bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromophore-assisted light inactivation and self-organization of microtubules and motors
Authors: Surrey Thomas , Elowitz Michael B. , Wolf Pierre-Etienne , Yang Feng , Nédélec François , Shokat Kevan , Leibler Stanislas
Published: July 2002

Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an
in vitro
system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.
Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia
Authors: Liu Fu-Chin , Graybiel Ann M.
Published: July 2002

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (
i
) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (
ii
) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (
iii
) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca
2+
influx by stimulation of L-type Ca
2+
channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.
CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex
Authors: Hsieh James J.-D. , Zhou Sifang , Chen Lin , Young David B. , Hayward S. Diane
Published: July 2002

CBF1 is a member of the CSL family of DNA binding factors, which mediate either transcriptional repression or transcriptional activation. CSL proteins play a central role in Notch signaling and in Epstein–Barr virus-induced immortalization. Notch is a transmembrane protein involved in cell-fate decisions, and the cytoplasmic domain of Notch (NotchIC) targets CBF1. The Epstein–Barr virus-immortalizing protein EBNA2 activates both cellular and viral gene expression by targeting CBF1 and mimicking NotchIC. We have examined the mechanism of CBF1-mediated repression and show that CBF1 binds to a unique corepressor, CBF1 interacting corepressor (CIR). A CIR homolog is encoded byCaenorhabditis elegans, indicating that CIR is evolutionarily conserved. Two CBF1 mutants that were unable to bind CIR did not function as repressors, suggesting that targeting of CIR to CBF1 is an important component of repression. When expressed as a Gal4 fusion protein, CIR repressed reporter gene expression. CIR binds to histone deacetylase and to SAP30 and serves as a linker between CBF1 and the histone deacetylase complex.
O
2
sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase
Authors: Archer Stephen L. , Reeve Helen L. , Michelakis Evangelos , Puttagunta Lakshmi , Waite Ross , Nelson Daniel P. , Dinauer Mary C. , Weir E. Kenneth
Published: July 2002

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O
2
-responsive K
+
channels. K
+
channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K
+
channels are not themselves “O
2
sensors” but rather respond to the reduced redox state created by hypoxic inhibition of candidate O
2
sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O
2
. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K
+
channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K
+
current but lack the specificity to identify the O
2
-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O
2
-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O
2
species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K
+
current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O
2
sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O
2
sensor in mice.
Genetic dissection of dome formation in a mammary cell line: Identification of two genes with opposing action
Authors: Zucchi Ileana , Montagna Cristina , Susani Lucia , Montesano Roberto , Affer Maurizio , Zanotti Simona , Redolfi Elena , Vezzoni Paolo , Dulbecco Renato
Published: July 2002

In this work, we extend the study of the genes controlling the formation of domes in the rat mammary cell line LA7 under the influence of DMSO. The role of therat8gene has already been demonstrated. We have now studied two additional genes. The first, called133, is the rat ortholog of the human epithelial membrane protein 3 (EMP3), a member of the peripheral myelin protein 22 (PMP22)/EMP/lens-specific membrane protein 20 (MP20) gene family that encodes for tetratransmembrane proteins; it is expressed in the LA7 line in the absence of DMSO but not in its presence. The second gene is the β subunit of the amiloride-sensitive Na+channel. Studies with antisense oligonucleotides show that the formation of domes is under the control of all three genes: the expression ofrat8is required for both their formation and their persistence; the expression of the Na+channel β subunit is required for their formation; and the expression of gene133blocks the expression of the Na+channel genes, thus preventing formation of the domes. The formation of these structures is also accompanied by the expression of α6β1integrin, followed by that of E-cadherin and cytokeratin 8. It appears, therefore, that dome formation requires the activity of the Na+channel and therat8-encoded protein and is under the negative control of gene133. DMSO induces dome formation by blocking this control.
Tyrosine replacement in P-selectin glycoprotein ligand-1 affects distinct kinetic and mechanical properties of bonds with P- and L-selectin
Authors: Ramachandran Vishwanath , Nollert Matthias U. , Qiu Haiying , Liu Wen-Jun , Cummings Richard D. , Zhu Cheng , McEver Rodger P.
Published: July 2002

Selectins are adhesion molecules that initiate tethering and
rolling of leukocytes on the vessel wall. Rolling requires rapid
formation and breakage of selectin–ligand bonds that must have
mechanical strength to resist premature dissociation by the forces
applied in shear flow. P- and L-selectin bind to the N-terminal region
of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. To
define determinants on PSGL-1 that contribute to the kinetic and
mechanical properties of bonds with selectins, we compared rolling of
transfected preB cells expressing P- or L-selectin on transfected cell
monolayers expressing wild-type PSGL-1 or PSGL-1 constructs with
substitutions in targeted N-terminal residues. Rolling through P- or
L-selectin required a Thr or Ser at a specific position on PSGL-1, the
attachment site for an essential O-glycan, but required only one of
three nearby Tyr residues, which are sites for Tyr-SO
3
formation. The adhesive strengths and numbers of cells rolling through
P- or L-selectin were similar on wild-type PSGL-1 and on each of the
three PSGL-1 constructs containing only a single Tyr. However, the
cells rolled more irregularly on the single-Tyr forms of PSGL-1.
Analysis of the lifetimes of transient tethers on limiting densities of
PSGL-1 revealed that L-selectin dissociated faster from single-Tyr than
wild-type PSGL-1 at all shears examined. In sharp contrast, P-selectin
dissociated faster from single-Tyr than wild-type PSGL-1 at higher
shear but not at lower shear. Thus, tyrosine replacements in PSGL-1
affect distinct kinetic and mechanical properties of bonds with P- and
L-selectin.
A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy
Authors: Burton Edward A. , Tinsley Jonathon M. , Holzfeind Paul J. , Rodrigues Nanda R. , Davies Kay E.
Published: July 2002

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated
in vivo
. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.
Development of an
in vitro
reconstitution assay for glucose transporter 4 translocation
Authors: Inoue Gen , Cheatham Bentley , Kahn C. Ronald
Published: July 2002

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an
in vitro
reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca
2+
-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5′-[γ-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted
in vitro
to a limited extent by using isolated membranes. This association appears to involve protein–protein interactions among the SNARE (soluble
N
-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.
FOG-2: A novel GATA-family cofactor related to multitype zinc-finger proteins Friend of GATA-1 and U-shaped
Authors: Tevosian Sergei G. , Deconinck Anne E. , Cantor Alan B. , Rieff Heather I. , Fujiwara Yuko , Corfas Gabriel , Orkin Stuart H.
Published: July 2002

GATA factors are transcriptional regulatory proteins that play critical roles in the differentiation of multiple cell types in both vertebrates and invertebrates. Recent evidence suggests that the biological activities of both mammalian and
Drosophila
GATA factors are controlled in part by physical interaction with multitype zinc-finger proteins, Friend of GATA-1 (FOG) and U-shaped (Ush), respectively. Here we describe a new FOG-related polypeptide, designated FOG-2, that is likely to participate in differentiation mediated by GATA factors in several tissues. Expression of FOG-2 mRNA differs from that of FOG and is largely restricted to heart, neurons, and gonads in the adult. Somewhat broader expression is evident during mouse embryonic development. Similar to FOG and Ush, FOG-2 protein interacts specifically with the amino finger of GATA factors in the yeast two-hybrid system and in mammalian cells. Remarkably, though FOG-2 is quite divergent from FOG in its primary sequence, forced expression of FOG-2 rescues terminal erythroid maturation of FOG
−/−
hematopoietic cells. Thus, members of the FOG family of cofactors share highly specific association with GATA factors and are substantially interchangeable with respect to some aspects of function
in vivo
. The interaction of GATA and FOG family members constitutes an evolutionarily conserved paradigm for transcriptional control in differentiation and organogenesis.
Opposing effects of protein kinase C and protein kinase A on metabotropic glutamate receptor signaling: Selective desensitization of the inositol trisphosphate/Ca
2+
pathway by phosphorylation of the receptor-G protein-coupling domain
Authors: Francesconi Anna , Duvoisin Robert M.
Published: July 2002

Signaling by the metabotropic glutamate receptor 1α (mGluR1α) can lead to the accumulation of inositol 1,4,5-trisphosphate (Ins
P
3
) and cAMP and to the modulation of K
+
and Ca
2+
channel opening. At present, very little is known about how these different actions are integrated and eventually turned off. Unraveling the molecular mechanisms underlying these functions is crucial for understanding mGluR-mediated regulation of synaptic transmission. It has been shown that receptor-induced activation of the Ins
P
3
pathway is subject to feedback inhibition mediated by protein kinase C (PKC). In this study, we provide evidence for a differential regulation by PKC and protein kinase A of two distinct mGluR1α-dependent signaling pathways. PKC activation selectively inhibits agonist-dependent stimulation of the Ins
P
3
pathway but does not affect receptor signaling via cAMP. In contrast, protein kinase A potentiates agonist-independent signaling of the receptor via Ins
P
3
. Furthermore, we demonstrate that the selectivity of PKC action on receptor signaling rests on phosphorylation of a threonine residue located in the G protein-interacting domain of the receptor. Modification at Thr
695
selectively disrupts mGluR1α–G
q/11
interaction without affecting signaling through G
s
. Together, these data provide insight on the mechanisms by which selective down-regulation of a specific receptor-dependent signaling pathway can be achieved and on how cross-talk between different second messenger cascades may contribute to fine-tune short- and long-term receptor activity.
Monopoles and fractional vortices in chiral superconductors
Authors: Volovik G. E.
Published: July 2002

I discuss two exotic objects that must be experimentally identified in chiral superfluids and superconductors. These are (
i
) the vortex with a fractional quantum number (
N
= 1/2 in chiral superfluids, and
N
= 1/2 and
N
= 1/4 in chiral superconductors), which plays the part of the Alice string in relativistic theories and (
ii
) the hedgehog in the ^
l
field, which is the counterpart of the Dirac magnetic monopole. These objects of different dimensions are topologically connected. They form the combined object that is called a nexus in relativistic theories. In chiral superconductors, the nexus has magnetic charge emanating radially from the hedgehog, whereas the half-quantum vortices play the part of the Dirac string. Each half-quantum vortex supplies the fractional magnetic flux to the hedgehog, representing 1/4 of the “conventional” Dirac string. I discuss the topological interaction of the superconductor's nexus with the ‘t Hooft–Polyakov magnetic monopole, which can exist in Grand Unified Theories. The monopole and the hedgehog with the same magnetic charge are topologically confined by a piece of the Abrikosov vortex. Such confinement makes the nexus a natural trap for the magnetic monopole. Other properties of half-quantum vortices and monopoles are discussed as well, including fermion zero modes.
Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles
Authors: Fischer Thierry , Elenko Eric , Wan Lei , Thomas Gary , Farquhar Marilyn Gist
Published: July 2002

GAIP (G α interacting protein) is a member of the RGS (regulators of G protein signaling) family and accelerates the turnover of GTP bound to Gαi, Gαq, and Gα13. There are two pools of GAIP—a soluble and a membrane-anchored pool. The membrane-anchored pool is found on clathrin-coated vesicles (CCVs) and pits in rat liver and AtT-20 pituitary cells. By treatment of a GAIP-enriched rat liver fraction with alkaline phosphatase, we found that membrane-bound GAIP is phosphorylated. By immunoprecipitation carried out on [32P]orthophosphate-labeled AtT-20 pituitary cells stably expressing GAIP,32P-labeling was associated exclusively with the membrane pool of GAIP. Phosphoamino acid analysis revealed that phosphorylation of GAIP occurred largely on serine residues. Recombinant GAIP could be phosphorylated at its N terminus with purified casein kinase 2 (CK2). It could also be phosphorylated by isolated CCVsin vitro. Phosphorylation was Mn2+-dependent, using both purified CK2 and CCVs. Ser-24 was identified as one of the phosphorylation sites. Our results establish that GAIP is phosphorylated and that only the membrane pool is phosphorylated, suggesting that GAIP can be regulated by phosphorylation events taking place at the level of clathrin-coated pits and vesicles.
Identifying Prenatal Alcohol Use: Screening Instruments Versus Clinical Predictors
Authors: Chang Grace , Goetz Margaret Ann , Wilkins‐Haug Louise , Berman Susan
Published: July 2002

The purpose of this study is to compare the accuracy of screening instruments with clinical predictors in the identification of prenatal alcohol use. 350 women initiating prenatal care at the Brigham and Women's Hospital (Boston, MA) completed the T‐ACE, AUDIT, and SMAST. The predictive accuracy of each was compared using Receiver Operating Characteristic (ROC) curve analysis. The T‐ACE, AUDIT, and clinical predictors alone correctly identified 65 to 70% of current drinkers, whereas the SMAST alone performed only slightly better than chance. The predictive ability of the T‐ACE was further improved with the addition of clinical predictors.
Mitochondrial Association of a Plus End–Directed Microtubule Motor Expressed during Mitosis in Drosophila
Authors: Pereira Andrea J. , Dalby Brian , Stewart Russell J. , Doxsey Stephen J. , Goldstein Lawrence S.B.
Published: July 2002

The kinesin superfamily is a large group of proteins (kinesin-like proteins [KLPs]) that share sequence similarity with the microtubule (MT) motor kinesin. Several members of this superfamily have been implicated in various stages of mitosis and meiosis. Here we report our studies on KLP67A of Drosophila. DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain. To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction. We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types. In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern. Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled. These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle.
Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum
Authors: Scheel Jochen , Pepperkok Rainer , Lowe Martin , Griffiths Gareth , Kreis Thomas E.
Published: July 2002

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.
ICAMs Redistributed by Chemokines to Cellular Uropods as a Mechanism for Recruitment of T Lymphocytes
Authors: Pozo Miguel Angel del , Cabañas Carlos , Montoya María C. , Ager Ann , Sánchez-Mateos Paloma , Sánchez-Madrid Francisco
Published: July 2002

The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte–endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5–10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti– ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.
Gelsolin Modulates Phospholipase C Activity In Vivo through Phospholipid Binding
Authors: Sun Hui-qiao , Lin Keng-mean , Yin Helen L.
Published: July 2002

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ during agonist stimulation. A physiologically relevant rise in Ca2+ increases their affinity for PIP2 and can promote significant interactions with PIP2 in activated cells. This may impact divergent PIP2- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase Cγ (PLCγ) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP2 in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLCβ. Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLCβ, and only gelsolin domains containing PIP2-binding sites were effective. Inhibition was mitigated by increasing PIP2 concentration in a manner consistent with competition between gelsolin and PLCβ for PIP2. Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLCγ, although they inhibited PLCγ less than PLCβ. Our findings indicate that as PIP2 level and availability change during signaling, cross talk between PIP2-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.
Neocentromere-mediated Chromosome Movement in Maize
Authors: Yu Hong-Guo , Hiatt Evelyn N. , Chan Annette , Sweeney Mary , Dawe R. Kelly
Published: July 2002

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences.
Activation of a Retroviral Membrane Fusion Protein: Soluble Receptor-induced Liposome Binding of the ALSV Envelope Glycoprotein
Authors: Hernandez Lorraine D. , Peters Reuben J. , Delos Sue E. , Young John A.T. , Agard David A. , White Judith M.
Published: July 2002

It is not known how membrane fusion proteins that function at neutral pH, for example the human immunodeficiency virus envelope (Env) glycoprotein and intracellular fusion machines, are activated for target bilayer binding. We have addressed this question using a soluble oligomeric form of an avian retroviral Env glycoprotein (API) and soluble forms of its receptor. Binding of soluble receptor to API induces API to bind to liposomes composed of phosphatidylcholine and cholesterol at neutral pH. Liposome binding only occurs at fusion permissive temperatures (T > 20°C), is complete between 2 to 5 min at 37°C, and is stable to high salt, carbonate, and urea. Liposome binding is mediated by the ectodomain of the transmembrane subunit of API, and a mutant with a Val to Glu substitution in the Env fusion peptide (located in the ectodomain of the transmembrane subunit) shows significantly reduced liposome binding. Moreover, under conditions of equivalent binding to API, a mutant receptor that does not support infection (Zingler, K., and J.A.T. Young. 1996. J. Virol. 70:7510–7516) does not induce significant liposome binding. Our results indicate that a highly specific interaction between an avian retroviral Env and its receptor activates the retroviral glycoprotein for target bilayer binding at neutral pH in much the same way as low pH activates the influenza hemagglutinin. Our findings are discussed in terms of the mechanisms of viral and cellular fusion proteins that function at neutral pH.
Thyroid Hormone Induces Apoptosis in Primary Cell Cultures of Tadpole Intestine: Cell Type Specificity and Effects of Extracellular Matrix
Authors: Su Yuan , Shi Yufang , Stolow Melissa A. , Shi Yun-Bo
Published: July 2002

Thyroid hormone (T3 or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T3 can enhance the proliferation of both cell types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis.
L-Selectin Ligands That Are O-glycoprotease Resistant and Distinct from MECA-79 Antigen are Sufficient for Tethering and Rolling of Lymphocytes on Human High Endothelial Venules
Authors: Clark Rachael A. , Fuhlbrigge Robert C. , Springer Timothy A.
Published: July 2002

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)–containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease–resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by ∼60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.
Dystrophic Muscle in Mice Chimeric for Expression of α5 Integrin
Authors: Taverna Daniela , Disatnik Marie-Helene , Rayburn Helen , Bronson Roderick T. , Yang Joy , Rando Thomas A. , Hynes Richard O.
Published: July 2002

α5-deficient mice die early in embryogenesis (Yang et al., 1993). To study the functions of α5 integrin later in mouse embryogenesis and during adult life we generated α5 −/−;+/+ chimeric mice. These animals contain α5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that α5 −/− cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (Taverna et al., 1998). Histological analysis of α5 −/−;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration–regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of α5 −/− ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is α5-integrin-dependent.
Elasticity Measurements Show the Existence of Thin Rigid Cores Inside Mitotic Chromosomes
Authors: Houchmandzadeh Bahram , Dimitrov Stefan
Published: July 2002

Chromosome condensation is one of the most critical steps during cell division. However, the structure of condensed mitotic chromosomes is poorly understood. In this paper we describe a new approach based on elasticity measurements for studying the structure of in vitro assembled mitotic chromosomes in Xenopus egg extract. The approach is based on a unique combination of measurements of both longitudinal deformability and bending rigidity of whole chromosomes. By using specially designed micropipettes, the chromosome force–extension curve was determined. Analysis of the curvature fluctuation spectrum allowed for the measurement of chromosome bending ridigity. The relationship between the values of these two parameters is very specific: the measured chromosome flexibility was found to be 2,000 times lower than the flexibility calculated from the experimentally determined Young modulus. This requires the chromosome structure to be formed of one or a few thin rigid elastic axes surrounded by a soft envelope. The properties of these axes are well-described by models developed for the elasticity of titin-like molecules. Additionally, the deformability of in vitro assembled chromosomes was found to be very similar to that of native somatic chromosomes, thus demonstrating the existence of an essentially identical structure.
Type 3 and Type 1 Ryanodine Receptors Are Localized in Triads of the Same Mammalian Skeletal Muscle Fibers
Authors: Flucher Bernhard E. , Conti Antonio , Takeshima Hiroshi , Sorrentino Vincenzo
Published: July 2002

The type 3 ryanodine receptor (RyR3) is a ubiquitous calcium release channel that has recently been found in mammalian skeletal muscles. However, in contrast to the skeletal muscle isoform (RyR1), neither the subcellular distribution nor the physiological role of RyR3 are known. Here, we used isoform-specific antibodies to localize RyR3 in muscles of normal and RyR knockout mice. In normal hind limb and diaphragm muscles of young mice, RyR3 was expressed in all fibers where it was codistributed with RyR1 and with the skeletal muscle dihydropyridine receptor. This distribution pattern indicates that RyR3 is localized in the triadic junctions between the transverse tubules and the sarcoplasmic reticulum. During development, RyR3 expression declined rapidly in some fibers whereas other fibers maintained expression of RyR3 into adulthood. Comparing the distribution of RyR3-containing fibers with that of known fiber types did not show a direct correlation. Targeted deletion of the RyR1 or RyR3 gene resulted in the expected loss of the targeted isoform, but had no adverse effects on the expression and localization of the respective other RyR isoform. The localization of RyR3 in skeletal muscle triads, together with RyR1, is consistent with an accessory function of RyR3 in skeletal muscle excitation–contraction coupling.
Single Channel Properties and Regulated Expression of Ca2+ Release-Activated Ca2+ (Crac) Channels in Human T Cells
Authors: Fomina Alla F. , Fanger Christopher M. , Kozak J. Ashot , Cahalan Michael D.
Published: July 2002

Although the crucial role of Ca2+ influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca2+ channels in normal human T lymphocytes. The use of Na+ as the permeant ion in divalent-free solution permitted Ca2+ release-activated Ca2+ (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca2+ store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca2+ in the micromolar range, selective Ca2+ permeation in the millimolar range, and inactivation that depended upon intracellular Mg2+ ions. The number of CRAC channels per cell increased greatly from ∼15 in resting T cells to ∼140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to ∼60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 μM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca2+ influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca2+ influx in human resting T cells, and that the expression of CRAC channels increases ∼10-fold during activation, resulting in enhanced Ca2+ signaling.
Src Homology 2 Protein Tyrosine Phosphatase (SHPTP2)/Src Homology 2 Phosphatase 2 (SHP2) Tyrosine Phosphatase Is a Positive Regulator of the Interleukin 5 Receptor Signal Transduction Pathways Leading to the Prolongation of Eosinophil Survival
Authors: Pazdrak Konrad , Adachi Tetsuya , Alam Rafeul
Published: July 2002

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5–stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with β common (βc) chain of the IL-5 receptor (IL-5βcR). The association of SHPTP2 with IL-5βcR was reconstituted using a synthetic phosphotyrosine-containing peptide, βc 605–624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612→ F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated βc receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.
Autopathogenic T Helper Cell Type 1 (Th1) and Protective Th2 Clones Differ in Their Recognition of the Autoantigenic Peptide of Myelin Proteolipid Protein
Authors: Das Mercy Prabhu , Nicholson Lindsay B. , Greer Judith M. , Kuchroo Vijay K.
Published: July 2002

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139–151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139–151 generated either by immunization with the PLP 139–151 peptide with anti– B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139–151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139–151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.