bluuebunny/crossref_metadata_2025 huggingface dataset:
Constraining the subunit order of rod cyclic nucleotide-gated channels reveals a diagonal arrangement of like subunits
Authors: He Yejun , Ruiz MariaLuisa , Karpen Jeffrey W.
Published: July 2002

Retinal rod cyclic nucleotide-gated channels are composed of α and β subunits. We have explored possible subunit arrangements by expressing tandemly linked dimers of both subunits and examining their responses to three different modulating agents. Channels formed from either α-β or β-α heterodimers had functional properties similar to those formed from coexpressed α and β monomers and to native channels. These results point to an α-β-α-β arrangement. To ensure that heterodimers had not flipped around, we coexpressed α-α dimers with an excess of either β monomers or β-β dimers. Our data indicate that heteromultimers do not form efficiently in an α-α-β-β arrangement. Thus, we propose that native rod cyclic nucleotide-gated channels are arranged with like subunits diagonally opposed: α-β-α-β.
The salicylate-derived mycobactin siderophores of
Mycobacterium tuberculosis
are essential for growth in macrophages
Authors: De Voss James J. , Rutter Kerry , Schroeder Benjamin G. , Su Hua , Zhu YaQi , Barry Clifton E.
Published: July 2002

Mycobacterium tuberculosis
is an important pathogen of mammals that relies on 2-hydroxyphenyloxazoline-containing siderophore molecules called mycobactins for the acquisition of iron in the restrictive environment of the mammalian macrophage. These compounds have been proposed to be biosynthesized through the action of a cluster of genes that include both nonribosomal peptide synthase and polyketide synthase components. One of these genes encodes a protein, MbtB, that putatively couples activated salicylic acid with serine or threonine and then cyclizes this precursor to the phenyloxazoline ring system. We have used gene replacement through homologous recombination to delete the
mbtB
gene and replace this with a hygromycin-resistance cassette in the virulent strain of
M. tuberculosis
H37Rv. The resulting mutant is restricted for growth in iron-limited media but grows normally in iron-replete media. Analysis of siderophore production by this organism revealed that the biosynthesis of all salicylate-derived siderophores was interrupted. The mutant was found to be impaired for growth in macrophage-like THP-1 cells, suggesting that siderophore production is required for virulence of
M. tuberculosis
. These results provide conclusive evidence linking this genetic locus to siderophore production.
Two populations of kainate receptors with separate signaling mechanisms in hippocampal interneurons
Authors: Rodríguez-Moreno Antonio , López-García Juan C. , Lerma Juan
Published: July 2002

Consistent with the epileptogenic and deleterious effects of the potent neurotoxin kainate, the activation of kainate receptors reduces the synaptic inhibition induced by the amino acid γ-aminobutyric acid (GABA). Extrapolating from these data led to the conclusion that kainate receptors are located presynaptically. However, kainate directly depolarizes the inhibitory interneurons, causing them to fire repeatedly. This effect might indirectly decrease the size of inhibitory postsynaptic currents recorded from pyramidal cells and places in doubt the presynaptic location for kainate receptors. Here we show that both effects, membrane depolarization and the reduction of inhibitory potentials, can be dissociated by several means, particularly by the natural agonist of kainate receptors, glutamate. Indeed, when applied at low concentrations, glutamate inhibited GABA release without affecting the firing rate of GABA interneurons. These results indicate that CA1 interneurons contain two populations of kainate receptors, each with different agonist sensitivity and coupled to distinct signaling pathways.
Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10)
Authors: Kotenko Sergei V. , Saccani Simona , Izotova Lara S. , Mirochnitchenko Olga V. , Pestka Sidney
Published: July 2002

We identified a viral IL-10 homolog encoded by an ORF (UL111a) within the human cytomegalovirus (CMV) genome, which we designated cmvIL-10. cmvIL-10 can bind to the human IL-10 receptor and can compete with human IL-10 for binding sites, despite the fact that these two proteins are only 27% identical. cmvIL-10 requires both subunits of the IL-10 receptor complex to induce signal transduction events and biological activities. The structure of the cmvIL-10 gene is unique by itself. The gene retained two of four introns of the IL-10 gene, but the length of the introns was reduced. We demonstrated that cmvIL-10 is expressed in CMV-infected cells. Thus, expression of cmvIL-10 extends the range of counter measures developed by CMV to circumvent detection and destruction by the host immune system.
Caenorhabditis elegans
embryonic axial patterning requires two recently discovered posterior-group Hox genes
Authors: Van Auken Kimberly , Weaver Daniel C. , Edgar Lois G. , Wood William B.
Published: July 2002

Hox genes encode highly conserved transcription factors that control regional identities of cells and tissues along the developing anterior–posterior axis, probably in all bilaterian metazoans. However, in invertebrate embryos other than
Drosophila
, Hox gene functions remain largely unknown except by inference from sequence similarities and expression patterns. Recent genomic sequencing has shown that
Caenorhabditis elegans
has three Hox genes of the posterior paralog group [Ruvkun, G. & Hobert, O. (1998)
Science
282, 2033–2041]. However, only one has been previously identified genetically, and it is not required for embryonic development [Chisholm, A. (1991)
Development (Cambridge, U.K.)
111, 921–932]. Herein, we report identification of the remaining two posterior paralogs as the
nob-1
gene and the neighboring
php-3
gene. Elimination of
nob-1
and
php-3
functions causes gross embryonic defects in both posterior patterning and morphogenetic movements of the posterior hypodermis, as well as posterior-to-anterior cell fate transformations and lethality. The only other Hox gene essential for embryogenesis is the
labial
/
Hox1
homolog
ceh-13
, required for more anterior patterning [Brunschwig, K., Wittmann, C., Schnabel, R., Burglin, T. R., Tobler, H. & Muller, F. (1999)
Development (Cambridge, U.K.)
126, 1537–1546]. Therefore, essential embryonic patterning in
C. elegans
requires only Hox genes of the anterior and posterior paralog groups, raising interesting questions about evolution of the medial-group genes.
Characterization of Rny1, the
Saccharomyces cerevisiae
member of the T
2
RNase family of RNases: Unexpected functions for ancient enzymes?
Authors: MacIntosh Gustavo C. , Bariola Pauline A. , Newbigin Ed , Green Pamela J.
Published: July 2002

The T
2
family of nonspecific endoribonucleases (EC
3.1.27.1
) is a widespread family of RNases found in every organism
examined thus far. Most T
2
enzymes are secretory RNases and
therefore are found extracellularly or in compartments of the
endomembrane system that would minimize their contact with cellular
RNA. Although the biological functions of various T
2
RNases
have been postulated on the basis of enzyme location or gene expression
patterns, the cellular roles of these enzymes are generally unknown. In
the present work, we characterized Rny1, the only T
2
RNase
in
Saccharomyces cerevisiae
. Rny1 was found to be an
active, secreted RNase whose gene expression is controlled by heat
shock and osmotic stress. Inactivation of
RNY1
leads to
unusually large cells that are temperature-sensitive for growth. These
phenotypes can be complemented not only by
RNY1
but also
by both structurally related and unrelated secretory RNases.
Additionally, the complementation depends on RNase activity. When
coupled with a recent report on the effect of specific RNAs on membrane
permeability [Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I. &
Yarus, M. (1999)
Proc. Natl. Acad. Sci. USA
96,
10649–10654], our work suggests an unexpected role for Rny1 and
possibly other secretory RNases. These enzymes may regulate membrane
permeability or stability, a hypothesis that could present an
alternative perspective for understanding their functions.
Better for Everyone? Travel Experiences and Transport Exclusion
Authors: Hine Julian , Mitchell Fiona
Published: July 2002

Many groups within society continue to experience, and be subjected to, what can only be termed 'transport disadvantage'. Traditionally, recognition of the barriers experienced by these groups has been overlooked. The paper explores the reasons why this is the case with reference to the problematical perspectives and methodologies of transport policy-makers and planners. Personal accounts of individuals are used to demonstrate that 'transport disadvantage' is experienced by many different groups within society and is a social concern that must be included as a priority target for public policy. The importance of considering such travel experiences becomes apparent and is paramount to the development of barrier-free living.
Developmental Aspects of Sustained Attention among 2- to 6-year-old Children
Authors: Sarid Miriam , Breznitz Zvia
Published: July 2002

This study examined sustained attention in children aged 2-6 years in two settings; during free play and during a structured test. Subjects were 48 nursery schoolchildren and 47 kindergarteners. In the free play setting, linear and quadratic relations with age were found for ability to sustain attention as measured by duration of play time and number of attended activities. Linear and quadratic relations were also found for distractibility as measured by the number of pauses in play. Older children tended to return to a previously attended activity following a break, whereas younger children did not. The ability to sustain attention increased until the age of 4 years, after which a plateau in development appeared. Results from the structured test showed no significant differences between age groups on the time spent attending to pictures, but revealed a significant linear trend for the ability to recall items from pictures. It is suggested that free play may be a sensitive measure of preschool-aged children’s ability to sustain attention.
The Community Reinforcement Approach to the Treatment of Substance Use Disorders
Authors: Smith Jane Ellen , Meyers Robert J. , Miller William R.
Published: July 2002

Empirical support is presented for the Community Reinforcement Approach (CRA), a broad‐spectrum cognitive‐behavioral treatment for substance use disorders. At the core of CRA is the belief that an individual's environment can play a powerful role in encouraging or discouraging drinking and drug use. Consequently, it attempts to rearrange contingencies so that sober behavior is more rewarding than substance‐abusing behavior. Originally tested in the early 1970s with a small sample of alcohol‐dependent inpatients, it has repeatedly proven to be successful over the years with larger, diverse populations. Empirical backing is also presented for a new variant of CRA that works through family members to engage treatment‐resistant individuals into substance abuse treatment.
Staff Communication With People With Intellectual Disabilities: The Impact Of A Work‐Based Training Programme
Authors: Purcell Margaret , McConkey Roy , Morris Irene
Published: July 2002

Previous studies have identified changes that front‐line service staff could usefully make to enhance their communications and those of their clients who have intellectual disabilities. These were incorporated into a training programme delivered in the workplace that involved a self‐selected group of 24 experienced staff working either in small‐scale residential settings and day centres. Analysis of video‐recordings made before and after training showed that although most clients had become more active communicators (particularly when they were engaged in shared activities with staff), significant changes in staff behaviours as a whole were not observed. However increased responsiveness from staff did correlate significantly with increases in the client's communication acts. In addition, qualitative reports from staff and tutors pinpointed specific changes that staff had made. The difficulties of evaluating changes in staff‐client communications are discussed and four features for training staff in communication are identified as essential; namely, it should be work‐based, client focused, mentor‐guided, and effective strategies documented and shared.
Contribution of finger tracing to the recognition of Chinese characters
Authors: Yim‐Ng Yuet‐Yee , Varley Rosemary , Andrade Jackie
Published: July 2002

Finger tracing is a simulation of the act of writing without the use of pen and paper. It is claimed to help in the processing of Chinese characters, possibly by providing additional motor coding. In this study, blindfolded subjects were equally good at identifying Chinese characters and novel visual stimuli through passive movements made with the index finger of the preferred hand and those made with the last finger of that hand. This suggests that finger tracing provides a relatively high level of coding specific to individual characters, but non‐specific to motor effectors. Beginning each stroke from the same location, i.e. removing spatial information, impaired recognition of the familiar characters and the novel nonsense figures. Passively tracing the strokes in a random sequence also impaired recognition of the characters. These results therefore suggest that the beneficial effect of finger tracing on writing or recall of Chinese characters is mediated by sequence and spatial information embedded in the motor movements, and that proprioceptive channel may play a part in mediating visuospatial information. Finger tracing may be a useful strategy for remediation of Chinese language impairments.
Free Radical‐Induced Protein Degradation of Erythrocyte Membrane is Influenced by the Localization of Radical Generation
Authors: Celedón Gloria , González Gustavo , Lissi Eduardo A. , Hidalgo Gissela
Published: July 2002

We have investigated the role of the localization of free radical generation in erythrocyte membrane proteins degradation. The extracellular radical producer 2,2'‐azobis(2‐amidinopropane) hydrochloride induced a greater loss of band‐3 protein in comparison with spectrin whereas the intracellular radical initiator cysteine induced the reverse effect. However, a large extent of main‐chain fragmentation was observed for both proteins under the action of cysteine‐derived radicals. The results show that the relative localization of the radical generation has an important influence on the degradation of specific proteins of the erythrocyte membrane.
Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor
Authors: Schmidt Anne , Hannah Matthew J. , Huttner Wieland B.
Published: July 2002

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.
The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation
Authors: Kalies Kai-Uwe , Rapoport Tom A. , Hartmann Enno
Published: July 2002

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the β subunit (Sec61β) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61β is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61β kinetically facilitates cotranslational translocation, but is not essential for it.

Using chemical cross-linking we show that Sec61β not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61β and SPC25 and between Sec61β and Sec61α depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.
Positive Charges Determine the Topology and Functionality of the Transmembrane Domain in the Chloroplastic Outer Envelope Protein Toc34
Authors: May Timo , Soll Jürgen
Published: July 2002

The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation Ncyto-Cin. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid residues NH2- and COOH-proximal of the hydrophobic α-helix indicates that a double-positive charge at the cytosolic side of the transmembrane α-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence–dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.
Polyunsaturated Fatty Acids Inhibit T Cell Signal Transduction by Modification of Detergent-insoluble Membrane Domains
Authors: Stulnig Thomas M. , Berger Markus , Sigmund Thomas , Raederstorff Daniel , Stockinger Hannes , Waldhäusl Werner
Published: July 2002

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs.
Cloning and Characterization of a Potassium-Dependent Sodium/Calcium Exchanger in Drosophila
Authors: Haug-Collet K. , Pearson B. , Webel R. , Szerencsei R.T. , Winkfein R.J. , Schnetkamp P.P.M. , Colley N.J.
Published: July 2002

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH2 terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal–ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.
Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion
Authors: Lu Q.L. , Morris G.E. , Wilton S.D. , Ly T. , Artem'yeva O.V. , Strong P. , Partridge T.A.
Published: July 2002

Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.
Partitioning of the 2-μm Circle Plasmid of Saccharomyces cerevisiae
Authors: Velmurugan Soundarapandian , Yang Xian-Mei , Chan Clarence S.-M. , Dobson Melanie , Jayaram Makkuni
Published: July 2002

The efficient partitioning of the 2-μm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-μm–derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-μm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.
Conditional Ablation of β1 Integrin in Skin
Authors: Raghavan Srikala , Bauer Christoph , Mundschau Gina , Li Qingqin , Fuchs Elaine
Published: July 2002

The major epidermal integrins are α3β1 and hemidesmosome-specific α6β4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for αβ1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of α6 or β4 results in loss of hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of α3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate β1 in skin epithelium, we have uncovered biological roles for αβ1 integrins not predicted from either the α3 knockout or from in vitro studies. In contrast to α3 null mice, β1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization, hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that β1's minor partners in skin are important, and together, αβ1 integrins are required not only for extracellular matrix assembly but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle morphogenesis. However, β1 downregulation does not provide the trigger to terminally differentiate.
Equine Herpesvirus Protein E10 Induces Membrane Recruitment and Phosphorylation of Its Cellular Homologue, Bcl-10
Authors: Thome Margot , Gaide Olivier , Micheau Olivier , Martinon Fabio , Bonnet David , Gonzalez Montserrat , Tschopp Jürg
Published: July 2002

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH2-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-κB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10–mediated NF-κB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor–associated factor (TRAF)6. Moreover, v-E10–induced NF-κB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10–induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-κB activation.
Angiomotin
Authors: Troyanovsky Boris , Levchenko Tetyana , Månsson Göran , Matvijenko Olga , Holmgren Lars
Published: July 2002

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type–specific manner. We have used a construct encoding the kringle domains 1–4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.
Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin α8β1 in the embryonic kidney
Authors: Brandenberger Ralph , Schmidt Andrea , Linton James , Wang Denan , Backus Carey , Denda Sumiko , Müller Ullrich , Reichardt Louis F.
Published: July 2002

The epithelial–mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin α8β1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin α8β1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, α8β1-AP detects a novel ligand of 70–90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin α8β1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas α8β1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by α8β1-AP and forms a complex with α8β1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating α8β1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.
CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro
Authors: Stepanek Laurie , Sun Qi Lun , Wang Jun , Wang Cong , Bixby John L.
Published: July 2002

Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.
Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue
Authors: Denis Valérie , Cyert Martha S.
Published: July 2002

Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca2+ from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca2+ release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca2+ in vivo. Vacuolar Ca2+, which is the major intracellular Ca2+ store in yeast, is required for this response, whereas extracellular Ca2+ is not. We aimed to identify the channel responsible for this regulated vacuolar Ca2+ release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca2+ release. After this release, low cytosolic Ca2+ is restored and vacuolar Ca2+ is replenished through the activity of Vcx1p, a Ca2+/H+ exchanger. These studies reveal a novel mechanism of internal Ca2+ release and establish a new function for TRP channels.
HLA Class I Binding Motifs Derived from Random Peptide Libraries Differ at the COOH Terminus from Those of Eluted Peptides
Authors: Davenport Miles P. , Smith Katherine J. , Barouch Dan , Reid Scott W. , Bodnar Wanda M. , Willis Anthony C. , Hunt Donald F. , Hill Adrian V.S.
Published: July 2002

Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant β2-microglobulin (β2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.
Early HIV-1 Envelope-specific Cytotoxic T Lymphocyte Responses in Vertically Infected Infants
Authors: Pikora Cheryl A. , Sullivan John L. , Panicali Dennis , Luzuriaga Katherine
Published: July 2002

High frequencies of cytotoxic T lymphocyte precursors (CTLp) recognizing HIV-1 laboratory strain gene products have been detected in adults within weeks of primary infection. In contrast, HIV-1–specific CTLp are uncommonly detected in infants younger than 6 mo. To address the hypothesis that the use of target cells expressing laboratory strain env gene products might limit the detection of HIV-1 env-specific CTLp in early infancy, recombinant vaccinia vectors (vv) expressing HIV-1 env genes from early isolates of four vertically infected infants were generated. The frequencies of CTLp recognizing target cells infected with vv-expressing env gene products from early isolates and HIV-1 IIIB were serially measured using limiting dilution followed by in vitro stimulation with mAb to CD3. In one infant, the detection of early isolate env-specific CTLp preceded the detection of IIIB-specific CTLp. CTLp recognizing HIV-1 IIIB and infant isolate env were detected by 6 mo of age in two infants. In a fourth infant, HIV-1 IIIB env and early isolate env-specific CTLp were simultaneously detected at 12 mo of age. These results provide evidence that young infants can generate HIV-1–specific CTL responses and provide support for the concept of neonatal vaccination to prevent HIV-1 transmission. However, the early predominance of type-specific CTL detected in some young infants suggests that the use of vaccines based on laboratory strains of HIV-1 may not protect against vertical infection.
Intracellular Antimicrobial Activity in the Absence of Interferon-γ: Effect of Interleukin-12 in Experimental Visceral Leishmaniasis in Interferon-γ Gene-disrupted Mice
Authors: Taylor Alice P. , Murray Henry W.
Published: July 2002

Despite permitting uncontrolled intracellular visceral infection for 8 wk, interferon-γ (IFN-γ) gene knockout (GKO) mice infected with Leishmania donovani proceeded to reduce liver parasite burdens by 50% by week 12. This late-developing IFN-γ–independent antileishmanial mechanism appeared to be dependent largely on endogenous tumor necrosis factor-α (TNF-α): L. donovani infection induced TNF-α mRNA expression in parasitized GKO livers and neutralization of TNF-α reversed control at week 12. 7 d of treatment of infected GKO mice with interleukin-12 (IL-12) readily induced leishmanicidal activity and also partially restored the near-absent tissue granulomatous response, observations that for the first time expand the antimicrobial repertoire of IL-12 to include IFN-γ–independent effects. The action of IL-12 against L. donovani was TNF-α dependent and required the activity of inducible nitric oxide synthase. These results point to the presence of an IFN-γ–independent antimicrobial mechanism, mediated by TNF-α, which remains quiescent until activated late in the course of experimental visceral leishmaniasis. However, as judged by the effect of exogenous IL-12 this quiescent mechanism can readily be induced to rapidly yield enhanced intracellular antimicrobial activity.
RANTES and Monocyte Chemoattractant Protein–1 (MCP-1) Play an Important Role in the Inflammatory Phase of Crescentic Nephritis, but Only MCP-1 Is Involved in Crescent Formation and Interstitial Fibrosis
Authors: Lloyd Clare M. , Minto Andrew W. , Dorf Martin E. , Proudfoot Amanda , Wells Timothy N.C. , Salant David J. , Gutierrez-Ramos Jose-Carlos
Published: July 2002

The involvement of chemokines in inflammation is well established, but their functional role in disease progression, and particularly in the development of fibrosis, is not yet understood. To investigate the functional role that the chemokines monocyte chemoattractant protein–1 (MCP-1) and RANTES play in inflammation and the progression to fibrosis during crescentic nephritis we have developed and characterized a murine model for this syndrome. Significant increases in T-lymphocytes and macrophages were observed within glomeruli and interstitium, paralleled by an induction of mRNA expression of MCP-1 and RANTES, early after disease initiation. Blocking the function of MCP-1 or RANTES resulted in significant decreases in proteinuria as well as in numbers of infiltrating leukocytes, indicating that both MCP-1 and RANTES (regulated upon activation in normal T cells expressed and secreted) play an important role in the inflammatory phase of crescentic nephritis. In addition, neutralization of MCP-1 resulted in a dramatic decrease in both glomerular crescent formation and deposition of type I collagen. These results highlight a novel role for MCP-1 in crescent formation and development of interstitial fibrosis, and indicate that in addition to recruiting inflammatory cells this chemokine is critically involved in irreversible tissue damage.
Macrophage Microbicidal Mechanisms In Vivo: Reactive Nitrogen versus Oxygen Intermediates in the Killing of Intracellular Visceral Leishmania donovani
Authors: Murray Henry W. , Nathan Carl F.
Published: July 2002

To determine the relative contributions of respiratory burst–derived reactive oxygen intermediates (ROI) versus reactive nitrogen intermediates (RNI) to macrophage-mediated intracellular host defense, mice genetically deficient in these mechanisms were challenged with Leishmania donovani, a protozoan that selectively parasitizes visceral tissue macrophages. During the early stage of liver infection at wk 2, both respiratory burst–deficient gp91phox−/− (X-linked chronic granulomatous disease [X-CGD]) mice and inducible nitric oxide synthase (iNOS) knockout (KO) mice displayed comparably increased susceptibility. Thereafter, infection was unrestrained in mice lacking iNOS but was fully controlled in X-CGD mice. Mononuclear cell influx into infected liver foci in X-CGD and iNOS KO mice was also overtly impaired at wk 2. However, granuloma assembly in parasitized tissue eventually developed in both hosts but with divergent effects: mature granulomas were functionally active (leishmanicidal) in X-CGD mice but inert in iNOS-deficient animals. These results suggest that (a) ROI and RNI probably act together in the early stage of intracellular infection to regulate both tissue recruitment of mononuclear inflammatory cells and the initial extent of microbial replication, (b) RNI alone are necessary and sufficient for eventual control of visceral infection, and (c) although mature granulomas have traditionally been associated with control of such infections, these structures fail to limit intracellular parasite replication in the absence of iNOS.
Interleukin 9–induced In Vivo Expansion of the B-1 Lymphocyte Population
Authors: Vink Anne , Warnier Guy , Brombacher Frank , Renauld Jean-Christophe
Published: July 2002

The activity of interleukin (IL)-9 on B cells was analyzed in vivo using transgenic mice that constitutively express this cytokine. These mice show an increase in both baseline and antigen-specific immunoglobulin concentrations for all isotypes tested. Analysis of B cell populations showed a specific expansion of Mac-1+ B-1 cells in the peritoneal and pleuropericardial cavities, and in the blood of IL-9 transgenic mice. In normal mice, the IL-9 receptor was found to be expressed by CD5+ as well as CD5− B-1 cells, and repeated injections of IL-9 resulted in accumulation of B-1 cells in the peritoneal cavity, as observed in transgenic animals. Unlike other mouse models, such as IL-5 transgenic mice, in which expansion of the B-1 population is associated with high levels of autoantibodies, IL-9 did not stimulate the production of autoantibodies in vivo, and most of the expanded cells were found to belong to the B-1b subset (IgM+Mac-1+CD5−). In addition, we found that these IL-9–expanded B-1b cells do not share the well-documented antibromelain-treated red blood cell specificity of CD5+ B-1a cells. The increase of antigen-specific antibody concentration in immunized mice suggests that these B-1 cells are directly or indirectly involved in antibody responses in IL-9 transgenic mice.
Distinct Role of Antigen-Specific T Helper Type 1 (Th1) and Th2 Cells in Tumor Eradication in Vivo
Authors: Nishimura Takashi , Iwakabe Kenji , Sekimoto Masashi , Ohmi Yasushi , Yahata Takashi , Nakui Minoru , Sato Takehito , Habu Sonoko , Tashiro Hiroyuki , Sato Marimo , Ohta Akio
Published: July 2002

The role of T helper type 1 (Th1) and Th2 cells in tumor immunity was investigated using Th cells induced from ovalbumin (OVA)-specific T cell receptor transgenic mice. Although Th1 cells exhibited stronger cytotoxicity than Th2 cells, both cell types completely eradicated tumors when transferred into mice bearing A20 tumor cells transfected with the OVA gene (A20-OVA). Th1 cells eradicated the tumor mass by inducing cellular immunity, whereas Th2 cells destroyed the tumor by inducing tumor necrosis. Both Th1 and Th2 cells required CD8+ T cells to eliminate tumors, and neither of these cells were able to completely eliminate A20-OVA tumors from T and B cell–deficient RAG2−/− mice. Mice cured from tumors by Th1 and Th2 cell therapy rejected A20-OVA upon rechallenge, but CD8+ cytotoxic T lymphocytes were induced only from spleen cells prepared from cured mice by Th1 cell therapy. Moreover, we demonstrated that Th1 and Th2 cells used distinct adhesion mechanisms during tumor eradication: the leukocyte function-associated antigen (LFA)-1–dependent cell–cell adhesion step was essential for Th1 cell therapy, but not for Th2 cell therapy. These findings demonstrated for the first time the distinct role of antigen-specific Th1 and Th2 cells during eradication of established tumors in vivo.