bluuebunny/crossref_metadata_2025 huggingface dataset:
Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE
Authors: Darsow Tamara , Burd Christopher G. , Emr Scott D.
Published: July 2002

The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Δ cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein–protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7–amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor–dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein–specific sorting signal in a t-SNARE protein, and suggest that AP-3–dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3–dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.
A Morphogenesis Checkpoint Monitors the Actin Cytoskeleton in Yeast
Authors: McMillan John N. , Sia Rey A.L. , Lew Daniel J.
Published: July 2002

A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I. Reed. 1995. J. Cell Biol. 129:739– 749). The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Mol. Biol. Cell. 7:1657– 1666). In this report, we have investigated the nature of the defect(s) that trigger this checkpoint. A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact. Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation. In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle. However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.
Reconstitution of Mammary Gland Development In Vitro: Requirement of c-met and c-erbB2 Signaling for Branching and Alveolar Morphogenesis
Authors: Niemann Catherin , Brinkmann Volker , Spitzer Eva , Hartmann Guido , Sachs Martin , Naundorf Helga , Birchmeier Walter
Published: July 2002

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met–mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.
The Septins Are Required for the Mitosis-specific Activation of the Gin4 Kinase
Authors: Carroll Christopher W. , Altman Roger , Schieltz David , Yates John R. , Kellogg Douglas
Published: July 2002

In budding yeast, a protein kinase called Gin4 is specifically activated during mitosis and functions in a pathway initiated by the Clb2 cyclin to control bud growth. We have used genetics and biochemistry to identify additional proteins that function with Gin4 in this pathway, and both of these approaches have identified members of the septin family. Loss of septin function produces a phenotype that is very similar to the phenotype caused by loss of Gin4 function, and the septins are required early in mitosis to activate Gin4 kinase activity. Furthermore, septin mutants display a prolonged mitotic delay at the short spindle stage, consistent with a role for the septins in the control of mitotic events. Members of the septin family bind directly to Gin4, demonstrating that the functions of Gin4 and the septins must be closely linked within the cell. These results demonstrate that the septins in budding yeast play an integral role in the mitosis-specific regulation of the Gin4 kinase and that they carry out functions early in mitosis.
Induction of Filopodia by Direct Local Elevation of Intracellular Calcium Ion Concentration
Authors: Lau Pak-ming , Zucker Robert S. , Bentley David
Published: July 2002

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from ∼60 nM to ∼1 μM within the illuminated zone, and then returned to resting level in ∼10–15 s. New filopodia arose in this area within 1–5 min, and persisted for ∼15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.
Conformational Requirements for Glycoprotein Reglucosylation in the Endoplasmic Reticulum
Authors: Trombetta E. Sergio , Helenius Ari
Published: July 2002

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.
Keratins Turn over by Ubiquitination in a Phosphorylation-Modulated Fashion
Authors: Ku Nam-On , Omary M. Bishr
Published: July 2002

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.
Interferon (IFN)-α Activation of Human Blood Mononuclear Cells In Vitro and In Vivo for Nitric Oxide Synthase (NOS) Type 2 mRNA and Protein Expression: Possible Relationship of Induced NOS2 to the Anti–Hepatitis C Effects of IFN-α In Vivo
Authors: Sharara Ala I. , Perkins Douglas J. , Misukonis Mary A. , Chan Stanley U. , Dominitz Jason A. , Weinberg J. Brice
Published: July 2002

Although researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) γ and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-α serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-α caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-α also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-α therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-α treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-α–treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-α–enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-α treatment of cells in vitro or administration of IFN-α to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-α in hepatitis C infection.
Modulation of Natural Killer Cell Cytotoxicity in Human Cytomegalovirus Infection: The Role of Endogenous Class I Major Histocompatibility Complex and a Viral Class I Homolog
Authors: Leong Clement C. , Chapman Tara L. , Bjorkman Pamela J. , Formankova Danuska , Mocarski Edward S. , Phillips Joseph H. , Lanier Lewis L.
Published: July 2002

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell–mediated cytotoxicity.
Induction of HIV-1 Replication in Latently Infected CD4+ T Cells Using a Combination of Cytokines
Authors: Chun Tae-Wook , Engel Delphine , Mizell Stephanie B. , Ehler Linda A. , Fauci Anthony S.
Published: July 2002

Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1–infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4+ T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4+ T cells derived from HIV-infected individuals who are antiretroviral therapy–naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-α, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.
B Cell Antigen Receptor Engagement Inhibits Stromal Cell–derived Factor (SDF)-1α Chemotaxis and Promotes Protein Kinase C (PKC)-induced Internalization of CXCR4
Authors: Guinamard Rodolphe , Signoret Nathalie , Ishiai Masamichi , Marsh Mark , Kurosaki Tomohiro , Ravetch Jeffrey V.
Published: July 2002

The entry of B lymphocytes into secondary lymphoid organs is a critical step in the development of an immune response, providing a site for repertoire shaping, antigen-induced activation and selection. These events are controlled by signals generated through the B cell antigen receptor (BCR) and are associated with changes in the migration properties of B cells in response to chemokine gradients. The chemokine stromal cell–derived factor (SDF)-1α is thought to be one of the driving forces during those processes, as it is produced inside secondary lymphoid organs and induces B lymphocyte migration that arrests upon BCR engagement. The signaling pathway that mediates this arrest was genetically dissected using B cells deficient in specific BCR-coupled signaling components. BCR-induced inhibition of SDF-1α chemotaxis was dependent on Syk, BLNK, Btk, and phospholipase C (Plc)γ2 but independent of Ca2+ mobilization, suggesting that the target of BCR stimulation was a protein kinase C (PKC)-dependent substrate. This target was identified as the SDF-1α receptor, CXCR4, which undergoes PKC- dependent internalization upon BCR stimulation. Mutation of the internalization motif SSXXIL in the COOH terminus of CXCR4 resulted in B cells that constitutively expressed this receptor upon BCR engagement. These studies suggest that one pathway by which BCR stimulation results in inhibition of SDF-1α migration is through PKC-dependent downregulation of CXCR4.
Dendritic Cell Maturation Overrules H-2d–Mediated Natural Killer T (Nkt) Cell Inhibition
Authors: Ikarashi Yoshinori , Mikami Rumiko , Bendelac Albert , Terme Magali , Chaput Nathalie , Terada Masahiro , Tursz Thomas , Angevin Eric , Lemonnier François A. , Wakasugi Hiro , Zitvogel Laurence
Published: July 2002

Given the broad expression of H-2 class Ib molecules on hematopoietic cells, antigen presentation pathways among CD1d expressing cells might tightly regulate CD1d-restricted natural killer T (NKT) cells. Bone marrow–derived dendritic cells (BM-DCs) and not adherent splenocytes become capable of triggering NK1.1+/T cell receptor (TCR)int hepatic NKT cell activation when (a) immature BM-DCs lack H-2Db−/− molecules or (b) BM-DCs undergo a stress signal of activation. In such conditions, BM-DCs promote T helper type 1 predominant CD1d-restricted NKT cell stimulation. H-2 class Ia–mediated inhibition involves more the direct H-2Db presentation than the indirect Qa-1b pathway. Such inhibition can be overruled by B7/CD28 interactions and marginally by CD40/CD40L or interleukin 12. These data point to a unique regulatory role of DCs in NKT cell innate immune responses and suggest that H-2 class Ia and Ib pathways differentially control NKT cell recognition of DC antigens.
Local Control Model of Excitation–Contraction Coupling in Skeletal Muscle
Authors: Stern Michael D. , Pizarro Gonzalo , Ríos Eduardo
Published: July 2002

This is a quantitative model of control of Ca2+ release from the sarcoplasmic reticulum in skeletal muscle, based on dual control of release channels (ryanodine receptors), primarily by voltage, secondarily by Ca2+ (Ríos, E., and G. Pizarro. 1988. NIPS. 3:223–227). Channels are positioned in a double row array of between 10 and 60 channels, where exactly half face voltage sensors (dihydropyridine receptors) in the transverse (t) tubule membrane (Block, B.A., T. Imagawa, K.P. Campbell, and C. Franzini-Armstrong. 1988. J. Cell Biol. 107:2587–2600). We calculate the flux of Ca2+ release upon different patterns of pulsed t-tubule depolarization by explicit stochastic simulation of the states of all channels in the array. Channels are initially opened by voltage sensors, according to an allosteric prescription (Ríos, E., M. Karhanek, J. Ma, A. González. 1993. J. Gen. Physiol. 102:449–482). Ca2+ permeating the open channels, diffusing in the junctional gap space, and interacting with fixed and mobile buffers produces defined and changing distributions of Ca2+ concentration. These concentrations interact with activating and inactivating channel sites to determine the propagation of activation and inactivation within the array. The model satisfactorily simulates several whole-cell observations, including kinetics and voltage dependence of release flux, the “paradox of control,” whereby Ca2+-activated release remains under voltage control, and, most surprisingly, the “quantal” aspects of activation and inactivation (Pizarro, G., N. Shirokova, A. Tsugorka, and E. Ríos. 1997. J. Physiol. 501:289–303). Additionally, the model produces discrete events of activation that resemble Ca2+ sparks (Cheng, H., M.B. Cannell, and W.J. Lederer. 1993. Science (Wash. DC). 262:740–744). All these properties result from the intersection of stochastic channel properties, control by local Ca2+, and, most importantly, the one dimensional geometry of the array and its mesoscopic scale. Our calculations support the concept that the release channels associated with one face of one junctional t-tubule segment, with its voltage sensor, constitute a functional unit, termed the “couplon.” This unit is fundamental: the whole cell behavior can be synthesized as that of a set of couplons, rather than a set of independent channels.
Regulation of Deactivation by an Amino Terminal Domain in Human Ether-à-go-go –related Gene Potassium Channels
Authors: Wang Jinling , Trudeau Matthew C. , Zappia Angelina M. , Robertson Gail A.
Published: July 2002

Abnormalities in repolarization of the cardiac ventricular action potential can lead to life-threatening arrhythmias associated with long QT syndrome. The repolarization process depends upon the gating properties of potassium channels encoded by the human ether-à-go-go–related gene (HERG), especially those governing the rate of recovery from inactivation and the rate of deactivation. Previous studies have demonstrated that deletion of the NH2 terminus increases the deactivation rate, but the mechanism by which the NH2 terminus regulates deactivation in wild-type channels has not been elucidated. We tested the hypothesis that the HERG NH2 terminus slows deactivation by a mechanism similar to N-type inactivation in Shaker channels, where it binds to the internal mouth of the pore and prevents channel closure. We found that the regulation of deactivation by the HERG NH2 terminus bears similarity to Shaker N-type inactivation in three respects: (a) deletion of the NH2 terminus slows C-type inactivation; (b) the action of the NH2 terminus is sensitive to elevated concentrations of external K+, as if its binding along the permeation pathway is disrupted by K+ influx; and (c) N-ethylmaleimide, covalently linked to an aphenotypic cysteine introduced within the S4–S5 linker, mimics the N deletion phenotype, as if the binding of the NH2 terminus to its receptor site were hindered. In contrast to N-type inactivation in Shaker, however, there was no indication that the NH2 terminus blocks the HERG pore. In addition, we discovered that separate domains within the NH2 terminus mediate the slowing of deactivation and the promotion of C-type inactivation. These results suggest that the NH2 terminus stabilizes the open state and, by a separate mechanism, promotes C-type inactivation.
Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein
Authors: Molokanova Elena , Savchenko Alexei , Kramer Richard H.
Published: July 2002

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.
Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution
Authors: Lacampagne Alain , Ward Christopher W. , Klein Michael G. , Schneider Martin F.
Published: July 2002

Discrete Ca2+ release events (Ca2+ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca2+ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca2+ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca2+ ions are being released by the sarcoplasmic reticulum Ca2+ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca2+ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.
Impaired Calcium Release in Cerebellar Purkinje Neurons Maintained in Culture
Authors: Womack Mary D. , Walker Jeffery W. , Khodakhah Kamran
Published: July 2002

Cerebellar Purkinje neurons demonstrate a form of synaptic plasticity that, in acutely prepared brain slices, has been shown to require calcium release from the intracellular calcium stores through inositol trisphosphate (InsP3) receptors. Similar studies performed in cultured Purkinje cells, however, find little evidence for the involvement of InsP3 receptors. To address this discrepancy, the properties of InsP3- and caffeine-evoked calcium release in cultured Purkinje cells were directly examined. Photorelease of InsP3 (up to 100 μM) from its photolabile caged analogue produced no change in calcium levels in 70% of cultured Purkinje cells. In the few cells where a calcium increase was detected, the response was very small and slow to peak. In contrast, the same concentration of InsP3 resulted in large and rapidly rising calcium responses in all acutely dissociated Purkinje cells tested. Similar to InsP3, caffeine also had little effect on calcium levels in cultured Purkinje cells, yet evoked large calcium transients in all acutely dissociated Purkinje cells tested. The results demonstrate that calcium release from intracellular calcium stores is severely impaired in Purkinje cells when they are maintained in culture. Our findings suggest that cultured Purkinje cells are an unfaithful experimental model for the study of the role of calcium release in the induction of cerebellar long term depression.
Heterologous Facilitation of G Protein-Activated K+ Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase
Authors: Müllner Carmen , Vorobiov Dimitry , Bera Amal Kanti , Uezono Yasuhito , Yakubovich Daniel , Frohnwieser-Steinecker Bibiane , Dascal Nathan , Schreibmayer Wolfgang
Published: July 2002

To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K+ channels (GIRK), acetylcholine (ACh)-evoked K+ current, IKACh, was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of IKACh was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited IKACh was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 μM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by β-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing β2-adrenergic receptors, m2 -receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30–60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the β2-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the β-adrenergic receptor (β2AR) by PKA was excluded by using a mutant β2AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the α subunit of G proteins (Gαs) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Gαs, GIRK currents were inhibited, presumably due to sequestration of the β/γ subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, β-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from β2AR.
Identification of a Peptide Toxin fromGrammostola spatulataSpider Venom That Blocks Cation-Selective Stretch-Activated Channels
Authors: Suchyna Thomas M. , Johnson Janice H. , Hamer Katherine , Leykam Joseph F. , Gage Douglas A. , Clemo Henry F. , Baumgarten Clive M. , Sachs Frederick
Published: July 2002

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (∼45 pS at −100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.
Conjoint Control of Hippocampal Place Cell Firing by Two Visual Stimuli
Authors: Fenton André A. , Csizmadia Gyorgy , Muller Robert U.
Published: July 2002

To better understand how hippocampal place cell activity is controlled by sensory stimuli, and to further elucidate the nature of the environmental representation provided by place cells, we have made recordings in the presence of two distinct visual stimuli under standard conditions and after several manipulations of these stimuli. In line with a great deal of earlier work, we find that place cell activity is constant when repeated recordings are made in the standard conditions in which the centers of the two stimuli, a black card and a white card, are separated by 135° on the wall of a cylindrical recording chamber. Rotating the two stimuli by 45° causes equal rotations of place cell firing fields. Removing either card and rotating the other card also causes fields to rotate equally, showing that the two stimuli are individually salient. Increasing or decreasing the card separation (card reconfiguration) causes a topological distortion of the representation of the cylinder floor such that field centers move relative to each other. We also found that either kind of reconfiguration induces a position-independent decrease in the intensity of place cell firing. We argue that these results are not compatible with either of two previously stated views of the place cell representation; namely, a nonspatial theory in which each place cell is tuned to an arbitrarily selected subset of available stimuli or a rigid map theory. We propose that our results imply that the representation is map-like but not rigid; it is capable of undergoing stretches without altering the local arrangement of firing fields.
Role of Aquaporin Water Channels in Airway Fluid Transport, Humidification, and Surface Liquid Hydration
Authors: Song Yuanlin , Jayaraman Sujatha , Yang Baoxue , Matthay Michael A. , Verkman A.S.
Published: July 2002

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport–related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54–56% efficient in wild-type mice, and reduced by only 3–4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3–5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 ± 5 μm and [Na+] was 115 ± 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by ∼40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption.
Compact weakly symmetric spaces and spherical pairs
Authors: Nguyêñ H.
Published: July 2002

Let

(
G
,
H
)

(G,H)

be a spherical pair and assume that

G
G

is a connected compact simple Lie group and

H
H

a closed subgroup of

G
G

. We prove in this paper that the homogeneous manifold

G

/

H

G/H

is weakly symmetric with respect to

G
G

and possibly an additional fixed isometry

μ
\mu

. It follows that M. Krämer’s classification list of such spherical pairs also becomes a classification list of compact weakly symmetric spaces. In fact, our proof involves a case-by-case study of the isotropy representations of all spherical pairs on Krämer’s list.
Representation of feedback operators for parabolic control problems
Authors: King Belinda
Published: July 2002

In this paper we present results on existence and regularity of integral representations of feedback operators arising from parabolic control problems. The existence of such representations is important for the design of low order compensators and in the placement of sensors. This paper extends earlier results of J. A. Burns and B. B. King to problems withNNspatial dimensions.
Almost everywhere convergence of lacunary partial sums of Vilenkin-Fourier series
Authors: Young Wo-Sang
Published: July 2002

We prove that if

f
∈

L

p

,

p
>
1

f\in L^{p},\;p>1

, and

{

n

k

}

\{n_{k}\}

is any lacunary sequence of positive integers, then the sequence of

n

k

n_{k}

th partial sums of Vilenkin-Fourier series of

f
f

converges almost everywhere to

f
f

.
The Lusin-Privalov theorem for subharmonic functions
Authors: Gardiner Stephen
Published: July 2002

This paper establishes a generalization of the Lusin-Privalov radial uniqueness theorem which applies to subharmonic functions in all dimensions. In particular, it answers a question of Rippon by showing that no subharmonic function on the upper half-space can have normal limit−∞-\inftyat every boundary point.
Fixed point iteration for pseudocontractive maps
Authors: Chidume C. , Moore Chika
Published: July 2002

Let

K
K

be a compact convex subset of a real Hilbert space,

H
H

;

T
:
K
→
K

T:K\rightarrow K

a continuous pseudocontractive map. Let
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="left-brace a Subscript n Baseline right-brace comma left-brace b Subscript n Baseline right-brace comma left-brace c Subscript n Baseline right-brace comma left-brace a Subscript n Superscript Super Superscript prime Superscript Baseline right-brace comma left-brace b Subscript n Superscript prime Baseline right-brace">

{

a

n

}
,
{

b

n

}
,
{

c

n

}
,
{

a

n

′

}
,
{

b

n

′

}

\{a_{n}\}, \{b_{n}\}, \{c_{n}\}, \{a_{n}^{’}\}, \{b_{n}^{’}\}

and

{

c

n

′

}

\{c_{n}^{’}\}

be real sequences in [0,1] satisfying appropriate conditions. For arbitrary

x

1

∈
K
,

x_{1}\in K,

define the sequence

{

x

n

}

n
=
1

∞

\{x_{n}\}_{n=1}^{\infty }

iteratively by
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="x Subscript n plus 1 Baseline equals a Subscript n Baseline x Subscript n Baseline plus b Subscript n Baseline upper T y Subscript n Baseline plus c Subscript n Baseline u Subscript n Baseline semicolon y Subscript n Baseline equals a Subscript n Superscript Super Superscript prime Superscript Baseline x Subscript n Baseline plus b Subscript n Superscript Super Superscript prime Superscript Baseline upper T x Subscript n Baseline plus c Subscript n Superscript Super Superscript prime Superscript Baseline v Subscript n Baseline comma n greater-than-or-equal-to 1 comma">

x

n
+
1

=

a

n

x

n

+

b

n

T

y

n

+

c

n

u

n

;

y

n

=

a

n

′

x

n

+

b

n

′

T

x

n

+

c

n

′

v

n

,
n
≥
1
,

x_{n+1} = a_{n}x_{n} + b_{n}Ty_{n} + c_{n}u_{n}; y_{n} = a_{n}^{’}x_{n} + b_{n}^{’}Tx_{n} + c_{n}^{’}v_{n}, n\geq 1,

where

{

u

n

}
,
{

v

n

}

\{u_{n}\}, \{v_{n}\}

are arbitrary sequences in

K
K

. Then,

{

x

n

}

n
=
1

∞

\{x_{n}\}_{n=1}^{\infty }

converges strongly to a fixed point of

T
T

. A related result deals with the convergence of

{

x

n

}

n
=
1

∞

\{x_{n}\}_{n=1}^{\infty }

to a fixed point of

T
T

when

T
T

is Lipschitz and pseudocontractive. Our theorems also hold for the slightly more general class of continuous hemicontractive nonlinear maps.
A generalization of Kelley’s theorem for 𝐶-spaces
Authors: Levin Michael , Rogers James
Published: July 2002

We prove that if an open mapf:X⟶Yf:X \longrightarrow Yof compactaXXandYYhas perfect fibers andYYis aCC-space, then there exists a00-dimensional compact subset ofXXintersecting each fiber offf. This is a stronger version of a well-known theorem of Kelley. Applications of this result and related topics are discussed.
Milnor classes of local complete intersections
Authors: Brasselet J.-P. , Lehmann D. , Seade J. , Suwa T.
Published: July 2002

LetVVbe a compact local complete intersection defined as the zero set of a section of a holomorphic vector bundle over the ambient space. For each connected componentSSof the singular setSing⁡(V)\operatorname {Sing}(V)ofVV, we define the Milnor classμ∗(V,S)\mu _{*}(V,S)in the homology ofSS. The difference between the Schwartz-MacPherson class and the Fulton-Johnson class ofVVis shown to be equal to the sum ofμ∗(V,S)\mu _{*}(V,S)over the connected componentsSSofSing⁡(V)\operatorname {Sing}(V). This is done by proving Poincaré-Hopf type theorems for these classes with respect to suitable tangent frames. The00-degree componentμ0(V,S)\mu _{0}(V,S)coincides with the Milnor numbers already defined by various authors in particular situations. We also give an explicit formula forμ∗(V,S)\mu _{*}(V,S)whenSSis a non-singular component andVVsatisfies the Whitney condition alongSS.
Simple families of Thue inequalities
Authors: Lettl Günter , Pethő Attila , Voutier Paul
Published: July 2002

We use the hypergeometric method to solve three families of Thue inequalities of degree 3, 4 and 6, respectively, each of which is parametrized by an integral parameter. We obtain bounds for the solutions, which are astonishingly small compared to similar results which use estimates of linear forms in logarithms.
On the distribution of the power generator
Authors: Friedlander John , Shparlinski Igor
Published: July 2002

We present a new method to study the power generator of pseudorandom numbers modulo a Blum integermm. This includes as special cases the RSA generator and the Blum–Blum–Shub generator. We prove the uniform distribution of these, provided that the periodt≥m3/4+δt\ge m^{3/4 + \delta }with fixedδ>0\delta > 0and, under the same condition, the uniform distribution of a positive proportion of the leftmost and rightmost bits. This sharpens and generalizes previous results which dealt with the RSA generator, provided the periodt≥m23/24+δt\ge m^{23/24 + \delta }. We apply our results to deduce that the period of the binary sequence of the rightmost bit has exponential length.
Optimal, quasi-optimal and superlinear band-Toeplitz preconditioners for asymptotically ill-conditioned positive definite Toeplitz systems
Authors: Serra Stefano
Published: July 2002

In this paper we are concerned with the solution ofn×nn\times nHermitian Toeplitz systems with nonnegative generating functionsff. The preconditioned conjugate gradient (PCG) method with the well–known circulant preconditioners fails in the case whereffhas zeros. In this paper we consider as preconditioners band–Toeplitz matrices generated by trigonometric polynomialsggof fixed degreell. We use different strategies of approximation offfto devise a polynomialggwhich has some analytical properties offf, is easily computable and is such that the corresponding preconditioned system has a condition number bounded by a constant independent ofnn. For each strategy we analyze the cost per iteration and the number of iterations required for the convergence within a preassigned accuracy. We obtain different estimates ofllfor which the total cost of the proposed PCG methods is optimal and the related rates of convergence are superlinear. Finally, for the most economical strategy, we perform various numerical experiments which fully confirm the effectiveness of approximation theory tools in the solution of this kind of linear algebra problems.
Separation of variables and the computation of Fourier transforms on finite groups, I
Authors: Maslen David , Rockmore Daniel
Published: July 2002

This paper introduces new techniques for the efficient computation of a Fourier transform on a finite group. We present a divide and conquer approach to the computation. The divide aspect uses factorizations of group elements to reduce the matrix sum of products for the Fourier transform to simpler sums of products. This is the separation of variables algorithm. The conquer aspect is the final computation of matrix products which we perform efficiently using a special form of the matrices. This form arises from the use of subgroup-adapted representations and their structure when evaluated at elements which lie in the centralizers of subgroups in a subgroup chain. We present a detailed analysis of the matrix multiplications arising in the calculation and obtain easy-to-use upper bounds for the complexity of our algorithm in terms of representation theoretic data for the group of interest. Our algorithm encompasses many of the known examples of fast Fourier transforms. We recover the best known fast transforms for some abelian groups, the symmetric groups and their wreath products, and the classical Weyl groups. Beyond this, we obtain greatly improved upper bounds for the general linear and unitary groups over a finite field, and for the classical Chevalley groups over a finite field. We also apply these techniques to obtain analogous results for homogeneous spaces. This is part I of a two part paper. Part II will present a refinement of these techniques which yields further savings.