Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Maintenance of delay of gratification by four chimpanzees (Pan troglodytes): the effects of delayed reward visibility, experimenter presence, and extended delay intervals

Previous research in our laboratory has demonstrated that chimpanzees can delay gratification by inhibiting consumption of available food items for as long as 3 min as an experimenter transfers additional food items from a transparent container to a bowl placed in front of the subject. In this study, we examined the influence of the visibility of the food source, as well as the presence of the experimenter, on four chimpanzees' self-control in this paradigm. In Experiment 1 an experimenter transferred 15 preferred food items between a distant opaque container and a bowl placed in front of the subject. In Experiment 2 we tested the chimpanzees with an automated system that (in the absence of the experimenter) transferred up to 36 highly preferred food items from a universal food dispenser to a container located either inside or outside of the subject's enclosure. There were no differences in self-directed behaviors or attentiveness to the food items between the self-imposed and externally imposed delay conditions. A final experiment with the automated paradigm indicated that individuals could delay gratification for up to 11min in order to obtain all 36 food items.     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Establishing a Mechanistic Basis for the Large Kinetic Steps of the NS3 Helicase

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to ∼11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.The DEx(H/D) proteins represent a large and ubiquitous family of putative RNA helicases (1, 2). The members of this highly conserved protein family are present in the vast majority of organisms, from viruses to humans, and are involved in virtually every known aspect of RNA metabolism (3). Despite the importance of these proteins, only a small fraction of them has been well characterized, and the mechanism of their action is poorly understood. Although they are usually called “RNA helicases” because of the NTP-dependent RNA unwinding activity displayed by many DEx(H/D) proteins in vitro, other activities, such as ribonucleoprotein remodeling and RNA strand annealing, have been described for several proteins of the family, and these activities may be relevant to their functions in vivo (2, 4-6). The physiological functions and targets of most DEx(H/D) proteins remain unknown.The nonstructural protein 3 (NS3) from hepatitis C virus (HCV)³ possesses robust RNA and DNA unwinding activities and is a prototypical member of the DEx(H/D) family of ATPase proteins (7). NS3 is an essential component of the HCV replication machinery, and it is an important drug target for anti-HCV therapy. NS3 is one of the most exhaustively studied RNA helicases, and a wealth of structural and biochemical information is available for this enzyme (8-10). Ensemble and single molecule studies have established that NS3 is a processive helicase, capable of making multiple unwinding steps of well defined size without dissociating from the substrate (11, 12). This stepping behavior involves alternating pauses and rapid unwinding events and is similar to the behavior displayed by cytoskeletal motor proteins. Despite these important findings, the molecular mechanism of NS3 unwinding remains largely obscure. The pauses are likely to be caused by the necessity to reset the conformation of the helicase-RNA complex after each unwinding step, consistent with an inch-worm model of unwinding. However, the mechanism by which pauses are triggered and the role of ATP in this process are not understood. The number of base pairs unwound by NS3 per step appears to be one of the largest reported for helicases to date, and it significantly exceeds the footprint of a monomeric NS3 bound to an RNA substrate (6-8 nt) (8). The structural basis for these large steps is unknown, although it has been suggested that it may be related to the oligomeric state of NS3 in its active form (12).Kinetic methods have long been employed to study helicase activity, and they provide essential tools for dissecting the mechanism of helicase function (13-17). One of the fundamental parameters that can be measured is the kinetic step size, which is defined as the number of base pairs unwound per rate-limiting step. The kinetic step size is often reflected in a periodic mode of unwinding that is commonly displayed by processive helicases that make multiple repetitive unwinding steps before dissociating from the nucleic acid substrate (13).To characterize the mechanism of RNA unwinding by NS3 and to develop a physical explanation for its unusual stepping behavior, we systematically studied the dependence of the NS3 kinetic step size on duplex stability and ionic conditions. We find that the step size of NS3 shows no dependence on duplex stability or sequence, in contrast to its processivity and unwinding rate constant, which have been previously shown to strongly depend on duplex stability (18, 19). However, we observe that the apparent kinetic step size of RNA unwinding by NS3 is sensitive to monovalent salt concentration and is reduced to ∼11 bp under low salt conditions. We address this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas NS3 oligomers are capable of processive unwinding, we find that monomeric NS3 is an active helicase that possesses low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3 and are likely to account for the changes in kinetic step size of NS3 under low salt conditions. Combining these results with data from previous studies, we propose a physical unwinding mechanism that explains the kinetic step size and the diverse kinetic behaviors that are displayed by NS3.     

Influence of inverse dynamics methods on the calculation of inter-segmental moments in vertical jumping and weightlifting

Background  

A vast number of biomechanical studies have employed inverse dynamics methods to calculate inter-segmental moments during movement. Although all inverse dynamics methods are rooted in classical mechanics and thus theoretically the same, there exist a number of distinct computational methods. Recent research has demonstrated a key influence of the dynamics computation of the inverse dynamics method on the calculated moments, despite the theoretical equivalence of the methods. The purpose of this study was therefore to explore the influence of the choice of inverse dynamics on the calculation of inter-segmental moments.     

Comparison of the conventional culture,the manual fluorescent MGIT system and the automated fluorescent MGIT 960 culture system for the detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> ssp. <Emphasis Type="Italic">avium</Emphasis> in tissues of naturally infected hens

Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold’s and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).     

Ensemble attribute profile clustering: discovering and characterizing groups of genes with similar patterns of biological features

Background  

Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.