral movement. However, there currently are limited in vivo data to support this distinction. Methods. Electromyographic activity was recorded in both the deep and superficial multifidus, transversus abdominis, erector spinae, and deltoid using selective intramuscular electrodes and surface electrodes during single and repetitive arm movements. The latency of electromyographic onset in each muscle during single movements and the pattern of electromyographic activity during repetitive movements were compared between muscles. Results. With single arm movements, the onset of electromyography in the erector spinae and superficial multifidus relative to the deltoid was dependent on the direction of movement, but the onset in the deep multifidus and transversus abdominis was not. With repetitive arm movements, peaks in superficial multifidus and erector spinae electromyography occurred only during flexion for most subjects, whereas peaks in deep multifidus electromyography occurred during movement in both directions. Conclusions. The deep and superficial fibers of the multifidus are differentially active during single and repetitive movements of the arm. The data from this study support the hypothesis that the superficial multifidus contributes to the control of spine orientation, and that the deep multifidus has a role in controlling intersegmental motion. (C) 2002 Lippincott Williams & Wilkins, Inc.

Ductile fabrics in the zone of active oblique convergence near the Alpine Fault, New Zealand: identifying the neotectonic overprint

The mid-crustal Alpine Schist in central Southern Alps, New Zealand has been exhumed during the past similar to3 m.y. on the hanging wall of the oblique-slip Alpine Fault. These rocks underwent ductile deformation during their passage through the similar to 150-km-wide Pacific-Australia plate boundary zone. Likely to be Cretaceous in age, peak metamorphism predates the largely Pliocene and younger oblique convergence that continues to uplift the Southern Alps today. Late Cenozoic ductile deformation constructively reinforced a pre-existing fabric that was well oriented to accommodate a dextral-transpressive overprint. Quartz microstructures below a recently exhumed brittle-ductile transition zone reflect a late Cenozoic increment of ductile strain that was distributed across deeper levels of the Pacific Plate. Deformation was transpressive, including a dextral-normal shear component that bends and rotates a delaminated panel of Pacific Plate crust onto the oblique footwall ramp of the Alpine Fault. Progressive ductile shear in mylonites at the base of the Pacific Plate overprints earlier fabrics in a dextral-reverse sense, a deformation that accompanies translation of the schists up the Alpine Fault. Ductile shear along that structure affects not only the 12-km-thick section of Alpine mylonites, but is distributed across several kilometres of overlying nonmylonitic rocks. (C) 2001 Elsevier Science Ltd. All rights reserved.

Surface-active phospholipid in total hip arthroplasty

This is the first report of surface-active phospholipid as the boundary lubricant in total hip arthroplasty. Aspirate and rinsings from the bearing surfaces of 25 revision operations and one primary surgery were analyzed from 3 weeks to 26 years postoperatively. All samples contained substantial amounts of surface-active phospholipids ranging from 14 to 4186 μg. These findings indicate that synoviocytes continue producing the lubricant in significant quantities after arthroplasty surgery independent of the type of joint replacement and its fixation. Surface-active phospholipid was found on all bearing surfaces analyzed including polyethylene, stainless steel, chrome cobalt, alumina, zirconia, and titanium.