bluuebunny/crossref_metadata_2025 huggingface dataset:
The
in situ
spatial arrangement of the influenza A virus matrix protein M1 assessed by tritium bombardment
Authors: Shishkov Alexander V. , Goldanskii Vitalii I. , Baratova Ludmila A. , Fedorova Natalia V. , Ksenofontov Alexander L. , Zhirnov Oleg P. , Galkin Alexander V.
Published: July 2002

Intact influenza A virions were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the matrix protein M1 was analyzed to determine the
in situ
accessibility of its tryptic fragments. These data were combined with the previously reported x-ray crystal structure of the M1 fragment 2–158 [Sha, B. & Luo, M. (1997)
Nat. Struct. Biol.
4, 239–244] and the predicted topology of the C domain (159–252) to propose a model of M1 arrangement in the virus particle.
Phosphorylated tau can promote tubulin assembly
Authors: Tseng Huang-Chun , Lu Quan , Henderson Eric , Graves Donald J.
Published: July 2002

Phosphorylation can affect the function of microtubule-associated protein tau. Here, the human brain tau with 441 amino acids was phosphorylated by cyclic-AMP-dependent protein kinase (PKA) or glycogen synthase kinase-3β. PKA-phosphorylated tau (2.7 mol phosphates/mol) does not promote tubulin assembly as judged by spectrophotometric and atomic force microscopy measurements, unless trimethylamine N-oxide (TMAO), a natural occurring osmolyte, is included in these assays. TMAO is also found to promote tubulin assembly of glycogen synthase kinase-3β-phosphorylated tau (1.6 mol phosphates/mol). TMAO does not act by causing a chemical dephosphorylation of phosphorylated tau, but it acts to overcome the functional deficit caused by phosphorylation. PKA-phosphorylated tau binds to tubulin in the presence of TMAO and lowers the critical concentration of tubulin needed for assembly. From these data, we conclude that PKA-phosphorylated tau retains the ability to bind tubulin and promote tubulin assembly. TMAO is required, however, to sensitize the reaction. Possible uses of TMAO in relation to studies of tubulin assembly
in vitro
, in intact cells, and in relation to Alzheimer’s disease are presented in this report.
Nuclear hourglass technique: An approach that detects electrically open nuclear pores in
Xenopus laevis
oocyte
Authors: Danker T. , Schillers H. , Storck J. , Shahin V. , Krämer B. , Wilhelmi M. , Oberleithner H.
Published: July 2002

Nuclear pore complexes (NPCs) mediate both active transport and
passive diffusion across the nuclear envelope (NE). Determination of NE
electrical conductance, however, has been confounded by the lack of an
appropriate technical approach. The nuclear patch clamp technique is
restricted to preparations with electrically closed NPCs, and
microelectrode techniques fail to resolve the extremely low input
resistance of large oocyte nuclei. To address the problem, we have
developed an approach for measuring the NE electrical conductance of
Xenopus
laevis
oocyte nuclei. The method
uses a tapered glass tube, which narrows in its middle part to 2/3 of
the diameter of the nucleus. The isolated nucleus is sucked into the
narrow part of the capillary by gentle fluid movement, while the
resulting change in electrical resistance is monitored. NE electrical
conductance was unexpectedly large (7.9 ± 0.34
S/cm
2
). Evaluation of NPC density by atomic force
microscopy showed that this conductance corresponded to 3.7 ×
10
6
NPCs. In contrast to earlier conclusions drawn from
nuclear patch clamp experiments, NPCs were in an electrically
“open” state with a mean single NPC electrical conductance of
1.7 ± 0.07 nS. Enabling or blocking of active NPC transport
(accomplished by the addition of cytosolic extracts or gp62-directed
antibodies) revealed this large NPC conductance to be independent of
the activation state of the transport machinery located in the center
of NPCs. We conclude that peripheral channels, which are presumed to
reside in the NPC subunits, establish a high ionic permeability that is
virtually independent of the active protein transport mechanism.
Identification of a cytochrome
b
-type NAD(P)H oxidoreductase ubiquitously expressed in human cells
Authors: Zhu Hao , Qiu Huawei , Yoon Hae-Won Patricia , Huang Shuning , Bunn H. Franklin
Published: July 2002

Cytochrome
b
-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome
b
-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome
b
5 (b5) and
b
5 reductase (b5R) domains. A genetic approach, using
blast
searches against
dbest
for FAD-, NAD(P)H-binding sequences followed by reverse transcription–PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome
b
with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome
c
reduction
in vitro
. The properties of this protein make it a plausible candidate oxygen sensor.
Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: Implications for molecular mimicry in autoimmune disease
Authors: Misko Ihor S. , Cross Simone M. , Khanna Rajiv , Elliott Suzanne L. , Schmidt Christopher , Pye Stephanie J. , Silins Sharon L.
Published: July 2002

The immunodominant, CD8+cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein–Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide—RSKFRQIV—located in a serine/threonine kinase and a bacterial peptide—RRKYKQII—located inStaphylococcus aureusreplication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted αβ TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8+CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.
Enhancement of fluid filtration across tumor vessels: Implication for delivery of macromolecules
Authors: Netti Paolo A. , Hamberg Leena M. , Babich John W. , Kierstead Diane , Graham Wendy , Hunter George J. , Wolf Gerald L. , Fischman Alan , Boucher Yves , Jain Rakesh K.
Published: July 2002

Cancer therapies using genes and other macromolecules might realize their full clinical potential if they could be delivered to tumor tissue in optimal quantities. Unfortunately, the compromised circulation within tumors poses a formidable resistance to adequate and uniform penetration of these agents. Previously, we have proposed elevated interstitial fluid pressure (IFP) as a major physiological barrier to delivery of macromolecules. Here we postulate that modulation of tumor microvascular pressure (MVP) and associated changes in IFP would enhance macromolecular delivery into a solid tumor. To test our hypothesis, we altered tumor MVP by either periodic injection or continuous infusion of angiotensin II (AII) and measured the resulting changes in IFP and uptake of macromolecules. We used the nicotinyl hydrazine derivative of human polyclonal IgG (HYNIC-IgG) as a nonspecific macromolecule and CC49 antibody as a specific macromolecule. We found that both chronic and periodic modulation of tumor MVP enhances transvascular fluid filtration, leading to a 40% increase in total uptake of the specific antibody within 4 hr of its administration. Conversely, neither continuous nor periodic infusion of AII induced any increase in uptake of nonspecific antibodies. Strategies to improve delivery of macromolecules and limitations of this approach are identified.
Mouse
numb
is an essential gene involved in cortical neurogenesis
Authors: Zhong Weimin , Jiang Ming-Ming , Schonemann Marcus D. , Meneses Juanito J. , Pedersen Roger A. , Jan Lily Yeh , Jan Yuh Nung
Published: July 2002

During neurogenesis of the mammalian neocortex, neural progenitor cells divide to generate daughter cells that either become neurons or remain as progenitor cells. The mouse
numb
(
m-numb
) gene encodes a membrane-associated protein that is asymmetrically localized to the apical cell membrane of dividing cortical progenitor cells and may be segregated to only the apical daughter cell that has been suggested to remain as a progenitor cell. To examine
m-numb
function during neural development, we generated a loss-of-function mutant allele of
m-numb
. Mice homozygous for this mutation exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain and die around embryonic day 11.5 (E11.5). These findings suggest that
m-numb
is an essential gene that plays a role in promoting progenitor cell fate during cortical neurogenesis.
Miniaturized metalloproteins: Application to iron–sulfur proteins
Authors: Lombardi Angela , Marasco Daniela , Maglio Ornella , Di Costanzo Luigi , Nastri Flavia , Pavone Vincenzo
Published: July 2002

The miniaturization process applied to rubredoxins generated a
class of peptide-based metalloprotein models, named METP
(
m
iniaturized
e
lectron
t
ransfer
p
rotein). The crystal structure
of
Desulfovibrio vulgaris
rubredoxin was selected as a
template for the construction of a tetrahedral
(S
γ
-Cys)
4
iron-binding site. Analysis of the
structure showed that a sphere of 17 Å in diameter, centered on the
metal, circumscribes two unconnected approximately C
2
symmetry related β-hairpins, each containing the
-Cys-(Aaa)
2
-Cys- sequence. These observations provided a
starting point for the design of an undecapeptide, which self assembles
in the presence of tetrahedrally coordinating metal ions. The METP
peptide was synthesized in good yield by standard methodologies.
Successful assembly of the METP peptide with Co(II), Zn(II),
Fe(II/III), in the expected 2:1 stoichiometry, was proven by
UV-visible and circular dichroism spectroscopies. UV-visible analysis
of the metal complexes indicated the four Cys ligands tetrahedrally
arrange around the metal ion, as designed. Circular dichroism
measurements on both the free and metal-bound forms revealed that the
metal coordination drives the peptide chain to fold into a turned
conformation. NMR characterization of the Zn(II)-METP complex fully
supported the structure of the designed model. These results prove that
METP reproduces the main features of rubredoxin.
Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity
Authors: Axworthy D. B. , Reno J. M. , Hylarides M. D. , Mallett R. W. , Theodore L. J. , Gustavson L. M. , Su F.-M. , Hobson L. J. , Beaumier P. L. , Fritzberg A. R.
Published: July 2002

A covalent conjugate (NR-LU-10/SA) was prepared between streptavidin (SA) and NR-LU-10, a mAb that binds an antigen expressed on the surface of most human carcinomas. NR-LU-10/SA was injected into nude mice bearing human tumor xenografts. Injection of biotinylated galactosyl-human serum albumin reduced the circulating levels of conjugate by 95%. Subsequent administration of
90
Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin achieved peak uptake at the tumor within 2 hr while >80% of the radioactivity was eliminated in the urine. A single dose of 600–800 μCi of
90
Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin produced cures in 10/10 mice with established (>200 mm
3
) s.c. human small cell lung or colon cancer xenografts and 8/10 cures in mice with human breast cancer xenografts without significant toxicity.
Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription
Authors: Llopis Juan , Westin Stefan , Ricote Mercedes , Wang Jiahong , Cho Charles Y. , Kurokawa Riki , Mullen Tina-Marie , Rose David W. , Rosenfeld Michael G. , Tsien Roger Y. , Glass Christopher K.
Published: July 2002

Members of the nuclear receptor superfamily are thought to activate transcription by recruitment of one or more recently identified coactivator complexes. Here we demonstrate that both peroxisome proliferator-activated receptor binding protein (PBP) and steroid receptor coactivator-1 (SRC-1) are required for ligand-dependent transcription of transiently transfected and chromosomally integrated reporter genes by the estrogen receptor (ER) and retinoic acid receptor (RAR). To examine ligand-dependent interactions between nuclear receptors and specific coactivators in living cells, these proteins were tagged with cyan (CFP) and yellow (YFP) mutants of the green fluorescent protein. Fluorescence resonance energy transfer (FRET) from the CFP to the YFP indicated interaction between the receptor and coactivator. CFP fusions to RAR or its ligand-binding domain exhibited rapid ligand-dependent FRET to YFP-tagged nuclear receptor interaction domains of the coactivators SRC-1 and PBP. The ER-ligand-binding domain, unlike RAR, also exhibited some basal interaction with coactivators in unstimulated cells that was abolished by the receptor antagonists tamoxifen or ICI182,780. Inhibition of FRET by tamoxifen but not ICI182,780 could be reversed by estradiol, whereas estradiol-enhanced FRET could not be inhibited by either antagonist, indicating that ligand effects can show varying degrees of hysteresis. These findings suggest that ligand-dependent transcriptional activities of the RAR and ER require concurrent or sequential recruitment of SRC-1 and PBP-containing coactivator complexes.
Coding Position in Short‐term Memory
Authors: Henson Richard N.A.
Published: July 2002

Many formal models of short‐term memory have recently been proposed (e.g. Anderson & Matessa, 1997; Brown, Preece, & Hulme, in press; Burgess & Hitch, 1992; Henson, 1998; Lee & Estes, 1981; Lewandowsky & Murdock, 1989; Murdock, 1995; Nairne, Neath, Serra, & Byun, 1997; Neath, 1993; Page & Norris, 1998). An important issue addressed by these models is the problem of serial order: that is, how people store and retrieve a novel sequence of items in the appropriate order. A popular solution to this problem is to assume that each item is coded for its position within a sequence. The present article discusses three different means of coding position. It is argued that the pattern of errors people make when they misrecall a sequence supports the hypothesis that position is coded relative to the start and the end of a sequence. Other evidence, however, suggests that positional coding is also sensitive to temporal factors. A new model is described that reconciles these two strands of evidence.
Ambulance attendance at heroin overdose in Melbourne: the establishment of a database of Ambulance Service records
Authors: DIETZE PAUL M. , CVETKOVSKI STEFAN , RUMBOLD GREG , MILLER PETER
Published: July 2002

This paper presents some of the preliminary data extracted from a database of non‐fatal heroin overdoses attended by ambulances in metropolitan Melbourne. The database has been developed in order to provide interested parties with reliable, high quality and timely data on heroin‐related harm. Heroin overdose victims were defined as those people who were unconscious at the time the ambulance arrived and subsequently responded positively to the administration of naloxone. There were 388 heroin overdoses attended by ambulances in Melbourne over the period 1 November 1997–31 January 1998. The majority of victims were male (74 %) with an estimated mean age of 27.1 years. The overdoses occurred most commonly during the afternoon and evening, with Thursdays and Fridays most prominent. Police attendance was low (12%) and the vast majority of victims were not transported to hospital. An analysis by postcode revealed that the overdoses tended to be concentrated around a number of locations which have identified street heroin markets. The findings are discussed in relation to the utility of the data as an indicator of heroin‐related harm within the community and for informing overdose prevention initiatives.
Attitudes and practices of general practitioners training to work with drug‐using patients
Authors: Jacka David , Clode Danielle , Patterson Sharon , Wyman Kim
Published: July 2002

This study assessed the current attitudes, knowledge and practices of 26 inner metropolitan GPs training to work with drug‐users. In comparison to nicotine, alcohol and benzodiazepine, illicit drugs (such as opiates and amphetamines) were associated with significantly lower GP confidence, self‐reported effectiveness and general knowledge. GPs were also less likely to ask their patients about illicit drug‐use than other types of drugs. Older GPs were more confident, believed themselves to be more effective but had less general knowledge about all drug types than GPs under 40. Female doctors also reported lower levels of confidence. Self‐reported effectiveness and confidence did not show any association with accuracy of general knowledge about drugs. Although most GPs were very positive about their role in working with drug‐users, GPs tended to associate wanting to work with drug‐users with professional responsibility, rather than personal interest. These findings suggest that training programmes for GPs in the area of drug‐use need to be tailored to account for differing knowledge bases, but also tackle the more difficult task of addressing attitudes and self‐perceptions of the role GPs play in dealing with drug‐use.
Adolescent Females with a Substance Use Disorder: Affiliations with Adult Male Sexual Partners
Authors: Mezzich Ada Castillo , Giancola Peter R. , Lu Sandy Y. , Parks Susan M. , Ratica Gary M. , Dunn Marija
Published: July 2002

Objective: To test the hypothesis that low socioeconomic status (SES), a disturbed parent‐daughter relationship, early sexual development, and antisocial behavior are risk factors in adolescent females affiliating with adult male sexual partners. To determine whether the relation between these risk factors and affiliating with adult male sexual partners is stronger in females with greater, rather than fewer, substance use disorders (SUD). Method: Subjects were 180 adolescent females with SUD and 87 normal controls (14–18 years of age). Results: The SUD group had a lower SES and more negative parent‐daughter interactions, and exhibited greater antisocial tendencies. Also, the SUD group showed a more frequent affiliation with adult male sexual partners. Chronological age, age of menarche (sexual development), antisocial behavior, and quality of the parent‐daughter relationship were significantly associated with affiliation with adult male sexual partners. Moreover, the number of SUD diagnoses enhanced the relation between the quality of the parent‐daughter relationship and antisocial behavior with affiliation with adult male sexual partners. Conclusions: From a prevention perspective, interventions directed at enhancing child rearing practices, communication skills, and involvement in children's needs and activities might result in improved parent‐child attachments that may attentuate young women's propensities to become involved in antisocial behavior and affiliate with adult sexual partners. Also, the risk imposed by an early sexual maturation may be offset by enhancing the female adolescent's social skills to select non‐deviant and supportive male partners.
Examining interaction in language therapy
Authors: Horton Simon , Byng Sally
Published: July 2002

Discussions around a ‘theory of therapy’ have prompted attempts to delineate the components of therapy for language impairment in aphasia. Here it is suggested that the therapy components associated with how language therapy is enacted need more precise specification, especially in relation to what is entailed by interaction between therapist and person with aphasia. Presented too is a system used for the analysis of interaction in language impairment therapy for aphasia and its possible applications are discussed. Components of the system are described and a sample analysis presented. The methodological difficulties entailed in making comparisons of therapy enactment are discussed. It is suggested furthermore that unless more is known about precisely defined interactions in therapy, it will be difficult to answer basic questions not only about how language therapy is being carried out, but also, subsequently, whether the ways in which therapy tasks are enacted have a significant impact on the outcome.
Enhancing communication in jargon aphasia: a small group study of writing therapy
Authors: Robson Jo , Marshall Jane , Chiat Shula , Pring Tim
Published: July 2002

People with jargon aphasia have severely disordered and incomprehensible speech that may be resistant to therapeutic intervention. In this study, we treated written output and examined whether it assisted communication for these clients. In stage one of the study, anagram sorting, delayed copying and lexical decision tasks were used to investigate the residual knowledge of written words in a group of ten people with jargon aphasia. Evidence of the presence of orthographic knowledge was taken as an indication that writing might be a useful focus for therapy. This hypothesis was explored in stage two with six clients. A personally useful vocabulary was selected for each, and copying, word completion and written picture‐naming tasks were used in therapy to improve written production of these words. The clients made progress in written naming. However, they showed little change on a ‘message’ assessment that tested their ability to use the written words to convey messages. Stage three targeted communicative writing. Here, three of the clients received ‘message therapy’, which encouraged them to relate treated words to functional messages and to communicate them to a partner. The clients improved on the message assessment and observation of their communication and reports from relatives suggested that they made functional use of writing in a range of communication settings.
Written communication in undifferentiated jargon aphasia: a therapy study
Authors: Robson Jo , Pring Tim , Marshall Jane , Morrison Sarah , Chiat Shula
Published: July 2002

A subject, R.M.M., with a 2‐year history of jargon aphasia is described. At the beginning of this study she had minimal meaningful spoken output and showed little awareness of her speech despite having relatively well‐preserved auditory comprehension. Her spoken output had proved resistant to earlier periods of therapy. In contrast, R.M.M.'s written output showed some ability to access orthographic information and monitoring of this modality was shown by an acute awareness of her errors. A 3‐stage therapy programme is described. This was designed to improve R.M.M.'s writing of single words and to encourage use of writing as an alternative means of communication. The initial stage of therapy aimed to increase R.M.M.'s access to written word forms by use of picture stimuli. She showed significant improvement in writing treated items in response to pictures both immediately after therapy and at re‐assessment 6 weeks later. Despite the acquisition of these skills, R.M.M. failed to use them in communicative contexts. A second stage of therapy replicated the results of the first and sought to facilitate R.M.M.'s functional use of her written vocabulary by asking her to write words to spoken questions. She again showed improved written naming of the treated items and could now produce written names appropriately in a questionnaire‐type assessment. Generalization of this ability extended to items that had not been trained in this way. Functional use of writing in everyday communication remained absent, however. The final stage of therapy made explicit the potential links between items which R.M.M. could now write and functional messages which they might convey. She again showed significant changes in the acquisition of new vocabulary and, encouragingly, progress was also seen in her use of the strategy in functional communication. R.M.M.'s speech is almost entirely incomprehensible. It has remained unchanged for 2 years and has not responded to therapy. Relatively wellpreserved auditory comprehension and good monitoring of written output allowed therapy to effectively target a small written vocabulary. Despite significant progress in the acquisition of new items, transfer of this skill to functional communication was initially absent. Further therapy which specifically targeted the impairment causing this failure was needed before functional use was seen. The potential for treating written output in cases of jargon aphasia which have been resistant to therapy for spoken language is discussed.
Imaging Signal Transduction in Living Cells with GFP‐Based Probes
Authors: Zaccolo Manuela , Pozzan Tullio
Published: July 2002

Since the cloning and the eterologous expression of the Green Fluorescence Protein (GFP), a number of applications have been reported where protein location within the cell or gene expression is revealed by fluorescent imaging of living cells. Modified GFPs, however, can now be exploited not only as a fluorescent reporter but also as a dynamic marker of intracellular signalling events, such as fluctuations in the levels of the second messengers Ca2+ and cAMP, or as a probe for detecting changes in pH in various cell compartments. These genetically manipulated GFPs allow monitoring of the biochemistry of the cell in real time and thus offer the possibility to gain a more precise view of the functioning of live cells.
Intrinsic Neuronal Determinants Locally Regulate Extrasynaptic and Synaptic Growth at the Adult Neuromuscular Junction
Authors: Caroni Pico , Aigner Ludwig , Schneider Corinna
Published: July 2002

Long-term functional plasticity in the nervous system can involve structural changes in terminal arborization and synaptic connections. To determine whether the differential expression of intrinsic neuronal determinants affects structural plasticity, we produced and analyzed transgenic mice overexpressing the cytosolic proteins cortical cytoskeleton–associated protein 23 (CAP-23) and growth-associated protein 43 (GAP-43) in adult neurons.

Like GAP-43, CAP-23 was downregulated in mouse motor nerves and neuromuscular junctions during the second postnatal week and reexpressed during regeneration. In transgenic mice, the expression of either protein in adult motoneurons induced spontaneous and greatly potentiated stimulus-induced nerve sprouting at the neuromuscular junction. This sprouting had transgene-specific features, with CAP-23 inducing longer, but less numerous sprouts than GAP-43. Crossing of the transgenic mice led to dramatic potentiation of the sprout-inducing activities of GAP-43 and CAP-23, indicating that these related proteins have complementary and synergistic activities. In addition to ultraterminal sprouting, substantial growth of synaptic structures was induced. Experiments with pre- and postsynaptic toxins revealed that in the presence of GAP-43 or CAP-23, sprouting was stimulated by a mechanism that responds to reduced transmitter release and may be independent of postsynaptic activation.

These results demonstrate the importance of intrinsic determinants in structural plasticity and provide an experimental approach to study its role in nervous system function.
Presentation of Integrins on Leukocyte Microvilli: A Role for the Extracellular Domain in Determining Membrane Localization
Authors: Abitorabi M. Abi , Pachynski Russell K. , Ferrando Ronald E. , Tidswell Mark , Erle David J.
Published: July 2002

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins α4β7, αLβ2, and αMβ2 were heterologously expressed in K562 cells. α4β7 initiated adhesion under flow and localized to microvilli, whereas β2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the α4β7 cytoplasmic and/or transmembrane domains with the homologous domains of αLβ2 or αMβ2. Unexpectedly, these chimeras efficiently mediated adhesion to the α4β7 ligand mucosal addressin cell adhesion molecule–1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of α4 and β2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins α4β1, α5β1, α6Aβ1, αvβ3, and αEβ7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.
A Nuclear Import Pathway for a Protein Involved in tRNA Maturation
Authors: Rosenblum Jonathan S. , Pemberton Lucy F. , Blobel Günter
Published: July 2002

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.
Agrin-Induced Acetylcholine Receptor Clustering Is Mediated by the Small Guanosine Triphosphatases Rac and Cdc42
Authors: Weston Christi , Yee Barry , Hod Eldad , Prives Joav
Published: July 2002

During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. Here, we investigated the role of Rac and Cdc42 in coupling agrin signaling to AChR clustering. We found that agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis.
Chromatin Assembly at Kinetochores Is Uncoupled from DNA Replication
Authors: Shelby Richard D. , Monier Karine , Sullivan Kevin F.
Published: July 2002

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric “state” on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [3H]thymidine we demonstrate that CENP-A–associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.
Dual Stimulation of Ras/Mitogen-Activated Protein Kinase and Rhoa by Cell Adhesion to Fibronectin Supports Growth Factor–Stimulated Cell Cycle Progression
Authors: Danen Erik H.J. , Sonneveld Petra , Sonnenberg Arnoud , Yamada Kenneth M.
Published: July 2002

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. Here, we show that a similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supported growth factor–induced, E2F-dependent S phase entry. Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21Cip/Waf occurred specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.
Agrin-Induced Phosphorylation of the Acetylcholine Receptor Regulates Cytoskeletal Anchoring and Clustering
Authors: Borges Lúcia S. , Ferns Michael
Published: July 2002

At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR β subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus forms of the β subunit in mouse Sol8 myotubes. We find that agrin-induced phosphorylation of the β subunit occurs only on cell surface AChR, and that AChR-containing tyrosine-minus β subunit is targeted normally to the plasma membrane. Surface AChR that is tyrosine phosphorylated is less detergent extractable than nonphosphorylated AChR, indicating that it is preferentially linked to the cytoskeleton. Consistent with this, we find that agrin treatment reduces the detergent extractability of AChR that contains tagged wild-type β subunit but not tyrosine-minus β subunit. In addition, agrin-induced clustering of AChR containing tyrosine-minus β subunit is reduced in comparison to wild-type receptor. Thus, we find that agrin-induced phosphorylation of AChR β subunit regulates cytoskeletal anchoring and contributes to the clustering of the AChR, and this is likely to play an important role in the postsynaptic localization of the receptor at the developing synapse.
The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis
Authors: Jarousse Nadine , Kelly Regis B.
Published: July 2002

One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the β sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein–protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage.
Regulation of the Receptor Specificity and Function of the Chemokine RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) by Dipeptidyl Peptidase IV (CD26)-mediated Cleavage
Authors: Oravecz Tamas , Pall Marina , Roderiquez Gregory , Gorrell Mark D. , Ditto Mary , Nguyen Nga Y. , Boykins Robert , Unsworth Edward , Norcross Michael A.
Published: July 2002

CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3–68) lacked the ability of native RANTES(1–68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3–68) showed a reduced activity, relative to that of RANTES(1–68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation–induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.
The Common Cytokine Receptor γ Chain Controls Survival of γ/δ T Cells
Authors: Malissen Marie , Pereira Pablo , Gerber David J. , Malissen Bernard , DiSanto James P.
Published: July 2002

We have investigated the role of common γ chain (γc)-signaling pathways for the development of T cell receptor for antigen (TCR)-γ/δ T cells. TCR-γ/δ–bearing cells were absent from the adult thymus, spleen, and skin of γc-deficient (γc−) mice, whereas small numbers of thymocytes expressing low levels of TCR-γ/δ were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-γ/δ development through induction of V-J (variable-joining) rearrangements at the TCR-γ locus. In contrast, we detected clearly TCR-γ rearrangements in fetal thymi from γc− mice (which fail to signal in response to IL-7) and reduced TCR-γ rearrangements in adult γc thymi. No gross defects in TCR-δ or TCR-β rearrangements were observed in γc− mice of any age. Introduction of productively rearranged TCR Vγ1 or TCR Vγ1/Vδ6 transgenes onto mice bearing the γc mutation did not restore TCR-γ/δ development to normal levels suggesting that γc-dependent pathways provide additional signals to developing γ/δ T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from γc− mice were dramatically reduced compared to γc+ transgenic littermates. We favor the concept that γc-dependent receptors are required for the maintenance of TCR-γ/δ cells and contribute to the completion of TCR-γ rearrangements primarily by promoting survival of cells committed to the TCR-γ/δ lineage.
Modulation of Proteasomal Activity Required for the Generation of a Cytotoxic T Lymphocyte–defined Peptide Derived from the Tumor Antigen MAGE-3
Authors: Valmori Danila , Gileadi Uzi , Servis Catherine , Dunbar P. Rod , Cerottini Jean-Charles , Romero Pedro , Cerundolo Vincenzo , Lévy Frédéric
Published: July 2002

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201–associated tumor peptide antigen MAGE-3271–279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271–279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271–279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271–279–specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymatic activities.
A Role for the Tec Family Tyrosine Kinase Txk in T Cell Activation and Thymocyte Selection
Authors: Sommers Connie L. , Rabin Ronald L. , Grinberg Alexander , Tsay Henry C. , Farber Joshua , Love Paul E.
Published: July 2002

Recent data indicate that several members of the Tec family of protein tyrosine kinases function in antigen receptor signal transduction. Txk, a Tec family protein tyrosine kinase, is expressed in both immature and mature T cells and in mast cells. By overexpressing Txk in T cells throughout development, we found that Txk specifically augments the phospholipase C (PLC)-γ1–mediated calcium signal transduction pathway upon T cell antigen receptor (TCR) engagement. Although Txk is structurally different from inducible T cell kinase (Itk), another Tec family member expressed in T cells, expression of the Txk transgene could partially rescue defects in positive selection and signaling in itk−/− mice. Conversely, in the itk+/+ (wild-type) background, overexpression of Txk inhibited positive selection of TCR transgenic thymocytes, presumably due to induction of cell death. These results identify a role for Txk in TCR signal transduction, T cell development, and selection and suggest that the Tec family kinases Itk and Txk perform analogous functions.
Regulation of Cytokine Signaling by B Cell Antigen Receptor and Cd40-Controlled Expression of Heparan Sulfate Proteoglycans
Authors: van der Voort Robbert , Keehnen Robert M.J. , Beuling Esther A. , Spaargaren Marcel , Pals Steven T.
Published: July 2002

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell–derived factor (SDF)-1α, even though the affinities of HGF and SDF-1α for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.