bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: Implications for morphogenesis and organization of encapsidated RNA
Authors: Zlotnick A. , Cheng N. , Stahl S. J. , Conway J. F. , Steven A. C. , Wingfield P. T.
Published: July 2002

The capsid protein of hepatitis B virus, consisting of an “assembly” domain (residues 1–149) and an RNA-binding “protamine” domain (residues 150–183), assembles from dimers into icosahedral capsids of two different sizes. The C terminus of the assembly domain (residues 140–149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell. We now have defined the location of this peptide in capsids assembled
in vitro
by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy. The labeled protein is unimpaired in its ability to form capsids. Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers. Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds. Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds. Capsids assembled by expressing the full-length protein in
Escherichia coli
package bacterial RNAs in amounts equivalent to the viral pregenome. Our density map of these capsids reveals a distinct inner shell of density—the RNA. The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.
smg
mutants affect the expression of alternatively spliced SR protein mRNAs in
Caenorhabditis elegans
Authors: Morrison Mike , Harris Kevin S. , Roth Mark B.
Published: July 2002

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the
smg
genes of
Caenorhabditis elegans
participate in this process.
smg
genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that
smg
genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of
smg
genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMP-dependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a
smg
background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.
Neuroprotective activity of a new class of steroidal inhibitors of the
N
-methyl-
d
-aspartate receptor
Authors: Weaver Charles E. , Marek Przemyslaw , Park-Chung Mijeong , Tam S. William , Farb David H.
Published: July 2002

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of
N
-methyl-
d
-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregn
a
n-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregn
a
nolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The
in vitro
and
in vivo
neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.
Spatial memory is related to hippocampal subcellular concentrations of calcium-dependent protein kinase C isoforms in young and aged rats
Authors: Colombo Paul J. , Wetsel William C. , Gallagher Michela
Published: July 2002

Relationships were examined between spatial learning and hippocampal concentrations of the α, β
2
, and γ isoforms of protein kinase C (PKC), an enzyme implicated in neuronal plasticity and memory formation. Concentrations of PKC were determined for individual 6-month-old (
n
= 13) and 24-month-old (
n
= 27) male Long–Evans rats trained in the water maze on a standard place-learning task and a transfer task designed for rapid acquisition. The results showed significant relationships between spatial learning and the amount of PKC among individual subjects, and those relationships differed according to age, isoform, and subcellular fraction. Among 6-month-old rats, those with the best spatial memory were those with the highest concentrations of PKCγ in the particulate fraction and of PKCβ
2
in the soluble fraction. Aged rats had increased hippocampal PKCγ concentrations in both subcellular fractions in comparison with young rats, and memory impairment was correlated with higher PKCγ concentrations in the soluble fraction. No age difference or correlations with behavior were found for concentrations of PKCγ in a comparison structure, the neostriatum, or for PKCα in the hippocampus. Relationships between spatial learning and hippocampal concentrations of calcium-dependent PKC are isoform-specific. Moreover, age-related spatial memory impairment is associated with altered subcellular concentrations of PKCγ and may be indicative of deficient signal transduction and neuronal plasticity in the hippocampal formation.
Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr
Authors: Lowell Clifford A. , Berton Giorgio
Published: July 2002

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61hck(Hck) and p58c-fgr(Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β2and β3integrins. To determine if the defective function ofhck−/−fgr−/−neutrophils observedin vitroalso results in impaired inflammatory responsesin vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. Thehck−/−fgr−/−mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injuryin vivo.
Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells
Authors: Olofsson Birgitta , Korpelainen Eija , Pepper Michael S. , Mandriota Stefano J. , Aase Karin , Kumar Vijay , Gunji Yuji , Jeltsch Michael M. , Shibuya Masabumi , Alitalo Kari , Eriksson Ulf
Published: July 2002

The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp
63
, Asp
64
, and Glu
67
to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B
186
dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.
staggerer
phenotype in retinoid-related orphan receptor α-deficient mice
Authors: Steinmayr Markus , André Elisabeth , Conquet François , Rondi-Reig Laure , Delhaye-Bouchaud Nicole , Auclair Nathalie , Daniel Hervé , Crépel Francis , Mariani Jean , Sotelo Constantino , Becker-André Michael
Published: July 2002

Retinoid-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a
lacZ
reporter gene encoding the enzyme β-galactosidase. In heterozygous RORα
+/−
mice we found β-galactosidase activity, indicative of RORα protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORα gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORα is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORα. Recently, it was reported that the ataxic mouse mutant
staggerer
(
sg
/
sg
) is caused by a deletion in the RORα gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORα
−/−
mice proves identity to
sg
/
sg
mice. Although the absence of RORα causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORα
−/−
mice display a normal phenotype. However, the pelage hair of both
sg/sg
and RORα
−/−
is significantly less dense and when shaved shows reluctance to regrow.
Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity
in vitro
Authors: Fischer Thierry , Elenko Eric , McCaffery J. Michael , DeVries Luc , Farquhar Marilyn Gist
Published: July 2002

Gα-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Gα subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Gα
i
proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5×) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Gα
i3
and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Gα
i3
protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Gα
i3
and that membrane-associated GAIP is capable of interacting with Gα
i3
. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.
CD8
+
minor histocompatibility antigen-specific cytotoxic T lymphocyte clones eliminate human acute myeloid leukemia stem cells
Authors: Bonnet Dominique , Warren Edus H. , Greenberg Philip D. , Dick John E. , Riddell Stanley R.
Published: July 2002

Effective immunotherapy for human leukemia based on infusions of T lymphocytes requires the identification of effector T cells that target the leukemic stem cell. The transplantation of human acute myeloid leukemia into nonobese diabetic/severe combined immune deficient (SCID) mice has identified a rare leukemic progenitor termed the SCID leukemia-initiating cell, which is present in low frequency in the leukemic population and is essential for establishing leukemic hematopoiesis. Thus, this transplant model may be ideally suited to identify effector T cells with antileukemic activity. We report that CD8
+
cytotoxic T lymphocyte (CTL) clones specific for minor histocompatibility antigens inhibit the engraftment of human acute myeloid leukemia cells in nonobese diabetic/SCID mice and demonstrate that this inhibition is mediated by direct CTL recognition of SCID leukemia-initiating cells. These results indicate that CD8
+
minor histocompatibility antigen-specific CTL may be mediators of the graft-versus-leukemia effect associated with allogeneic hematopoietic cell transplantation and provide an experimental model to identify and select T cell clones for immunotherapy to prevent or treat relapse after allogeneic hematopoietic cell transplantation.
Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy
Authors: Brock Roland , Vàmosi György , Vereb György , Jovin Thomas M.
Published: July 2002

Fluorescence correlation microscopy (FCM) was applied to characterize fusion proteins of the green fluorescent protein (GFP) on the cellular as well as molecular level within seconds in an integrated instrument. FCM combines the inherent sensitivity and high spatial resolution of fluorescence correlation spectroscopy with fluorescence imaging and micropositioning, thereby providing a spectrum of molecular information in the cellular context. Signatures of characteristic parameters derived from the autocorrelation functions served to distinguish a GFP fusion protein of the epidermal growth factor receptor from GFP fluorescence in the endoplasmic reticulum and cytoplasm. Diffusion constants measured for free transiently expressed GFP reproduced values reported previously with other techniques. The accessible concentration range extends from millions to only a few thousand molecules per cell, with single molecule detectability in the femtoliter detection volume. The detailed molecular characterization offered by FCM is fully compatible with automation in sample identification and detection, offering new possibilities for highly integrated high-throughput screening.
Ecosystem rooting depth determined with caves and DNA
Authors: Jackson R. B. , Moore L. A. , Hoffmann W. A. , Pockman W. T. , Linder C. R.
Published: July 2002

Belowground vertical community composition and maximum rooting depth of the Edwards Plateau of central Texas were determined by using DNA sequence variation to identify roots from caves 5–65 m deep. Roots from caves were identified by comparing their DNA sequences for the internal transcribed spacer (ITS) region of the 18S–26S ribosomal DNA repeat against a reference ITS database developed for woody plants of the region. Sequencing the ITS provides, to our knowledge, the first universal method for identifying plant roots. At least six tree species in the system grew roots deeper than 5 m, but only the evergreen oak,Quercus fusiformis, was found below 10 m. The maximum rooting depth for the ecosystem was ≈25 m.18O isotopic signatures for stem water ofQ. fusiformisconfirmed water uptake from 18 m underground. The availability of resources at depth, coupled with small surface pools of water and nutrients, may explain the occurrence of deep roots in this and other systems.
RB
-mediated suppression of spontaneous multiple neuroendocrine neoplasia and lung metastases in
Rb
+/−
mice
Authors: Nikitin Alexander Yu. , Juárez-Pérez María I. , Li Song , Huang Leaf , Lee Wen-Hwa
Published: July 2002

Alterations in pathways mediated by retinoblastoma susceptibility gene (
RB
) product are among the most common in human cancer. Mice with a single copy of the
Rb
gene are shown to develop a syndrome of multiple neuroendocrine neoplasia. The earliest Rb-deficient atypical cells were identified in the intermediate and anterior lobes of the pituitary, the thyroid and parathyroid glands, and the adrenal medulla within the first 3 months of postnatal development. These cells form gross tumors with various degrees of malignancy by postnatal day 350. By age of 380 days, 84% of
Rb
+/−
mice exhibited lung metastases from C-cell thyroid carcinomas. Expression of a human
RB
transgene in the
Rb
+/−
mice suppressed carcinogenesis in all tissues studied. Of particular clinical relevance, the frequency of lung metastases also was reduced to 12% in
Rb
+/−
mice by repeated i.v. administration of lipid-entrapped, polycation-condensed
RB
complementary DNA. Thus, in spite of long latency periods during which secondary alterations can accumulate, the initial loss of Rb function remains essential for tumor progression in multiple types of neuroendocrine cells. Restoration of RB function in humans may prove an effective general approach to the treatment of RB-deficient disseminated tumors.
Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids
Authors: Royer William E. , Strand Kristen , van Heel Marin , Hendrickson Wayne A.
Published: July 2002

Many annelids, including the earthworm
Lumbricus terrestris
, have giant cooperative respiratory proteins (molecular masses greater than 3.5 million Da) freely dissolved in the blood, rather than packaged in cells. These complexes, termed either erythrocruorins or hemoglobins, are assembled from many copies of both hemoglobin subunits and nonhemoglobin or “linker” subunits. In this paper, we present the crystal structure of
Lumbricus
erythrocruorin at 5.5-Å resolution, which reveals a remarkable hierarchical organization of 144 oxygen-binding hemoglobin subunits and 36 nonhemoglobin linker subunits. The hemoglobin chains arrange in novel dodecameric substructures. Twelve trimeric linker complexes project triple-stranded helical coiled-coil “spokes” toward the center of the complex; interdigitation of these spokes appears crucial for stabilization. The resulting complex of linker chains forms a scaffold on which twelve hemoglobin dodecamers assemble. This structure specifies the unique, self-limited assemblage of a highly cooperative single molecule.
Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients
Authors: Lacerra Giuseppina , Sierakowska Halina , Carestia Clementina , Fucharoen Suthat , Summerton James , Weller Dwight , Kole Ryszard
Published: July 2002

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant β-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of β-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2–654/β
E
, IVS2–745/IVS2–745, and IVS2–745/IVS2–1 genotypes. The maximal Hb A level for repaired IVS2–745 mutation was ≈30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective β-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.
Putting the process in developmental processes
Authors: Nesselroade John R. , McCollam Karen M. Schmidt
Published: July 2002

Several signs point to a strengthening of our capabilities for rigorously modelling developmental processes and other kinds of changes. The indicators of progress range from stronger formulations of “systems thinking” and definitions through measurement and design considerations to advanced mathematical representations, such as linear and nonlinear dynamical systems models. We believe these advances offer major improvements for the treatment of process and related concepts as they have evolved thus far largely within the meta-model of stability and equilibrium that has dominated much of science (Holling, 1973). These issues are summarised and some of the promising innovations that we believe will make the coming decades highly productive ones for the study of development will be discussed.
The curriculum knowledge of teachers: a review of the potential of large‐scale, electronic conference environments for professional development
Authors: Leach Jenny
Published: July 2002

This article presents an analysis of two large‐scale computer mediated conference environments, supporting teacher education programmes. The analysis draws on programme purposes and policies, log file data, user questionnaires, and conference observation. A review of the task of creating, developing and sustaining such environments for professional learning is presented, based in particular on the community of practice (COP) concept. This focus on a social view of learning is sustained in the final section of the paper, where a model is proposed for exploring the development of teacher professional knowledge. The model is exemplified by means of individual messages and discussion threads drawn from the conferences. The article concludes that professional learning in e‐environments is only sustainable through the gradual development of joint enterprises, which draw on the common, as well as distributed knowledge, of a range of participants, including newcomers and expert practitioners.
Differential Effects of Tri‐n‐Butylstannyl Benzoates on Induction of Apoptosis in K562 and MCF‐7 Cells
Authors: Ray Dipankar , Sarma Koushik Das , Antony Abraham
Published: July 2002

Tributyltin compounds have been shown to induce apoptosis by causing extracellular Ca2+ influx and generating reactive oxygen species (ROS). Several organotin compounds were reported to have differential cytotoxicity on various human cell lines depending on the length of the alkyl chain. In this report, the cytotoxic effects of three tri‐n‐butylstannyl (halo)benzoate compounds‐tri‐n‐butylstannyl benzoate (TBSB), tri‐n‐butylstannyl 2,6‐difluorobenzoate (TBSDFB) and tri‐n‐butylstannyl‐2‐iodobenzoate (TBSIB)‐were studied on lymphocytic cells of human leukemic K562 lineage and epithelial cells of human breast cancer MCF‐7 cells lineage. K562 cells were found to be more sensitive to these compounds than MCF‐7 cells. Although the induction of apoptosis by the above compounds in K562 cells resulted from the extracellular Ca2+ influx and the generation of ROS, the initial amount of extracellular Ca2+ influx was greater in TBSB‐treated K562 cells than the cells treated with either TBSDFB or TBSIB. Similarly, DNA fragmentation by endonucleases was observed as an early event in TBSB‐treated K562 cells, which might be correlated with the initially greater extracellular Ca2+ influx. In contrast, MCF‐7 cells were found to undergo apoptosis mainly because of the generation of ROS. The present results suggest that the differential effects of tributyltin compounds on induction of apoptosis in K562 and MCF‐7 cells are largely attributable to the extent of extracellular Ca2+ influx.
The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway
Authors: Piper Robert C. , Bryant Nia J. , Stevens Tom H.
Published: July 2002

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.
The Ras Target AF-6 is a Substrate of the Fam Deubiquitinating Enzyme
Authors: Taya Shinichiro , Yamamoto Takaharu , Kano Kyoko , Kawano Yoji , Iwamatsu Akihiro , Tsuchiya Tomoko , Tanaka Keiji , Kanai-Azuma Masami , Wood Stephen A. , Mattick John S. , Kaibuchi Kozo
Published: July 2002

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell–cell adhesions, and is thought to participate in cell–cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of ∼220 kD (p220) to investigate the function of AF-6 at cell–cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell–cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.
Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo
Authors: Wu Xufeng , Bowers Blair , Rao Kang , Wei Qin , Hammer John A.
Published: July 2002

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.
A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants
Authors: Hurt Ed , Hannus Stefan , Schmelzl Birgit , Lau Denise , Tollervey David , Simos George
Published: July 2002

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.
Similarities and Differences in RANTES- and (AOP)-RANTES–triggered Signals: Implications for Chemotaxis
Authors: Rodríguez-Frade José M. , Vila-Coro Antonio J. , Martín Ana , Nieto Marta , Sánchez-Madrid Francisco , Proudfoot Amanda E.I. , Wells Timothy N.C. , Martínez-A Carlos , Mellado Mario
Published: July 2002

Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein–coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Gαi as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.
Functions of c-Jun in Liver and Heart Development
Authors: Eferl Robert , Sibilia Maria , Hilberg Frank , Fuchsbichler Andrea , Kufferath Iris , Guertl Barbara , Zenz Rainer , Wagner Erwin F. , Zatloukal Kurt
Published: July 2002

Mice lacking the AP-1 transcription factor c-Jun die around embryonic day E13.0 but little is known about the cell types affected as well as the cause of embryonic lethality. Here we show that a fraction of mutant E13.0 fetal livers exhibits extensive apoptosis of both hematopoietic cells and hepatoblasts, whereas the expression of 15 mRNAs, including those of albumin, keratin 18, hepatocyte nuclear factor 1, β-globin, and erythropoietin, some of which are putative AP-1 target genes, is not affected. Apoptosis of hematopoietic cells in mutant livers is most likely not due to a cell-autonomous defect, since c-jun−/− fetal liver cells are able to reconstitute all hematopoietic compartments of lethally irradiated recipient mice. A developmental analysis of chimeras showed contribution of c-jun−/− ES cell derivatives to fetal, but not to adult livers, suggesting a role of c-Jun in hepatocyte turnover. This is in agreement with the reduced mitotic and increased apoptotic rates found in primary liver cell cultures derived from c-jun−/− fetuses. Furthermore, a novel function for c-Jun was found in heart development. The heart outflow tract of c-jun−/− fetuses show malformations that resemble the human disease of a truncus arteriosus persistens. Therefore, the lethality of c-jun mutant fetuses is most likely due to pleiotropic defects reflecting the diversity of functions of c-Jun in development, such as a role in neural crest cell function, in the maintenance of hepatic hematopoiesis and in the regulation of apoptosis.
Focal Adhesion Kinase Mediates the Integrin Signaling Requirement for Growth Factor Activation of Map Kinase
Authors: Renshaw Mark W. , Price Leo S. , Schwartz Martin Alexander
Published: July 2002

The mitogen-activated protein (MAP) kinase pathway is a critical regulator of cell growth, migration, and differentiation. Growth factor activation of MAP kinase in NIH 3T3 cells is strongly dependent upon integrin-mediated adhesion, an effect that contributes to the anchorage dependence of normal cell growth. We now show that expression of constructs that constitutively activate focal adhesion kinase (FAK) rescued the defect in serum activation of MAP kinase in suspended cells without directly activating MAP kinase. Dominant negative FAK blocked both the rescue of suspended cells by the activated construct and the serum activation of MAP kinase in adherent cells. MAP kinase in FAK−/− mouse embryo fibroblasts was adhesion-insensitive, and reexpression of FAK restored its adhesion dependence. MAP kinase activity in ras-transformed cells is still decreased in suspension, but expression of constructs that constitutively activate FAK enhanced their anchorage-independent growth without increasing adherent growth. V-src, which activates both Ras and FAK, induced MAP kinase activation that was insensitive to loss of adhesion, and that was blocked by a dominant negative FAK. These results demonstrate that FAK mediates the integrin requirement for serum activation of MAP kinase in normal cells, and that bypassing this mechanism contributes to anchorage-independent growth in transformed cells.
Role of Cell Surface Metalloprotease Mt1-Mmp in Epithelial Cell Migration over Laminin-5
Authors: Koshikawa Naohiko , Giannelli Gianluigi , Cirulli Vincenzo , Miyazaki Kaoru , Quaranta Vito
Published: July 2002

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the γ2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2− cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2− cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.
Analysis of Notch Lacking the Carboxyl Terminus Identified in Drosophila Embryos
Authors: Wesley Cedric S. , Saez Lino
Published: July 2002

The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. We report here that certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch lacking the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor we designate NΔCterm.
The Specificity for the Differentiation Blocking Activity of Carcinoembryonic Antigen Resides in Its Glycophosphatidyl-Inositol Anchor
Authors: Screaton Robert A. , DeMarte Luisa , Dráber Petr , Stanners Clifford P.
Published: July 2002

Ectopic expression of various members of the human carcinoembryonic antigen (CEA) family of intercellular adhesion molecules in murine myoblasts either blocks (CEA, CEACAM6) or allows (CEACAM1) myogenic differentiation. These surface glycoproteins form a subset of the immunoglobulin (Ig) superfamily and are very closely related, but differ in the precise sequence of their external domains and in their mode of anchorage to the cell membrane. CEA and CEACAM6 are glycophosphatidyl-inositol (GPI) anchored, whereas CEACAM1 is transmembrane (TM) anchored. Overexpression of GPI-linked neural cell adhesion molecule (NCAM) p125, also an adhesion molecule of the Ig superfamily, accelerates myogenic differentiation. The molecular requirements for the myogenic differentiation block were investigated using chimeric constructs in which the COOH-terminal hydrophobic domains of CEA, CEACAM1, and NCAM p125 were exchanged. The presence of the GPI signal sequence specifically from CEA in the chimeras was sufficient to convert both CEACAM1 and NCAM into differentiation-blocking proteins. Conversely, CEA could be converted into a neutral protein by exchanging its GPI anchor for the TM anchor of CEACAM1. Since the external domains of CEA, CEACAM1, and NCAM can all undergo homophilic interactions, and mutations in the self-adhesive domains of CEA abrogate its differentiation-blocking activity, the structural requirements for differentiation-inhibition are any self-adhesive domains attached to the specific GPI anchor derived from CEA. We therefore suggest that biologically significant functional information resides in the processed extreme COOH terminus of CEA and in the GPI anchor that it determines.
Developmental Regulation of  VDJ Recombination By the Core Fragment of the T Cell Receptor α Enhancer
Authors: Roberts Joseph L. , Lauzurica Pilar , Krangel Michael S.
Published: July 2002

The role of T cell receptor α enhancer (Eα) cis-acting elements in the developmental regulation of VDJ recombination at the TCR α/δ locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp Tα1,2 core enhancer fragment of the 1.4-kb Eα is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within Tα1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb Eα initiates at fetal day 16.5 and is strictly limited to αβ T cells, we find that rearrangement under the control of Tα1,2 initiates slightly earlier during ontogeny and occurs in both γδ and αβ T cells. We conclude that the core fragment of Eα can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the Eα core cannot account for the precise developmental onset of accessibility that is provided by the intact Eα. Rather, our data suggests a critical role for factors that bind Eα outside of the core Tα1,2 region in establishing the precise developmental onset of TCR α rearrangement in vivo.
Association with FcRγ Is Essential for Activation Signal through NKR-P1 (CD161) in Natural Killer (NK) Cells and NK1.1+ T Cells
Authors: Arase Noriko , Arase Hisashi , Park Seung Yong , Ohno Hiroshi , Ra Chisei , Saito Takashi
Published: July 2002

Natural killer (NK) cells exhibit cytotoxicity against variety of tumor cells and virus-infected cells without prior sensitization and represent unique lymphocytes involved in primary host defense. NKR-P1 is thought to be one of NK receptors mediating activation signals because cross-linking of NKR-P1 activates NK cells to exhibit cytotoxicity and IFN-γ production. However, molecular mechanism of NK cell activation via NKR-P1 is not well elucidated. In this study, we analyzed the cell surface complex associated with NKR-P1 on NK cells and found that NKR-P1 associates with the FcRγ chain which is an essential component of Fc receptors for IgG and IgE. The association between FcRγ and NKR-P1 is independent of Fc receptor complexes. Furthermore, NK cells from FcRγ-deficient mice did not show cytotoxicity or IFN-γ production upon NKR-P1 cross-linking. Similarly, NK1.1+ T cells from FcRγ-deficient mice did not produce IFN-γ upon NKR-P1 crosslinking. These findings demonstrate that the FcRγ chain plays an important role in activation of NK cells via the NKR-P1 molecule.
A New Monoclonal Antibody, mAb 4A12, Identifies a Role for the Glycosaminoglycan (GAG) Binding Domain of RANTES in the Antiviral Effect against HIV-1 and Intracellular Ca2+ Signaling
Authors: Burns Jennifer M. , Gallo Robert C. , DeVico Anthony L. , Lewis George K.
Published: July 2002

The β-chemokine RANTES (regulated on activation, normal T cell expressed and secreted) suppresses the infection of susceptible host cells by macrophage tropic strains of HIV-1. This effect is attributed to interactions of this chemokine with a 7-transmembrane domain receptor, CCR5, that is required for virus–cell fusion and entry. Here we identify domains of RANTES that contribute to its biological activities through structure–function studies using a new monoclonal antibody, mAb 4A12, isolated from mice immunized with recombinant human RANTES. This monoclonal antibody (mAb) blocked the antiviral activity of RANTES in infectivity assays with HIV-1Bal, and inhibited the mobilization of intracellular Ca2+ elicited by RANTES, yet recognized this chemokine bound to cell surfaces. Epitope mapping using limited proteolysis, reversed phase high-performance liquid chromatography, and mass spectrometry suggest that residues 55–66 of RANTES, which include the COOH-terminal α-helical region implicated as the glycosaminoglycan (GAG) binding domain, overlap the determinant recognized by mAb 4A12. This is supported by affinity chromatography studies, which showed that RANTES could be eluted specifically by heparin from a mAb 4A12 immunoaffinity matrix. Removal of cell surface GAGs by enzymatic digestion greatly reduced the ability of mAb 4A12 to detect RANTES passively bound on cell surfaces and abrogated the ability of RANTES to elicit an intracellular Ca2+ signal. Taken together, these studies demonstrate that the COOH-terminal α-helical region of RANTES plays a key role in GAG-binding, antiviral activity, and intracellular Ca2+ signaling and support a model in which GAGs play a key role in the biological activities of this chemokine.
B7-2 Expressed on EL4 Lymphoma Suppresses Antitumor Immunity by an Interleukin 4–dependent Mechanism
Authors: Stremmel C. , Greenfield E.A. , Howard E. , Freeman G.J. , Kuchroo V.K.
Published: July 2002

For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the “second signal” pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4–B7-1 cells are completely rejected in syngeneic mice. Unlike EL4–B7-1 cells, we find that EL4–B7-2 cells are not rejected but progressively grow in the mice. A B7-1– and B7-2–EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4–B7-1 tumor line that regressed in vivo. The EL4–B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti–B7-1 antibodies, anti–B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti–B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4–B7-2 and EL4–B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response.