bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12–rapamycinassociated protein
Authors: Peterson Randall T. , Desai Bimal N. , Hardwick James S. , Schreiber Stuart L.
Published: July 2002

The FKBP12–rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70
s6k
(the 70-kDa S6 kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70
s6k
in response to mitogenic stimuli. We present evidence that FRAP controls 4E-BP1 and p70
s6k
phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70
s6k
, and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70
s6k
but not with a mutated p70
s6k
that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A
in vitro
, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70
s6k
, whereas amino acid deprivation or rapamycin treatment inhibits FRAP’s ability to restrain the phosphatase.
Knowledge-based analysis of microarray gene expression data by using support vector machines
Authors: Brown Michael P. S. , Grundy William Noble , Lin David , Cristianini Nello , Sugnet Charles Walsh , Furey Terrence S. , Ares Manuel , Haussler David
Published: July 2002

We introduce a method of functionally classifying genes by using gene expression data from DNA microarray hybridization experiments. The method is based on the theory of support vector machines (SVMs). SVMs are considered a supervised computer learning method because they exploit prior knowledge of gene function to identify unknown genes of similar function from expression data. SVMs avoid several problems associated with unsupervised clustering methods, such as hierarchical clustering and self-organizing maps. SVMs have many mathematical features that make them attractive for gene expression analysis, including their flexibility in choosing a similarity function, sparseness of solution when dealing with large data sets, the ability to handle large feature spaces, and the ability to identify outliers. We test several SVMs that use different similarity metrics, as well as some other supervised learning methods, and find that the SVMs best identify sets of genes with a common function using expression data. Finally, we use SVMs to predict functional roles for uncharacterized yeast ORFs based on their expression data.
Escherichia coli
CspA-family RNA chaperones are transcription antiterminators
Authors: Bae Weonhye , Xia Bing , Inouye Masayori , Severinov Konstantin
Published: July 2002

CspA, the major cold-shock protein of
Escherichia coli
, is an RNA chaperone, which is thought to facilitate translation at low temperature by destabilizing mRNA structures. Here we demonstrate that CspA, as well as homologous RNA chaperones CspE and CspC, are transcription antiterminators.
In vitro
, the addition of physiological concentrations of recombinant CspA, CspE, or CspC decreased transcription termination at several intrinsic terminators and also decreased transcription pausing.
In vivo
, overexpression of cloned CspC and CspE at 37°C was sufficient to induce transcription of the
metY
-
rpsO
operon genes
nusA
,
infB
,
rbfA
, and
pnp
located downstream of multiple transcription terminators. Similar induction of downstream
metY
-
rpsO
operon genes was observed at cold shock, a condition to which the cell responds by massive overproduction of CspA. The products of
nusA
,
infB
,
rbfA
, and
pnp
—NusA, IF2, RbfA, and PNP—are known to be induced at cold shock. We propose that the cold-shock induction of
nusA
,
infB
,
rbfA
, and
pnp
occurs through transcription antitermination, which is mediated by CspA and other cold shock-induced Csp proteins.
Natural selection mapping of the warfarin-resistance gene
Authors: Kohn Michael H. , Pelz Hans-Joachim , Wayne Robert K.
Published: July 2002

In theory, genes under natural selection can be revealed by unique patterns of linkage disequilibrium (LD) and polymorphism at physically linked loci. However, given the effects of recombination and mutation, the physical extent and persistence of LD patterns in natural populations is uncertain. To assess the LD signature of selection, we survey variation in 26 microsatellite loci spanning an ≈32-cM region that includes the warfarin-resistance gene (
Rw
) in five wild rat populations having resistance levels between 0 and 95%. We find a high frequency of heterozygote deficiency at microsatellite loci in resistant populations, and a negative association between gene diversity (H) and resistance. Contrary to previous studies, these data suggest that directional rather than overdominant selection may predominate during periods of intense anticoagulant treatment. In highly resistant populations, extensive LD was observed over a chromosome segment spanning ≈14% of rat chromosome 1. In contrast, LD in a moderately resistant population was more localized and, in conjunction with likelihood ratios, allowed assignment of
Rw
to a 2.2-cM interval. Within this genomic window, a diagnostic marker,
D1Rat219
, assigned 91% of rats to the correct resistance category. These results further demonstrate that “natural selection mapping” in field populations can detect and map major fitness-related genes, and question overdominance as the predominant mode of selection in anticoagulant-resistant rat populations.
Retrograde axonal transport of herpes simplex virus: Evidence for a single mechanism and a role for tegument
Authors: Bearer E. L. , Breakefield X. O. , Schuback D. , Reese T. S. , LaVail J. H.
Published: July 2002

Herpes simplex virus type I (HSV) typically enters peripheral nerve terminals and then travels back along the nerve to reach the neuronal cell body, where it replicates or enters latency. To monitor axoplasmic transport of HSV, we used the giant axon of the squid,
Loligo pealei
, a well known system for the study of axoplasmic transport. To deliver HSV into the axoplasm, viral particles stripped of their envelopes by detergent were injected into the giant axon, thereby bypassing the infective process. Labeling the viral tegument protein, VP16, with green fluorescent protein allowed viral particles moving inside the axon to be imaged by confocal microscopy. Viral particles moved 2.2 ± 0.26 μm/sec in the retrograde direction, a rate comparable to that of the transport of endogenous organelles and of virus in mammalian neurons in culture. Electron microscopy confirmed that 96% of motile (stripped) viral particles had lost their envelope but retained tegument, and Western blot analysis revealed that these particles had retained protein from capsid but not envelope. We conclude that (
i
) HSV recruits the squid retrograde transport machinery; (
ii
) viral tegument and capsid but not envelope are sufficient for this recruitment; and (
iii
) the giant axon of the squid provides a unique system to dissect the viral components required for transport and to identify the cellular transport mechanisms they recruit.
The crystal structure of
d-
lactate dehydrogenase, a peripheral membrane respiratory enzyme
Authors: Dym Orly , Pratt Elizabeth Ann , Ho Chien , Eisenberg David
Published: July 2002

d
-Lactate dehydrogenase (
d
-LDH) of
Escherichia coli
is a peripheral membrane respiratory enzyme involved in electron transfer, located on the cytoplasmic side of the inner membrane.
d
-LDH catalyzes the oxidation of
d
-lactate to pyruvate, which is coupled to transmembrane transport of amino acids and sugars. Here we describe the crystal structure at 1.9 Å resolution of the three domains of
d
-LDH: the flavin adenine dinucleotide (FAD)-binding domain, the cap domain, and the membrane-binding domain. The FAD-binding domain contains the site of
d
-lactate reduction by a noncovalently bound FAD cofactor and has an overall fold similar to other members of a recently discovered FAD-containing family of proteins. This structural similarity extends to the cap domain as well. The most prominent difference between
d
-LDH and the other members of the FAD-containing family is the membrane-binding domain, which is either absent in some of these proteins or differs significantly. The
d
-LDH membrane-binding domain presents an electropositive surface with six Arg and five Lys residues, which presumably interacts with the negatively charged phospholipid head groups of the membrane. Thus,
d
-LDH appears to bind the membrane through electrostatic rather than hydrophobic forces.
CopA: An
Escherichia coli
Cu(I)-translocating P-type ATPase
Authors: Rensing Christopher , Fan Bin , Sharma Rakesh , Mitra Bharati , Rosen Barry P.
Published: July 2002

The
copA
gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in
Escherichia coli
. The
copA
gene was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis. The copper-sensitive phenotype could be rescued by complementation by a plasmid carrying
copA
from
E. coli
or
copB
from
Enterococcus hirae
. Expression of
copA
was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing
copA
exhibited ATP-coupled accumulation of copper, presumably as Cu(I). The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.
Intestinal trefoil factor confers colonic epithelial resistance to apoptosis
Authors: Taupin D. R. , Kinoshita K. , Podolsky D. K.
Published: July 2002

Intestinal trefoil factor (ITF) is an essential regulator of colonic epithelial restitution, the rapid migration of colonocytes over mucosal wounds. High levels of ITF are frequently present in colorectal cancers and derived cell lines. Mucosal restitution requires the detachment of epithelium from substrate, which would be expected to induce apoptosis. However, mice deficient in ITF showed an increase in colonocyte apoptosis unaccompanied by changes in expression of receptor-related (TNFR/Fas) or stress-related (Bcl-family) cell death regulators. An ITF-expressing colonic (HT-ITF1) cell line was resistant to apoptosis induced by serum starvation and ceramide. Exogenous ITF also protected another human colonic carcinoma-derived cell line (HCT116) and a nontransformed rat intestinal epithelial cell line (IEC-6) from apoptosis. This effect was abrogated by wortmannin and tyrphostin A25, indicating the potential involvement of phosphatidylinositol 3-kinase and epidermal growth factor (EGF) receptor activation. Expression of phosphorylated Akt, which lies downstream of phosphatidylinositol 3-kinase activation, was elevated in this HT-29-ITF line. p53-dependent cell death in the AGS human gastric cancer cell line after etoposide was similarly inhibited by transient expression of ITF but not a C-terminal truncation mutant of ITF, and it required functional phosphatidylinositol 3-kinase and EGF receptor. These findings support a central role for ITF in the maintenance of intestinal mucosal continuity, and conversely demonstrate the potential for ITF expression to confer resistance of colorectal tumors to therapy.
Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral
Authors: Baird Geoffrey S. , Zacharias David A. , Tsien Roger Y.
Published: July 2002

DsRed is a recently cloned 28-kDa fluorescent protein responsible
for the red coloration around the oral disk of a coral of the
Discosoma
genus. DsRed has attracted tremendous interest
as a potential expression tracer and fusion partner that would be
complementary to the homologous green fluorescent protein from
Aequorea
, but very little is known of the biochemistry
of DsRed. We now show that DsRed has a much higher extinction
coefficient and quantum yield than previously reported, plus excellent
resistance to pH extremes and photobleaching. In addition, its 583-nm
emission maximum can be further shifted to 602 nm by mutation of Lys-83
to Met. However, DsRed has major drawbacks, such as strong
oligomerization and slow maturation. Analytical ultracentrifugation
proves DsRed to be an obligate tetramer
in vitro
, and
fluorescence resonance energy transfer measurements and yeast
two-hybrid assays verify oligomerization in live cells. Also, DsRed
takes days to ripen fully from green to red
in vitro
or
in vivo
, and mutations such as Lys-83 to Arg prevent the
color change. Many potential cell biological applications of DsRed will
require suppression of the tetramerization and acceleration of the
maturation.
Identification of a transcriptional repressor related to the noncatalytic domain of histone deacetylases 4 and 5
Authors: Zhou Xianbo , Richon Victoria M. , Rifkind Richard A. , Marks Paul A.
Published: July 2002

Histone deacetylases (HDACs) are involved in regulating transcription by modifying the core histones of the nucleosome. To date, six HDACs have been identified in mammalian cells: the yeast RPD3 homologs HDAC1, 2, and 3 and the yeast HDA1 homologs HDAC4, 5, and 6. HDAC4 and HDAC5 contain a noncatalytic N-terminal domain. Herein, we report the identification of a protein HDRP (
HD
AC-
r
elated
p
rotein) that shares 50% identity in deduced amino acid sequence to the noncatalytic N-terminal domain of HDAC4 and 5. The steady-state levels of HDRP mRNA are high in human brain, heart, and skeletal muscle and low in the several other tissues. HDRP has an apparent molecular mass of approximately 75 kDa. HDRP does not possess intrinsic HDAC activity but forms complexes with both HDAC1 and HDAC3. HDRP represses both basal and activated transcription in transient transfection assays when tethered to DNA as a Gal4-fusion protein. HDAC inhibitors do not reverse transcriptional repression mediated by Gal4–HDRP. Thus, HDRP is a transcriptional repressor and can repress transcription in the presence of HDAC inhibitors.
The Cambrian “explosion”: Slow-fuse or megatonnage?
Authors: Conway Morris Simon
Published: July 2002

Clearly, the fossil record from the Cambrian period is an invaluable tool for deciphering animal evolution. Less clear, however, is how to integrate the paleontological information with molecular phylogeny and developmental biology data. Equally challenging is answering why the Cambrian period provided such a rich interval for the redeployment of genes that led to more complex bodyplans.
The “glycogen shunt” in exercising muscle: A role for glycogen in muscle energetics and fatigue
Authors: Shulman R. G. , Rothman D. L.
Published: July 2002

Stimulated by recent
13
C and
31
P NMR
studies of exercising muscle, we propose a model of the energetics of
contraction. Previous studies of energetics have followed energy
consumption. However, the rapidity of contraction, in 10–40 msec,
requires that energy be delivered rapidly, so that the muscle has power
requirements of rapid energy expenditure that are ultimately met by the
slower averaged consumption of carbon and oxygen from blood. We propose
that energy is supplied in milliseconds by glycogenolysis and that
between contractions, glycogenesis refills the pools. The energy for
glycogenesis is supplied by oxidative phosphorylation. This mechanism
utilizes the rapid conversion of glycogen phosphorylase, the
“fight-or-flight” enzyme, to its active form. Lactate is
necessarily generated by this pathway to serve as a time buffer between
fast and slow energy needs, which resolves the paradoxical generation
of lactate in well oxygenated tissue. Consequences of the glycogen
shunt are compatible with numerous biochemical and physiological
experiments. The model provides a possible mechanism for muscle
fatigue, suggesting that at low but nonzero glycogen concentrations,
there is not enough glycogen to supply millisecond energy needs.
The earliest archaeological maize (
Zea mays
L.) from highland Mexico: New accelerator mass spectrometry dates and their implications
Authors: Piperno D. R. , Flannery K. V.
Published: July 2002

Accelerator mass spectrometry age determinations of maize cobs
(
Zea mays
L.) from Guilá Naquitz Cave in Oaxaca,
Mexico, produced dates of 5,400 carbon-14 years before the present
(about 6,250 calendar years ago), making those cobs the oldest in the
Americas. Macrofossils and phytoliths characteristic of wild and
domesticated
Zea
fruits are absent from older strata
from the site, although
Zea
pollen has previously been
identified from those levels. These results, together with the modern
geographical distribution of wild
Zea mays,
suggest that
the cultural practices that led to
Zea
domestication
probably occurred elsewhere in Mexico. Guilá Naquitz Cave has now
yielded the earliest macrofossil evidence for the domestication of two
major American crop plants, squash (
Cucurbita pepo
) and
maize.
Towards an Interactive Acculturation Model: A Social Psychological Approach
Authors: Bourhis Richard Y. , Moïse Léna Céline , Perreault Stéphane , Senécal Sacha
Published: July 2002

The first part of this paper proposes a continuum of ideological premises that seeks to account for the broad range of immigrant integration policies adopted by Western democratic states. In the second part, a review of Social Psychological models of immigrant acculturation strategies demonstrates the need to explain more clearly the interactive nature of immigrant and host community relations. The Interactive Acculturation Model (IAM) presented next proposes that relational outcomes are the product of the acculturation orientations of both the host majority and immigrant groups as influenced by state integration policies. The model makes predictions regarding the acculturation combinations most likely to produce consensual, problematic, and conflictual relational outcomes between immigrants and members of the host community. Social psychological research is needed to test the validity of the IAM model empirically.
On ‘Systematic’ Reviews of Research Literatures: A ‘narrative’ response to Evans & Benefield
Authors: Hammersley Martyn
Published: July 2002

AbstractThe production of ‘systematic’ reviews of research findings has recently come to be treated as a priority in the field of education, and other areas too. Such reviews are believed to play an important role in making evidence from research available in a usable form to policy‐makers and practitioners. This article examines the assumptions about research, and about the task of reviewing, which are built into the concept of systematic review. In addition, attention is given to the likely consequences of the priority now being given to this type of review.
Black Women and Mental Health: Working towards Inclusive Mental Health Services
Authors: Wilson Melba
Published: July 2002

The position concerning the mental health of black and minority ethnic women in Britain is closely linked to that of their respective communities in general. Issues concerning inappropriate care and treatment; lack of access to services; and service delivery based on assumptions and stereotypes govern the way in which black women and men experience mental health care and treatment. This article discusses the specific nature of black women's position, within the wider context of black communities’ experience as a whole. While the term ‘black and minority ethnic communities’ covers a range of differing communities, the focus here is on the experiences of women from African, African-Caribbean and Asian communities. In relation to women in these communities, reference is made to issues concerning: (1) older women; (2) women of mixed heritage; (3) women's roles as care-givers; and (4) the position of refugees and asylum seekers. The article also explores and outlines the importance of self-help strategies including, e.g. befriending and counselling and the role of religion and spirituality in enabling many black people to manage their mental distress.
Subchronic Sodium Chlorate Exposure in Drinking Water Results in a Concentration-Dependent Increase in Rat Thyroid Follicular Cell Hyperplasia
Authors: Hooth Michelle J. , Deangelo Anthony B. , George Michael H. , Gaillard Elias T. , Travlos Gregory S. , Boorman Gary A. , Wolf Douglas C.
Published: July 2002

Chlorine dioxide (ClO 2) is an effective drinking water disinfectant, but sodium chlorate (NaClO3) has been identified as a potentially harmful disinfection by-product. Studies were performed to describe the development of thyroid lesions in animals exposed to NaClO3 in the drinking water. Male and female F344 rats and B6C3F1 mice were exposed to 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/L NaClO3 for 21 days. Additional male F344 rats were exposed to 0, 0.001, 0.01, 0.1, 1.0, or 2.0 g/L NaClO 3 for 90 days. Female F344 rats were exposed to 0, 0.5, 1.0, 2.0, 4.0, or 6.0 g/L of NaClO3 for 105 days. Thyroid tissues were processed by routine methods for light microscopic examination, and follicular cell hyperplasia was diagnosed using a novel method. Thyroid hormone levels were altered significantly after 4 and 21 days. NaClO3 treatment induced a concentration-dependen t increase in the incidence and severity of thyroid follicular cell hyperplasia. Male rats are more sensitive to the effects of NaClO3 treatment than females. Follicular cell hyperplasi a was not present in male or female B6C3F1 mice. These data can be used to estimate the human health risk that would be associated with using ClO 2, rather than chlorine, to disinfect drinking water.
Metabolic effects of the consumption of Areca catechu
Authors: Boucher B. J. , Mannan N.
Published: July 2002

Abstract Betel nut (Areca catechu) is chewed regularly by at least 10% of the world population, imported by immigrant users wherever they settle, and is the fourth most widely used addictive substance. It is thought, by users, to soothe the digestion and to be a stimulant and its use has a major role in social situations. Specific arecal alkaloids act as competitive inhibitors of GABA receptors and have widespread effects in the body, including actions on the brain, cardiovascular system, lungs, gut and pancreas. Nitrosated derivatives of arecal alkaloids, proven carcinogens inducing tumours throughout the upper gut and foregut derivatives in animals, are also associated with increased tumour risks in man. These nitrosated compounds are also diabetogenic in CD1 mice, producing a type 2 diabetes with obesity. Increased central obesity is found in association with betel usage in man as well as increases in circulating markers of inflammatory and cardiovascular damage. The effects of chronic betel usage in man are at least as diverse as those of smoking and the habit increases the risks of ill health.
Neurological aspects of areca and betel chewing
Authors: Chu Nai‐Shin
Published: July 2002

Abstract Betel quid chewing has been claimed to produce a sense of well‐being, euphoria, warm sensation of the body, sweating, salivation, palpitation, heightened alertness and increased capacity to work. These effects suggest that betel quid chewing affects predominantly the central and autonomic nervous systems. Several studies have been conducted to elucidate the central and autonomic effects of betel quid chewing. The results are: (1) betel quid chewing increased the heart rate with onset within 2 minutes, maximal effect within 4–6 minutes and an average duration of 16.8 minutes. The cardio‐acceleratory response was more prominent for fresh and occasional chewers than for habitual chewers; (2) betel quid chewing increased the skin temperature with onset and duration similar to a cardio‐acceleratory response. The hyperthermic effect was abolished by atropine and partly inhibited by propranolol. (3) Betel quid chewing had no effect on simple reaction time but shortened the choice reaction time. (4) Betel quid chewing produced widespread cortical desynchronization of EEG. (5) Chewing of one or two betel quids attenuated the sympathetic skin response while continued consumption of more than two betel quids affected the RR interval variation. (6) Plasma concentrations of noradrenaline and adrenaline were elevated during betel quid chewing. These studies have confirmed several effects claimed by betel quid users. The effects of betel quid chewing appeared to be habit‐related and dose‐dependent. Although arecoline has been thought to be responsible for several effects of betel quid chewing, the present data suggest a role also played by sympathetic activation.
Voice problems in female‐to‐male transsexuals
Authors: van Borsel J. , de Cuypere G. , Rubens R. , Destaerke B.
Published: July 2002

Voice therapy with the aim of raising a client's fundamental frequency is a common practice in the reassignment of male‐to‐female transsexuals. In female‐to‐male transsexuals on the other hand voice therapy is usually not considered since the administration of androgens is assumed automatically to change the voice in the direction of the desired sex. The present study examined whether the voice change in female‐to‐male transsexuals is indeed as straightforward as it is assumed. Results from a sample survey involving 16 female‐to‐male transsexuals and longitudinal data from two clients suggest that the voice change in female‐to‐male transsexuals is not always totally unproblematic. A voice assessment and some counselling before the hormone therapy are recommended.
Interaction between children with cerebral palsy and their mothers: the effects of speech intelligibility
Authors: Pennington Lindsay , McConachie Helen
Published: July 2002

Children with cerebral palsy and severe speech disorders have been described as passive communicators, whose conversation partners control and dominate interaction. This paper studies the effects of speech intelligibility on interaction between mothers and their children with cerebral palsy to examine if similar patterns of child passivity and adult domination are also observed in dyads in which children are physically dependent but naturally intelligible to their parents. Two groups, each of 20 children who had four‐limb cerebral palsy and who differed in their speech intelligibility, participated in the study. Children in Group 1 (whose interaction has been previously reported) were unintelligible to their parents out of context. Children in Group 2 had speech that was understood without situational cues. Children and their mothers were videotaped playing with a set of toys that had been shown to elicit from non‐disabled children a full range of the conversational moves and speech acts targeted in the study. Videotaped interaction was transcribed and coded at three levels to show conversation structure, the pragmatic functions expressed and the method of communication used. Percentage distribution of coding categories was compared across groups using coda. Results showed that verbally intelligible children initiated more conversations and used their communication for a wider range of functions than did non‐speaking children, for whom more restricted patterns of conversation were noted. Unexpectedly, few differences were observed between the two groups of mothers, who initiated and closed most exchanges. Implications for intervention are discussed.
A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier
Authors: Wong Vivian , Gumbiner Barry M.
Published: July 2002

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 μM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.
NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion
Authors: Barth Angela I.M. , Pollack Anne L. , Altschuler Yoram , Mostov Keith E. , Nelson W. James
Published: July 2002

β-Catenin is essential for the function of cadherins, a family of Ca2+-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH2-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH2-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH2-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH2-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH2 terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.
Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells
Authors: Kuliawat Regina , Klumperman Judith , Ludwig Thomas , Arvan Peter
Published: July 2002

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.
Delayed Development of Nervous System in Mice Homozygous for Disrupted Microtubule-associated Protein 1B (MAP1B) Gene
Authors: Takei Yosuke , Kondo Satoru , Harada Akihiro , Inomata Satomi , Noda Tetsuo , Hirokawa Nobutaka
Published: July 2002

Microtubule-associated protein 1B (MAP1B), one of the microtubule-associated proteins (MAPs), is a major component of the neuronal cytoskeleton. It is expressed at high levels in immature neurons during growth of their axons, which indicates that it plays a crucial role in neuronal morphogenesis and neurite extension. To better define the role of MAP1B in vivo, we have used gene targeting to disrupt the murine MAP1B gene. Heterozygotes of our MAP1B disruption exhibit no overt abnormalities in their development and behavior, while homozygotes showed a slightly decreased brain weight and delayed nervous system development. Our data indicate that while MAP1B is not essential for survival, it is essential for normal time course development of the murine nervous system. These conclusions are very different from those of a previous MAP1B gene–targeting study (Edelmann, W., M. Zervas, P. Costello, L. Roback, I. Fischer, A. Hammarback, N. Cowan, P. Davis, B. Wainer, and R. Kucherlapati. 1996. Proc. Natl. Acad. Sci. USA. 93: 1270–1275). In this previous effort, homozygotes died before reaching 8-d embryos, while heterozygotes showed severely abnormal phenotypes in their nervous systems. Because the gene targeting event in these mice produced a gene encoding a 571–amino acid truncated product of MAP1B, it seems likely that the phenotypes seen arise from the truncated MAP1B product acting in a dominant-negative fashion, rather than a loss of MAP1B function.
Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTPσ by Inducible Proteolytic Processing
Authors: Aicher Babette , Lerch Markus M. , Müller Thomas , Schilling James , Ullrich Axel
Published: July 2002

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPσ. PTPσ biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPσ underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCα. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPσ cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and β-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPσ in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell–cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPσ is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell–cell contacts.
Compartmentalized IgE Receptor–mediated Signal Transduction in Living Cells
Authors: Stauffer Thomas P. , Meyer Tobias
Published: July 2002

Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein–tagged SH2 domains to investigate where and when IgE receptor (FcεRI)–mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-γ1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-γ1, and other SH2 domains selectively suppressed the induction of IgE receptor–mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-γ1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-γ1.
Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome
Authors: Dictenberg Jason B. , Zimmerman Wendy , Sparks Cynthia A. , Young Aaron , Vidair Charles , Zheng Yixian , Carrington Walter , Fay Fredric S. , Doxsey Stephen J.
Published: July 2002

Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.
Rvs161p Interacts with Fus2p to Promote Cell Fusion in Saccharomyces cerevisiae
Authors: Brizzio Valeria , Gammie Alison E. , Rose Mark D.
Published: July 2002

FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects. Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization. Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion. First, several other endocytic mutants showed no cell fusion defects. Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis. The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization. The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion. The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion.
Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins, Mmm1p and Mdm10p
Authors: Boldogh Istvan , Vojtov Nikola , Karmon Sharon , Pon Liza A.
Published: July 2002

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.
Junctional Adhesion Molecule, a Novel Member of the Immunoglobulin Superfamily That Distributes at Intercellular Junctions and Modulates Monocyte Transmigration
Authors: Martìn-Padura Inés , Lostaglio Susan , Schneemann Markus , Williams Lisa , Romano Maria , Fruscella Paolo , Panzeri Carla , Stoppacciaro Antonella , Ruco Luigi , Villa Antonello , Simmons David , Dejana Elisabetta
Published: July 2002

Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane–membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.