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.

Wolbachia-mediated sperm modification is dependent on the host genotype in Drosophila

Estimates of Wolbachia density in the eggs, testes and whole flies of drosophilid hosts have been unable to predict the lack of cytoplasmic incompatibility (CI) expression in so-called mod(-) variants. Consequently, the working hypothesis has been that CI expression, although related to Wolbachia density, is also governed by unknown factors that are influenced by both host and bacterial genomes. Here, we compare the behaviour of the mod(-) over-replicating Wolbachia popcorn strain in its native Drosophila melanogaster host to the same strain transinfected into a novel host, namely Drosophila simulans. We report that (i) the popcorn strain is a close relative of other D. melanogaster infections, (ii) the mod(-) status of popcorn in D. melanogaster appears to result from its inability to colonize sperm bundles, (iii) popcorn is present in the bundles in D. simulans and induces strong CI expression, which demonstrates that the bacterial strain does not lack the genetic machinery for inducing CI and that there is host-species-specific control over Wolbachia tissue tropism, and (iv) infection of sperm bundles by the mod(-) D. simulans wCof strain indicates that there are several independent routes by which a strain can be a CI non-expressor.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na+,K+-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors. (C) 2001 Wiley-Liss, Inc.

Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal robes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y

Objective. The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. Materials and Methods. A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. Results. We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1 HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PYlo and PYhi subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the (PYRhHolo/sp)-Rh-lo-Ho-lo PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PYhi) We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. Conclusion. Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the (RhHolosp)-Ho-lo PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Preprocessing and time-frequency analysis of newborn EEG seizures

Neurological disease or dysfunction in newborn infants is often first manifested by seizures. Prolonged seizures can result in impaired neurodevelopment or even death. In adults, the clinical signs of seizures are well defined and easily recognized. In newborns, however, the clinical signs are subtle and may be absent or easily missed without constant close observation. This article describes the use of adaptive signal processing techniques for removing artifacts from newborn electroencephalogram (EEG) signals. Three adaptive algorithms have been designed in the context of EEG signals. This preprocessing is necessary before attempting a fine time-frequency analysis of EEG rhythmical activities, such as electrical seizures, corrupted by high amplitude signals. After an overview of newborn EEG signals, the authors describe the data acquisition set-up. They then introduce the basic physiological concepts related to normal and abnormal newborn EEGs and discuss the three adaptive algorithms for artifact removal. They also present time-frequency representations (TFRs) of seizure signals and discuss the estimation and modeling of the instantaneous frequency related to the main ridge of the TFR.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.

The BRCA2 372 HH genotype is associated with risk of breast cancer in Australian women under age 60 years

The BRCA2 N372H nonconservative amino acid substitution polymorphism appears to affect fetal survival in a sex-dependent manner, and the HH genotype was found to be associated with a 1.3-fold risk of breast cancer from pooling five case-control studies of Northern European women. We investigated whether the BR 2 N372H polymorphism was associated with breast cancer in Australian women using a population-based case-control design. The BRCA2 372 genotype was determined in 1397 cases under the age of 60 years at diagnosis of a first primary breast cancer and in 775 population-sampled controls frequency matched for age. Case-control analyses and comparisons of genotype distributions were conducted using logistic regression. All of the statistical tests were two-tailed. The HH genotype was independent of age and family history of breast cancer within cases and controls, and was more common in cases (9.2% versus 6.5%). It was associated with an increased risk of breast cancer, 1.47-fold unadjusted (95% confidence interval, 1.05-2.07; P = 0.02), and 1.42-fold (95% confidence interval, 1.00-2.02; P = 0.05) after adjusting for measured risk factors. This effect was still evident after excluding women with any non-Caucasian ancestry or the 33 cases known to have inherited a mutation in BRCA1 or BRCA2, and would explain similar to3% of breast cancer. The BRCA2 N372H polymorphism appears to be associated with a modest recessively inherited risk of breast cancer in Australian women. This result is consistent with the findings for Northern European women.

Sites of dental erosion are saliva-dependent

Acid demineralization of teeth causes occlusal erosion and attrition and associated non-carious cervical lesions at sites relatively unprotected by saliva. Associations of occlusal pathology and cervical lesions were looked for in 450 patients with toothwear, and 174 subjects with cervical lesions were identified. Associations of occlusal attrition, or erosion, or no wear, with cervical lesions at 72 buccal and lingual sites were recorded from epoxy resin replicas of the subjects' dentitions (3241 teeth). Criteria used to discriminate occlusal erosion from attrition; and shallow from grooved and wedge-shaped cervical lesions were delineated by scanning electron microscopy (SEM). In the absence of occlusal pathology, cervical lesions were very rare (

Polarization squeezing and continuous-variable polarization entanglement

A concept of polarization entanglement for continuous variables is introduced. For this purpose the Stokes-parameter operators and the associated Poincare sphere, which describe the quantum-optical polarization properties of light, are defined and their basic properties are reviewed. The general features of the Stokes operators are illustrated by evaluation of their means and variances for a range of simple polarization states. Some of the examples show polarization squeezing, in which the variances of one or more Stokes parameters are smaller than the coherent-state value. The main object of the paper is the application of these concepts to bright squeezed light. It is shown that a light beam formed by interference of two orthogonally polarized quadrature-squeezed beams exhibits squeezing in some of the Stokes parameters. Passage of such a primary polarization-squeezed beam through suitable optical components generates a pair of polarization-entangled light beams with the nature of a two-mode squeezed state. Implementation of these schemes using the double-fiber Sagnac interferometer provides an efficient method for the generation of bright nonclassical polarization states. The important advantage of these nonclassical polarization states for quantum communication is the possibility of experimentally determining all of the relevant conjugate variables of both squeezed and entangled fields using only linear optical elements followed by direct detection.