bluuebunny/crossref_metadata_2025 huggingface dataset:
Fiber diffraction of synthetic α-synuclein filaments shows amyloid-like cross-β conformation
Authors: Serpell Louise C. , Berriman John , Jakes Ross , Goedert Michel , Crowther R. Anthony
Published: July 2002

Filamentous inclusions made of α-synuclein constitute the defining neuropathological characteristic of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Rare familial cases of Parkinson's disease are associated with mutations A53T and A30P in α-synuclein. We report here the assembly properties and secondary structure characteristics of recombinant α-synuclein. Carboxy-terminally truncated human α-synuclein (1–87) and (1–120) showed the fastest rates of assembly, followed by human A53T α-synuclein, and rat and zebra finch α-synuclein. Wild-type human α-synuclein and the A30P mutant showed slower rates of assembly. Upon shaking, filaments formed within 48 h at 37°C. The related proteins β- and γ-synuclein only assembled after several weeks of incubation. Synthetic human α-synuclein filaments were decorated by an antibody directed against the carboxy-terminal 10 amino acids of α-synuclein, as were filaments extracted from dementia with Lewy bodies and multiple system atrophy brains. Circular dichroism spectroscopy indicated that α-synuclein undergoes a conformational change from random coil to β-sheet structure during assembly. X-ray diffraction and electron diffraction of the α-synuclein assemblies showed a cross-β conformation characteristic of amyloid.
Cadmium and iron transport by members of a plant metal transporter family in
Arabidopsis
with homology to
Nramp
genes
Authors: Thomine Sébastien , Wang Rongchen , Ward John M. , Crawford Nigel M. , Schroeder Julian I.
Published: July 2002

Metal cation homeostasis is essential for plant nutrition and resistance to toxic heavy metals. Many plant metal transporters remain to be identified at the molecular level. In the present study, we have isolated
AtNramp
cDNAs from
Arabidopsis
and show that these genes complement the phenotype of a metal uptake deficient yeast strain,
smf1
.
AtNramp
s show homology to the
Nramp
gene family in bacteria, yeast, plants, and animals. Expression of
AtNramp
cDNAs increases Cd
2+
sensitivity and Cd
2+
accumulation in yeast. Furthermore,
AtNramp3
and
AtNramp4
complement an iron uptake mutant in yeast. This suggests possible roles in iron transport in plants and reveals heterogeneity in the functional properties of
Nramp
transporters. In
Arabidopsis
,
AtNramps
are expressed in both roots and aerial parts under metal replete conditions. Interestingly,
AtNramp3
and
AtNramp4
are induced by iron starvation. Disruption of the
AtNramp3
gene leads to slightly enhanced cadmium resistance of root growth. Furthermore, overexpression of
AtNramp3
results in cadmium hypersensitivity of
Arabidopsis
root growth and increased accumulation of Fe, on Cd
2+
treatment. Our results show that
Nramp
genes in plants encode metal transporters and that
AtNramps
transport both the metal nutrient Fe and the toxic metal cadmium.
Multiplex allele-specific target amplification based on PCR suppression
Authors: Broude Natalia E. , Zhang Lingang , Woodward Karen , Englert David , Cantor Charles R.
Published: July 2002

We have developed a strategy for multiplex PCR based on PCR
suppression. PCR suppression allows DNA target amplification with only
one sequence-specific primer per target and a second primer that is
common for all targets. Therefore, an n-plex PCR would require only n +
1 primers. We have demonstrated uniform, efficient amplification of
targeted sequences in 14-plex PCR. The high specificity of suppression
PCR also provides multiplexed amplification with allele specificity.
Multiplexed PCR was used to develop assays for genotyping DNA samples
from cystic fibrosis-affected individuals. The new approach greatly
simplifies primer design, significantly increases the PCR multiplexing
level, and decreases the overall primer cost. In addition, this assay
is more readily amenable to automation and is therefore suitable for
high-throughput genetic diagnostics.
Behavioural genetics in the 21st century
Authors: Plomin Robert
Published: July 2002

Behavioural genetics will continue to flow into the mainstream of behavioural research as more behavioural scientists incorporate this approach in their research. Future research will go beyond simply asking whether and how much genetic factors influence behaviour to ask questions about development, about relations among traits, and about the interplay between nature and nurture. The identification of specific genes associated with behaviour will make it possible for behavioural scientists to ask more precise questions about how genotypes become phenotypes.
Selection of favourite idols and models among Chinese young people: A comparative study in Hong Kong and Nanjing
Authors: Yue Xiao Dong , Cheung Chau-kiu
Published: July 2002

This paper examines the conceptual differences between an idol and a model among a sample of young people in China. A questionnaire was specifically designed to measure young people’s criteria for idol worship and model learning using three pairs of contrasting constructs: idealism versus realism, romanticism versus rationalism, absolutism versus relativism. The questionnaire also asks each respondent to nominate up to three favourite idols and models in life. Atotal of 826 high school students and university students in Hong Kong and Nanjing completed the questionnaire. The results provide convergent support for the hypothesised conceptual differences between an idol and a model. Idealism, romanticism, and absolutism were more important in idol selection whereas realism, rationalism, and relativism were more important in model selection. Hong Kong young people selected significantly more idealism-romanticism-absolutism oriented celebrities whereas Nanjing young people selected significantly more realism-rationalism-relativism oriented celebrities.
Development of Chinese preschoolers’ understanding of biological phenomena: Growth and aliveness
Authors: Zhu Liqi , Fang Fuxi
Published: July 2002

Chinese preschoolers’ understandings of the biological phenomena “growth” and “aliveness” were investigated. Seventy-two 4-, 5-, and 6-year-old subjects with equal numbers of boys and girls in each age group were selected from different socioeconomic backgrounds. The same children participated in the three experiments reported in this study so that both individual and intra-individual differences could be explored. Multiple methods, including picture-choice, retrieval, and classification tasks were used. The results show that 6-year-old children could distinguish living and nonliving things on both the growth and aliveness tasks, even when tested by different methods, whereas 4- and 5-year-olds’ performance varied across tasks and methods. Children whose parents had higher levels of formal education performed better than their counterparts, but the difference declined as age increased.
Consistency and Inconsistency in Adolescents’ Moral Reasoning
Authors: Rique Júlio , Camino Cleonice
Published: July 2002

Authors in the cognitive developmental tradition (e.g. Damon & Hart, 1988; Snyder & Feldman, 1984; Turiel, 1983) have created models to explain transition between stages of development. This study introduces consistency and inconsistency of moral reasoning as patterns of moral thinking and presents implications for moral education. Consistency and inconsistency are determined by the level of “stage mixture” scored on the Defining Issues Test. A 2 × 2 quasi-experimental design was created. Participants were 52, 15-year-old male high school students. Moral judgement was the dependent variable. Consistency, inconsistency, and the experimental procedures were independent variables. Results showed a significant interaction between levels of consistency and educational methods ( P < .014), a developmental progression for the condition inconsistent experimental ( P < .004), and a predicted pattern of change for the condition consistent experimental ( P < .001). The authors concluded that consistency and inconsistency of moral reasoning require different assumptions for the promotion of moral development.
Resource Utilisation in Groups of Black South African Adolescents: Gender, Age, and Individual Differences
Authors: Liddell Christine , McConville Chris
Published: July 2002

This study uses a movie-viewing instrument to assess patterns of resource utilisation in South African township adolescents. The degree to which resource utilisation and other task behaviours were associated with gender, age, and individual differences form the focus. Boys used more gestures denoting dominant and subordinate status, were more physically aggressive, and were generally more coercive than girls. Older children shared the resource more equitably, showed more positive affect, and spent less time issuing directives. There were inequities in children’s access to the movie. However, neither on-task behaviours nor participants’ academic achievement were consistently associated with some children accessing the movie more than others.
Understanding the Distinction between Sensations and Physical Properties of Objects by Children and Adults
Authors: Subbotsky Eugene
Published: July 2002

The study addressed the phenomenon according to which even educated adult individuals often confuse sensations received from physical objects (such as colour, sound, taste, weight, temperature) with physical properties of the objects that initiated these sensations. This means that in many cases scientific education fails to achieve its goal, which is to create in an individual the physical-scientific view of the world. To examine this phenomenon four experiments were conducted with the aims to determine to what extent: (1) children of various ages and adults were able (a) to acknowledge the fact that sensations appear due to the work of the human mind or sense organs and are not physical properties of the objects that produce them; (b) to appreciate the difference between the mind and sense organs as contributors to the production of sensations (Exp. 1); (2) various types of training could enhance children’s and adults’ understanding that sensations do not belong to the physical properties and objects (Exps. 2-4).

The results of Experiment 1 conducted with 6- and 9-year-old children and adults did not reveal an age-related increase in the appreciation of the subjective character of sensations. However, 9-year-old children and adults (but not 6-year-old children) showed a diffentiated approach to various types of sensations, attributing pain mainly to the subject and softness-hardness mainly to the objects. Experiment 2 (direct explanation treatment), and Experiment 3 (cognitive conflict treatment) yielded no improvement in 6- and 9-year-old children’s appreciation of the subjective character of sensations and only partial improvement in adults. In Experiment 4 (social conflict treatment) adult subjects only were involved; the experiment showed a significant improvement in the subjects’ judgements about colour sensations and a marginal improvement in their judgements about weight sensations.
Parent-Adolescent Conflict: Possible Determinants and Consequences
Authors: Deković Maja
Published: July 2002

The first aim of this study was to examine the factors that might account for variations in the level of parent-adolescent conflict. These factors were grouped into two classes of variables: (1) personal characteristics of the adolescent, such as temperament and pubertal timing; and (2) parental child-rearing style. Second, we examined the relationship between the level of conflict, the quality of the parent-adolescent relationship, and adolescents’ and parents’ well-being. The sample consisted of 508 families with adolescents (12 to 18 years old). During a home visit, a battery of questionnaires was administered individually to mothers, fathers, and adolescents. The amount of conflict was related to the temperamental characteristics of the adolescent but not to pubertal timing or child-rearing style. With regard to the consequences of the parent-adolescent conflict, it appears that a higher level of conflict is a better predictor of parental, rather than adolescents’ well-being.
Age-related Differences in Driving: Are Older Drivers More Serial?
Authors: Hakamies-Blomqvist Liisa , Mynttinen Sami , Backman Martin , Mikkonen Valde
Published: July 2002

A measurement-instrumented car was used to test the hypothesis that older drivers organise their car-controlling movements in a more serial way in order to reduce the momentary mental workload produced by complex traffic situations. Forty-two older drivers (65+, mean age 67.5) and 36 young middle-aged drivers (26-49, mean age 36.8) drove a measurement-instrumented car around a 7.4 km route in normal traffic. The use of car controls (e.g. clutch, brake) was registered by sensors connected to a computer. In the most complex parts of the test route (e.g. during which three or more controls were in use simultaneously), the older drivers tended to use three controls rather than four or more, whereas the younger group had a greater percentage of four or more controls. This percentage was positively related to speed. However, although older drivers drove more slowly in general, in pairs matched for speed, they still drove in a less parallel manner. Irrespective of age, parallel use of controls was positively related to the level of skill as indicated by the amount of cumulated driving experience. The shift towards more serial operation of controls possibly represents an empirical demonstration of a compensatory mechanism allowing older drivers to maintain their level of performance.
Investing in Practical Intelligence: Ageing and Cognitive Efficiency among Executives
Authors: Colonia-Willner Regina
Published: July 2002

This paper has two objectives. (1) To review in part empirical evidence obtained in a recent study on practical intelligence in bank managers (Colonia-Willner, 1998). (2) To discuss the implications of these findings for the study of practical intelligence, expertise, and compensatory abilities. The first goal of the initial study was to determine whether managerial practical intelligence is preserved in older bank managers whereas differences in psychometric measures of reasoning are associated with age. The second was to verify whether practical intelligence measures better predict managerial job performance than traditional psychometric tests. Two hundred bank managers (43 experts and 157 nonexperts) participated in the initial study. Increased age appeared to be associated with lower performance in the Raven’s Advanced Progressive Matrices (Raven’s) and the Verbal Reasoning subtest of the Differential Aptitude Test (DAT), but less so with the Tacit Knowledge Inventory for Managers (TKIM). Although scoring lower on psychometric reasoning measures, the best performing older managers, on average, exhibited high levels of tacit knowledge, an instantiation of practical knowledge acquired in situations where information is not openly expressed. TKIM predicted managerial skill; DAT and Raven’s did not. These findings suggest: (1) that stabilisation of some aspects of intelligence may occur in old age; and (2) that domain-specific knowledge may contribute to compensation for age-related losses in functioning.
Age Relevance and Context Effects on Attributions across the Adult Lifespan
Authors: Blanchard-Fields Fredda , Baldi Renee , Stein Renee
Published: July 2002

Attributions for events with information varying on age relevance (teen, young, middle-aged, and older main characters) and context (family, work) were examined in young, middle-aged, and older adults. Participants rated the degree to which the causes of a negative outcome were a function of three dispositional dimensions of the primary character, two dimensions of situational factors, and a combination of these. They also wrote essays justifying their ratings. Overall and in support of previous research, older adults were more likely than younger adults to attribute the cause of the negative outcome to the primary character (Blanchard-Fields, 1994). This bias was not attenuated by the age-relevance factor. Instead, in the work context, older and middle-aged adults placed greater blame on main characters relevant to their age group for negative outcomes than did young adults. However, older adults were also more likely than middle-aged or young adults to attribute the cause of a negative outcome to situational factors. Age relevance and context influenced attributional ratings for all participants in that: (a) older characters were rated higher on external attributions and younger characters were rated higher on internal attributions; and (b) higher internal attributions were made for work situations than for family situations. Finally, young and middle-aged adults were more dialectical in justifying their causal attributions than older adults. Findings are discussed in terms of the degree to which a dispositional bias in older adults is influenced by the developmental relevance of one’s everyday context.
Children’s Reactions to Physical Disability: A Cross-national and Developmental Study
Authors: Crystal David S. , Watanabe Hirozumi , Chen Rusan
Published: July 2002

This study examined reactions to physical disability among 5th and 11th graders in the United States and Japan. Students were presented with a drawing of four children standing next to a child in a wheelchair, and told that this group was going swimming. Students were asked to describe and explain their feelings, first, as the disabled person, and then as a nondisabled group member. Although cultural differences in specific response categories were found in both hypothetical roles, on the whole, Japanese and American children appeared to be more similar than different in their reactions to physical disability. Patterns of developmental differences, however, varied by culture. Japanese adolescents were more likely than their younger peers to worry about imposing on others in the disabled role, whereas Japanese 5th graders were more likely than 11th graders to make empathic responses in the interactant role. In addition, there was a tendency for more American 11th than 5th graders to express embarrassment in the disabled role. Findings are discussed in terms of Goffman’s (1963) model of “stigma”, the individualism-collectivism paradigm, and theories of cognitive development.
Possible in vivo tolerance of human polymorphonuclear neutrophil to low‐grade exercise‐induced endotoxaemia
Authors: Camus G. , Nys M. , Poortmans J-R. , Venneman I. , Monfils T. , Deby-Dupont G. , Juchmés-Ferir A. , Deby C. , Lamy M. , Duchateau J.
Published: July 2002

To address the question of whether translocation of bacterial lipopolysaccharide (LPS) into the blood could be involved in the process of exercise‐induced polymorphonuclear neutrophil (PMN) activation, 12 healthy male subjects who took part in a sprint triathlon (1.5 km river swim, 40 km bicycle race, 10 km road race) were studied. While there was no detectable amount of endotoxin in the blood samples drawn at rest, exercise was followed by the appearance of circulating endotoxin molecules at the end of competition in four subjects, and after one and 24 h recovery in three and seven athletes, respectively. The concentrations of plasma granulocyte myeloperoxidase ([MPO]), were significantly higher immediately after exercise and one hour later than baseline values (P < 0.001). This variable returned to pre‐race levels the day after exercise, despite the presence of detectable amounts of LPS, at that time, in seven athletes. The absence of significant correlation (r = 0.26; P = 0.383) and temporal association between [MPO]and plasma endotoxin levels led us to conclude that endotoxaemia was not involved in the process of exercise‐induced PMN degranulation observed in our subjects.
Review Article: Safety Assessment of the Mycotoxin Cyclopiazonic Acid
Authors: Burdock G. A. , Flamm W. G.
Published: July 2002

Cyclopiazonic acid (CPA) is an indol-tetramic acid mycotoxin and is produced by the nearly ubiquitous molds, Aspergillus and Penicillium. CPA produced by these molds has been identified in a number of food sources (including, but not limited to, grain, legumes, meat, milk, and cheese) and from parasitic infections of man and other animals. Few incidents of CPA mycotoxicoses have been reported because of the benign nature of the intoxication, the small amounts present, and its effects may be disguised with concurrent aflatoxicosis (some toxicity data may have been generated using aflatoxin-contaminated CPA). CPA is absorbed in the gastrointestinal tract and following oral administration; it has a half-life of approximately 30 hours and is excreted largely unchanged in the urine and feces. Cyclopiazonic acid is not considered to be a potent acute toxin as its oral LD50 in rodents is in the range of 30 to 70 mg/kg. Multiple dose studies also show a range of effects in several species and among mammalian models, the pig appears to be the most sensitive with a no-observable-effect level (NOEL) in the range of 1.0 mg/kg/day. The preponderance of evidence from the rat and other test animals supports this dose as a defensible estimate of a no effect level. The target organs of CPA toxicity appear to be muscle, hepatic tissue, and spleen, with a localization in the former, although a more apparent toxic change in the latter two. The toxicity and symptoms of CPA poisoning can be attributed to its ability to alter normal intracellular calcium flux through its inhibition of the reticular form of the Ca2+-ATPase pump. CPA was not teratogenic in mice. CPA is not considered a carcinogen and the weight of evidence militates against its characterization as a mutagen. Despite CPA-induced pathological changes ascribed to the spleen or bursa of Fabricius, there does not appear to be an effect on the immune system. In vitro studies imply a potential immunomodulatory effect of CPA, but in all of those reports very high concentrations of CPA were required and none of these findings have been supported with in vivo studies. Therefore, based on a NOEL of 1 mg/kg/day and accounting for species variation, an appropriate acceptable daily intake (ADI) would be approximately 10 μg/kg/day or 700 μg/day. In the context of human exposure, if the uppermost limit of CPA found in cheese is 4 μg/g and the average individual consumes 50 g of cheese daily, this allows an intake of 200 μg, less than one third of a traditionally established ADI.
Chromosomes with Two Intact Axial Cores Are Induced by G2 Checkpoint Override: Evidence That DNA Decatenation Is not Required to Template the Chromosome Structure
Authors: Andreassen Paul R. , Lacroix Françoise B. , Margolis Robert L.
Published: July 2002

Here we report that DNA decatenation is not a physical requirement for the formation of mammalian chromosomes containing a two-armed chromosome scaffold. 2-aminopurine override of G2 arrest imposed by VM-26 or ICRF-193, which inhibit topoisomerase II (topo II)–dependent DNA decatenation, results in the activation of p34cdc2 kinase and entry into mitosis. After override of a VM-26–dependent checkpoint, morphologically normal compact chromosomes form with paired axial cores containing topo II and ScII. Despite its capacity to form chromosomes of normal appearance, the chromatin remains covalently complexed with topo II at continuous levels during G2 arrest with VM-26. Override of an ICRF-193 block, which inhibits topo II–dependent decatenation at an earlier step than VM-26, also generates chromosomes with two distinct, but elongated, parallel arms containing topo II and ScII. These data demonstrate that DNA decatenation is required to pass a G2 checkpoint, but not to restructure chromatin for chromosome formation. We propose that the chromosome core structure is templated during interphase, before DNA decatenation, and that condensation of the two-armed chromosome scaffold can therefore occur independently of the formation of two intact and separate DNA helices.
Structure of L-A Virus: A Specialized Compartment for the Transcription and Replication of Double-stranded RNA
Authors: Castón José R. , Trus Benes L. , Booy Frank P. , Wickner Reed B. , Wall Joseph S. , Steven Alasdair C.
Published: July 2002

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment—the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-Å resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10–15 Å across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40–45 Å, compared with 25–30 Å in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.
Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death
Authors: Miller Timothy M. , Moulder Krista L. , Knudson C. Michael , Creedon Douglas J. , Deshmukh Mohanish , Korsmeyer Stanley J. , Johnson Eugene M.
Published: July 2002

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.
Processing of Laminin-5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator
Authors: Goldfinger Lawrence E. , Stack M. Sharon , Jones Jonathan C.R.
Published: July 2002

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell– matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5–rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The α3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD α3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 β and γ chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5–rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 α3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.
Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells
Authors: Priddle Helen , Hemmings Lance , Monkley Susan , Woods Alison , Patel Bipin , Sutton Deborah , Dunn Graham A. , Zicha Daniel , Critchley David R.
Published: July 2002

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.
Blocking Cytochrome c Activity within Intact Neurons Inhibits Apoptosis
Authors: Neame Stephen J. , Rubin Lee L. , Philpott Karen L.
Published: July 2002

Cytochrome c has been shown to play a role in cell-free models of apoptosis. During NGF withdrawal–induced apoptosis of intact rat superior cervical ganglion (SCG) neurons, we observe the redistribution of cytochrome c from the mitochondria to the cytoplasm. This redistribution is not inhibited by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) but is blocked by either of the neuronal survival agents 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (CPT-cAMP) or cycloheximide. Moreover, microinjection of SCG neurons with antibody to cytochrome c blocks NGF withdrawal–induced apoptosis. However, microinjection of SCG neurons with cytochrome c does not alter the rate of apoptosis in either the presence or absence of NGF. These data suggest that cytochrome c is an intrinsic but not limiting component of the neuronal apoptotic pathway.
Centriole Disassembly In Vivo and Its Effect on Centrosome Structure and Function in Vertebrate Cells
Authors: Bobinnec Y. , Khodjakov A. , Mir L.M. , Rieder C.L. , Eddé B. , Bornens M.
Published: July 2002

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223–232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population.

During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis.

Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.
Aggresomes: A Cellular Response to Misfolded Proteins
Authors: Johnston Jennifer A. , Ward Cristina L. , Kopito Ron R.
Published: July 2002

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.
Regulated Actin Cytoskeleton Assembly at Filopodium Tips Controls Their Extension and Retraction
Authors: Mallavarapu Aneil , Mitchison Tim
Published: July 2002

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.
Characterization and Dynamics of Aggresome Formation by a Cytosolic Gfp-Chimera✪
Authors: García-Mata Rafael , Bebök Zsuzsa , Sorscher Eric J. , Sztul Elizabeth S.
Published: July 2002

Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883–1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481–490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end–directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.
Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast
Authors: Kirisako Takayoshi , Baba Misuzu , Ishihara Naotada , Miyazawa Kouichi , Ohsumi Mariko , Yoshimori Tamotsu , Noda Takeshi , Ohsumi Yoshinori
Published: July 2002

We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.
Cofilin Phosphorylation and Actin Cytoskeletal Dynamics Regulated by Rho- and Cdc42-Activated Lim-Kinase 2
Authors: Sumi Tomoyuki , Matsumoto Kunio , Takai Yoshimi , Nakamura Toshikazu
Published: July 2002

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.
A Specific Activation of the Mitogen-Activated Protein Kinase Kinase 1 (Mek1) Is Required for Golgi Fragmentation during Mitosis
Authors: Colanzi Antonino , Deerinck Thomas J. , Ellisman Mark H. , Malhotra Vivek
Published: July 2002

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal–regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.
Mammalian Sprouty-1 and -2 Are Membrane-Anchored Phosphoprotein Inhibitors of Growth Factor Signaling in Endothelial Cells
Authors: Impagnatiello Maria-Antonietta , Weitzer Stefan , Gannon Grainne , Compagni Amelia , Cotten Matt , Christofori Gerhard
Published: July 2002

Growth factor–induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1–4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor– and vascular endothelial growth factor–induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor–induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.
A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing
Authors: Tran P.T. , Marsh L. , Doye V. , Inoué S. , Chang F.
Published: July 2002

The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.