ClC-3 and IClswell are required for normal neutrophil chemotaxis and shape change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).     

The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2)

In the present study we describe the complete cytochrome P450 complement, the "CYPome," of Streptomyces coelicolor A3(2). Eighteen cytochromes P450 (CYP) are described, in contrast to the absence of CYPs in Escherichia coli, and the twenty observed in Mycobacterium tuberculosis. Here we confirm protein identity as cytochromes P450 by heterologous expression in E. coli and measurement of reduced carbon monoxide difference spectra. We also report on their arrangement in the linear chromosome and relatedness to other CYPs in the superfamily. The future development of manipulation of antibiotic pathways and the use of streptomycetes in bioremediation and biotransformations will involve many of the new CYP forms identified here.     

The cell cycle control protein cdc25C is present, and phosphorylated on serine 214 in the transition from germinal vesicle to metaphase II in human oocyte meiosis

Cdc25C is a dual specificity phosphatase essential for dephosphorylation and activation of cyclin-dependent kinase 1 (cdk1), a prerequisite step for mitosis in all eucaryotes. Cdc25C activation requires phosphorylation on at least six sites including serine 214 (S214) which is essential for metaphase/anaphase transit. Here, we have investigated S214 phosphorylation during human meiosis with the objectives of determining if this mitotic phosphatase cdc25C participates in final meiotic divisions in human oocytes. One hundred forty-eight human oocytes from controlled ovarian stimulation protocols were stained for immunofluorescence: 33 germinal vesicle (GV), 37 metaphase stage I (MI), and 78 unfertilized metaphase stage II (MII). Results were stage dependent, identical, independent of infertility type, or stimulation protocol. During GV stages, phospho-cdc25C is localized at the oocyte periphery. During early meiosis I (MI), phosphorylated cdc25C is no longer detected until onset of meiosis I. Here, phospho-cdc25C localizes on interstitial microtubules and at the cell periphery corresponding to the point of polar body expulsion. As the first polar body reaches the periphery, phosphorylated cdc25C is localized at the junction corresponding to the mid body position. On polar body expulsion, the interior signal for phospho-cdc25C is lost, but remains clearly visible in the extruded polar body. In atresic or damaged oocytes, the polar body no longer stains for phospho-cdc25C. Human cdc25C is both present and phosphorylated during meiosis I and localizes in a fashion similar to that seen during human mitotic divisions implying that the involvement of cdc25C is conserved and functional in meiotic cells.     

Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.     

Notch signalling in the regulation of peripheral T-cell function

The Notch signalling pathway plays a highly-conserved role in regulating the cellular differentiation and proliferation events that characterise pattern formation in the embryo. As cells in the embryo respond to environmental signals, similarly T-cells in the peripheral immune system must monitor their environment for antigens and respond accordingly by entering one of several potential differentiation pathways. Recent studies have identified a role for the Notch pathway in regulating the responses of T-cells in the periphery. In this review, we discuss these findings in the context of the Notch signalling pathway's role as an orchestrator of cellular differentiation, and propose a central role for Notch as a regulator of immune system function.     

Diet reveals links between morphology and foraging in a cryptic temperate reef fish

Predators select prey so as to maximize energy and minimize manipulation time. In order to reduce prey detection and handling time, individuals must actively select their foraging space (microhabitat) and populations exhibit morphologies that are best suited for capturing locally available prey. We explored how variation in diet correlates with habitat type, and how these factors influence key morphological structures (mouth gape, eye diameter, fin length, fin area, and pectoral fin ratio) in a common microcarnivorous cryptic reef fish species, the triplefin Helcogrammoides cunninghami. In a mensurative experiment carried out at six kelp‐dominated sites, we observed considerable differences in diet along 400 km of the Chilean coast coincident with variation in habitat availability and prey distributions. Triplefins preferred a single prey type (bivalves or barnacles) at northern sites, coincident with a low diversity of foraging habitats. In contrast, southern sites presented varied and heterogeneous habitats, where triplefin diets were more diverse and included amphipods, decapods, and cumaceans. Allometry‐corrected results indicated that some morphological structures were consistently correlated with different prey items. Specifically, large mouth gape was associated with the capture of highly mobile prey such as decapods, while small mouth gape was more associated with cumaceans and copepods. In contrast, triplefins that capture sessile prey such as hydroids tend to have larger eyes. Therefore, morphological structures co‐vary with habitat selection and prey usage in this species. Our study shows how an abundant generalist reef fish exhibits variable feeding morphologies in response to the distribution of potential habitats and prey throughout its range.     

Structural dynamics of myoglobin: ligand migration among protein cavities studied by Fourier transform infrared/temperature derivative spectroscopy.

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A(0) and A(3), which can be distinguished by their different CO bands near 1965 and 1933 cm(-1). They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C', in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C") has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at approximately 180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C', C", and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C') is severely restricted by introduction of the bulky Phe side chain at position 107.     

Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.