bluuebunny/crossref_metadata_2025 huggingface dataset:
Retention in Psychosocial Treatment of Cocaine Dependence: Predictors and Impact on Outcome
Authors: Siqueland Lynne , Crits‐Christoph Paul , Gallop Robert , Barber Jacques P. , Griffin Margaret L. , Thase Michael E. , Daley Denis , Frank Arlene , Gastfriend David R. , Blaine Jack , Connolly Mary Beth , Gladis Madeline
Published: July 2002

This report describes retention in treatment in the National Institute on Drug Abuse Collaborative Cocaine Treatment Study (CCTS), a multi‐site trial of four psychosocial treatments for 487 cocaine dependent patients. Younger, African‐American, and unemployed patients were retained in treatment for fewer days than their counterparts. African‐American patients who lived with a partner were retained in treatment for less time than if they lived alone. Higher psychiatric severity kept men in treatment longer but put women at risk for dropping out sooner. Patients who completed the full treatment used drugs less often than patients who dropped out, but outcome did not differ at each month. Patients in the drug counseling condition stayed in treatment for fewer days than patients in psychotherapy, but they were more likely to be abstinent after dropout. Patients with higher psychiatric severity were more at risk for continuing to use drugs after dropout.
The relative impact of waiting time and treatment entry on drug and alcohol use
Authors: Best David , Noble Alison , Ridge Gayle , Gossop Michael , Farrell Michael , Strang John
Published: July 2002

Abstract One hundred and twenty‐three treatment‐seeking substance misusers were recruited to a study assessing the early impact of treatment. Participants were interviewed at treatment entry and 3 and 6 months later, regardless of their treatment status (i.e. including those who had dropped out of treatment), while additional data were obtained from the two assessment interviews carried out prior to the initiation of treatment. Three consistent observations can be applied to both the opiate misuser (n = 61) and problem drinker samples (n = 62): (1) the period of pre‐treatment wait (mean of 8 weeks) was characterized by stable patterns of substance misuse with no significant 'spontaneous’improvement in indices of severity of drug or alcohol problems; (2) the period immediately following initiation of treatment was associated with substantial reductions in the quantity and frequency of substance use, an effect not influenced by the length of time for treatment initiation; (3) these benefits are maintained to 6 months after treatment initiation. The waiting period for treatment initiation does not seem to be characterized by significant changes in drug or alcohol use patterns, at least among those who made it into treatment, with clear and sustained improvements irrespective of the length of treatment wait.
Microbial metabolism of ethanol and acetaldehyde and clinical consequences
Authors: SALASPURO MIKKO
Published: July 2002

Abstract Many bacteria possess marked alcohol dehydrogenase activity and in the presence of ethanol they produce reactive and toxic acetaldehyde. Acetaldehyde production mediated by microbial alcohol dehydrogenases has been demonstrated in the oropharynx and bronchopulmonary washings. Also the most important gastric pathogen, Helicobacter pylori, and many skin bacteria associating with pathological dermatological conditions, possess alcohol dehydrogenase activity and produce acetaldehyde from ethanol. The most richly colonized site of the human body, however, is the large intestine, and therefore bacterial acetaldehyde production is most important in this organ. Alcohol ingested orally is transported to the colon by blood circulation and, after the distribution phase, intracolonic ethanol levels are equal to those in the blood. In the large bowel ethanol is oxidized by a bacteriocolonic pathway. In this pathway intracolonic ethanol is at first oxidized by bacterial alcohol dehydrogenase to acetaldehyde. Then acetaldehyde is oxidized either by colonic mucosal or bacterial aldehyde dehydrogenase to acetate. Part of intracolonic acetaldehyde may also be absorbed via the portal vein and metabolized in the liver. Bacteriocolonic pathway offers a new explanation for the disappearance of a part of ethanol calories. Due to the low aldehyde dehydrogenase activity of colonic mucosa acetaldehyde accumulates in the colon. Accordingly, during ethanol oxidation highest acetaldehyde levels of the body are found in the colon and not in the liver. High intracolonic acetaldehyde may contribute to the pathogenesis of alcohol‐induced diarrhoea. Acetaldehyde has been proven to be a carcinogen in experimental animals. It may therefore contribute to the increased risk of colon polyps and colon cancer found to be associated with heavy alcohol consumption in man. Intracolonic acetaldehyde may also be an important determinant of blood acetaldehyde level and a possible hepatotoxin. In addition to acetaldehyde, gut‐derived endotoxin is another potential candidate in the pathogenesis of alcohol‐related liver injury.
Phonological disruption and subsequent self‐correcting behaviour in Cantonese aphasic speakers
Authors: K‐S. Lee Raymond , Yiu Edwin M‐L. , Stonham John
Published: July 2002

This study investigated the phonological disruption and subsequent selfcorrecting behaviour in Cantonese aphasic speakers. The self‐correcting behaviour was investigated by examining the sequence of successive spontaneous attempts in producing a target sound. Five anomic, five non‐fluent aphasic and five normal control subjects were assessed by using a confrontation naming task which included monosyllabic, disyllabic and trisyllabic targets. All the aphasic subjects demonstrated successive phonological self‐corrections towards the target. Initial consonants were more vulnerable to phonological disruption and more resistant to self‐correction than vowels, lexical tones and final consonants.
A longitudinal perspective on the study of specific language impairment: the long term follow‐up of an Italian child
Authors: Cipriani Paola , Bottari Piero , Chilosi Anna Maria , Pfanner Lucia
Published: July 2002

Most of the literature on children with specific language impairment (SLI) is centred on the study of cross‐sectional samples and little is known on how language develops in these children‐that is, whether it occurs along steps and modes analogous to those observed in normal acquisition, the only difference being significant slowness, or following rather idiosyncratic courses leading to atypical results. This paper presents findings from a longitudinal study of the acquisition of formal aspects of Italian grammar in a child with severe SLI of the so‐called phonologic‐syntactic subtype or grammatical SLI. The analysis concerns a set of morphosyntactic phenomena sufficiently differentiated in terms of both grammatical properties and modes of acquisition in normal development, so as to offer a panorama that approximates to what can be thought of as an overall morphosyntactic competence: (1) free morphology (prepositions, clitics and articles); (2) bound morphology (verb inflection); (3) WH movement (questions and relative clauses) and (4) the evolution of complex sentences. These phenomena will be dealt with in four independent studies. This paper argues that each domain or even each sub‐domain presents its own set of properties, some of which are common to other domains but some of which are not. The results reveal the existence of important dissociations among the various domains and even within specific sub‐domains. These dissociations may be ascribed to deficits affecting specific properties of functors or constructions. The picture that emerges is one in which the deficit involves, rather than morphology in the strict sense of a bare set of paradigmatic forms, some properties or components of syntax which are involved in the use, not only of certain morphemes, but of certain extended syntactic constructions as well. This result will be checked against the most widely held current theoretical approaches to SLI.
Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products
Authors: Seaman Matthew N.J. , Marcusson Eric G. , Cereghino Joan Lin , Emr Scott D.
Published: July 2002

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.
Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization
Authors: Heald Rebecca , Tournebize Régis , Habermann Anja , Karsenti Eric , Hyman Anthony
Published: July 2002

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.
The Testicular Antiviral Defense System: Localization, Expression, and Regulation of 2′5′ Oligoadenylate Synthetase, Double-Stranded RNA-activated Protein Kinase, and Mx Proteins in the Rat Seminiferous Tubule
Authors: Dejucq Nathalie , Chousterman Suzanne , Jégou Bernard
Published: July 2002

Although the involvement of viruses in alterations of testicular function and in sexually transmitted diseases is well known, paradoxically, the testicular antiviral defense system has virtually not been studied. The well known antiviral activity of interferons (IFNs) occurs via the action of several IFN-induced proteins, among which the 2′5′ oligoadenylate synthetase (2′5′ A synthetase), the double-stranded RNA-activated protein kinase (PKR), and the Mx proteins are the best known. To explore the antiviral capacity of the testis and to study the testicular action of IFNs, we looked for the presence and regulation of these three proteins in isolated seminiferous tubule cells, cultured in the presence or in the absence of IFN α, IFN γ, or Sendai virus. In all conditions tested, the meiotic pachytene spermatocytes and the post-meiotic early spermatids lacked 2′5′ A synthetase, PKR, and Mx mRNAs and proteins. In contrast, Sertoli cells constitutively expressed these mRNAs and proteins, and their levels were greatly increased after IFN α or Sendai virus exposure. While peritubular cells were also able to markedly express 2′5′ A synthetase, PKR, and Mx mRNA and proteins after IFN α or viral exposure, only PKR was constitutively present in these cells. Interestingly, IFN γ had no effect on peritubular cells' 2′5′ A synthetase and Mx production but it enhanced Mx proteins in Sertoli cells. In conclusion, this study reveals that the seminiferous tubules are particularly well equipped to react to a virus attack. The fact that the two key tubular elements of the blood–testis barrier, namely, Sertoli and peritubular cells, were found to assume this protection allows the extension of the concept of blood–testis barrier to the testicular antiviral defense.
Neurofilaments and Orthograde Transport Are Reduced in Ventral Root Axons of Transgenic Mice that Express Human SOD1 with a G93A Mutation
Authors: Zhang Bin , Tu Pang-hsien , Abtahian Farhad , Trojanowski John Q. , Lee Virginia M.-Y.
Published: July 2002

Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly93 → Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS). Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals. Since the mechanism whereby mutations in SOD1 lead to MND remains enigmatic, we asked whether NF inclusions in motor neurons compromise axonal transport during the onset and progression of MND in a line of mice that contained ∼30% fewer copies of the transgene than the original G93A (Gurney et al., 1994). The onset of MND was delayed in these mice compared to the original G93A mice, but they developed the same neuropathologic abnormalities seen in the original G93A mice, albeit at a later time point with fewer vacuoles and more NF inclusions. Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness. By ∼200 d, both fast and slow axonal transports were impaired in the ventral roots of these mice coincidental with the appearance of NF inclusions and vacuoles in the axons and perikarya of vulnerable motor neurons. This is the first demonstration of impaired axonal transport in a mouse model of ALS, and we infer that similar impairments occur in authentic ALS. Based on the temporal correlation of these impairments with the onset of motor weakness and the appearance of NF inclusions and vacuoles in vulnerable motor neurons, the latter lesions may be the proximal cause of motor neuron dysfunction and degeneration in the G93A mice and in FALS patients with SOD1 mutations.
The Regulation of Reactive Oxygen Species Production during Programmed Cell Death
Authors: Tan Shirlee , Sagara Yutaka , Liu Yuanbin , Maher Pamela , Schubert David
Published: July 2002

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5–10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200–400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5–10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1β–converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.
A Human Dynamin-related Protein Controls the Distribution of Mitochondria
Authors: Smirnova Elena , Shurland Dixie-Lee , Ryazantsev Sergey N. , van der Bliek Alexander M.
Published: July 2002

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.
Regulation of the Cell Cycle by Focal Adhesion Kinase
Authors: Zhao Ji-He , Reiske Heinz , Guan Jun-Lin
Published: July 2002

In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant ΔC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in ΔC14. Biochemical analyses indicated that FAK mutant ΔC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKΔC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that ΔC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.
Endothelial Claudin
Authors: Morita Kazumasa , Sasaki Hiroyuki , Furuse Mikio , Tsukita Shoichiro
Published: July 2002

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6–specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell–cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face–associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell–specific component of TJ strands.
The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae
Authors: Wiederkehr Andreas , Avaro Sandrine , Prescianotto-Baschong Cristina , Haguenauer-Tsapis Rosine , Riezman Howard
Published: July 2002

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling.
Glutamate Slows Axonal Transport of Neurofilaments in Transfected Neurons
Authors: Ackerley Steven , Grierson Andrew J. , Brownlees Janet , Thornhill Paul , Anderton Brian H. , Leigh P. Nigel , Shaw Christopher E. , Miller Christopher C.J.
Published: July 2002

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein–tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases.
Tracking Col1a1 RNA in Osteogenesis Imperfecta
Authors: Johnson Carol , Primorac Dragan , McKinstry Monique , McNeil John , Rowe David , Lawrence Jeanne Bentley
Published: July 2002

This study illuminates the intra-nuclear fate of COL1A1 RNA in osteogenesis imperfecta (OI) Type I. Patient fibroblasts were shown to carry a heterozygous defect in splicing of intron 26, blocking mRNA export. Both the normal and mutant allele associated with a nuclear RNA track, a localized accumulation of posttranscriptional RNA emanating to one side of the gene. Both tracks had slightly elongated or globular morphology, but mutant tracks were cytologically distinct in that they lacked the normal polar distribution of intron 26. Normal COL1A1 RNA tracks distribute throughout an SC-35 domain, from the gene at the periphery. Normally, almost all 50 COL1A1 introns are spliced at or adjacent to the gene, before mRNA transits thru the domain. Normal COL1A1 transcripts may undergo maturation needed for export within the domain such as removal of a slow-splicing intron (shown for intron 24), after which they may disperse. Splice-defective transcripts still distribute thru the SC-35 domain, moving ∼1–3 μm from the gene. However, microfluorimetric analyses demonstrate mutant transcripts accumulate to abnormal levels within the track and domain. Hence, mutant transcripts initiate transport from the gene, but are impeded in exit from the SC-35 domain. This identifies a previously undefined step in mRNA export, involving movement through an SC-35 domain. A model is presented in which maturation and release for export of COL1A1 mRNA is linked to rapid cycling of metabolic complexes within the splicing factor domain, adjacent to the gene. This paradigm may apply to SC-35 domains more generally, which we suggest may be nucleated at sites of high demand and comprise factors being actively used to facilitate expression of associated loci.
P120 Catenin Regulates the Actin Cytoskeleton via Rho Family Gtpases
Authors: Noren Nicole K. , Liu Betty P. , Burridge Keith , Kreft Bertolt
Published: July 2002

Cadherins are calcium-dependent adhesion molecules responsible for the establishment of tight cell–cell contacts. p120 catenin (p120ctn) binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which has been implicated in regulating cell motility. It has previously been shown that overexpression of p120ctn induces a dendritic morphology in fibroblasts (Reynolds, A.B., J. Daniel, Y. Mo, J. Wu, and Z. Zhang. 1996. Exp. Cell Res. 225:328–337.). We show here that this phenotype is suppressed by coexpression of cadherin constructs that contain the juxtamembrane region, but not by constructs lacking this domain. Overexpression of p120ctn disrupts stress fibers and focal adhesions and results in a decrease in RhoA activity. The p120ctn-induced phenotype is blocked by dominant negative Cdc42 and Rac1 and by constitutively active Rho-kinase, but is enhanced by dominant negative RhoA. p120ctn overexpression increased the activity of endogenous Cdc42 and Rac1. Exploring how p120ctn may regulate Rho family GTPases, we find that p120ctn binds the Rho family exchange factor Vav2. The behavior of p120ctn suggests that it is a vehicle for cross-talk between cell–cell junctions and the motile machinery of cells. We propose a model in which p120ctn can shuttle between a cadherin-bound state and a cytoplasmic pool in which it can interact with regulators of Rho family GTPases. Factors that perturb cell–cell junctions, such that the cytoplasmic pool of p120ctn is increased, are predicted to decrease RhoA activity but to elevate active Rac1 and Cdc42, thereby promoting cell migration.
Nicotinic Acid Adenine Dinucleotide Phosphate (Naadp+) Is an Essential Regulator of T-Lymphocyte Ca2+-Signaling
Authors: Berg Ingeborg , Potter Barry V.L. , Mayr Georg W. , Guse Andreas H.
Published: July 2002

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP+) dose-dependently stimulated intracellular Ca2+-signaling. At a concentration of 10 nM NAADP+ evoked repetitive and long-lasting Ca2+-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca2+-peak followed by trains of smaller Ca2+-oscillations was observed. Higher concentrations of NAADP+ (1 and 10 μM) gradually reduced the initial Ca2+-peak, and a complete self-inactivation of Ca2+-signals was seen at 100 μM. The effect of NAADP+ was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4,5-trisphosphate– and cyclic adenosine diphosphoribose–mediated Ca2+-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP+. Most importantly, microinjection of a self-inactivating concentration of NAADP+ completely abolished subsequent stimulation of Ca2+-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP+ Ca2+-release system is essential for T-lymphocyte Ca2+-signaling.
The Supramolecular Organization of Fibrillin-Rich Microfibrils
Authors: Baldock Clair , Koster Abraham J. , Ziese Ulrike , Rock Matthew J. , Sherratt Michael J. , Kadler Karl E. , Shuttleworth C. Adrian , Kielty Cay M.
Published: July 2002

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-Å resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of ∼100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.
Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo
Authors: Xu Jingsong , Rodriguez Dorothy , Petitclerc Eric , Kim Jenny J. , Hangai Masanori , Yuen S. Moon , Davis George E. , Brooks Peter C.
Published: July 2002

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of α1β1 integrin binding and the gain of αvβ3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.
Calcineurin-dependent nuclear import of the transcription factor Crz1p requires Nmd5p
Authors: Polizotto Renée S. , Cyert Martha S.
Published: July 2002

Calcineurin is a conserved Ca2+/calmodulin-specific serine-threonine protein phosphatase that mediates many Ca2+-dependent signaling events. In yeast, calcineurin dephosphorylates Crz1p, a transcription factor that binds to the calcineurin-dependent response element, a 24-bp promoter element. Calcineurin-dependent dephosphorylation of Crz1p alters Crz1p nuclear localization. This study examines the mechanism by which calcineurin regulates the nuclear localization of Crz1p in more detail. We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import. We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin. Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.
Determinant Spreading of  T Helper Cell 2 (Th2) Responses to Pancreatic Islet Autoantigens
Authors: Tian Jide , Lehmann Paul V. , Kaufman Daniel L.
Published: July 2002

The nature (Th1 versus Th2) and dynamics of the autoimmune response during the development of insulin-dependent diabetes mellitus (IDDM) and after immunotherapy are unclear. Here, we show in nonobese diabetic (NOD) mice that the autoreactive T cell response starts and spreads as a pure Th1 type autoimmunity, suggesting that a spontaneous Th1 cascade underlies disease progression. Surprisingly, induction of antiinflammatory Th2 responses to a single β cell antigen (βCA) resulted in the spreading of Th2 cellular and humoral immunity to unrelated βCAs in an infectious manner and protection from IDDM. The data suggest that both Th1 and Th2 autoimmunity evolve in amplificatory cascades by generating site-specific, but not antigen-specific, positive feedback circuits. Determinant spreading of Th2 responses may be a fundamental mechanism underlying antigen-based immunotherapeutics, explaining observations of infectious tolerance and providing a new theoretical framework for therapeutic intervention.
Degradation of Mouse Invariant Chain: Roles of Cathepsins S and D and the Influence of Major Histocompatibility Complex Polymorphism
Authors: Villadangos José A. , Riese Richard J. , Peters Christoph , Chapman Harold A. , Ploegh Hidde L.
Published: July 2002

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II–peptide complexes and, second, that most class II–associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.
Treponema pallidum Major Sheath Protein Homologue Tpr K Is a Target of Opsonic Antibody and the Protective Immune Response
Authors: Centurion-Lara Arturo , Castro Christa , Barrett Lynn , Cameron Caroline , Mostowfi Maryam , Van Voorhis Wesley C. , Lukehart Sheila A.
Published: July 2002

We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.
Fcα Receptor (Cd89) Mediates the Development of Immunoglobulin a (Iga) Nephropathy (Berger's Disease)
Authors: Launay Pierre , Grossetête Béatrice , Arcos-Fajardo Michelle , Gaudin Emmanuelle , Torres Sonia P. , Beaudoin Lucie , Patey-Mariaud de Serre Natacha , Lehuen Agnès , Monteiro Renato C.
Published: July 2002

The pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN), the most prevalent form of glomerulonephritis worldwide, involves circulating macromolecular IgA1 complexes. However, the molecular mechanism(s) of the disease remain poorly understood. We report here the presence of circulating soluble FcαR (CD89)-IgA complexes in patients with IgAN. Soluble CD89 was identified as a glycoprotein with a 24-kD backbone that corresponds to the expected size of CD89 extracellular domains. To demonstrate their pathogenic role, we generated transgenic (Tg) mice expressing human CD89 on macrophage/monocytes, as no CD89 homologue is found in mice. These mice spontaneously developed massive mesangial IgA deposition, glomerular and interstitial macrophage infiltration, mesangial matrix expansion, hematuria, and mild proteinuria. The molecular mechanism was shown to involve soluble CD89 released after interaction with IgA. This release was independent of CD89 association with the FcRγ chain. The disease was induced in recombination activating gene (RAG)2−/− mice by injection of serum from Tg mice, and in severe combined immunodeficiency (SCID)-Tg mice by injection of patients' IgA. Depletion of soluble CD89 from serum abolished this effect. These results reveal the key role of soluble CD89 in the pathogenesis of IgAN and provide an in vivo model that will be useful for developing new treatments.
An N-Acetylated Natural Ligand of Human Histocompatibility Leukocyte Antigen (Hla)-B39
Authors: Yagüe Jesús , Alvarez Iñaki , Rognan Didier , Ramos Manuel , Vázquez Jesús , de Castro José A. López
Published: July 2002

Sequence-independent interactions involving the free peptidic NH2 terminus are thought to be an essential feature of peptide binding to classical major histocompatibility complex (MHC) class I proteins. Challenging this paradigm, a natural Nα-acetylated ligand of human histocompatibility leukocyte antigen (HLA)-B39 was identified in this study. It matched the NH2-terminal sequence of two human helicases, was resistant to aminopeptidase M, and was produced with high yield from a synthetic 30 mer with the sequence of the putative parental protein by the 20S proteasome. This is the first reported natural ligand of classical MHC class I antigens that has a blocked NH2 terminus.
Cloning of the Murine Thymic Stromal Lymphopoietin (Tslp) Receptor
Authors: Park Linda S. , Martin Unja , Garka Kirsten , Gliniak Brian , Di Santo James P. , Muller Werner , Largaespada David A. , Copeland Neal G. , Jenkins Nancy A. , Farr Andrew G. , Ziegler Steven F. , Morrissey Philip J. , Paxton Raymond , Sims John E.
Published: July 2002

The cellular receptor for murine thymic stromal lymphopoietin (TSLP) was detected in a variety of murine, but not human myelomonocytic cell lines by radioligand binding. cDNA clones encoding the receptor were isolated from a murine T helper cell cDNA library. TSLP receptor (TSLPR) is a member of the hematopoietin receptor family. Transfection of TSLPR cDNA resulted in only low affinity binding. Cotransfection of the interleukin 7 (IL-7)Rα chain cDNA resulted in conversion to high affinity binding. TSLP did not activate cells from IL-7Rα−/− mice, but did activate cells from γc−/− mice. Thus, the functional TSLPR requires the IL-7Rα chain, but not the γc chain for signaling.
Differential T Cell Function and Fate in Lymph Node and Nonlymphoid Tissues
Authors: Harris Nicola L. , Watt Victoria , Ronchese Franca , Le Gros Graham
Published: July 2002

The functions and fate of antigen-experienced T cells isolated from lymph node or nonlymphoid tissues were analyzed in a system involving adoptive transfer of in vitro–activated T cells into mice. Activated T cells present in the lymph nodes could be stimulated by antigen to divide, produce effector cytokines, and migrate to peripheral tissues. By contrast, activated T cells that had migrated into nonlymphoid tissues (lung and airway) produced substantial effector cytokines upon antigen challenge, but were completely unable to divide or migrate back to the lymph nodes. Therefore, activated T cells can undergo clonal expansion in the lymph node, but are recruited and retained as nondividing cells in nonlymphoid tissues. These distinct regulatory events in lymph node and nonlymphoid tissues reveal simple key mechanisms for both inducing and limiting T cell immunity.
Ion Conduction through C-Type Inactivated Shaker Channels
Authors: Starkus John G. , Kuschel Lioba , Rayner Martin D. , Heinemann Stefan H.
Published: July 2002

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel.
Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F
Authors: Starkus John G. , Kuschel Lioba , Rayner Martin D. , Heinemann Stefan H.
Published: July 2002

C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.
Novel Gating Mechanism of Polyamine Block in the Strong Inward Rectifier K Channel Kir2.1
Authors: Lee Jong-Kook , John Scott A. , Weiss James N.
Published: July 2002

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.