bluuebunny/crossref_metadata_2025 huggingface dataset:
Noise shaping in populations of coupled model neurons
Authors: Mar D. J. , Chow C. C. , Gerstner W. , Adams R. W. , Collins J. J.
Published: July 2002

Biological information-processing systems, such as populations of sensory and motor neurons, may use correlations between the firings of individual elements to obtain lower noise levels and a systemwide performance improvement in the dynamic range or the signal-to-noise ratio. Here, we implement such correlations in networks of coupled integrate-and-fire neurons using inhibitory coupling and demonstrate that this can improve the system dynamic range and the signal-to-noise ratio in a population rate code. The improvement can surpass that expected for simple averaging of uncorrelated elements. A theory that predicts the resulting power spectrum is developed in terms of a stochastic point-process model in which the instantaneous population firing rate is modulated by the coupling between elements.
Atomic force microscopy captures length phenotypes in single proteins
Authors: Carrion-Vazquez Mariano , Marszalek Piotr E. , Oberhauser Andres F. , Fernandez Julio M.
Published: July 2002

We use single-protein atomic force microscopy techniques to detect length phenotypes in an Ig module. To gain amino acid resolution, we amplify the mechanical features of a single module by engineering polyproteins composed of up to 12 identical repeats. We show that on mechanical unfolding, mutant polyproteins containing five extra glycine residues added to the folded core of the module extend 20 Å per module farther than the wild-type polyproteins. By contrast, similar insertions near the N or C termini have no effect. Hence, our atomic force microscopy measurements readily discriminate the location of the insert and measure its size with a resolution similar to that of NMR and x-ray crystallography.
Identification of the von Hippel–Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex
Authors: Iwai Kazuhiro , Yamanaka Koji , Kamura Takumi , Minato Nagahiro , Conaway Ronald C. , Conaway Joan W. , Klausner Richard D. , Pause Arnim
Published: July 2002

Mutations of von Hippel–Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B–elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.
Dynamics of tremor-related oscillations in the human globus pallidus: A single case study
Authors: Hurtado José M. , Gray Charles M. , Tamas Laszlo B. , Sigvardt Karen A.
Published: July 2002

Physiological evidence indicates that the resting tremor of
Parkinson’s disease originates in oscillatory neural activity in the
forebrain, but it is unknown whether that activity is globally
synchronized or consists of parallel, independently oscillating
circuits. In the present study, we used dual microelectrodes to record
tremor-related neuronal activity from eight sites in the internal
segment of the globus pallidus (GPi) from an awake Parkinson’s disease
patient undergoing stereotaxic pallidotomy. We utilized spectral
analysis to evaluate the temporal correlations between multiunit
activity at spatially separated sites and between neural and limb
electromyographic activity. We observed that some GPi neural pairs
oscillated synchronously at the tremor frequency, whereas other neural
pairs oscillated independently. Additionally, we found that GPi
tremor-related activity at a given site could fluctuate between states
of synchronization and independence with respect to upper limb tremor.
Consistent with this finding, some paired recording sites within GPi
showed periods of transient synchronization. These observations support
the hypothesis of independent tremor-generating circuits whose coupling
can fluctuate over time.
Molecular identification of human G-substrate, a possible downstream component of the cGMP-dependent protein kinase cascade in cerebellar Purkinje cells
Authors: Endo Shogo , Suzuki Masako , Sumi Mariko , Nairn Angus C. , Morita Ryoji , Yamakawa Kazuhiro , Greengard Paul , Ito Masao
Published: July 2002

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exists almost exclusively in cerebellar Purkinje cells, where it is possibly involved in the induction of long-term depression. A G-substrate cDNA was identified by screening expressed sequence tag databases from a human brain library. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr-119) with amino acid sequences [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-substrate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chromosome 7p15 by radiation hybrid panel analysis.
In vitro
translation products of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE which was close to that of purified rabbit G-substrate (23 kDa). Bacterially expressed human G-substrate is a heat-stable and acid-soluble protein that cross-reacts with antibodies raised against rabbit G-substrate. Recombinant human G-substrate was phosphorylated efficiently by cGMP-dependent protein kinase exclusively at Thr residues, and it was recognized by antibodies specific for rabbit phospho-G-substrate. The amino acid sequences surrounding the sites of phosphorylation in G-substrate are related to those around Thr-34 and Thr-35 of the dopamine- and cAMP-regulated phosphoprotein DARPP-32 and inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein kinase inhibited protein phosphatase 2A more effectively than protein phosphatase 1, suggesting a distinct role as a protein phosphatase inhibitor.
Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin
Authors: Shriver Zachary , Sundaram Mallikarjun , Venkataraman Ganesh , Fareed Jawed , Linhardt Robert , Biemann Klaus , Sasisekharan Ram
Published: July 2002

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from
Flavobacterium heparinum
, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.
Double trans-chromosomic mice: Maintenance of two individual human chromosome fragments containing Ig heavy and κ loci and expression of fully human antibodies
Authors: Tomizuka Kazuma , Shinohara Tokuyuki , Yoshida Hitoshi , Uejima Hiroshi , Ohguma Atsuko , Tanaka Sonoko , Sato Kaoru , Oshimura Mitsuo , Ishida Isao
Published: July 2002

The use of a human chromosome or its fragment as a vector for animal transgenesis may facilitate functional studies of large human genomic regions. We describe here the generation and analysis of double trans-chromosomic (Tc) mice harboring two individual human chromosome fragments (hCFs). Two transmittable hCFs, one containing the Ig heavy chain locus (IgH, ≈1.5 Mb) and the other the κ light chain locus (Igκ, ≈2 Mb), were introduced into a mouse strain whose endogenousIgHandIgκloci were inactivated. In the resultant double-Tc/double-knockout mice, substantial proportion of the somatic cells retained both hCFs, and the rescue in the defect of Ig production was shown by high level expression of human Ig heavy and κ chains in the absence of mouse heavy and κ chains. In addition, serum expression profiles of four human Ig γ subclasses resembled those seen in humans. They mounted an antigen-specific human antibody response upon immunization with human serum albumin, and human serum albumin-specific human monoclonal antibodies with various isotypes were obtained from them. These results represent a generation of mice with “humanized” loci by using the transmittable hCFs, which suggest that the Tc technology may allow for the humanization of over megabase-sized, complex loci in mice or other animals. Such animals may be useful not only for studyingin vivofunctions of the human genome but also for obtaining various therapeutic products.
The spatial and temporal representation of a tone on the guinea pig basilar membrane
Authors: Nilsen K. E. , Russell I. J.
Published: July 2002

In the mammalian cochlea, the basilar membrane's (BM) mechanical responses are amplified, and frequency tuning is sharpened through active feedback from the electromotile outer hair cells (OHCs). To be effective, OHC feedback must be delivered to the correct region of the BM and introduced at the appropriate time in each cycle of BM displacement. To investigate when OHCs contribute to cochlear amplification, a laser-diode interferometer was used to measure tone-evoked BM displacements in the basal turn of the guinea pig cochlea. Measurements were made at multiple sites across the width of the BM, which are tuned to the same characteristic frequency (CF). In response to CF tones, the largest displacements occur in the OHC region and phase lead those measured beneath the outer pillar cells and adjacent to the spiral ligament by about 90°. Postmortem, responses beneath the OHCs are reduced by up to 65 dB, and all regions across the width of the BM move in unison. We suggest that OHCs amplify BM responses to CF tones when the BM is moving at maximum velocity. In regions of the BM where OHCs contribute to its motion, the responses are compressive and nonlinear. We measured the distribution of nonlinear compressive vibrations along the length of the BM in response to a single frequency tone and estimated that OHC amplification is restricted to a 1.25- to 1.40-mm length of BM centered on the CF place.
Traces of learning in the auditory localization pathway
Authors: Knudsen Eric I. , Zheng Weimin , DeBello William M.
Published: July 2002

One of the fascinating properties of the central nervous system is
its ability to learn: the ability to alter its functional properties
adaptively as a consequence of the interactions of an animal with the
environment. The auditory localization pathway provides an opportunity
to observe such adaptive changes and to study the cellular mechanisms
that underlie them. The midbrain localization pathway creates a
multimodal map of space that represents the nervous system's
associations of auditory cues with locations in visual space. Various
manipulations of auditory or visual experience, especially during early
life, that change the relationship between auditory cues and locations
in space lead to adaptive changes in auditory localization behavior and
to corresponding changes in the functional and anatomical properties of
this pathway. Traces of this early learning persist into adulthood,
enabling adults to reacquire patterns of connectivity that were learned
initially during the juvenile period.
Active segregation by the
Bacillus subtilis
partitioning system in
Escherichia coli
Authors: Yamaichi Yoshiharu , Niki Hironori
Published: July 2002

Bacterial genes required for proper partitioning consist of two
transacting genes that encode proteins and a cis-acting gene that
functions like a centromere. Plasmids actively partitioning by means of
these genes migrate from midcell to the cell quarters and are tethered
to these sites until the cells divide. Previously the partitioning
genes were mainly found on plasmids and phages in
Escherichia
coli
. However, progress in genome sequencing reveals that
partitioning genes are ubiquitous in many bacterial plasmids and
chromosomes. Each homologue of the two transacting genes belongs to a
family, ParA or ParB. Moreover, phylogenic analysis of members of the
ParA and ParB families indicates that each member falls into a
chromosomal group or an extrachromosomal group. It is known that the
parAB
genes in the chromosomal group are located on
relatively conserved chromosomal regions in several bacterial species.
This suggests that the
parAB
genes were transferred from
a chromosome to plasmids and phages, so the genes have diverged among
bacterial species. To support this possibility, we show that the
Bacillus subtilis
Soj and Spo0J members of the ParAB
families are responsible for the specific localization of plasmids at
cell quarters in
E. coli
and can function as partition
proteins. Host factors to tether actively partitioning plasmids at
subcellular sites may be conserved in Gram-negative and Gram-positive
bacteria so that phages and plasmids with the ParAB partitioning system
can be stably inherited in host cells across bacterial species.
Characterization of Mbb1, a nucleus-encoded tetratricopeptide-like repeat protein required for expression of the chloroplast
psbB
/
psbT
/
psbH
gene cluster in
Chlamydomonas reinhardtii
Authors: Vaistij Fabián E. , Boudreau Eric , Lemaire Stéphane D. , Goldschmidt-Clermont Michel , Rochaix Jean-David
Published: July 2002

Genetic analysis has revealed that the accumulation of several
chloroplast mRNAs of the green alga
Chlamydomonas
reinhardtii
requires specific nucleus-encoded functions. To
gain insight into this process, we have cloned the nuclear gene
encoding the Mbb1 factor by genomic rescue of a mutant specifically
deficient in the accumulation of the mRNAs of the
psbB
/
psbT
/
psbH
chloroplast transcription unit. Mbb1 is a soluble protein in the
stromal phase of the chloroplast. It consists of 662 amino acids with a
putative chloroplast-transit peptide at its N-terminal end. A striking
feature is the presence of 10 tandemly arranged tetratricopeptide-like
repeats that account for half of the protein sequence and are thought
to be involved in protein–protein interactions. The Mbb1 protein seems
to have a homologue in higher plants and is part of a 300-kDa complex
that is associated with RNA. This complex is most likely involved in
psbB
mRNA processing, stability, and/or translation.
Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells
Authors: Lutz Pierre G. , Moog-Lutz Christel , Coumau-Gatbois Edith , Kobari Ladan , Di Gioia Yolande , Cayre Yvon E.
Published: July 2002

Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.
Feminist Contestations of Institutional Domains in Iran
Authors: Povey Elaheh Rostami
Published: July 2002

Iranian Feminists outside Iran are divided on women's positions in Iran under the Islamic state. Some have argued that the process of Islamization has marginalized women. Others have argued that the dynamic nature of Shari'a interpretation and the debate among religious scholars in Iran have shaped the indigenous forms of feminist consciousness, feminisms and women's involvement in the process of change. This paper, based on field research, is challenging both views. It will be argued that the contradictions of the Islamic state and institutions led to the process of feminist consciousness. In the period 1990–2000, Muslim and secular feminists in Iran have found their own ways of coming together, making demands and pressurizing the State and institutions to reform laws and regulations in favour of women's rights. But women are divided by the nature of their diversity. As their alliance has challenged the limitation of the Islamic state, the breakdown of their alliance (2000–2001), could have a great impact not only on gender relations, but also on the process of democratization and secularization.
‘Drunken Tans’: Representations of Sex and Violence in the Anglo-Irish War (1919–21)
Authors: Ryan Louise
Published: July 2002

War is a highly gendered experience which is both informed by and informs constructions of masculinity and femininity. The dominant depiction of masculine heroes and feminine victims simplifies the complex intersections of militarism, nationalism and gendered roles and identities. Focusing on a case study of the Anglo-Irish War or War of Independence (1919–1921), this paper examines how violence against women, especially sexual violence, was written about and reported in ways which framed representations of Irish and British masculinity and Irish femininity.

In addition, by analysing a range of varied sources including newspapers, autobiographical accounts and recorded testimonies, this paper attempts to assess the extent to which violence against women formed a key aspect of military practice in the war. In conclusion, I engage with some of the difficulties faced by researchers today in exploring evidence of gendered violence in specific historical, cultural and militarized contexts.
Cracks in the Feminist Mirror?
Authors: Humphrey Jill C.
Published: July 2002

This article is an offshoot of a research project on lesbian and gay self-organization in the UK's public sector union UNISON. The site upon which lesbians and gay men ‘work together’ is a complex and contradictory one, located at the juncture of several pathways – women's and men's movements, gendered politics and sexual politics, purist ghettos and queer rainbows. The UNISON group furnishes an ideal site for a case-study of sexual and gendered dynamics in lesbian-and-gay politics by dint of institutional arrangements whereby separate spaces for lesbians and gay men respectively encourage reflexivity in working through these hyphens. While reflections from the lesbian feminist mirror have been active ingredients in the ongoing resocialization of gay men, it is argued that reflections from a gay male mirror will also be necessary if we are to nurture greater wisdom and justice in gender relations. Since the lesbian and gay group co-exists with other self-organized groups for women, black people and disabled people within a broader pro-socialist movement, the potential for working across other sets of differences is also noteworthy. Nevertheless, it can be demonstrated that a sustained coalition between lesbians and gay men is not sufficient in bringing about coalitions with other oppressed minorities or majorities, and may even be at loggerheads with them.
Theorizing the Politics of ‘Islamic Feminism’
Authors: Mojab Shahrzad
Published: July 2002

This article examines developments in ‘Islamic feminism’, and offers a critique of feminist theories, which construct it as an authentic and indigenous emancipatory alternative to secular feminisms. Focusing on Iranian theocracy, I argue that the Islamization of gender relations has created an oppressive patriarchy that cannot be replaced through legal reforms. While many women in Iran resist this religious and patriarchal regime, and an increasing number of Iranian intellectuals and activists, including Islamists, call for the separation of state and religion, feminists of a cultural relativist and postmodernist persuasion do not acknowledge the failure of the Islamic project. I argue that western feminist theory, in spite of its advances, is in a state of crisis since (a) it is challenged by the continuation of patriarchal domination in the West in the wake of legal equality between genders, (b) suspicious of the universality of patriarchy, it overlooks oppressive gender relations in non-western societies and (c) rejecting Eurocentrism and racism, it endorses the fragmentation of women of the world into religious, national, ethnic, racial and cultural entities with particularist agendas.
Literacy, schooling and ideology in a multilingual state
Authors: Blackledge Adrian
Published: July 2002

In this article I argue that current government policy for teaching literacy in schools in England is part of a broader ideology of homogeneity which is visible in other dominant‐culture institutions. Despite the multilingual nature of England and its schools, the dominant discourse is one which values English at the expense of other languages, leading to the ‘misrecognition’ (Bourdieu, 1998b: 131), by majority and minority language speakers alike, of English as the sole language of symbolic capital. While the first part of this article outlines the monolingual ideology of current literacy policy in schools in England, and locates this in the context of a similar ideology evident in discourses beyond education, I also suggest that this process of symbolic domination is not inevitable. Schools need not be sites of social and cultural reproduction. They can challenge existing relations of power in society, and put in place structures and practices which question and even reverse the coercive relations between majority‐culture schools and minority‐culture communities and their children. I propose some ways forward in the development of structures which can enable schools to become sites of social and cultural transformation, rather than reproduction.
Aging of Human Skin: Review of a Mechanistic Model and First Experimental Data
Authors: Giacomoni Paolo U. , Declercq Lieve , Hellemans Lieveke , Maes Daniel
Published: July 2002

The physical, chemical, and biochemical factors that accelerate skin aging have been proposed to activate a self‐maintained micro‐inflammatory process, one of the expected end results of which is an imbalance in the turnover of macromolecules in the dermis. Surface peroxides are recognized as controllable factors of skin aging, and their accumulation is attributed to environmentally induced impairment of defense enzymes. Topical application of antioxidants decreases the rate at which skin elasticity and skin thickness are modified.
Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the αvβ3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth
Authors: Jones Peter Lloyd , Crack Julie , Rabinovitch Marlene
Published: July 2002

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a β3 integrin– dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and “rescues” cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with αvβ3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC β3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating β3 integrin ligands in type I collagen. In turn, αvβ3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.
Growth and Muscle Defects in Mice Lacking Adult Myosin Heavy Chain Genes
Authors: Acakpo-Satchivi Leslie J.R. , Edelmann Winfried , Sartorius Carol , Lu Brian D. , Wahr Philip A. , Watkins Simon C. , Metzger Joseph M. , Leinwand Leslie , Kucherlapati Raju
Published: July 2002

The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.
Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells
Authors: Gradin Helena Melander , Larsson Niklas , Marklund Ulrica , Gullberg Martin
Published: July 2002

Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.
Differential Localization of VE- and N-Cadherins in Human Endothelial Cells: VE-Cadherin Competes with N-Cadherin for Junctional Localization
Authors: Navarro Pilar , Ruco Luigi , Dejana Elisabetta
Published: July 2002

The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell–cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of α- and β-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin–dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg621–Pro702) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg621–Pro702 sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.
Microinjection of Anti-coilin Antibodies Affects the Structure of Coiled Bodies
Authors: Almeida Fátima , Saffrich Rainer , Ansorge Wilhelm , Carmo-Fonseca Maria
Published: July 2002

The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within ∼6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for ∼24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human β-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.
Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells
Authors: Saoudi Yasmina , Fotedar Rati , Abrieu Ariane , Dorée Marcel , Wehland Jürgen , Margolis Robert L. , Job Didier
Published: July 2002

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
Mgm101p Is a Novel Component of the Mitochondrial Nucleoid That Binds DNA and Is Required for the Repair of Oxidatively Damaged Mitochondrial DNA
Authors: Meeusen Shelly , Tieu Quinton , Wong Edith , Weiss Eric , Schieltz David , Yates John R. , Nunnari Jodi
Published: July 2002

Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.
Drosophila roadblock and Chlamydomonas Lc7
Authors: Bowman Aaron B. , Patel-King Ramila S. , Benashski Sharon E. , McCaffery J. Michael , Goldstein Lawrence S.B. , King Stephen M.
Published: July 2002

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97–amino acid polypeptide that is 57% identical (70% similar) to the 105–amino acid Chlamydomonas outer arm dynein–associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.
ErbB2 Is Necessary for Induction of Carcinoma Cell Invasion by Erbb Family Receptor Tyrosine Kinases
Authors: Spencer Kathryn S.R. , Graus-Porta Diana , Leng Jie , Hynes Nancy E. , Klemke Richard L.
Published: July 2002

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.
Keratin-Dependent, Epithelial Resistance to Tumor Necrosis Factor-Induced Apoptosis
Authors: Caulin Carlos , Ware Carl F. , Magin Thomas M. , Oshima Robert G.
Published: July 2002

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8−) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are ∼100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH2-terminal kinase (JNK) intracellular signaling and NFκB activation. Furthermore, K8− and K18− mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.
The Glycosylphosphatidyl Inositol-Anchored Adhesion Molecule F3/Contactin Is Required for Surface Transport of Paranodin/Contactin-Associated Protein (Caspr)
Authors: Faivre-Sarrailh Catherine , Gauthier France , Denisenko-Nehrbass Natalia , Le Bivic André , Rougon Geneviève , Girault Jean-Antoine
Published: July 2002

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100–insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.
Cyk-4
Authors: Jantsch-Plunger Verena , Gönczy Pierre , Romano Alper , Schnabel Heinke , Hamill Danielle , Schnabel Ralf , Hyman Anthony A. , Glotzer Michael
Published: July 2002

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.
Plasmin-Sensitive Dibasic Sequences in the Third Fibronectin-like Domain of L1–Cell Adhesion Molecule (Cam) Facilitate Homomultimerization and Concomitant Integrin Recruitment
Authors: Silletti Steve , Mei Fang , Sheppard Dean , Montgomery Anthony M.P.
Published: July 2002

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including αvβ3 and α5β1. A pep- tide derived from the putative C-C′ loop of FN3 (GSQRKHSKRHIHKDHV852) also forms trimeric complexes and supports αvβ3 and α5β1 binding. Substitution of the dibasic RK841 and KR845 sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK↓HSK↓RH846. The integrin α9β1, which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell–cell contact. We propose that distal receptor ligation events at the cell–cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.
Functional Analysis of a Human Homologue of the Drosophila Actin Binding Protein Anillin Suggests a Role in Cytokinesis
Authors: Oegema Karen , Savoian Matthew S. , Mitchison Timothy J. , Field Christine M.
Published: July 2002

We have characterized a human homologue of anillin, a Drosophila actin binding protein. Like Drosophila anillin, the human protein localizes to the nucleus during interphase, the cortex following nuclear envelope breakdown, and the cleavage furrow during cytokinesis. Anillin also localizes to ectopic cleavage furrows generated between two spindles in fused PtK1 cells. Microinjection of antianillin antibodies slows cleavage, leading to furrow regression and the generation of multinucleate cells. GFP fusions that contain the COOH-terminal 197 amino acids of anillin, which includes a pleckstrin homology (PH) domain, form ectopic cortical foci during interphase. The septin Hcdc10 localizes to these ectopic foci, whereas myosin II and actin do not, suggesting that anillin interacts with the septins at the cortex. Robust cleavage furrow localization requires both this COOH-terminal domain and additional NH2-terminal sequences corresponding to an actin binding domain defined by in vitro cosedimentation assays. Endogenous anillin and Hcdc10 colocalize to punctate foci associated with actin cables throughout mitosis and the accumulation of both proteins at the cell equator requires filamentous actin. These results indicate that anillin is a conserved cleavage furrow component important for cytokinesis. Interactions with at least two other furrow proteins, actin and the septins, likely contribute to anillin function.