bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison In Vivo Study of Genotoxic Action of High- Versus Very Low Dose-Rate γ-Irradiation
Authors: Osipov A. N. , Klokov D. Y. , Elakov A. L. , Rozanova O. M. , Zaichkina S. I. , Aptikaeva G. F. , Akhmadieva A. Kh.
Published: October 2004

The aim of the present study was to compare genotoxicity induced by high- versus very low dose-rate exposure of mice to γ-radiation within a dose range of 5 to 61 cGy using the single-cell gel electrophoresis (comet) assay and the micronucleus test. CBA/lac male mice were irradiated at a dose rate of 28.2 Gy/h (high dose rate) or 0.07 mGy/h (very low dose rate). The comet assay study on spleen lymphocytes showed that very low dose-rate irradiation resulted in a statistically significant increase in nucleoid relaxation (DNA breaks), starting from a dose of 20 cGy. Further prolongation of exposure time and, hence, increase of a total dose did not, however, lead to further increase in the extent of nucleoid relaxation. Doses of 20 and 61 cGy were equal in inducing DNA breaks in mouse spleen lymphocytes as assayed by the comet assay. Of note, the level of DNA damage by 20–61 cGy doses of chronic irradiation (0.07 mGy/h) was similar to that an induced by an acute (28.2 Gy/h) dose of 14 cGy. The bone marrow micronucleus test revealed that an increase in polychromatic erythrocytes with micronuclei over a background level was induced by very low-level γ-irradiation with a dose of 61 cGy only, with the extent of the cytogenetic effect being similar to that of 10 cGy high-dose-rate exposure. In summary, presented results support the hypothesis of the nonlinear threshold nature of mutagenic action of chronic low dose-rate irradiation.
Low-Dose Radiation and Genotoxic Chemicals Can Protect against Stochastic Biological Effects
Authors: Scott Bobby R. , Walker Dale M. , Walker Vernon E.
Published: October 2004

A protective apoptosis-mediated (PAM) process that is turned on in mammalian cells by low-dose photon (X and γ) radiation and appears to also be turned on by the genotoxic chemical ethylene oxide is discussed. Because of the PAM process, exposure to low-dose photon radiation (and possibly also some genotoxic chemicals) can lead to a reduction in the risk of stochastic effects such as problematic mutations, neoplastic transformation (an early step in cancer occurrence), and cancer. These findings indicate a need to revise the current low-dose risk assessment paradigm for which risk of cancer is presumed to increase linearly with dose (without a threshold) after exposure to any amount of a genotoxic agent such as ionizing radiation. These findings support a view seldom mentioned in the past, that cancer risk can actually decrease, rather than increase, after exposure to low doses of photon radiation and possibly some other genotoxic agents. The PAM process (a form of natural protection) may contribute substantially to cancer prevention in humans and other mammals. However, new research is needed to improve our understanding of the process. The new research could unlock novel strategies for optimizing cancer prevention and novel protocols for low-dose therapy for cancer. With low-dose cancer therapy, normal tissue could be spared from severe damage while possibly eliminating the cancer.
Low Doses of Gamma-Radiation Induce Nonlinear Dose Responses in Mammalian and Plant Cells
Authors: Zaichkina S. I. , Rozanova O. M. , Aptikaeva G. F. , Achmadieva A. Ch. , Klokov D. Y.
Published: October 2004

The percentage of cells with chromosome aberrations or micronuclei induced by low doses of acute (dose rate of 47 cGy/min) or chronic (dose rate of 0.01 cGy/min) gamma-irradiation was studied in vitro in Chinese hamster fibroblasts, human lymphocytes, and Vicia faba seeds and seedlings. The sensitivity of the indicated biological entities to low doses was greater than expected based on linear extrapolation from higher doses. The dose-response curves for cytogenetic damage that were obtained were nonlinear when evaluated over the full range of the doses used. At very low doses, the dose-response curves appeared linear, followed by a plateau region at intermediate doses. At high doses the dose response curves again appeared linear with a slope different from that for the low-dose region. There was no statistically significant difference between the yields of cells with micronuclei induced by low doses of acute versus chronic irradiation. Similar data were obtained both for human lymphocyte culture and for roots and seeds of Vicia faba. Our experiments revealed that the dose range over which the plateau occurs depends on the type of cells irradiated. We have also shown that the modifying effects of the repair inhibitor caffeine and the radioprotector mercaptoethylenamine (MEA) are absent at low doses of gamma irradiation and that caffeine increased the number of cells with cytogenetic damage when evaluated over the plateau region. In the presence of MEA, the upper end of the plateau region was extended from just above 1 Gy to about 2 Gy. We therefore provide direct evidence that a plateau exists in the dose-response curve for the indicated radiation-induced stochastic effects. Furthermore, our results suggest that, for low linear energy transfer radiation, the induction of DNA repair occurs only after a threshold level of cytogenetic damage and that the higher yield of cytogenetic damage per unit dose at low radiation doses is attributable to an insignificant contribution or the absence of DNA repair processes.
Roles of p-ERM and Rho–ROCK signaling in lymphocyte polarity and uropod formation
Authors: Lee Jong-Hwan , Katakai Tomoya , Hara Takahiro , Gonda Hiroyuki , Sugai Manabu , Shimizu Akira
Published: October 2004

Front–rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton–dependent manner. Rho-associated coiled coil–containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho–ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane “posteriority” in the induction of the uropod in T lymphocytes.
UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II
Authors: Lord Matthew , Pollard Thomas D.
Published: October 2004

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.
Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development
Authors: Colognato Holly , Ramachandrappa Shwetha , Olsen Inger M. , ffrench-Constant Charles
Published: October 2004

Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with α6β1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive αVβ3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.
A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them
Authors: Carman Christopher V. , Springer Timothy A.
Published: October 2004

The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel “cuplike” transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1– and vascular cell adhesion molecule-1–enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the “transmigratory cup”, by which the endothelium provides directional guidance to leukocytes for extravasation.
The critical role of cyclin D2 in adult neurogenesis
Authors: Kowalczyk Anna , Filipkowski Robert K. , Rylski Marcin , Wilczynski Grzegorz M. , Konopacki Filip A. , Jaworski Jacek , Ciemerych Maria A. , Sicinski Piotr , Kaczmarek Leszek
Published: October 2004

Adult neurogenesis (i.e., proliferation and differentiation of neuronal precursors in the adult brain) is responsible for adding new neurons in the dentate gyrus of the hippocampus and in the olfactory bulb. We describe herein that adult mice mutated in the cell cycle regulatory gene Ccnd2, encoding cyclin D2, lack newly born neurons in both of these brain structures. In contrast, genetic ablation of cyclin D1 does not affect adult neurogenesis. Furthermore, we show that cyclin D2 is the only D-type cyclin (out of D1, D2, and D3) expressed in dividing cells derived from neuronal precursors present in the adult hippocampus. In contrast, all three cyclin D mRNAs are present in the cultures derived from 5-day-old hippocampi, when developmental neurogenesis in the dentate gyrus takes place. Thus, our results reveal the existence of molecular mechanisms discriminating adult versus developmental neurogeneses.
Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis
Authors: Wright Kevin M. , Linhoff Michael W. , Potts Patrick Ryan , Deshmukh Mohanish
Published: October 2004

Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c–dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c–induced apoptosis in naïve pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.
The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi
Authors: Gillingham Alison K. , Tong Amy Hin Yan , Boone Charles , Munro Sean
Published: October 2004

Rud3p is a coiled-coil protein of the yeast cis-Golgi. We find that Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p. In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal “GRIP-related Arf-binding” (GRAB) domain. Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi. The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and γ-tubulin (Rios, R.M, A. Sanchis, A.M. Tassin, C. Fedriani, and M. Bornens. 2004. Cell. 118:323–335). In contrast, we find that this region binds to the Golgi in a GRAB domain–dependent manner, suggesting that GMAP-210 may not link the Golgi to γ-tubulin and centrosomes.
Getting the message across, STAT! Design principles of a molecular signaling circuit
Authors: Vinkemeier Uwe
Published: October 2004

The STAT transcription factors, usually referred to as “latent cytoplasmic proteins,” have experienced a fundamental reevaluation of their dynamic properties. This review focuses on recent studies that have identified continuous transport factor–independent nucleocytoplasmic cycling of STAT1, STAT3, and STAT5 as a basic principle of cytokine signaling. In addition, molecular mechanisms that modulate flux rates or cause retention were recognized, and together these findings have provided novel insight into the rules of cellular signal processing.
Coordinating cytoskeletal tracks to polarize cellular movements
Authors: Kodama Atsuko , Lechler Terry , Fuchs Elaine
Published: October 2004

For many years after the discovery of actin filaments and microtubules, it was widely assumed that their polymerization, organization, and functions were largely distinct. However, in recent years it has become increasingly apparent that coordinated interactions between microtubules and filamentous actin are involved in many polarized processes, including cell shape, mitotic spindle orientation, motility, growth cone guidance, and wound healing. In the past few years, significant strides have been made in unraveling the intricacies that govern these intertwined cytoskeletal rearrangements.
Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis
Authors: Weis Sara , Cui Jianhua , Barnes Leo , Cheresh David
Published: October 2004

VEGF is unique among angiogenic growth factors because it disrupts endothelial barrier function. Therefore, we considered whether this property of VEGF might contribute to tumor cell extravasation and metastasis. To test this, mice lacking the Src family kinases Src or Yes, which maintain endothelial barrier function in the presence of VEGF, were injected intravenously with VEGF-expressing tumor cells. We found a dramatic reduction in tumor cell extravasation in lungs or livers of mice lacking Src or Yes. At the molecular level, VEGF compromises the endothelial barrier by disrupting a VE-cadherin–β-catenin complex in lung endothelium from wild-type, but not Yes-deficient, mice. Disrupting the endothelial barrier directly with anti–VE-cadherin both amplifies metastasis in normal mice and overcomes the genetic resistance in Yes-deficient mice. Pharmacological blockade of VEGF, VEGFR-2, or Src stabilizes endothelial barrier function and suppresses tumor cell extravasation in vivo. Therefore, disrupting Src signaling preserves host endothelial barrier function providing a novel host-targeted approach to control metastatic disease.
Cell differentiation
Authors: Lindholm Dan , Arumäe Urmas
Published: October 2004

The molecular mechanisms by which differentiated cells combat cell death and injury have remained unclear. In the current issue, it has been shown in neurons that cell differentiation is accompanied by a decrease in Apaf-1 and the activity of the apoptosome with an increased ability of the inhibitor of apoptosis proteins (IAPs) to sustain survival (Wright et al., 2004). These results, together with earlier ones, deepen our understanding of how cell death and the apoptosome are regulated during differentiation and in tumor cells.
Polyvalent Cations Constitute the Voltage Gating Particle in Human Connexin37 Hemichannels
Authors: Puljung Michael C. , Berthoud Viviana M. , Beyer Eric C. , Hanck Dorothy A.
Published: October 2004

Connexins oligomerize to form intercellular channels that gate in response to voltage and chemical agents such as divalent cations. Historically, these are believed to be two independent processes. Here, data for human connexin37 (hCx37) hemichannels indicate that voltage gating can be explained as block/unblock without the necessity for an independent voltage gate. hCx37 hemichannels closed at negative potentials and opened in a time-dependent fashion at positive potentials. In the absence of polyvalent cations, however, the channels were open at relatively negative potentials, passing current linearly with respect to voltage. Current at negative potentials could be inhibited in a concentration-dependent manner by the addition of polyvalent cations to the bathing solution. Inhibition could be explained as voltage-dependent block of hCx37, with the field acting directly on polyvalent cations, driving them through the pore to an intracellular site. At positive potentials, in the presence of polyvalent cations, the field favored polyvalent efflux from the intracellular blocking site, allowing current flow. The rate of appearance of current depended on the species and valence of the polyvalent cation in the bathing solution. The rate of current decay upon repolarization depended on the concentration of polyvalent cations in the bathing solution, consistent with deactivation by polyvalent block, and was rapid (time constants of tens of milliseconds), implying a high local concentration of polyvalents in or near the channel pore. Sustained depolarization slowed deactivation in a flux-dependent, voltage- and time-independent fashion. The model for hCx37 voltage gating as polyvalent block/unblock can be expanded to account for observations in the literature regarding hCx37 gap junction channel behavior.
Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels
Authors: Luvisetto Siro , Fellin Tommaso , Spagnolo Michele , Hivert Bruno , Brust Paul F. , Harpold Michael M. , Stauderman Kenneth A. , Williams Mark E. , Pietrobon Daniela
Published: October 2004

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary β1b, β2e, β3a, and β4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463–474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four β subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the β subtype. The type of β subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; β3a promotes the fast mode, whereas β4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a β subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.
Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels
Authors: Fellin Tommaso , Luvisetto Siro , Spagnolo Michele , Pietrobon Daniela
Published: October 2004

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445–461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.
Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter
Authors: Ehnes Colin , Forster Ian C. , Kohler Katja , Bacconi Andrea , Stange Gerti , Biber Jürg , Murer Heini
Published: October 2004

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.
Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na+/Pi Cotransporter
Authors: Ehnes Colin , Forster Ian C. , Bacconi Andrea , Kohler Katja , Biber Jürg , Murer Heini
Published: October 2004

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.
Invariant Aspartic Acid in Muscle Nicotinic Receptor Contributes Selectively to the Kinetics of Agonist Binding
Authors: Lee Won Yong , Sine Steven M.
Published: October 2004

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant αD89 forms a highly conserved interdomain contact near αT148, αW149, and αT150. Patch-clamp recordings show that the mutation αD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither αT148L, αT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor αD89. However substituting a negative charge at αT148, but not at αT150, counteracts the effect of αD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring αW149 as principal hydrogen bond donors for αD89. The specific effect of αD89N on ACh association suggests that interdomain hydrogen bonding positions αW149 for optimal interaction with ACh.
Long Term Endocrine Regulation of Nucleoside Transporters in Rat Intestinal Epithelial Cells
Authors: Aymerich Ivette , Pastor-Anglada Marçal , Casado F. Javier
Published: October 2004

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-α) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-α induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.
Altered Inactivation of Ca2+ Current and Ca2+ Release in Mouse Muscle Fibers Deficient in the DHP receptor γ1 subunit
Authors: Ursu Daniel , Schuhmeier Ralph Peter , Freichel Marc , Flockerzi Veit , Melzer Werner
Published: October 2004

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit γ1 have previously been studied using coexpression with the cardiac α1C polypeptide in nonmuscle cells and primary-cultured myotubes of γ1-deficient mice. Data from single adult muscle fibers of γ−/− mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from γ+/+ and γ−/− mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of −80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage–dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in γ−/− compared with γ+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the γ1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.
Integration of Human Papillomavirus Type 11 in Recurrent Respiratory Papilloma‐Associated Cancer
Authors: Reidy Patrick M. , Dedo Herbert H. , Rabah Raja , Field Jayson B. , Mathog Robert H. , Gregoire Lucie , Lancaster Wayne D.
Published: October 2004

Objectives/Hypothesis: The main objective was to demonstrate that human papillomavirus (HPV) type 11 is an aggressive virus that plays a significant role in the development of laryngeal cancer in patients with a history of recurrent respiratory papillomatosis (RRP). We have done so by preliminary investigation into the molecular mechanism underlying the malignant transformation of RRP to invasive squamous cell carcinoma.

Study Design: An experimental, nonrandomized, retrospective study using tissue specimens from nine patients with a history of RRP that progressed to laryngeal or bronchogenic cancer was performed.

Methods: DNA and RNA were extracted from 20 formalin‐fixed, paraffin‐embedded specimens from six patients with a history of early onset RRP and laryngeal cancer and from three patients with early onset RRP and bronchogenic cancer. Polymerase chain reaction (PCR) was performed on DNA to determine the HPV type in each specimen. Reverse‐transcriptase PCR specific for virus transcripts was performed on RNA to determine whether the viral genome was integrated into the host genome.

Results: HPV‐11 but not HPV‐6, 16, or 18 was found in all of the laryngeal and bronchogenic cancers in patients with a history of early onset RRP in this study. RNA, sufficiently intact for examination, was obtained from seven patients. Analysis of HPV 11 transcripts revealed integration of the viral genome in three of seven patients.

Conclusions: HPV type 6 and 11 are considered “low‐risk” viruses and are not associated with genital cancers, as are HPV types 16 and 18. However, our data suggests that HPV type 11 is an aggressive virus in laryngeal papilloma that should be monitored in patients with RRP.
Overexpression of ErbB‐2 Protein in Human Middle Ear Cholesteatomas
Authors: Sakamoto Takashi , Kondo Kenji , Yamasoba Tatsuya , Suzuki Mitsuya , Sugasawa Masashi , Kaga Kimitaka
Published: October 2004

Objective: The purpose of this study is to verify the hypothesis that ErbB‐2 protein is overexpressed in human middle ear cholesteatomas and to elucidate the relationship between overexpression of ErbB‐2 protein, cell proliferation, and apoptosis.

Study Design: Prospective review of 20 patients between 2001 and 2003 with middle ear cholesteatoma.

Methods: Middle ear cholesteatoma matrix and retroauricular skin were immunostained with anti‐ErbB‐2, Ki‐67, and single‐stranded DNA (ssDNA) antibody. The distribution of immunoreactivity to these antibodies and labeling indices were compared between cholesteatoma and retroauricular skin.

Results: In matrix of middle ear cholesteatoma, ErbB‐2 and ssDNA were expressed in the keratinocytes of all layers and Ki‐67 was expressed in the keratinocytes of the basal, lower spinous, and occasionally granular layer. In retroauricular skin, ErbB‐2 and Ki‐67 were expressed in the keratinocytes of the basal and occasionally lower spinous layer and ssDNA was expressed in the keratinocytes of all layers. Labeling indices against anti‐ErbB‐2, Ki‐67, and ssDNA antibody were significantly greater in cholesteatoma as compared with retroauricular skin.

Conclusions: In cases of cholesteatoma, ErbB‐2 protein was overexpressed and cell proliferation and apoptosis of keratinocytes were accelerated. ErbB‐2 protein could modulate terminal differentiation and apoptosis in the keratinocytes of all layers in cholesteatoma matrix and cell proliferation in the keratinocytes of the basal and lower spinous layer in normal skin.
Could Valerian Have Been the First Anticonvulsant?
Authors: Eadie Mervyn J.
Published: October 2004

Summary:  Purpose: To assess the available evidence for the belief that valerian, highly recommended in the past for treating epilepsy, possessed real anticonvulsant effectiveness.

Methods: Review of available literature.

Results: In 1592, Fabio Colonna, in his botanical classic Phytobasanos, reported that taking powdered valerian root cured his own epilepsy. Subsequent reports of valerian's anticonvulsant effectiveness appeared. By the late 18th and early 19th centuries, it was often regarded as the best available treatment for the disorder. Valerian preparations yield isovaleric acid, a substance analogous to valproic acid and likely to possess anticonvulsant properties, as isovaleramide does. In favorable circumstances, high valerian doses can be calculated to have sometimes provided potentially effective amounts of anticonvulsant substance for epilepsy patients.

Conclusions: Valerian probably did possess the potential for an anticonvulsant effect, but the uncertain chemical composition and content of valerian preparations, and their odor and taste, made it unlikely that they could ever prove satisfactory in widespread use.
VARIABILITY IN NITRATE UPTAKE KINETICS IN THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1
Authors: Dortch Quay , Thompson Peter A. , Harrison Paul J.
Published: October 2004

Short‐term (1–9 min) nitrate uptake kinetics were measured in Thalassiosira pseudonana (Hust.) Hasle & Heimdal grown in nitrate‐limited, ammonium‐limited, and nitrate‐sufficient continuous cultures. For all cultures, maximal nitrate uptake rates did not develop until approximately 3 min after nitrate addition; thereafter, nitrate uptake rates remained constant or declined slightly. The Ks and Vmax for the nitrate‐limited cultures were higher at any growth rate than those for the ammonium‐limited or nitrate‐sufficient cultures. Thus, much higher nitrate concentrations would be required to saturate nitrate uptake in nitrate‐limited Thalassiosira pseudonana than is usually considered necessary. The lack of data for other species grown under a range of environmental conditions makes it difficult to generalize about the effect of preconditioning on nitrate uptake kinetics.
ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1
Authors: Reed Daniel C. , Neushul Michael , Ebeling Alfred W.
Published: October 2004

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non‐aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm−2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ∼ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm−2. Total sporophyte production (i.e. number · cm−2) for both species, however, was greatest at intermediate densities of spore settlement (∼ 50 spores · mm−2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm−2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non‐aerated cultures was probably caused by nutrient limitation.
RELATIONSHIP BETWEEN THE NUMBER OF DIVIDING AND NONDIVIDING CELLS OF CYANOBACTERIA IN NORTH ATLANTIC PICOPLANKTON1
Authors: Li William K. W. , Dickie Paul M.
Published: October 2004

In the North Atlantic over a wide geographic region that includes various oceanic regimes and a temperature range from 10 to 22° C, an increase in the number of nondividing Synechococcus cells (X) was generally accompanied by a greater‐than‐proportional increase in the number of dividing cells (Y). As a result, the fraction of dividing cells (FDC = Y · (Y + X)−1) was positively related to population size (Y + X). Recognizing that FDC is generally greater in a rapidly growing population than in a slowly growing one, our empirical finding implies a positive correlation between specific growth rate and standing stock for Synechococcus. One notable exception occurred during winter (T < 5°C) in a eutrophic coastal embayment when a decrease in cell abundance was not matched by a decrease in FDC.
FISCHERELLIN, A NEW ALLELOCHEMICAL FROM THE FRESHWATER CYANOBACTERIUM FISCHERELLA MUSCICOLA1
Authors: Gross Elisabeth M. , Wolk C. Peter , Jüttner Friedrich
Published: October 2004

The benthic cyanobacterium Fischerella muscicola (Thur.) Gom. UTEX 1829 produces a secondary metabolite, fischerellin, that strongly inhibits other cyanobacteria and to a lesser extent members of the Chlorophyceae. Eubacteria are not affected. The major active compound is lipophilic and exhibits a molecular ion at m/z 408. It is heat‐ and acid‐stable but decomposes in 1 M sodium hydroxide (80° C. 1 h). Fischerellin inhibits the photosynthetic but not the respiratory electron transport of cyanobacteria and chlorophytes. Its site of action is located in PS II. Two other species of Fischerella also produce fischerellin, indicating that the synthesis of such allelochemicals might be characteristic of the genus.
The vertical distribution of juvenile salmon (Oncorhynchus spp.) and associated fishes in the Columbia River plume
Authors: Emmett Robert L. , Brodeur Richard D. , Orton Philip M.
Published: October 2004

Simultaneous trawling at surface and at depth at one location off the Columbia River, Oregon, in June 2000 identified the depth distribution of juvenile salmonids and associated fishes. Juvenile salmon off the Columbia River were distributed primarily near the surface, within the upper 12 m. Highest densities of subyearling chinook salmon (Oncorhynchus tshawytscha) off the Columbia River were associated with high surface currents and decreasing tidal levels, with time of day possibly a co‐factor. Densities of yearling chinook salmon increased with higher turbidity. Pacific herring (Clupea pallasi) was the most abundant and commonly caught forage fish, with density increasing at night, probably related to diel vertical migration. Catches of juvenile salmonids were not associated with catches of forage fishes. Daytime surface trawling appears to be an appropriate method for assessing the distribution and abundance of juvenile salmonids in marine habitats.
Influence of mesoscale physical forcing on trophic pathways and fish larvae retention in the central Cantabrian Sea
Authors: González‐Quirós Rafael , Pascual Ananda , Gomis Damiá , Anadón Ricardo
Published: October 2004

This study was carried out over the central Cantabrian shelf during a post‐bloom phase in May. Late phases of the spring phytoplankton bloom, which are characterized by high crustacean mesozooplankton biomass, have been proposed to be a particular case for high‐energy flow towards large metazoans. The overall objective of the study is to analyse the influence of mesoscale physical forcing on primary production patterns, its implications on food web pathways, and on larval fish distribution during a period of intense spawning of Sardina pilchardus and Scomber scombrus. Physical and biological variables of different trophic levels show coupled cross‐shelf and along‐shelf heterogeneity. Quasi‐geostrophic analysis and other indirect approaches, such as the depth of the slope salinity maximum, reveal predictable patterns of vertical instabilities associated with mesoscale physical forcing that enhance production of large‐size phytoplankton. The latter is expected to enhance the energy flow towards higher trophic levels at a time of high mesozooplankton biomass. Distributions of S. pilchardus and S. scombrus eggs and larvae indicate retention related to the coastal salinity front and the overall eastward circulation pattern. The observed mesoscale physical processes may favour survival of early stages of fish by their influence on the energy flow of primary production towards higher trophic levels and larval retention at the coast.
An individual‐based model of the early life history of mackerel (Scomber scombrus) in the eastern North Atlantic, simulating transport, growth and mortality
Authors: Bartsch J. , Coombs S. H.
Published: October 2004

The main purpose of this paper is to provide the core description of the modelling exercise within the Shelf Edge Advection Mortality And Recruitment (SEAMAR) programme. An individual‐based model (IBM) was developed for the prediction of year‐to‐year survival of the early life‐history stages of mackerel (Scomber scombrus) in the eastern North Atlantic. The IBM is one of two components of the model system. The first component is a circulation model to provide physical input data for the IBM. The circulation model is a geographical variant of the HAMburg Shelf Ocean Model (HAMSOM). The second component is the IBM, which is an i‐space configuration model in which large numbers of individuals are followed as discrete entities to simulate the transport, growth and mortality of mackerel eggs, larvae and post‐larvae. Larval and post‐larval growth is modelled as a function of length, temperature and food distribution; mortality is modelled as a function of length and absolute growth rate. Each particle is considered as a super‐individual representing 106 eggs at the outset of the simulation, and then declining according to the mortality function. Simulations were carried out for the years 1998–2000. Results showed concentrations of particles at Porcupine Bank and the adjacent Irish shelf, along the Celtic Sea shelf‐edge, and in the southern Bay of Biscay. High survival was observed only at Porcupine and the adjacent shelf areas, and, more patchily, around the coastal margin of Biscay. The low survival along the shelf‐edge of the Celtic Sea was due to the consistently low estimates of food availability in that area.