Temperature-Dependent Expression of a Squid Kv1 Channel in Sf9 Cells and Functional Comparison with the Native Delayed Rectifier

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) α-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (I _K) if cultured at the standard postinfection temperature of 27°C. At 18°C, less SqKv1A protein is produced than at 27°C, but cells with I _K currents are much more numerous and can survive for at least 20 days postinfection (vs. ∼5 days at 27°C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (∼3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of I _K inactivation in 20 mm external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL I _K is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.     

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.     

Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.     

Assessing transportation by the covariance of species with comments on contagious and random distributions

The paleoecologist must be able to distinguish a transported assemblage from an in situ one. A method is proposed to assess whether the post-mortem transport of individuals affected their observed spatial (horizontal) distribution. The spatial distribution of a species can be random or contagious. The spatial distribution of individuals of a species in the death assemblage produced by the cumulation of many temporally discrete inputs will be random if the individual inputs are random and contagious if the inputs are contagious. The spatial distribution patterns of several species should not covary in the absence of physical disturbance regardless of their own distributions, however. The degree of covariance between individuals of several species of similar hydrodynamic propensity is dependent on the amount and intensity of postmortem movement. The more species that covary, and the larger the size classes that covary, the more likely that transportation played an important role in the species' distribution patterns. Conversely, the absence of covariance suggests that, for at least some species, biological factors determined the species' spatial distributions. Similarly, covariance of vertical distribution patterns might suggest homogenization. by bioturbational or physical mixing.     

5-lipoxygenase and FLAP

The initial steps in the biosynthesis of leukotrienes from arachidonic acid are carried out by the enzyme 5-lipoxygenase (5-LO). In intact cells, the helper protein 5-LO activating protein (FLAP) is necessary for efficient enzyme utilization of endogenous substrate. The last decade has witnessed remarkable progress in our understanding of these two proteins. Here we review the molecular and cellular aspects of the expression, function, and regulation of 5-LO and FLAP.     

Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice

Lactoferrin (Lf) is an iron-binding protein of external secretions and neutrophil secondary granules with antimicrobial and immunomodulatory activities. To further define these properties of Lf, we have investigated the response to Staphylococcus aureus infection in transgenic mice carrying a functional human Lf gene. The transgenic mice cleared bacteria significantly better than congenic littermates, associated with a trend to reduced incidence of arthritis, septicemia, and mortality. We identified two pathways by which S. aureus clearance was enhanced. First, human Lf directly inhibited the growth of S. aureus LS-1 in vitro. Second, S. aureus-infected transgenic mice exhibited enhanced Th1 immune polarization. Thus, spleen cells from infected transgenic mice produced higher levels of TNF-alpha and IFN-gamma and less IL-5 and IL-10 upon stimulation ex vivo with the exotoxin toxic shock syndrome toxin-1 compared with congenic controls. To confirm that these effects of Lf transgene expression could occur in the absence of live bacterial infection, we also showed that Lf-transgenic DBA/1 mice exhibited enhanced severity of collagen-induced arthritis, an established model of Th1-induced articular inflammation. Higher levels of stainable iron in the spleens of transgenic mice correlated with human Lf distribution, but all other parameters of iron metabolism did not differ between transgenic mice and wild-type littermates. These results demonstrate that human Lf can mediate both antimicrobial and immunomodulatory activities with downstream effects on the outcome of immune pathology in infectious and inflammatory disease.     

Intracellular concentration measurements in adherent cells: a comparison of import efficiencies of cell-permeable peptides

A protocol was developed for performing intracellular concentration measurements in flat adherent tissue culture cells by fluorescence correlation microscopy (FCM). Determination of the number of molecules in the confocal detection volume had to account for background fluorescence caused by molecules adsorbed to the surface of the measurement chamber. Such a background signal leads to a decrease in the amplitude of the autocorrelation function, and thereby to the calculation of an erroneously high number of molecules. Because of the spatial heterogeneity of the background intensity, a method was devised by which its contribution to the total fluorescence could be determined directly from each individual autocorrelation measurement. This method was applied to a comparison of the import efficiencies of different cell-permeable peptides at nanomolar concentrations. The Antennapedia homeodomain-derived peptide penetratin was imported about three times as efficient as the basic fibroblast growth factor-derived MTS peptide. Both peptides equilibrated between cytoplasm and nucleus. A relatively high mobility of these molecules inside the cells indicated that they may be rapidly degraded by cytosolic proteases. Based on these results, it will be possible to determine intracellular concentrations of inhibitors linked to import peptides directly by FCM at nanomolar concentrations and to optimise such constructs for proteolytic stability.     

Cells respond to and bind countin,a component of a multisubunit cell number counting factor

In Dictyostelium discoideum counting factor (CF), a secreted approximately 450-kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups, with an EC(50) of approximately 3 ng/ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1-min exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway. (125)I-Labeled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have approximately 53 cell-surface sites that bind countin with a K(D) of approximately 1.5 ng/ml or 60 pm. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities.     

A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import

Cell-permeable peptides were evaluated for a quantitatively controlled import of small molecules. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochemical characteristics of the fluorophores, the import efficiencies for analogues labelled at different positions within the sequence of the import peptides showed a strong positive correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were observed. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the determination of the intracellular concentrations for each construct individually.