bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional genomics: Probing plant gene function and expression with transposons
Authors: Martienssen Robert A.
Published: July 2002

Transposable elements provide a convenient and flexible means to
disrupt plant genes, so allowing their function to be assessed. By
engineering transposons to carry reporter genes and regulatory signals,
the expression of target genes can be monitored and to some extent
manipulated. Two strategies for using transposons to assess gene
function are outlined here: First, the PCR can be used to identify
plants that carry insertions into specific genes from among pools of
heavily mutagenized individuals (site-selected transposon mutagenesis).
This method requires that high copy transposons be used and that a
relatively large number of reactions be performed to identify
insertions into genes of interest. Second, a large library of plants,
each carrying a unique insertion, can be generated. Each insertion site
then can be amplified and sequenced systematically. These two methods
have been demonstrated in maize,
Arabidopsis
, and
other plant species, and the relative merits of each are discussed in
the context of plant genome research.
Mutagenesis of conserved lysine residues in bacteriophage T5 5′-3′ exonuclease suggests separate mechanisms of endoand exonucleolytic cleavage
Authors: Garforth Scott J. , Ceska Thomas A. , Suck Dietrich , Sayers Jon R.
Published: July 2002

Efficient cellular DNA replication requires the activity of a 5′-3′ exonuclease. These enzymes are able to hydrolyze DNA⋅DNA and RNA⋅DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5′-3′ exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8–10 Å. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5′-3′ exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.
On the classification of binary shifts of minimal commutant index
Authors: Price Geoffrey L.
Published: July 2002

We provide a complete classification up to conjugacy of the binary shifts of commutant index 2 on the hyperfinite
II
1
factor. There is a natural correspondence between the conjugacy classes of these shifts and polynomials over
GF
(2) satisfying a certain duality condition.
Intragenic suppressors of Hsp70 mutants: Interplay between the ATPase- and peptide-binding domains
Authors: Davis Julie E. , Voisine Cindy , Craig Elizabeth A.
Published: July 2002

ATP hydrolysis and polypeptide binding, the two key activities of Hsp70 molecular chaperones, are inherent properties of different domains of the protein. The coupling of these two activities is critical because the bound nucleotide determines, in part, the affinity of Hsp70s for protein substrate. In addition, cochaperones of the Hsp40 (DnaJ) class, which stimulate Hsp70 ATPase activity, have been proposed to play an important role in promoting efficient Hsp70 substrate binding. Because little is understood about this functional interaction between domains of Hsp70s, we investigated mutations in the region encoding the ATPase domain that acted as intragenic suppressors of a lethal mutation (I485N) mapping to the peptide-binding domain of the mitochondrial Hsp70 Ssc1. Analogous amino acid substitution in the ATPase domain of the
Escherichia coli
Hsp70 DnaK had a similar intragenic suppressive effect on the corresponding I462T temperature-sensitive peptide-binding domain mutation. I462T protein had a normal basal ATPase activity and was capable of nucleotide-dependent conformation changes. However, the reduced affinity of I462T for substrate peptide (and DnaJ) is likely responsible for the inability of I462T to function
in vivo
. The suppressor mutation (D79A) appears to partly alleviate the defect in DnaJ ATPase stimulation caused by I462T, suggesting that alteration in the interaction with DnaJ may alter the chaperone cycle to allow productive interaction with polypeptide substrates. Preservation of the intragenic suppression phenotypes between eukaryotic mitochondrial and bacterial Hsp70s suggests that the phenomenon studied here is a fundamental aspect of the function of Hsp70:Hsp40 chaperone machines.
Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development
Authors: Vaudry David , Gonzalez Bruno J. , Basille Magali , Fournier Alain , Vaudry Hubert
Published: July 2002

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are present in the external granule cell layer of the rat cerebellum during postnatal development.
In vitro
studies have shown that PACAP promotes cell survival and neurite outgrowth on immature cerebellar granule cells in primary culture. In the present study, we have investigated the effect of PACAP on the development of the cerebellar cortex of 8-day-old rats. Incubation of cultured granule cells for 12 or 18 h with PACAP provoked a significant increase in the rate of incorporation of [
3
H]thymidine in cultured granule cells, suggesting that PACAP could stimulate the proliferation of granule cells. After 96 h of treatment,
in vivo
administration of PACAP provoked a transient increase in the number of granule cells in the molecular layer and in the internal granule cell layer. In contrast, PACAP did not affect the number of Purkinje cells. The augmentation of the number of granule cells evoked by PACAP was significantly inhibited by the PACAP receptor antagonist PACAP(6–38). Administration of PACAP also caused a significant increase in the volume of the cerebellar cortex. The present study provides evidence that PACAP can act
in vivo
as a trophic factor during rat brain development. Our data indicate that PACAP increases proliferation and/or inhibits programmed cell death of granule cells, as well as stimulating neuronal migration from the external granule cell layer toward the internal granule cell layer.
RNA aptamers as effective protein antagonists in a multicellular organism
Authors: Shi Hua , Hoffman Bryan E. , Lis John T.
Published: July 2002

RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms
in vivo
. We demonstrate that
Drosophila
B52 protein can be specifically inhibited
in vitro
and
in vivo
by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52
in vivo
and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.
Hyers–Ulam stability of monomial functional equations on a general domain
Authors: Gilányi Attila
Published: July 2002

In the present paper the Hyers–Ulam stability of monomial functional equations for functions defined on a power-associative, power-symmetric groupoid is proved.
Groucho and dCtBP mediate separate pathways of transcriptional repression in the
Drosophila
embryo
Authors: Zhang Hailan , Levine Michael
Published: July 2002

The precellular
Drosophila
embryo contains ≈10 well characterized transcriptional repressors. At least half are short-range repressors that must bind within 100 bp of either upstream activators or the core transcription complex to inhibit (or quench) gene expression. The two long-range repressors can function over distances of 1 kilobase or more to silence transcription. Previous studies have shown that three of the five short-range repressors interact with a common corepressor protein, dCtBP. In contrast, the two long-range repressors, Hairy and Dorsal, recruit a different corepressor protein, Groucho. Hairy also was shown to interact with dCtBP, thereby raising the possibility that Groucho and dCtBP are components of a common corepressor complex. To investigate this issue, we have misexpressed wild-type and mutant forms of Hairy in transgenic embryos. Evidence is presented that Hairy-mediated repression depends on the Groucho interaction sequence (WRPW) but not the weak dCtBP motif (PLSLV) present in the native protein. Conversion of the PLSLV motif into an optimal dCtBP interaction sequence (PLDLS) disrupts the activity of an otherwise normal Hairy protein. These results suggest that dCtBP and Groucho mediate separate pathways of transcriptional repression and that the two proteins can inhibit one another when both bind the same repressor.
Spatiotemporal activity patterns of rat cortical neurons predict responses in a conditioned task
Authors: Villa Alessandro E. P. , Tetko Igor V. , Hyland Brian , Najem Abdellatif
Published: July 2002

Precise and repeated spike-train timings within and across neurons define spatiotemporal patterns of activity. Although the existence of these patterns in the brain is well established in several species, there has been no direct evidence of their influence on behavioral output. To address this question, up to 15 neurons were recorded simultaneously in the auditory cortex of freely moving rats while animals waited for acoustic cues in a Go/NoGo task. A total of 235 significant patterns were detected during this interval from an analysis of 13 hr of recording involving over 1 million spikes. Of particular interest were 129 (55%) patterns that were significantly associated with the type of response the animal made later, independent of whether the response was that prompted by the cue because the response occurred later and the cue was chosen randomly. Of these behavior-predicting patterns, half (59/129) were associated with an enhanced tendency to go in response to the stimulus, and for 11 patterns of this subset, trials including the pattern were followed by significantly faster reaction time than those lacking the pattern. The remaining behavior-predicting patterns were associated with an enhanced NoGo tendency. Overall mean discharge rates did not vary across trials. Hence, these data demonstrate that particular spatiotemporal patterns predict future behavioral responses. Such presignal activity could form templates for extracting specific sensory information, motor programs prespecifying preference for a particular act, and/or some intermediate, associative brain process.
Role of calpain in adipocyte differentiation
Authors: Patel Yashomati M. , Lane M. Daniel
Published: July 2002

Evidence is presented that the calcium-activated protease, calpain,
is required for differentiation of 3T3-L1 preadipocytes into adipocytes
induced by methylisobutylxanthine (a cAMP phosphodiesterase inhibitor),
dexamethasone, and insulin. Calpain is expressed by preadipocytes and
its level falls during differentiation. Exposure of preadipocytes to
the calpain inhibitor
N-
acetyl-Leu-Leu-norleucinal or
overexpression of calpastatin, a specific endogenous
inhibitor of calpain, blocks expression of adipocyte-specific genes,
notably the CCAAT/enhancer-binding protein (C/EBP)α gene, and
acquisition of the adipocyte phenotype. The inhibitor disrupts the
differentiation-inducing effect of methylisobutylxanthine (by means of
the cAMP-signaling pathway), but is without effect on differentiation
induced by dexamethasone or insulin.
N-
acetyl-Leu-Leu-norleucinal, or overexpression of
calpastatin, inhibits reporter gene expression mediated by the
C/EBPα gene promoter by preventing C/EBPβ, a transcriptional
activator of the C/EBPα gene, from binding to the promoter. These
findings implicate calpain in the transcriptional activation of the
C/EBPα gene, a process required for terminal adipocyte
differentiation.
Structural analysis at 2.2 Å of orthorhombic crystals presents the asymmetry of the allophycocyanin–linker complex, AP⋅L
C
7.8
, from phycobilisomes of
Mastigocladus laminosus
Authors: Reuter Wolfgang , Wiegand Georg , Huber Robert , Than Manuel E.
Published: July 2002

An electrophoretically purified allophycocyanin–linker complex,
AP⋅L
C
7.8
, from phycobilisomes of
Mastigocladus laminosus
has been crystallized in the
orthorhombic space group P2
1
2
1
2
1
.
Cryocrystallographic x-ray measurements enabled the structural analysis
of the complex at a resolution of 2.2 Å. The asymmetric unit contains
two side-to-side associated “trimeric” (αβ)
3
allophycocyanin complexes comprising the linker polypeptide in a
defined orientation inside the trimer. The linker representing a
protein fold related to the prosegment of procarboxypeptidase A is in
contact with only two of the three β-subunits and directly interacts
with the corresponding chromophores of these proteins. In addition to a
modulation of the chromophores’ spectral properties, the linker
polypeptide attracts the αβ-subcomplexes, thereby bringing the
β-chromophores closer together. These results will enable
interpretations of energy-transfer mechanisms within phycobiliproteins.
Ca2+-induced inhibition of the cardiac Ca2+channel depends on calmodulin
Authors: Qin Ning , Olcese Riccardo , Bransby Michael , Lin Tony , Birnbaumer Lutz
Published: July 2002

Ca2+-induced inhibition of α1Cvoltage-gated Ca2+channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+has been a matter of some controversy, as previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of α1Cand/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Previously, using site directed mutagenesis, we have shown that disruption of the EF-hand motif does not remove Ca2+inhibition. We now show that the IQ motif binds CaM and that disruption of this binding activity prevents Ca2+inhibition. We propose that Ca2+entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+inhibition.
A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division
Authors: Cai Shi-Ying , Babbitt Roger W. , Marchesi Vincent T.
Published: July 2002

A new ubiquitin-processing protease (Ubp-M) has been identified in mammalian cells that is phosphorylated at the onset of mitosis and dephosphorylated during the metaphase/anaphase transition. The carboxyl-terminal domain of this 823-aa protein can be phosphorylated
in vitro
with either extracts of mitotic cells or purified cdc-2/cyclin B complexes. Recombinant Ubp-M is able to deubiquitinate histone H2A
in vitro
, and the phosphorylated form is also enzymatically active. Wild-type Ubp-M, transiently expressed as green fluorescent protein-fusion proteins, localizes in the cytoplasm of cultured cells, but mutant forms, lacking an active-site cysteine, associate closely with mitotic chromosomes during all stages of cell division and remain within the nucleus during the postmitotic period. Cells transfected with plasmids containing mutant Ubp-M genes stop dividing and eventually undergo apoptosis. Ubp-M may deubiquitinate one or more critical proteins that are involved in the condensation of mitotic chromosomes, possibly acting selectively on histones H2A and H2B, the major ubiquitinated proteins of chromatin.
Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia
Authors: Martin David A. , Zheng Lixin , Siegel Richard M. , Huang Baohua , Fisher Galen H. , Wang Jin , Jackson Christine E. , Puck Jennifer M. , Dale Janet , Straus Stephen E. , Peter Marcus E. , Krammer Peter H. , Fesik Stephen , Lenardo Michael J.
Published: July 2002

Heterozygous mutations in the CD95 (APO-1/Fas) receptor occur in most individuals with autoimmune lymphoproliferative syndrome (ALPS) and dominantly interfere with apoptosis by an unknown mechanism. We show that local or global alterations in the structure of the cytoplasmic death domain from nine independent ALPS CD95 death-domain mutations result in a failure to bind the FADD/MORT1 signaling protein. Despite heterozygosity for the abnormal allele, lymphocytes from ALPS patients showed markedly decreased FADD association and a loss of caspase recruitment and activation after CD95 crosslinking. These data suggest that intracytoplasmic CD95 mutations in ALPS impair apoptosis chiefly by disrupting death-domain interactions with the signaling protein FADD/MORT1.
Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis
Authors: Bridgen Anne , Weber Friedemann , Fazakerley John K. , Elliott Richard M.
Published: July 2002

Bunyamwera virus (family
Bunyaviridae
, genus
Bunyavirus
) contains a tripartite negative-sense RNA
genome. The smallest RNA segment, S, encodes the nucleocapsid protein N
and a nonstructural protein, NSs, in overlapping reading frames. We
have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse
genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a
smaller plaque size and generated titers of virus approximately 1 log
lower. In mammalian cells, the mutant expressed greatly increased
levels of N protein; significantly, the marked inhibition of host cell
protein synthesis shown by wt virus was considerably impaired by
BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed
BALB/c mice with a slower time course than wt and exhibited a reduced
cell-to-cell spread, and titers of virus in the brain were lower. In
addition, the abrogation of NSs expression changed Bunyamwera virus
from a noninducer to an inducer of an interferon-β promoter. These
results suggest that, although not essential for growth in tissue
culture or in mice, the bunyavirus NSs protein has several functions in
the virus life cycle and contributes to viral pathogenesis.
Testing the Idea of Distinct Storage Mechanisms in Memory
Authors: Laming Donald
Published: July 2002

This paper sets out quantitative foundations for testing the idea that memory is served by two distinct storage mechanisms, a short‐term and a separate long‐term store, using data from the single‐trial free‐recall experiment by Murdock and Okada (1970). In single‐trial free recall one can observe which word is recalled next and how long that recall takes, but that is all. So justification for two separate stores must turn either on the probabilities of recall, or on the latencies, for particular serial positions in the stimulus list.

(a) Nearly one‐third of all recalls after the first are successors (Word n+1 immediately following recall of Word n). So recall is dominated, not by absolute serial position (as a short‐term store would require), but by position relative to the preceding recall.

(b) To a first approximation, all first recalls have the same latency distribution except for Word 1. A common latency distribution is compatible with a single store. An explanation is offered why Word 1 should take longer, in which Word 1 is the second retrieval from a common store.

The idea of two separate stores appears to lack experimental support.
Stroboscopic articulography using fast magnetic resonance imaging
Authors: Mathiak Klaus , Klose Uwe , Ackermann Hermann , Hertrich Ingo , Kincses Wilhelm‐Emil , Grodd Wolfgang
Published: July 2002

A method to display dynamic aspects of vocal tract configuration during speech production by means of fast magnetic resonance imaging is presented. Data acquisition during repetitive movement relies on a stroboscopy‐like procedure. The time resolution achieved is 120 images s −1 in a selected plane. As compared with other techniques of kinematic measurements of speech motor processes, this procedure allows for visualization of the temporal and spatial coordination of all relevant articulators, e.g. the entire tongue, the velum and the lower vocal tract. As an example, the method was applied to repetitions of stop consonant‐vowel‐nasal syllables.
Novel Cytokine-independent Induction of Endothelial Adhesion Molecules Regulated by Platelet/Endothelial Cell Adhesion Molecule (CD31)
Authors: Litwin Marek , Clark Katherine , Noack Leanne , Furze Jill , Berndt Michael , Albelda Steven , Vadas Mathew , Gamble Jennifer
Published: July 2002

Tumor necrosis factor–α, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell–cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule–1, and intercellular adhesion molecule–1. In contrast, ECs failed to express E-selectin when plated on poly-l-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 ± 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 ± 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co– culture of subconfluent ECs with PECAM-1– coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin–expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.
Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium
Authors: Eddy R.J. , Han J. , Condeelis J.S.
Published: July 2002

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.
A SecY Homologue Is Required for the Elaboration of the Chloroplast Thylakoid Membrane and for Normal Chloroplast Gene Expression
Authors: Roy Laura M. , Barkan Alice
Published: July 2002

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and ΔpH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.
A Functional GTPase Domain, but not its Transmembrane Domain, is Required for Function of the SRP Receptor β-subunit
Authors: Ogg Stephen C. , Barz Wolfgang P. , Walter Peter
Published: July 2002

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRα and SRβ, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRβ. This notion is supported (a) by Srp102p's sequence similarity to SRβ; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRα homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.
Relationship between Contact Inhibition and Intranuclear S100c of Normal Human Fibroblasts
Authors: Sakaguchi Masakiyo , Miyazaki Masahiro , Inoue Yusuke , Tsuji Toshiya , Kouchi Hirosuke , Tanaka Toshio , Yamada Hidenori , Namba Masayoshi
Published: July 2002

Many lines of evidence indicate that neoplastic transformation of cells occurs by a multistep process. For neoplastic transformation of normal human cells, they must be first immortalized and then be converted into neoplastic cells. It is well known that the immortalization is a critical step for the neoplastic transformation of cells and that the immortal phenotype is recessive. Thus, we investigated proteins downregulated in immortalized cells by two-dimensional gel electrophoresis. As a result, S100C, a Ca2+-binding protein, was dramatically downregulated in immortalized human fibroblasts compared with their normal counterparts. When the cells reached confluence, S100C was phosphorylated on threonine 10. Then the phosphorylated S100C moved to and accumulated in the nuclei of normal cells, whereas in immortalized cells it was not phosphorylated and remained in the cytoplasm. Microinjection of the anti-S100C antibody into normal confluent quiescent cells induced DNA synthesis. Furthermore, when exogenous S100C was compelled to localize in the nuclei of HeLa cells, their DNA synthesis was remarkably inhibited with increase in cyclin-dependent kinase inhibitors such as p16Ink4a and p21Waf1. These data indicate the possible involvement of nuclear S100C in the contact inhibition of cell growth.
Phosphorylation of the Vesicle-Tethering Protein P115 by a Casein Kinase II–Like Enzyme Is Required for Golgi Reassembly from Isolated Mitotic Fragments
Authors: Dirac-Svejstrup A. Barbara , Shorter James , Waters M. Gerard , Warren Graham
Published: July 2002

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH2-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide–sensitive fusion protein)–catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of t-SNARE (trans–soluble NSF attachment protein [SNAP] receptor) complexes.
Distinct Ras Effector Pathways Are Involved in FcεR1 Regulation of the Transcriptional Activity of Elk-1 and NFAT in Mast Cells
Authors: Turner Helen , Cantrell Doreen A.
Published: July 2002

Activation of Ras GTPases is a conserved feature of antigen receptor signaling, including FcεR1 activation of mast cells. Antigenic cross-linking of the FcεR1 on mast cells results in secretion of allergic mediators and induction of immediate early and cytokine genes. Here we examine the role of Ras in coupling the FcεR1 to transcriptional regulation. The transcription factors Elk-1, an immediate early gene regulator and the nuclear factor of activated T cells (NFAT), in the context of the IL-4 gene, are identified as Ras targets in mast cells. Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1. We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells. Activation of the “classical” Ras/Raf-1/MEK/ ERK cascade is necessary and sufficient for FcεR1 induction of Elk-1. Ras function is required, but not sufficient for FcεR1 induction of NFAT. However, activation or inhibition of Ras markedly shifts the antigen dose-response for FcεR1 induction of NFAT. The effector pathway for Ras activation of NFAT is not Raf-1/MEK. We identify that the Rac-1 GTPase is critical in FcεR1 regulation of NFAT, acting either in parallel with or as an effector of Ras. These data place Ras in a crucial position in mast cells, regulating disparate nuclear targets. Moreover, we identify that two GTPases, Ras and Rac-1, are important regulators of NFAT, and therefore of cytokine expression in mast cells.
Ku70 Is Required for DNA Repair but Not for T Cell Antigen Receptor Gene Recombination In Vivo
Authors: Ouyang Honghai , Nussenzweig Andre , Kurimasa Akihiro , Soares Vera da Costa , Li Xiaoling , Cordon-Cardo Carlos , Li Wen-hui , Cheong Nge , Nussenzweig Michel , Iliakis George , Chen David J. , Li Gloria C.
Published: July 2002

Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70−/−). Like Ku80−/− mice, Ku70−/− mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80−/− mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8− and CD4−CD8+ T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.
Natural Infection of a Homozygous Δ24 CCR5 Red-capped Mangabey with an R2b-Tropic Simian Immunodeficiency Virus
Authors: Chen Zhiwei , Kwon Douglas , Jin Zhanqun , Monard Simon , Telfer Paul , Jones Morris S. , Lu Chang Y. , Aguilar Roberto F. , Ho David D. , Marx Preston A.
Published: July 2002

A homozygous 24-bp deletion (Δ24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Δ24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Δ24CCR5/ Δ24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Δ24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by β-chemokines. At 86.6%, the Δ24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Δ24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Δ24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Δ24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.
Integrin-mediated Ras–Extracellular Regulated Kinase (ERK) Signaling Regulates Interferon γ Production in Human Natural Killer Cells
Authors: Mainiero Fabrizio , Gismondi Angela , Soriani Alessandra , Cippitelli Marco , Palmieri Gabriella , Jacobelli Jordan , Piccoli Mario , Frati Luigi , Santoni Angela
Published: July 2002

Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 integrin–mediated Ras activation. In addition, we demonstrate that ligation of β1 integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors.
Deficiency in CD22, a B Cell–specific Inhibitory Receptor, Is Sufficient to Predispose to Development of High Affinity Autoantibodies
Authors: O'Keefe Theresa L. , Williams Gareth T. , Batista Facundo D. , Neuberger Michael S.
Published: July 2002

CD22 is a B cell–specific transmembrane glycoprotein that acts to dampen signals generated through the B cell antigen receptor (BCR): B cells from CD22-deficient mice give increased Ca2+ fluxes on BCR ligation. Here we show that this B cell hyperresponsiveness correlates with the development of autoantibodies. After the age of eight months, CD22-deficient mice developed high titers of serum IgG directed against double-stranded DNA; these antibodies were of multiclonal origin, somatically mutated, and high affinity. Increased titers of antibodies to cardiolipin and myeloperoxidase were also noted. The results demonstrate that a single gene defect exclusive to B lymphocytes is, without additional contrivance, sufficient to trigger autoantibody development in a large proportion of aging animals. Thus, CD22 might have evolved specifically to regulate B cell triggering thresholds for the avoidance of autoimmunity.
Restricted Immunoglobulin Variable Region (Ig V) Gene Expression Accompanies Secondary Rearrangements of Light Chain Ig V Genes in Mouse Plasmacytomas
Authors: Diaw Lena , Siwarski David , Coleman Allen , Kim Jennifer , Jones Gary M. , Dighiero Guillaume , Huppi Konrad
Published: July 2002

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (VL) and heavy (VH) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the VH-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both κ and λ light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.
Notch1 Deficiency Dissociates the Intrathymic Development of Dendritic Cells and T Cells
Authors: Radtke Freddy , Ferrero Isabel , Wilson Anne , Lees Rosemary , Aguet Michel , MacDonald H. Robson
Published: July 2002

Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1−/− BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1−/− T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.
Mast Cells Can Amplify Airway Reactivity and Features of Chronic Inflammation in an Asthma Model in Mice
Authors: Williams Cara M.M. , Galli Stephen J.
Published: July 2002

The importance of mast cells in the development of the allergen-induced airway hyperreactivity and inflammation associated with asthma remains controversial. We found that genetically mast cell–deficient WBB6F1-W/Wv mice that were sensitized to ovalbumin (OVA) without adjuvant, then challenged repetitively with antigen intranasally, exhibited much weaker responses in terms of bronchial hyperreactivity to aerosolized methacholine, lung tissue eosinophil infiltration, and numbers of proliferating cells within the airway epithelium than did identically treated WBB6F1-+/+ normal mice. However, W/Wv mice that had undergone selective reconstitution of tissue mast cells with in vitro–derived mast cells of congenic +/+ mouse origin exhibited airway responses that were very similar to those of the +/+ mice. By contrast, W/Wv mice that were sensitized with OVA emulsified in alum and challenged with aerosolized OVA exhibited levels of airway hyperreactivity and lung tissue eosinophil infiltration that were similar to those of the corresponding +/+ mice. Nevertheless, these W/Wv mice exhibited significantly fewer proliferating cells within the airway epithelium than did identically treated +/+ mice. These results show that, depending on the “asthma model” investigated, mast cells can either have a critical role in, or not be essential for, multiple features of allergic airway responses in mice.
FAS Ligand, Bcl-2, Granulocyte Colony-Stimulating Factor, and p38 Mitogen-Activated Protein Kinase
Authors: Villunger Andreas , O'Reilly Lorraine A. , Holler Nils , Adams Jerry , Strasser Andreas
Published: July 2002

The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2–controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.
Involvement of the TCR Cβ FG Loop in Thymic Selection and T Cell Function
Authors: Sasada Tetsuro , Touma Maki , Chang Hsiu-Ching , Clayton Linda K. , Wang Jia-huai , Reinherz Ellis L.
Published: July 2002

The asymmetric disposition of T cell receptor (TCR) Cβ and Cα ectodomains creates a cavity with a side-wall formed by the rigid Cβ FG loop. To investigate the significance of this conserved structure, we generated loop deletion (βΔFG) and βwt transgenic (tg) mice using the TCR β subunit of the N15 CTL. N15βwt and N15βΔFG H-2b animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4−CD8− double-negative (DN) compartment. N15βΔFG facilitates transition from DN to CD4+8+ double-positive (DP) thymocytes in recombinase activating gene (RAG)-2−/− mice, showing that pre-TCR function remains. N15βΔFG animals possess ∼twofold more CD8+ single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Vα repertoire observed in N15βΔFG mice may confound the deletion's effect, we crossed N15αβ TCR tg RAG-2−/− with N15βΔFG tg RAG-2−/− H-2b mice to generate N15αβ RAG-2−/− and N15αβ.βΔFG RAG-2−/− littermates. N15αβ.βΔFG RAG-2−/− mice show an 8–10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15αβ, N15αβ.βΔFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cβ FG loop facilitates negative selection of thymocytes and activation of T cells.
Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells
Authors: Strübing Carsten , Clapham David E.
Published: July 2002

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.