bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential ligand-dependent protein–protein interactions between nuclear receptors and a neuronal-specific cofactor
Authors: Greiner Erich F. , Kirfel Jutta , Greschik Holger , Huang Dongya , Becker Peter , Kapfhammer Josef P. , Schüle Roland
Published: July 2002

Nuclear receptors are transcription factors that require multiple protein–protein interactions to regulate target gene expression. We have cloned a 27-kDa protein, termed NIX1 (neuronal interacting factor X 1), that directly binds nuclear receptors
in vitro
and
in vivo
. Protein–protein interaction between NIX1 and ligand-activated or constitutive active nuclear receptors, including retinoid-related orphan receptor β (RORβ) (NR1F2), strictly depends on the conserved receptor C-terminal activation function 2 (AF2-D). NIX1 selectively binds retinoic acid receptor (RAR) (NR1A) and thyroid hormone receptor (TR) (NR1B) in a ligand-dependent manner, but does not interact with retinoid X receptor (RXR) (NR2B) or steroid hormone receptors. Interestingly, NIX1 down-regulates transcriptional activation by binding to ligand-bound nuclear receptors. A 39-aa domain within NIX1 was found to be necessary and sufficient for protein–protein interactions with nuclear receptors. Northern blot analysis demonstrates low-abundance RNA messages only in brain and neuronal cells.
In situ
hybridization and immunohistochemistry revealed that NIX1 expression is restricted to the central nervous system and could be confined to neurons in the dentate gyrus of the hippocampus, the amygdala, thalamic, and hypothalamic regions. In summary, protein–protein interactions between the neuronal protein NIX1 and ligand-activated nuclear receptors are both specific and selective. By suppressing receptor-mediated transcription, NIX1 implements coregulation of nuclear receptor functions in brain.
T cell homeostasis in patients with rheumatoid arthritis
Authors: Koetz Kerstin , Bryl Ewa , Spickschen Kay , O'Fallon W. Michael , Goronzy Jörg J. , Weyand Cornelia M.
Published: July 2002

The immune system is equipped with an extremely large spectrum of structurally diverse receptors to recognize all potential antigens. This fundamental principle of receptor diversity is no longer upheld in patients with rheumatoid arthritis (RA), who have a marked contraction of the T cell receptor repertoire. In this study, the ability of RA patients to produce T cells and to maintain T cell homeostasis was examined. CD4 T cells containing T cell receptor rearrangement excision circles (TREC) were substantially reduced in RA patients; TREC levels in young adult patients matched those of controls 20 years older. Increased self-replication of T cells in RA was indicated by age-inappropriate erosion of telomeres in circulating T cells with almost complete attrition of telomeric reserves in patients 20–30 yr of age. The degree of telomere loss was not related to disease duration or the use of disease-modifying medication and was most pronounced in CD4+CD45ROnull(naive) T cells. The loss of TREC-positive T cells could be a consequence of a primary defect in peripheral T cell homeostasis. Alternatively, RA patients may have impaired thymic function with the increased turnover of peripheral T cells being a secondary compensatory event.
A family of peptidoglycan recognition proteins in the fruit flyDrosophila melanogaster
Authors: Werner Thomas , Liu Gang , Kang Daiwu , Ekengren Sophia , Steiner Håkan , Hultmark Dan
Published: July 2002

Peptidoglycans from bacterial cell walls trigger immune responses in insects and mammals. A peptidoglycan recognition protein, PGRP, has been cloned from moths as well as vertebrates and has been shown to participate in peptidoglycan-mediated activation of prophenoloxidase in the silk moth. Here we report thatDrosophilaexpresses 12PGRPgenes, distributed in 8 chromosomal loci on the 3 major chromosomes. By analyzing cDNA clones and genomic databases, we grouped them into two classes:PGRP-SA, SB1, SB2, SC1A, SC1B, SC2,andSD, with short transcripts and short 5′-untranslated regions; andPGRP-LA, LB, LC, LD,andLE, with long transcripts and long 5′-untranslated regions. The predicted structures indicate that the first group encodes extracellular proteins and the second group, intracellular and membrane-spanning proteins. MostPGRPgenes are expressed in all postembryonic stages. Peptidoglycan injections strongly induce five of the genes. Transcripts from the differentPGRPgenes were found in immune competent organs such as fat body, gut, and hemocytes. We demonstrate that at leastPGRP-SAandSC1Bcan bind peptidoglycan, and a function in immunity is likely for this family.
Completing the heterotrimer: Isolation and characterization of an
Arabidopsis thaliana
G protein γ-subunit cDNA
Authors: Mason Michael G. , Botella José R.
Published: July 2002

Heterotrimeric G proteins consist of three subunits (α, β, and
γ). α- and β- subunits have been previously cloned in plants, but
the γ-subunit has remained elusive. To isolate the γ-subunit of a
plant heterotrimeric G protein an
Arabidopsis thaliana
yeast two-hybrid library was screened by using a tobacco G-β-subunit
as the bait protein. One positive clone (
AGG1
) was
isolated several times; it displays significant homology to the
conserved domains of mammalian γ-subunits. The predicted AGG1 protein
sequence contains all of the typical characteristics of mammalian
γ-subunits such as small size (98 amino acids, 10.8 kDa), presence of
a C-terminal CAAX box to direct isoprenyl modification, and an
N-terminal α-helix region capable of forming a coiled-coil
interaction with the β-subunit. Northern and Southern analyses showed
that
AGG1
is a single-copy gene in
Arabidopsis
with a similar expression pattern to the
Arabidopsis
β-subunit,
AGB1
[Weiss,
C. A., Garnaat, C. W., Mukai, K., Hu, Y. & Ma, H. (1994)
Proc. Natl. Acad. Sci. USA
91, 9554–9558]. By using
the yeast two-hybrid system, we show that AGG1 strongly interacts with
tobacco and
Arabidopsis
β-subunits. The
in
vivo
results have been confirmed by using
in
vitro
methods to prove the interaction between AGG1 and the
Arabidopsis
β-subunit. As previously observed in
mammalian systems, both the coiled-coil domain and the WD repeat
regions of the β-subunit are essential for AGG1 interaction. Also in
agreement with previous observations, the removal of the N-terminal
α-helix of the AGG1 greatly reduces but does not completely block the
interaction.
Heritable basis for choice of group size in a colonial bird
Authors: Brown Charles R. , Brown Mary Bomberger
Published: July 2002

Sizes of most kinds of animal groups vary considerably within a population, with group size often causing direct effects on the fitness of group members. Although the consequences of varying group size have been well studied, the causes of variation in group size remain poorly known for most animals. Groups might vary in size because different individuals perform better in differently sized groups and thus have genetic predispositions to choose large or small groups. We examined whether heritable variation for choice of group size exists in the cliff swallow (Petrochelidon pyrrhonota), a colonial bird that nests in colonies ranging from 2 to 3,700 nests. Parent–offspring regressions showed significant heritabilities for choice of colony size under natural conditions. Partial cross-fostering experiments showed that individuals reared in colonies of sizes different from those of their birth returned to breed the next year in colonies that matched their birth colony in size and actively avoided those similar to their rearing colony, suggesting that choice of colony size is genetically based. Common environmental effects, maternal effects, and philopatry did not explain these results. Variation in group size probably results in part from a polymorphism in genetic preferences within the population, and the range in colony sizes is maintained by natural selection on the type of bird occupying each site.
The erosion of citizenship
Authors: Turner Bryan S.
Published: July 2002

The Marshallian paradigm of social citizenship has been eroded because the social and economic conditions that supported postwar British welfare consensus have been transformed by economic and technological change. This article argues that effective entitlement was based on participation in work, war and reproduction, resulting in three types of social identity: worker‐citizens, warrior‐citizens and parent‐citizens. The casualization of labour and the technological development of war have eroded work and war as routes to active citizenship. Social participation through reproduction remains important, despite massive changes to marriage and family as institutions. In fact the growth of new reproductive technologies have reinforced the normative dominance of marriage as a social relation. These rights of reproduction are described as ‘reproductive citizenship’. The article also considers the role of voluntary associations in Third‐Way strategies as sources of social cohesion in societies where social capital is in decline, and argues that the voluntary sector is increasingly driven by an economic logic of accumulation. With the erosion of national citizenship, Marshall's three forms of rights (legal, political and social) have been augmented by rights that are global, namely environmental, aboriginal and cultural rights. These are driven by global concerns about the relationship between environment, community and body such that the quest for social security has been replaced by concerns for ontological security.
Efficient Urbanisation: Economic Performance and the Shape of the Metropolis
Authors: Cervero Robert
Published: July 2002

The influences of urban form and transport infrastructure on economic performance show up in several contemporary policy debates, notably 'sprawl versus compact city' and in the developing world, the future of mega-cities. This paper probes these relationships using two scales of analysis. At the macro scale, an econometric analysis using data across 47 US metropolitan areas reveals that employment densities and urban primacy are positively associated with worker productivity, suggesting the presence of agglomeration economies. Congested freeways are shown to be a consequence of strong economic performance. An intrametropolitan analysis using data on sub-districts of the San Francisco Bay Area generally reinforces the findings of the macro-scale analysis. In the Bay Area, labour productivity appears to increase with size of labour-marketshed and high accessibility between residences and firms. Higher employment density and well-functioning infrastructure also contribute positively to economic performance.
Social Cohesion, Social Capital and the Neighbourhood
Authors: Forrest Ray , Kearns Ade
Published: July 2002

In current theoretical and policy debates concerning social cohesion, the neighbourhood has re-emerged as an important setting for many of the processes which supposedly shape social identity and life-chances. It is in this context of a renewal of interest in local social relations and particularly the deployment of notions of social capital that this paper offers a critical review of a wide-ranging literature. The paper explores initially and briefly the idea that societies face a new crisis of social cohesion and outlines the key dimensions of societal cohesion. The core of the paper is then devoted to an examination of where the contemporary residential neighbourhood fits into these wider debates, particularly in relation to the interaction between social cohesion and social capital. In this context, some of the key debates around the concept of social capital are outlined. In moving beyond abstraction, the paper also shows how social capital can be broken down into relevant domains for policy action at the neighbourhood level and how concepts such as social cohesion and social capital can be operationalised for research purposes.
Disentangling Area Effects: Evidence from Deprived and Non-deprived Neighbourhoods
Authors: Atkinson Rowland , Kintrea Keith
Published: July 2002

This paper focuses on the question of whether it is worse to be poor in a poor area or in an area which is more socially mixed; in short, does living in a deprived area compound the disadvantage experienced by its residents, and do area effects contribute to social exclusion? The idea of social areas having direct or mediated effects on the lives of their residents continues to interest and challenge academic and policy debates on the effect of concentrated poverty and on the creation of more mixed and, thereby, more sustainable neighbourhood forms. However, area effects remain contentious and British research evidence is scant. Following a review of the theoretical and empirical understandings of the relationship between households and neighbourhoods, the paper presents survey data from a comparative study of deprived and socially mixed neighbourhoods in Glasgow and Edinburgh. These data provide evidence that supports the area effects thesis, in particular in relation to area reputation and employment. The paper concludes that, with certain caveats, living in areas of geographically concentrated poverty creates additional problems for residents.
Growing Up in Amsterdam: Differentiation and Segregation in Children's Daily Lives
Authors: Karsten Lia
Published: July 2002

So far, children have not gained much attention in the field of segregation studies. In the research reported here, the notion of segregation is related children's daily life paths in the public domain. Given the local dimension of children's everyday lives and the growing number of 'black' and 'white' schools, it is apparent that children deserve a higher place on the agenda of segregation studies. Drawing on research carried out in five different Amsterdam neighbourhoods, this paper addresses children's time-space behaviour—after-school time. The central question is whether differentiation and segregation form vital dimensions in Amsterdam childhoods. Special attention has been given to children's orientation towards the public domain, their membership of leisure clubs and their freedom of movement. Results show that differences among children growing up in Amsterdam are big. At first sight, there seems to be a sharp divide between Amsterdam children with Dutch parents and Amsterdam children with a Turkish/ Moroccan or Surinamese/Antillean background. However, incorporating gender and class into the analyses, the picture becomes less clear. Ethnicity is a far more complex and dynamic concept than is sometimes argued. However, on the geographical scale of the neighbourhood, we must conclude that in three out of the five studied areas the contours of segregated childhoods are evidently clear. The material presented in this paper is based on observational studies and interviews in public playgrounds and a survey with 454 schoolchildren (7-12 years of age) and 214 parents.
Research Mathematicians as Learners—and what mathematics education can learn from them
Authors: Burton Leone
Published: July 2002

Abstract A theoretical model describing coming to know mathematics was matched against the descriptions gathered from interviews with 70 practising research mathematicians. The model proved to be a good framework for understanding their epistemological practices and a number of pedagogical implications of wider application are drawn from the results.
Evaluating the effects of a peer support programme on adolescents' knowledge, attitudes and use of alcohol and tobacco
Authors: WEBSTER ROSEMARY A. , HUNTER MICHAEL , KEATS JOHN A.
Published: July 2002

This study was designed to evaluate the effects of a peer support programme on adolescents' knowledge, attitudes and use of alcohol and tobacco. Year 7 students (average age 12 years) from three schools who offered the programme (n = 169) and from three schools without the programme (n = 157) completed a self‐report assessment. Perceptions of their parents' and friends' use of alcohol and tobacco and attitudes towards the participants' use of these substances as well as the participants' own attitudes (preferences and norms) and use of the substances were assessed on three occasions; pre‐intervention, post‐intervention and at 6 months follow‐up. There were no significant effects of the programme on participants' knowledge, attitudes and use of alcohol and tobacco. Over time, participants in both groups reported increased enjoyment of alcohol, increased use of alcohol and tobacco and more lenient attitudes towards these substances. In conclusion, the peer support programme failed to show any positive influence on adolescents' knowledge, attitudes and use of alcohol and tobacco.
Topical glucocorticoids and the skin‐mechanisms of action: an update
Authors: Ahluwalia A.
Published: July 2002

The topical glucocorticoids (GCs) represent the treatment of choice for many types of inflammatory dermatoses. Despite the extensive use of this class of drugs as first line therapy the mechanism of their action is uncertain. It is clear that the multiplicity of actions of the topical GCs is an important facet of their scope in the treatment of dermal disorders. The aim of this update is to review past and current theories regarding how these agents might work. Current understanding of the molecular mechanism s of GC action has advanced significantly over the past decade with the realisation that multiple systems are responsible for transduction of GC effects at a molecular level. The two primary modes of action are via interaction directly with DNA or indirectly through modulation of specific transcription factors: the endpoint in both cases being modulation of specific protein synthesis. Both of these mechanisms will be discussed. In particular this review will concentrate on the possibility that a GC‐inducible protein, termed lipocortin 1, may have a significant role to play in the anti‐inflammatory actions of these drugs. Additionally it has become apparent that several inflammatory enzymes induced in inflamm ation are sites of inhibitory action of the GCs, and the possibility that this occurs in the skin will be discussed paying particular attention to the inducible phospholipase A2, nitric oxide synthase and cyclooxygenase systems.
Is Bipolar Disorder a Risk for Cigarette Smoking in ADHD Youth?
Authors: Wilens Timothy E. , Biederman Joseph , Milberger Sharon , Hahesy Amy L. , Goldman Sarah , Wozniak Janet , Spencer Thomas J.
Published: July 2002

Despite emerging literature linking juvenile bipolar disorder (BPD) and substance abuse, little is known about a link between BPD and cigarette smoking. To this end, we evaluated the association between BPD and cigarette smoking in youth. Subjects were 31 bipolar adolescents derived from a cohort of boys with DSM‐III‐R ADHD (N = 128) and non‐ADHD comparisons (N = 109) followed prospectively for 4 years into mid‐adolescence. Information on cigarette smoking was obtained in a standardized manner blind to the proband's clinical status. Logistic regression models were used to determine risk for smoking at follow‐up. BPD was associated with a higher risk for cigarette smoking in mid‐adolescence, which was largely accounted for by conduct disorder. The developmental onset of BPD in adolescence (age 13–18 years) conferred a greater risk for cigarette smoking compared to those youths with the onset of their BPD prepubertally (≤ 12 years; odds ratio = 10.8, p < 0.01), even after controlling for conduct disorder and other confounds. The naturalistic treatment of BPD with combined counseling and pharmacotherapy appeared to reduce the risk for cigarette smoking. BPD, particularly when it onsets in adolescence, is a significant risk factor for the early initiation of cigarette smoking in these ADHD youths. These data coupled with the literature strongly suggest that juveniles with BPD need to be carefully monitored for the early initiation of cigarette smoking and substance abuse.
Areca nut‐abuse liability, dependence and public health
Authors: Winstock A.
Published: July 2002

Abstract To define a dependence syndrome may convey attention upon a public health problems hitherto relatively ignored. It may, however, stigmatize substance use, especially when western diagnostic criteria are applied to essentially culture‐bound substances. However, when the pattern of use of a substance results in significant personal harm, then whether dependence exists or not is less important than developing an appropriate response, if its use is associated with significant morbidity and mortality. Such is the case with areca nut, the fourth most commonly used drug in the world after tobacco, alcohol and caffeine. In this paper I will explore its use and properties with respect to its abuse and dependence liability and consider the associated implications for public health.
Low Dose‐Rate Interstitial Brachytherapy in Soft Tissue Sarcomas
Authors: Rosenblatt Edward , Meushar Netanel , Eidelman Mark , Kuten Abraham
Published: July 2002

Purpose. To assess the effectiveness of Ir‐192 interstitial brachytherapy
as an adjunct to wide local excision as a functionsaving strategy for soft tissue sarcomas.

Subjects and methods. From September 1993 to April 1998, 20
consecutive patients diagnosed with soft tissue sarcomas were treated with a combination
of wide local excision and interstitial brachytherapy. In 16 patients brachytherapy was done
as an intraoperative procedure, while in four, the implant was performed post‐operatively
under local anesthesia. Eleven of the 20 patients also received external beam radiotherapy
following the implant.

Results. After a mean follow‐up of 27 months (4–54) the local control rate
for all 20 patients was 85% (17/20). In the 16 patients who had an intra‐operative implant,
local control was 94% (15/16). In the four patients who underwent a post‐operative implant,
local control was 50% (2/4). Actuarial 5‐year survival was 90%. There were three cases (15%)
of severe local complications.

Conclusions. Wide local excision followed by low dose rate intersitital
brachytherapy have yielded a 85% local control rate in 20 patients with soft tissue sarcomas.
Local control rates were higher when the implants were done as an intra‐operative procedure
than as a post‐operative one.
Therapy Outcome Measures In Speech And Language Therapy: Comparing Performance Between Different Providers
Authors: Enderby Pamela M , John Alexandra
Published: July 2002

Background: The use of outcome measures to monitor improved quality of care has been advocated for 20 years but has only achieved prominence with the increasing resource pressures and related changed in health service provision in the past 6 years.

Objective: This paper describes the development of an approach to outcome measurement suitable for all patients receiving speech and language therapy. The measure, which is based on rating the dimensions of impairment, disability, handicap and well‐being, is tested to assess whether it can usefully be used to compare the services of different providers.

Method: Five trusts volunteered for the study. Service descriptions suggest that these services are typical for the purposes of providing speech and language therapy. Twenty‐five therapists were trained to use the Therapy Outcome Measure (TOM); their reliability was assessed and they provided prospective data on clients with speech and language impairments related to dysphasia, stammering and dysphonia.

Results: The study provides evidence indicating the differences in the types of patients being referred to different providers of speech and language therapy. Different services have different impacts on the number and type of domains and that services discharge patients at different points in their recovery.

Discussion: Different outcomes by different providers may be associated with different referral policies, base populations, skills and work policies of therapists. Differences in outcomes associated with certain therapy services can initiate the task of analysing attributions and progress endeavours to provide equitable quality of care which is the philosophy underpinning the move towards benchmarking in health service delivery.
Carbon Nanotubes: A Future Material of Life
Authors: Chen P. , Lin J. , Tan K. L.
Published: July 2002

The synthesis and applications of carbon nanotubes, a new form of crystalline carbon, are reviewed. In particular, the results obtained in the authors' laboratory are summarized, including the preparation of highly crystalline multiwalled carbon nanotubes; also discussed are the valence band and core level structures, the nonlinear optical limiting behavior, the band gap measurement, and the hydrogen uptake by carbon nanotubes, which has been greatly enhanced by alkali‐doping.
Substrate Recognition by Osteoclast Precursors Induces C-src/Microtubule Association
Authors: Abu-Amer Yousef , Ross F. Patrick , Schlesinger Paul , Tondravi M. Mehrdad , Teitelbaum Steven L.
Published: July 2002

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src−/− osteopetrotic mice to form ruffled membranes indicates pp60c-src (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.
Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation
Authors: Sakakibara Akira , Furuse Mikio , Saitou Mitinori , Ando-Akatsuka Yuhko , Tsukita Shoichiro
Published: July 2002

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest Mr were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher Mr bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest Mr band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher Mr bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower Mr bands of occludin (non- or less phosphorylated form) but was specific to the higher Mr bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower Mr bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.
The Yeast v-SNARE Vti1p Mediates Two Vesicle Transport Pathways through Interactions with the t-SNAREs Sed5p and Pep12p
Authors: Mollard Gabriele Fischer von , Nothwehr Steven F. , Stevens Tom H.
Published: July 2002

Membrane traffic in eukaryotic cells requires that specific v-SNAREs on transport vesicles interact with specific t-SNAREs on target membranes. We identified a novel Saccharomyces cerevisiae v-SNARE (Vti1p) encoded by the essential gene, VTI1. Vti1p interacts with the prevacuolar t-SNARE Pep12p to direct Golgi to prevacuolar traffic. vti1-1 mutant cells missorted and secreted the soluble vacuolar hydrolase carboxypeptidase Y (CPY) rapidly and reversibly when vti1-1 cells were shifted to the restrictive temperature. However, overexpression of Pep12p suppressed the CPY secretion defect exhibited by vti1-1 cells at 36°C. Characterization of a second vti1 mutant, vti1-11, revealed that Vti1p also plays a role in membrane traffic at a cis-Golgi stage. vti1-11 mutant cells displayed a growth defect and accumulated the ER and early Golgi forms of both CPY and the secreted protein invertase at the nonpermissive temperature. Overexpression of the yeast cis-Golgi t-SNARE Sed5p suppressed the accumulation of the ER form of CPY but did not lead to CPY transport to the vacuole in vti1-11 cells. Overexpression of Sed5p allowed growth in the absence of Vti1p. In vitro binding and coimmunoprecipitation studies revealed that Vti1p interacts directly with the two t-SNAREs, Sed5p and Pep12p. These data suggest that Vti1p plays a role in cis-Golgi membrane traffic, which is essential for yeast viability, and a nonessential role in the fusion of Golgi-derived vesicles with the prevacuolar compartment. Therefore, a single v-SNARE can interact functionally with two different t-SNAREs in directing membrane traffic in yeast.
Talin-Null Cells of Dictyostelium Are Strongly Defective in Adhesion to Particle and Substrate Surfaces and Slightly Impaired in Cytokinesis
Authors: Niewöhner Jens , Weber Igor , Maniak Markus , Müller-Taubenberger Annette , Gerisch Günther
Published: July 2002

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.
Dictyostelium RasG Is Required for Normal Motility and Cytokinesis, But Not Growth
Authors: Tuxworth Richard I. , Cheetham Janet L. , Machesky Laura M. , Spiegelmann George B. , Weeks Gerald , Insall Robert H.
Published: July 2002

RasG is the most abundant Ras protein in growing Dictyostelium cells and the closest relative of mammalian Ras proteins. We have generated null mutants in which expression of RasG is completely abolished. Unexpectedly, RasG− cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG− cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.
CD36 Mediates the In Vitro Inhibitory Effects of Thrombospondin-1 on Endothelial Cells
Authors: Dawson David W. , Pearce S. Frieda A. , Zhong Ruiqin , Silverstein Roy L. , Frazier William A. , Bouck Noël P.
Published: July 2002

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase–CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM∅, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.
Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes
Authors: Mundel Peter , Heid Hans W. , Mundel Thomas M. , Krüger Meike , Reiser Jochen , Kriz Wilhelm
Published: July 2002

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level.

In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.
Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells
Authors: Takaishi Kenji , Sasaki Takuya , Kotani Hirokazu , Nishioka Hideo , Takai Yoshimi
Published: July 2002

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.
The Arabidopsis KNOLLE Protein Is a Cytokinesis-specific Syntaxin
Authors: Lauber Martina H. , Waizenegger Irene , Steinmann Thomas , Schwarz Heinz , Mayer Ulrike , Hwang Inwhan , Lukowitz Wolfgang , Jürgens Gerd
Published: July 2002

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H+-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.
Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum
Authors: Nicchitta Christopher V. , Zheng Tianli
Published: July 2002

A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation.

In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome–membrane junction by the nascent chain is presented.
Vac8p, a Vacuolar Protein with Armadillo Repeats, Functions in both Vacuole Inheritance and Protein Targeting from the Cytoplasm to Vacuole
Authors: Wang Yong-Xu , Catlett Natalie L. , Weisman Lois S.
Published: July 2002

During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells. This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p. A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration. Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance. Both myristoylation and palmitoylation are required for complete Vac8p localization. Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential. Thus, palmitoylation appears to play a more direct role in vacuole inheritance. Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats. Arm repeats are thought to mediate protein–protein interactions, and many Arm proteins have multiple functions. This is also true for Vac8p. In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole. Mutant analysis demonstrates that Vac8p functions separately in these two processes. Vac8p cosediments with actin filaments. Vac8p is related to β-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton. In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.
Xgrip109: A γ Tubulin–Associated Protein with an Essential Role in γ Tubulin Ring Complex (γTuRC) Assembly and Centrosome Function
Authors: Martin Ona C. , Gunawardane Ruwanthi N. , Iwamatsu Akihiro , Zheng Yixian
Published: July 2002

Previous studies indicate that γ tubulin ring complex (γTuRC) can nucleate microtubule assembly and may be important in centrosome formation. γTuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to γ tubulin. We found that one γTuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homologue, Spc98, is a spindle–pole body component that interacts with γ tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. We show that Xgrip109 is a centrosomal protein that directly interacts with γ tubulin. We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted γTuRC and for the recruitment of γ tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome.
An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle
Authors: Shima David T. , Cabrera-Poch Noemí , Pepperkok Rainer , Warren Graham
Published: July 2002

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.