bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation
Authors: Barnakov Alexander N. , Barnakova Ludmila A. , Hazelbauer Gerald L.
Published: July 2002

The mechanistic basis of sensory adaptation and gradient sensing in bacterial chemotaxis is reversible covalent modification of transmembrane chemoreceptors, methylation, and demethylation at specific glutamyl residues in their cytoplasmic domains. These reactions are catalyzed by a dedicated methyltransferase CheR and a dedicated methylesterase CheB. The esterase is also a deamidase that creates certain methyl-accepting glutamyls by hydrolysis of glutamine side chains. We investigated the action of CheB and its activated form, phospho-CheB, on a truncated form of the aspartate receptor of
Escherichia coli
that was missing the last 5 aa of the intact receptor. The deleted pentapeptide is conserved in several chemoreceptors in enteric and related bacteria. The truncated receptor was much less efficiently demethylated and deamidated than intact receptor, but essentially was unperturbed for kinase activation or transmembrane signaling. CheB bound specifically to an affinity column carrying the isolated pentapeptide, implying that in the intact receptor the pentapeptide serves as a docking site for the methylesterase/deamidase and that the truncated receptor was inefficiently modified because the enzyme could not dock. It is striking that the same pentapeptide serves as an activity-enhancing docking site for the methyltransferase CheR, the other enzyme involved in adaptational covalent modification of chemoreceptors. A shared docking site raises the tantalizing possibility that relative rates of methylation and demethylation could be influenced by competition between the two enzymes at that site.
Optical recording of signal-mediated protein transport through single nuclear pore complexes
Authors: Keminer Oliver , Siebrasse Jan-Peter , Zerf Katja , Peters Reiner
Published: July 2002

Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of
Xenopus
oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate
in vivo
conditions as closely as possible, a
Xenopus
egg extract was applied to the cytosolic side and a
Xenopus
oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, “patched” NPCs depended on signal sequences, extracts, and metabolic energy, as
in vivo
. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were ≈2 dimers/s for the import protein and ≈4 dimers/s for the export protein (≈15 μM substrate concentration, 22–24°C), in good agreement with
in vivo
rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms.
The STAR protein QKI-6 is a translational repressor
Authors: Saccomanno Lisa , Loushin Carrie , Jan Eric , Punkay Eric , Artzt Karen , Goodwin Elizabeth B.
Published: July 2002

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse,
quaking
proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in
Caenorhabditis elegans
, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for
tra-2
and
GLI
elements), present in the 3′ untranslated region of the sex-determining gene
tra-2.
This gene promotes female development, and repression of
tra-2
translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits
tra-2
translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both
in vitro
and
in vivo
, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in
C. elegans
wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene
tra-3
results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of
tra-2
. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.
Duplicated genes evolve independently after polyploid formation in cotton
Authors: Cronn Richard C. , Small Randall L. , Wendel Jonathan F.
Published: July 2002

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (
Gossypium hirsutum
) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.
Copper binding to the prion protein: Structural implications of four identical cooperative binding sites
Authors: Viles John H. , Cohen Fred E. , Prusiner Stanley B. , Goodin David B. , Wright Peter E. , Dyson H. Jane
Published: July 2002

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with
K
d
≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.
Functional differences between memory and naive CD8 T cells
Authors: Cho Bryan K. , Wang Changyu , Sugawa Satoshi , Eisen Herman N. , Chen Jianzhu
Published: July 2002

To determine how murine memory and naive T cells differ, we generated large numbers of long-lived memory CD8+T cells and compared them to naive cells expressing the same antigen-specific receptor (T cell receptor; TCR). Although both populations expressed similar levels of TCR and CD8, on antigen stimulationin vitromemory T cells down-regulated their TCR faster and more extensively and secreted IFN-γ and IL-2 faster than naive T cells. Memory cells were also larger, and when freshly isolated from mice they contained perforin and killed target cells without having to be restimulated. They further differed from naive cells in requiring IL-15 for proliferation and in having a greater tendency to undergo apoptosisin vitro. On antigen stimulationin vivo, however, they proliferated more rapidly than naive cells. These findings suggest that, unlike naive T cells, CD8 memory T cells are intrinsically programmed to rapidly express their effector functionsin vivowithout having to undergo clonal expansion and differentiation.
Presenilin-1 regulates the neuronal threshold to excitotoxicity both physiologically and pathologically
Authors: Grilli M. , Diodato E. , Lozza G. , Brusa R. , Casarini M. , Uberti D. , Rozmahel R. , Westaway D. , St George-Hyslop P. , Memo M. , Ongini E.
Published: July 2002

A direct pathophysiological role of Familial Alzheimer's
Disease (FAD)-associated Presenilin 1 (PS1) mutations in neuronal
vulnerability remains a controversial matter. We evaluated the
relationship between PS1 and excitotoxicity in four different
experimental models of neurotoxicity by using primary neurons from
(
i
) transgenic (tg) mice overexpressing a human
FAD-linked PS1 variant (L286V mutation), (
ii
) tg mice
overexpressing human wild-type (wt) PS1, (
iii
) PS1
knockout mice, and (
iv
) wt mice in which PS1 gene
expression was knocked down by antisense treatment. We found that
primary neurons overexpressing mutated PS1 showed an increased
vulnerability to both excitotoxic and hypoxic-hypoglycemic damage when
compared with neurons obtained from either mice overexpressing human wt
PS1 or in wt mice. In addition, reduced excitotoxic damage was obtained
in neurons in which PS1 expression was absent or diminished. Data
obtained in
in vivo
experimental models of
excitotoxicity partially supported the
in vitro
observations. Accelerated neuronal death was demonstrated in the
hippocampus of mice overexpressing mutated PS1 after peripheral
administration of kainic acid in comparison with wt animals. However,
measurement of the infarct volume after middle cerebral artery
occlusion did not show significant difference between the two animal
groups. The results altogether suggest that expression of FAD-linked
PS1 variants increases the vulnerability of neurons to a specific type
of damage in which excitotoxicity plays a relevant role. In addition,
they support the view that reduction of endogenous PS1
expression results in neuroprotection.
A computationally directed screen identifying interacting coiled coils from
Saccharomyces cerevisiae
Authors: Newman John R. S. , Wolf Ethan , Kim Peter S.
Published: July 2002

Computational methods can frequently identify protein-interaction
motifs in otherwise uncharacterized open reading frames. However, the
identification of candidate ligands for these motifs (e.g., so that
partnering can be determined experimentally in a directed manner) is
often beyond the scope of current computational capabilities. One
exception is provided by the coiled-coil interaction motif, which
consists of two or more α helices that wrap around each other: the
ligands for coiled-coil sequences are generally other coiled-coil
sequences, thereby greatly simplifying the motif/ligand recognition
problem. Here, we describe a two-step approach to identifying
protein–protein interactions mediated by two-stranded coiled coils
that occur in
Saccharomyces cerevisiae
. Coiled coils
from the yeast genome are first predicted computationally, by using the
multicoil
program, and associations between coiled coils
are then determined experimentally by using the yeast two-hybrid assay.
We report 213 unique interactions between 162 putative coiled-coil
sequences. We evaluate the resulting interactions, focusing on
associations identified between components of the spindle pole body
(the yeast centrosome).
The
Ath5
proneural genes function upstream of
Brn3
POU domain transcription factor genes to promote retinal ganglion cell development
Authors: Liu Wei , Mo Zeqian , Xiang Mengqing
Published: July 2002

During retinogenesis, the
Xenopus
basic
helix–loop–helix transcription factor Xath5 has been shown to promote
a ganglion cell fate. In the developing mouse and chicken retinas, gene
targeting and overexpression studies have demonstrated critical roles
for the
Brn3
POU domain transcription factor genes in
the promotion of ganglion cell differentiation. However, the genetic
relationship between
Ath5
and
Brn3
genes
is unknown. To understand the genetic regulatory network(s) that
controls retinal ganglion cell development, we analyzed the
relationship between
Ath5
and
Brn3
genes
by using a gain-of-function approach in the chicken embryo. We found
that during retinogenesis, the chicken
Ath5
gene
(
Cath5
) is expressed in retinal progenitors and in
differentiating ganglion cells but is absent in terminally
differentiated ganglion cells. Forced expression of both
Cath5
and the mouse
Ath5
gene
(
Math5
) in retinal progenitors activates the expression
of
cBrn3c
following central-to-peripheral and
temporal-to-nasal gradients. As a result, similar to the Xath5 protein,
both Cath5 and Math5 proteins have the ability to promote the
development of ganglion cells. Moreover, we found that forced
expression of all three
Brn3
genes also can stimulate
the expression of
cBrn3c
. We further found that Ath5 and
Brn3 proteins are capable of transactivating a
Brn3b
promoter. Thus, these data suggest that the expression of
cBrn3c
in the chicken and
Brn3b
in the
mouse is initially activated by Ath5 factors in newly generated
ganglion cells and later maintained by a feedback loop of Brn3 factors
in the differentiated ganglion cells.
Islam in Post-Communist Bulgaria: An Aborted “Clash of Civilizations”?
Authors: Ragaru Nadege
Published: July 2002

During the first years of the Bulgarian transition to democracy, all indicators seemed to point towards an impending explosion of interethnic hatred. Located at the crossroads of Islam and Christianity, this predominantly Orthodox country harbors a 13.1% strong Muslim minority, which was subjected to forcible assimilation under communist rule. The assimilation policy reached a climax in 1984–1985, when around 800,000 Bulgarian Turks were forced to renounce their Turkish-Arabic names in favor of Slavic patronyms within the framework of the so-called “Revival Process,” a campaign that aimed at precipitating the unification of the Bulgarian nation. Far from achieving the intended result, the authorities' move not only fostered a reassertion of distinct ethnic and religious identification among the Turks, but also succeeded in durably upsetting intercommunitarian relationships. Significantly, the Communist Party's announcement on 29 December 1989 that it would restore Muslim rights met with sharp resistance in mixed areas, where large-scale Bulgarian protests rapidly gathered momentum. Against this background, in 1990–1991, few analysts would have predicted that Bulgaria could avoid religious conflict, especially as the country was faced with growing regional instability and a belated shift to a market economy—two conditions often said to be conducive to the exacerbation of ethnic tensions.
Tugan-Baranovsky as a Pioneer of Trade Cycle Analysis
Authors: Barnett Vincent
Published: July 2002

Mikhail Ivanovich Tugan-Baranovsky (1865–1919) has not unjustly been calle the greatest Russian economist of all time (Jasny 1972, p. 159). This neglecte the fact that he was born near Kharkov and towards the end of his life came to see the Ukraine as his homeland, but the evaluation itself is not so far from the truth. However, opinion about the precise importance of Tugan-Baranovsky work to the development of trade cycle analysis has varied widely. J. M. Keynes and A. H. Hansen were both highly respectful of Tugan's contribution. For example, in the Treatise on Money, Keynes wrote in regards to business cycle theory that he was “in strong sympathy with the school of writers … of which Tugan-Baranovski was the first and most original” (Keynes 1930, vol. 2, p. 100 In his 1951 work, Business Cycles and National Income, Hansen was enthusiastic describing Tugan as “cutting his way though the jungle to a new outlook” (Hansen 1951, p. 281). This suggests that some aspects of both British an American Keynesianism might have originated in Tugan's work, or at least bee influenced by it. W. W. Rostow was also impressed by Tugan's approach, statin that it “took business cycle analysis some distance beyond Juglar and Marx” (Rostow 1990, p. 261).
Kaposi's Sarcoma‐Associated Herpesvirus: From Cell Biology to Pathogenesis
Authors: Sharp Tyson V. , Boshoff Chris
Published: July 2002

Kaposi's sarcoma‐associated herpesvirus (KSHV or HHV‐8) is linked to the etiopathogenesis of Kaposi's sarcoma, a plasmablastic variant of Castleman's disease and primary effusion lymphoma. KSHV is related to a number of non‐human primate viruses. Only a limited number of KSHV proteins are expressed in tumor cells. Here we discuss the putative role of these proteins in KSHV pathogenesis.
Topology of the Shaker Potassium Channel Probed with Hydrophilic Epitope Insertions
Authors: Shih Theodore M. , Goldin Alan L.
Published: July 2002

The structure of the Shaker potassium channel has been modeled as passing through the cellular membrane eight times with both the NH2 and COOH termini on the cytoplasmic side (Durrell, S.R., and H.R. Guy. 1992. Biophys. J. 62:238–250). To test the validity of this model, we have inserted an epitope consisting of eight hydrophilic amino acids (DYKDDDDK) in predicted extracellular and intracellular loops throughout the channel. The channels containing the synthetic epitope were expressed in Xenopus oocytes, and function was examined by two-electrode voltage clamping. All of the mutants containing insertions in putative extracellular regions and the NH2 and COOH termini expressed functional channels, and most of their electrophysiological properties were similar to those of the wild-type channel. Immunofluorescent staining with a monoclonal antibody against the epitope was used to determine the membrane localization of the insert in the channels. The data confirm and constrain the model for the transmembrane topology of the voltage-gated potassium channel.
SOI1 Encodes a Novel, Conserved Protein That Promotes TGN–Endosomal Cycling of Kex2p and Other Membrane Proteins by Modulating the Function of Two TGN Localization Signals
Authors: Brickner Jason H. , Fuller Robert S.
Published: July 2002

Localization of yeast Kex2 protease to the TGN requires a signal (TLS1) in its cytosolic tail (C-tail). Mutation of TLS1 results in rapid transit of Kex2p to the vacuole. Isolation of suppressors of the Tyr713Ala mutation in TLS1 previously identified three SOI genes. SOI1, cloned by complementation of a sporulation defect, encodes a novel, hydrophilic 3,144-residue protein with homologues in Caenorhabditis elegans, Drosophila melanogaster, and humans. Epitope-tagged Soi1p existed in a detergent-insensitive, sedimentable form. Deletion of SOI1 impaired TGN localization of wild-type Kex2p and a fusion protein containing the C-tail of Ste13p, and also caused missorting of carboxypeptidase Y and accelerated vacuolar degradation of the Vps10p sorting receptor. Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-α-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p. These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC). This hypothesis was confirmed in several ways. Soi1p was shown to be required for optimal function of TLS1. Suppression of the Tyr713Ala mutation by mutation of SOI1 was shown to be caused by activation of a second signal (TLS2) in the Kex2p C-tail. TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step. We propose that Soi1p promotes cycling of TGN membrane proteins between the TGN and PVC by antagonizing a TGN retention signal (TLS2) and facilitating the function of a retrieval signal (TLS1) that acts at the PVC.
Mal3, the Fission Yeast Homologue of the Human APC-interacting Protein EB-1 Is Required for Microtubule Integrity and the Maintenance of Cell Form
Authors: Beinhauer Jens D. , Hagan Iain M. , Hegemann Johannes H. , Fleig Ursula
Published: July 2002

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.
Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells
Authors: Job Christy , Lagnado Leon
Published: July 2002

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.

Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K+, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca2+ influx through L-type Ca2+ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca2+ influx and accelerated F-actin breakdown on removal of Ca2+.

To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca2+ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.
Dynamic Localization of CLIP-170 to Microtubule Plus Ends Is Coupled to Microtubule Assembly
Authors: Diamantopoulos Georgios S. , Perez Franck , Goodson Holly V. , Batelier Gérard , Melki Ronald , Kreis Thomas E. , Rickard Janet E.
Published: July 2002

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.
Chromosome Movement in Mitosis Requires Microtubule Anchorage at Spindle Poles
Authors: Gordon Michael B. , Howard Louisa , Compton Duane A.
Published: July 2002

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.
The Localization of Human Cyclins B1 and B2 Determines Cdk1 Substrate Specificity and Neither Enzyme Requires Mek to Disassemble the Golgi Apparatus
Authors: Draviam Viji Mythily , Orrechia Simona , Lowe Martin , Pardi Ruggero , Pines Jonathon
Published: July 2002

In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin–CDKs underlies the ability of cyclin B1–CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2–CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH2 terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin–CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.
Metaphase Arrest with Centromere Separation in polo Mutants of Drosophila
Authors: Donaldson Mary M. , Tavares Álvaro A.M. , Ohkura Hiroyuki , Deak Peter , Glover David M.
Published: July 2002

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20–50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with γ-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase–anaphase checkpoint.
Abelson kinase regulates epithelial morphogenesis in Drosophila
Authors: Grevengoed Elizabeth E. , Loureiro Joseph J. , Jesse Traci L. , Peifer Mark
Published: July 2002

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and α-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.
Kinetics and Extent of T Cell Activation as Measured with the Calcium Signal
Authors: Wülfing Christoph , Rabinowitz Joshua D. , Beeson Craig , Sjaastad Michael D. , McConnell Harden M. , Davis Mark M.
Published: July 2002

We have characterized the calcium response of a peptide–major histocompatibility complex (MHC)-specific CD4+ T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell–antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide–MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.
CXCR4/fusin Is Not a Species-specific Barrier in Murine Cells for HIV-1 Entry
Authors: Tachibana Kazunobu , Nakajima Toshihiro , Sato Akihiko , Igarashi Kenji , Shida Hisatoshi , Iizasa Hisashi , Yoshida Nobuaki , Yoshie Osamu , Kishimoto Tadamitsu , Nagasawa Takashi
Published: July 2002

Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.
Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors
Authors: O'Herrin Sean M. , Lebowitz Michael S. , Bieler Joan G. , al-Ramadi Basel K. , Utz Ursula , Bothwell Alfred L.M. , Schneck Jonathan P.
Published: July 2002

Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.
A Role for Neutral Sphingomyelinase-mediated Ceramide Production in T Cell Receptor–induced Apoptosis and Mitogen-activated Protein Kinase–mediated Signal Transduction
Authors: Tonnetti Laura , Verí Maria-Concetta , Bonvini Ezio , D'Adamio Luciano
Published: July 2002

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses. Moreover, specific inactivation of neutral sphingomyelinase by antisense RNA inhibited IL-2 production and mitogen-activated protein kinase activation after TCR triggering. These results suggest that ceramide production by activation of neutral sphingomyelinase is an essential component of the TCR signaling machinery.
Induction and Suppression of an Autoimmune Disease by Oligomerized T Cell Epitopes
Authors: Falk Kirsten , Rötzschke Olaf , Santambrogio Laura , Dorf Martin E. , Brosnan Celia , Strominger Jack L.
Published: July 2002

T cell epitope peptides derived from proteolipid protein (PLP139–151) or myelin basic protein (MBP86–100) induce experimental autoimmune encephalomyelitis (EAE) in “susceptible” strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund's adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the peptide epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10.S mice, “resistant” to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50 μg of the PLP139–151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139–151 peptide accelerated rather than retarded the progression of disease.
Macrophage Inflammatory Protein 3α Is Expressed at Inflamed Epithelial Surfaces and Is the Most Potent Chemokine Known in Attracting Langerhans Cell Precursors
Authors: Dieu-Nosjean Marie-Caroline , Massacrier Catherine , Homey Bernhard , Vanbervliet Béatrice , Pin Jean-Jacques , Vicari Alain , Lebecque Serge , Dezutter-Dambuyant Colette , Schmitt Daniel , Zlotnik Albert , Caux Christophe
Published: July 2002

Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3α plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3α was the most potent chemokine inducing the selective migration of in vitro–generated CD34+ hematopoietic progenitor cell–derived LC precursors and skin LCs in accordance with the restricted MIP-3α receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3α was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3α. (c) In vivo, MIP-3α was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3α upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3α was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1β plus tumor necrosis factor α) or T cell signals. Results of this study suggest a major role of MIP-3α in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.
A Transcription Function for the T Cell–Specific Adapter (Tsad) Protein in T Cells
Authors: Marti Francesc , Post Nicholas H. , Chan Elena , King Philip D.
Published: July 2002

T cell–specific adapter (TSAd) protein is an Src homology 2 (SH2) domain–containing adapter molecule implicated in T cell receptor for antigen (TCR)-mediated interleukin 2 (IL-2) secretion in T cells. Here, we demonstrate that a substantial fraction of TSAd is found in the T cell nucleus. Nuclear import of TSAd is an active process that depends on TSAd SH2 domain recognition of a phosphotyrosine-containing ligand. Importantly, we show that TSAd can act as a potent transcriptional activator in T cells. Furthermore, the TSAd SH2 domain appears to be essential for this transcription-activating function independent of its role in nuclear import. Biochemical analyses suggest that a single TSAd SH2 domain ligand of 95–100 kD may be involved in these processes. Consistent with a role as a transcription activator, cotransfection of TSAd with an IL-2 promoter–reporter gene construct results in a considerable upregulation of IL-2 promoter activity. Further, we show that this augmentation requires a functional TSAd SH2 domain. However, TSAd does not appear to modulate the activity of the major recognized IL-2 gene transcription factors, nuclear factor κB (NF-κB), nuclear factor of activated T cells (NFAT), or activator protein 1 (AP-1). These findings point to the function of TSAd as a novel transcription-regulatory protein in T cells and illustrate the importance of the TSAd SH2 domain in this role.
T Helper 1 and T Helper 2 Cells Are Pathogenic in an Antigen-specific Model of Colitis
Authors: Iqbal Nuzhat , Oliver James R. , Wagner Frederic H. , Lazenby Audrey J. , Elson Charles O. , Weaver Casey T.
Published: July 2002

Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2−/− TCR transgenic mice were transferred into RAG-2−/− recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2−/− T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.
Properties of the Mutant Ser-460-Cys Implicate This Site in a Functionally Important Region of the Type Iia Na+/Pi Cotransporter Protein
Authors: Lambert Georg , Forster Ian C. , Stange Gerti , Biber Jürg , Murer Heini
Published: July 2002

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na+/Pi cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 μM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of Pi transport. Alkylation in 100 mM Na+ by either cationic {[2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA} or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the Pi response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na+ resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating Pi suppressed the slippage current equally, which indicated that Pi binding could occur without cotransport. Pre–steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na+ dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na+/Pi cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.