bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the insulin-like developmental pathway of
Caenorhabditis elegans
by a homolog of the
PTEN
tumor suppressor gene
Authors: Gil Elad B. , Malone Link Elizabeth , Liu Leo X. , Johnson Carl D. , Lees Jacqueline A.
Published: July 2002

The human
PTEN
tumor suppressor gene is mutated in a wide variety of sporadic tumors. To determine the function of PTEN
in vivo
we have studied a
PTEN
homolog in
Caenorhabditis elegans
. We have generated a strong loss-of-function allele of the
PTEN
homolog and shown that the deficient strain is unable to enter dauer diapause. An insulin-like phosphatidylinositol 3-OH kinase (PI3′K) signaling pathway regulates dauer-stage entry. Mutations in either the
daf-2
insulin receptor-like (IRL) gene or the
age-1
encoded PI3′K catalytic subunit homolog cause constitutive dauer formation and also affect the life span, brood size, and metabolism of nondauer animals. Strikingly, loss-of-function mutations in the
age-1
PI3′K and
daf-2
IRL genes are suppressed by loss-of-function mutations in the
PTEN
homolog. We establish that the
PTEN
homolog is encoded by
daf-18
, a previously uncloned gene that has been shown to interact genetically with the DAF-2 IRL AGE-1 PI3′K signaling pathway. This interaction provides clear genetic evidence that PTEN acts to antagonize PI3′K function
in vivo
. Given the conservation of the PI3′K signaling pathway between
C. elegans
and mammals, the analysis of
daf-18 PTEN
mutant nematodes should shed light on the role of human PTEN in the etiology of metabolic disease, aging, and cancer.
A model of human phenylalanine metabolism in normal subjects and in phenylketonuric patients
Authors: Kaufman Seymour
Published: July 2002

The derivation of a quantitative model of phenylalanine metabolism in humans is described. The model is based on the kinetic properties of pure recombinant human phenylalanine hydroxylase and on estimates of the
in vivo
rates of phenylalanine transamination and protein degradation. Calculated values for the steady-state concentration of blood phenylalanine, rate of clearance of phenylalanine from the blood after an oral load of the amino acid, and dietary tolerance of phenylalanine all agree well with data from normal as well as from phenylketonuric patients and obligate heterozygotes. These calculated values may help in the decision about the degree of restriction of phenylalanine intake that is necessary to achieve a satisfactory clinical outcome in classical patients and in those with milder forms of the disease.
Establishment of an animal–bacterial association: Recruiting symbiotic vibrios from the environment
Authors: Nyholm Spencer V. , Stabb Eric V. , Ruby Edward G. , McFall-Ngai Margaret J.
Published: July 2002

While most animal–bacterial symbioses are reestablished each successive generation, the mechanisms by which the host and its potential microbial partners ensure tissue colonization remain largely undescribed. We used the model association between the squidEuprymna scolopesandVibrio fischerito examine this process. This light organ symbiosis is initiated whenV. fischericells present in the surrounding seawater enter pores on the surface of the nascent organ and colonize deep epithelia-lined crypts. We discovered that when newly hatched squid were experimentally exposed to natural seawater, the animals responded by secreting a viscous material from the pores of the organ. Animals maintained in filtered seawater produced no secretions unless Gram-negative bacteria, either living or dead, were reintroduced. The viscous material bound only lectins that are specific for eitherN-acetylneuraminic acid orN-acetylgalactosamine, suggesting that it was composed of a mucus-containing matrix. Complex ciliated fields on the surface of the organ produced water currents that focused the matrix into a mass that was tethered to, and suspended above, the light organ pores. WhenV. fischericells were introduced into the seawater surrounding the squid, the bacteria were drawn into its fluid-filled body cavity during ventilation and were captured in the matrix. After residing as an aggregate for several hours, the symbionts migrated into the pores and colonized the crypt epithelia. This mode of infection may be an example of a widespread strategy by which aquatic hosts increase the likelihood of successful colonization by rarely encountered symbionts.
Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor
Authors: Floriano Wely B. , Vaidehi Nagarajan , Goddard William A. , Singer Michael S. , Shepherd Gordon M.
Published: July 2002

The prevailing paradigm for G protein-coupled receptors is that each receptor is narrowly tuned to its ligand and closely related agonists. An outstanding problem is whether this paradigm applies to olfactory receptor (ORs), which is the largest gene family in the genome, in which each of 1,000 different G protein-coupled receptors is believed to interact with a range of different odor molecules from the many thousands that comprise “odor space.” Insights into how these interactions occur are essential for understanding the sense of smell. Key questions are: (i) Is there a binding pocket? (ii) Which amino acid residues in the binding pocket contribute to peak affinities? (iii) How do affinities change with changes in agonist structure? To approach these questions, we have combined single-cell PCR results [Malnic, B., Hirono, J., Sato, T. & Buck, L. B. (1999)Cell96, 713–723] and well-established molecular dynamics methods to model the structure of a specific OR (OR S25) and its interactions with 24 odor compounds. This receptor structure not only points to a likely odor-binding site but also independently predicts the two compounds that experimentally best activate OR S25. The results provide a mechanistic model for olfactory transduction at the molecular level and show how the basic G protein-coupled receptor template is adapted for encoding the enormous odor space. This combined approach can significantly enhance the identification of ligands for the many members of the OR family and also may shed light on other protein families that exhibit broad specificities, such as chemokine receptors and P450 oxidases.
Hair cell recovery in mitotically blocked cultures of the bullfrog saccule
Authors: Baird Richard A. , Burton Miriam D. , Fashena David S. , Naeger Rebecca A.
Published: July 2002

Hair cells in many nonmammalian vertebrates are regenerated by the
mitotic division of supporting cell progenitors and the differentiation
of the resulting progeny into new hair cells and supporting cells.
Recent studies have shown that nonmitotic hair cell recovery after
aminoglycoside-induced damage can also occur in the vestibular organs.
Using hair cell and supporting cell immunocytochemical markers, we have
used confocal and electron microscopy to examine the fate of damaged
hair cells and the origin of immature hair cells after gentamicin
treatment in mitotically blocked cultures of the bullfrog saccule.
Extruding and fragmenting hair cells, which undergo apoptotic
cell death, are replaced by scar formations. After losing their
bundles, sublethally damaged hair cells remain in the sensory
epithelium for prolonged periods, acquiring supporting cell-like
morphology and immunoreactivity. These modes of damage appear to be
mutually exclusive, implying that sublethally damaged hair cells repair
their bundles. Transitional cells, coexpressing hair cell and
supporting cell markers, are seen near scar formations created by the
expansion of neighboring supporting cells. Most of these cells have
morphology and immunoreactivity similar to that of sublethally damaged
hair cells. Ultrastructural analysis also reveals that most immature
hair cells had autophagic vacuoles, implying that they originated from
damaged hair cells rather than supporting cells. Some transitional
cells are supporting cells participating in scar formations. Supporting
cells also decrease in number during hair cell recovery, supporting the
conclusion that some supporting cells undergo phenotypic conversion
into hair cells without an intervening mitotic event.
High-resolution structure of hair-cell tip links
Authors: Kachar Bechara , Parakkal Marianne , Kurc Mauricio , Zhao Yi-dong , Gillespie Peter G.
Published: July 2002

Transduction-channel gating by hair cells apparently requires a
gating spring, an elastic element that transmits force to the channels.
To determine whether the gating spring is the tip link, a filament
interconnecting two stereocilia along the axis of mechanical
sensitivity, we examined the tip link's structure at high resolution
by using rapid-freeze, deep-etch electron microscopy. We found that the
tip link is a right-handed, coiled double filament that usually forks
into two branches before contacting a taller stereocilium; at the other
end, several short filaments extend to the tip link from the shorter
stereocilium. The structure of the tip link suggests that it is either
a helical polymer or a braided pair of filamentous macromolecules and
is thus likely to be relatively stiff and inextensible. Such behavior
is incompatible with the measured elasticity of the gating spring,
suggesting that the gating spring instead lies in series with the
helical segment of the tip link.
Implications of the
Tribolium Deformed
mutant phenotype for the evolution of Hox gene function
Authors: Brown Susan , DeCamillis Mark , Gonzalez-Charneco Karen , Denell Margaret , Beeman Richard , Nie Wensheng , Denell Robin
Published: July 2002

Among insects, the genetic regulation of regional identities in the postoral head or gnathal segments (mandibular, maxillary, and labial) is best understood in the fly
Drosophila melanogaster
. In part, normal gnathal development depends on
Deformed (Dfd)
and
Sex combs reduced (Scr)
, genes in the split
Drosophila
homeotic complex. The gnathal segments of
Dfd
and
Scr
mutant larvae are abnormal but not homeotically transformed. In the red flour beetle,
Tribolium castaneum
, we have isolated loss-of-function mutations of the
Deformed
ortholog. Mutant larvae display a strong transformation of mandibular appendages to antennae. The maxillary appendages, normally composed of an endite and a telopodite, develop only the telopodite in mutant larvae. We previously reported that mutations in the beetle
Scr
and
Antennapedia
orthologs cause the labial and thoracic appendages, respectively, to be transformed to antennae. Moreover, a deficiency of most of the beetle homeotic complex causes all gnathal (as well as thoracic and abdominal) segments to develop antennae. These and other observations are consistent with the hypothesis that ancestral insect homeotic gene functions have been modified considerably during the evolution of the highly specialized maggot head. One of the ancestral homeobox genes that arose close to the root of the Eumetazoa appears to have given rise to
Dfd, Scr
, and the Antennapedia homeobox-class homeotic genes. Evidence from both
Tribolium
and
Drosophila
suggests that this ancestral gene served to repress anterior development as well as confer a trunk-specific identity.
Crystal structure of a DEAD box protein from the hyperthermophile
Methanococcus jannaschii
Authors: Story Randall M. , Li Hong , Abelson John N.
Published: July 2002

We have determined the structure of a DEAD box putative RNA
helicase from the hyperthermophile
Methanococcus
jannaschii
. Like other helicases, the protein contains two
α/β domains, each with a recA-like topology. Unlike other
helicases, the protein exists as a dimer in the crystal. Through an
interaction that resembles the dimer interface of insulin, the
amino-terminal domain's 7-strand β-sheet is extended to 14 strands
across the two molecules. Motifs conserved in the DEAD box family
cluster in the cleft between domains, and many of their functions can
be deduced by mutational data and by comparison with other helicase
structures. Several lines of evidence suggest that motif III
Ser-Ala-Thr may be involved in binding RNA.
Industrialization, class structure, and social mobility in postwar Japan 1
Authors: Ishida Hiroshi
Published: July 2002

This study examines intergenerational class mobility in Japan using cross‐national comparisons with Western nations and cross‐temporal comparisons of five national surveys conducted in postwar Japan. Cross‐national comparisons highlight the similarity in relative mobility pattern between Japan and Western nations and at the same time the Japanese distinctiveness in absolute mobility rates especially regarding the demographic character of the Japanese manual working class. The results of cross‐temporal comparisons of mobility pattern report some systematic trends in total mobility, inflow and outflow rates, reflecting the Japanese experience of late but rapid industrialization. The pattern of association between class origin and class destination, however, was stable in postwar Japan. It is therefore the combination of distinctive absolute mobility rates and similar relative mobility rates that characterizes the Japanese mobility pattern in comparison with the Western experience. Furthermore, Japan's distinctive pattern of postwar social mobility is characterized by a combination of rapidly changing absolute mobility rates and comparatively stable relative mobility rates.
Report on IUPsyS — UN activities
Authors: Sabourin Michel
Published: July 2002

One of the most important conferences ever to be organized by the United Nations, the World Conference against Racism, Racial Discrimination, Xenophobia and Related Intolerance, will be held in South Africa, from August 31 to September 7, 2001. The UN Commission on Human Rights is acting as the Preparatory Committee for this World Conference. The purpose of the preparatory process is to raise public awareness of racism and racial discrimination globally and to focus on the action needed to fight it. Prior to the Conference, two preparatory meetings are planned. The first was held from 1 to 5 May 2000 in Geneva and the second will be in May/June 2001, also in Geneva. At the first Preparatory Committee meeting, a number of organizational decisions were taken, including adoption of the provisional agenda for the Conference and its draft rules of procedure. A voluntary fund has been established to provide additional resources for the preparatory process and the World Conference itself.

In the next paragraphs, information concerning the background of this important conference is provided by the Department of Public Information of the UN to help a better comprehension of the issues. No doubt that the IUPsyS will have to find ways it can become more deeply involved in the process and participate actively in the actual deliberations that will take place in South Africa, next August, with the goal of eventually submitting recommendations.
Oral cues improve subject‐verb agreement in written French
Authors: Largy Pierre , Fayol Michel
Published: July 2002

The aim of this study was to test the impact of an audible marker on the production of subject‐verb agreements. Earlier studies have shown that educated French‐speaking adults make subject‐verb agreement errors when writing as soon as a secondary task demands their attention. One hypothesis is that these errors occur primarily because in French many of the written inflections of the verbal plural are silent. However, errors of the same type have been reported in spoken English: in configurations such as “the dog of the neighbours arrive(s)”, arrive agrees with the noun closest to the verb rather than with the subject. The current experiment compares the production of subject‐verb agreements in written French depending on whether the singular/plural opposition is audible (finit/finissent) or not (chante/chantent). After having changed the tense of the verb, adult subjects had to recall, in writing, sentences which had been read aloud to them and which shared the same start (La flamme de la bougie = the flame of the candle) but contained different verbs matched for semantic plausibility and frequency, and either possessing (éblouir = to blind) or not possessing (éclairer = to illuminate) an audible singular/plural opposition. The results show that the presence of an audible marker reduces the error frequency and makes the agreement easier to manage. A chronometric study suggests that it is the competition between concurrent markers (e.g., ‐e, ‐s, ‐ent) that causes difficulties with regular verbs and that this competition is resolved at the very last moment, at the point when the marker is transcribed.
Legal Empowerment of the Neighbourhood
Authors: Poindexter Georgette C.
Published: July 2002

This article examines the inefficacy of the present legal form of local government in the US. This inefficacy manifests itself by providing no solid legal mooring for the power of local government while contributing to regional economic inequalities. In an attempt to address this problem, the author suggests realigning local government to the neighbourhood level while constructing a new tier of government to mitigate the negative economic consequences of such realignment.
Obsolescence and the Process of Creative Reconstruction
Authors: Bryson John R.
Published: July 2002

What is built and where it is built is largely determined by the activities and perceptions of global investment capital. Comparatively limited work has been undertaken into the property markets of regional cities as well as into the process of building obsolescence, refurbishment and valorisation. This paper explores the dynamics of the property development process in relation to land rent theory in marginal development locations. It argues that the debate over land rent theory, between academics who want to retain the canonical dogma and those who focus on landownership, is misplaced. What is required is a combination of these approaches. Increasingly, what is built and where it is built reflects the varied nature of property interests, as well as the actions of the local and national state. The argument is supported by a case study of Nottingham's office market.
Subject requirement and predication
Authors: Åfarli T. A. , Eide K. M.
Published: July 2002

In this paper, we argue that the subject requirement or Extended Projection Principle (EPP) is naturally derived from predication, not in the standard syntax-based manner of Rothstein (1983), Chomsky (1986) or Heycock (1991), but in a full-blooded semantic manner within the selective mentalist Grammar Semantics of Bouchard (1995). We propose that the EPP is the effect of a basic proposition-forming operation of natural language, which is induced by a predication operator (Bowers 1993). On our analysis, insertion of an expletive in the subject position or raising to the subject position are therefore ultimately analysed as semantically motivated processes, triggered by the operation of proposition formation.
Classification of Industries by Level of Technology: An Appraisal and some Implications
Authors: Carroll Peter , Pol Eduardo , Robertson Paul L.
Published: July 2002

Modern growth theory acknowledges that a country's economic prosperity depends in large part on its capacity for technological innovation. Empirical evidence, however, supports the view that not all sectors are equally innovative. As a result, it seems desirable from a public policy perspective to identify and promote sectors displaying both a high innovation rate and, in an increasingly competitive international economy, a high degree of international competitiveness. It is frequently argued that the high-tech industry sectors, in contrast to low-tech sectors, satisfy both conditions, with the clear implication that public policy should be directed to enhancing the performance of high-tech sectors. This approach raises at least two important issues. The first is whether such classifications can be meaningfully constructed given both the intractability of the concepts involved and the difficulties in data collection. A second issue is the basic assumption that policy emphasis should be placed on technology-intensive industries because they have a greater impact on growth. In this paper, we argue that while it may be possible to construct indices of technological intensity that are useful for some purposes, the ones that are currently proposed do not, in fact, address questions of economic growth and firm performance very well. In part, this is a reflection of the technicalities involved in formulating and operationalising the indices, but it also reflects problems in the underlying premise, namely technology-intensive sectors are more growth-inducing than low-tech sectors. We call, therefore, for the adoption of a more sophisticated and detailed approach that would provide a sensible classification of industries and new policy insights.
IT Investment Strategy for Development: An 'Instrumental Analysis' Based on the Tasmanian and New Brunswick Information Technology Strategies
Authors: Courvisanos Jerry
Published: July 2002

In April 1997, Tasmania (Australia) adopted the reputably successful New Brunswick (Canada) industrial strategy to build an information technology (IT) industry of significance. The strategy aims to overcome isolation in small regional economies and structurally change from declining natural resource industries. Both plans reject neo-classical economics-based industry policy, opting instead for a strong state-based investment planning approach. An analytical framework is set out, using Adolph Lowe's 'Instrumental Analysis', to examine implementation of both IT strategies. Implications of this analysis are drawn for any attempts at developing IT regional plans and, more generally, as a guide for broad strategic-based national industrial strategies.
Liver transplantation for alcoholic liver disease
Authors: Pageaux Georges‐Philippe , Perney Pascal , Larrey Dominique
Published: July 2002

Although increasing numbers of alcoholic patients are being referred to liver transplant centres, liver transplantation for alcoholic liver disease still remains controversial, essentially because we are in an era of organ shortage. In fact, the main issue is the likelihood of relapse and its influence on outcome, because it is the possibility of returning to alcohol use that separates patients with alcoholic liver disease from those with other forms of chronic liver disease. In all proposed clinical guidelines of indications for referral and assessment for liver transplantation for alcoholic liver disease, the authors emphasize the risk of alcoholism recurrence and, thus, a multidisciplinary approach is required to select patients who are likely to comply with follow‐up and not return to a damaging pattern of alcohol consumption after transplantation. It emerges from all clinical studies that when we take into account the usual criteria of success for liver transplantation, i.e. patient and graft survival, rejection rate and infection rate, alcoholic liver disease is a good indication for liver transplantation. Predictive factors for alcoholic relapse after liver transplantation have been assessed in numerous studies, often with contradictory results making these difficult to analyse and compare. Several predictive factors for alcoholic relapse have been studied: length of abstinence before transplantation, associated psychiatric problems, social conditions, associated drug addiction, age. Abstinence after transplantation is the goal, but the necessary treatment for alcoholic disease can result in considerable improvement, even when complete abstinence is not achieved. Finally, the good results obtained with liver transplantation for alcoholic liver disease should help us to educate the general population about alcoholic disease.
A putative sigma1 receptor antagonist NE‐100 attenuates the discriminative stimulus effects of ketamine in rats
Authors: Narita Minoru , Yoshizawa Kazumi , Aoki Kazue , Takagi Misa , Miyatake Mayumi , Suzuki Tsutomu
Published: July 2002

Ketamine, one of the dissociative anaesthetic agents, has been shown to produce psychotomimetic effects. It has been well documented that activation of sigma receptors is responsible for the pathogenesis of some psychiatric disorders. In the present study, the effects of NE‐100, a putative sigma 1 receptor antagonist, was investigated in rats trained to discriminate between ketamine (5 mg/kg, i.p.) from saline under a fixed‐ratio 10 food‐reinforced procedure. Here we report for the first time that NE‐100 (1 mg/kg) produced a shift to the right in the dose–response curve for ketamine's discriminative stimulus effects. These results suggest that the sigma 1 receptor is, at least in part, involved in the discriminative stimulus effects of ketamine.
The Early Reading Screening Instrument: a method for identifying kindergarteners at risk for learning to read
Authors: Lombardino Linda J. , Morris Darrell , Mercado Laurie , DeFillipo Frank , Sarisky Carol , Montgomery Ann
Published: July 2002

This study was designed to provide speech‐language pathologists and educators with a method for identifying children at risk for reading failure. The Early Reading Screening Instrument (ERSI) was given to 149 end‐of‐the‐year kindergarten children. Half of the sample was tested 1 year later with standardized reading measures. Total ERSI scores from the kindergarten children strongly correlated with reading skills in first grade. Reading comprehension in first grade was the skill most strongly predicted by the subjects' total ERSI scores. The word recognition and invented spelling subtests of the ERSI were the best variables to be selected as predictors of first grade word analysis, word identification, and passage comprehension skills. A split‐half reliability study of the ERSI showed that a shortened form could be used with the same degree of predictive validity. The ERSI or similar tools can be used to assess reading readiness in kindergarten children so that we can promptly begin an intervention to prevent or, at the very least, to diminish risk for reading failure. For screening reading readiness across different languages or cultures, the content of the four ERSI subtests can easily be modified.
Induction of Neurite Outgrowth through Contactin and Nr-CAM by Extracellular Regions of Glial Receptor Tyrosine Phosphatase β
Authors: Sakurai Takeshi , Lustig Marc , Nativ Moshe , Hemperly John J. , Schlessinger Joseph , Peles Elior , Grumet Martin
Published: July 2002

Receptor protein tyrosine phosphatase β (RPTPβ) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (βC) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, βCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than βCF. βS had no effect by itself, but it potentiated neurite growth when mixed with βCF. Neurite outgrowth induced by βCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound βCFS. NIH3T3 cells transfected to express βCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPβ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.
Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology
Authors: Gebbink Martijn F.B.G. , Kranenburg Onno , Poland Mieke , Horck Francis P.G. van , Houssa Brahim , Moolenaar Wouter H.
Published: July 2002

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth.
Presentation of a T Cell Receptor Antagonist Peptide by Immunoglobulins Ablates Activation of  T Cells by a Synthetic Peptide or Proteins Requiring Endocytic Processing
Authors: Legge Kevin L. , Min Booki , Potter Nicholas T. , Zaghouani Habib
Published: July 2002

T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell–mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (IgPLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR–specific T cells may be the operative mechanism for Ig-PLP-LR–mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell–mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.
T Cell Receptor (TCR) Mini-Gene mRNA Expression Regulated by Nonsense Codons: A Nuclear-associated Translation-like Mechanism
Authors: Li Shulin , Leonard Deana , Wilkinson Miles F.
Published: July 2002

Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-β gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-β exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.
Several Carcinoembryonic Antigens (CD66) Serve as Receptors for Gonococcal Opacity Proteins
Authors: Chen Tie , Grunert Fritz , Medina-Marino Andrew , Gotschlich Emil C.
Published: July 2002

Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLaCEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >>CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.
A Novel Antioxidant Gene from Mycobacterium tuberculosis
Authors: Ehrt Sabine , Shiloh Michael U. , Ruan Jia , Choi Michael , Gunzburg Stuart , Nathan Carl , Xie Qiao-wen , Riley Lee W.
Published: July 2002

Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.
Alteration of a Single Hydrogen Bond between Class II Molecules and Peptide Results in Rapid Degradation of Class II Molecules after Invariant Chain Removal
Authors: Ceman Stephanie , Wu Shenhong , Jardetzky Theodore S. , Sant Andrea J.
Published: July 2002

To characterize the importance of a highly conserved region of the class II β chain, we introduced an amino acid substitution that is predicted to eliminate a hydrogen bond formed between the class II molecule and peptide. We expressed the mutated β chain with a wild-type α chain in a murine L cell by gene transfection. The mutant class II molecule (81βH−) assembles normally in the endoplasmic reticulum and transits the Golgi complex. When invariant chain (Ii) is coexpressed with 81βH−, the class II–Ii complex is degraded in the endosomes. Expression of 81βH− in the absence of Ii results in a cell surface expressed molecule that is susceptible to proteolysis, a condition reversed by incubation with a peptide known to associate with 81βH−. We propose that 81βH− is protease sensitive because it is unable to productively associate with most peptides, including classII–associated invariant chain peptides. This model is supported by our data demonstrating protease sensitivity of peptide-free wild-type I-Ad molecules. Collectively, our results suggest both that the hydrogen bonds formed between the class II molecule and peptide are important for the integrity and stability of the complex, and that empty class II molecules are protease sensitive and degraded in endosomes. One function of DM may be to insure continuous groove occupancy of the class II molecule.
Expression of Recombination Activating Genes in Germinal Center B Cells: Involvement of Interleukin 7 (IL-7) and the IL-7 Receptor
Authors: Hikida Masaki , Nakayama Yasunori , Yamashita Yumi , Kumazawa Yoshio , Nishikawa Shin-Ichi , Ohmori Hitoshi
Published: July 2002

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD+ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.
Endotoxin-tolerant Mice Have Mutations in Toll-like Receptor 4 (Tlr4)
Authors: Qureshi Salman T. , Larivière Line , Leveque Gary , Clermont Sophie , Moore Karen J. , Gros Philippe , Malo Danielle
Published: July 2002

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.
Dramatic Rise in Plasma Viremia after CD8+ T Cell Depletion in Simian Immunodeficiency Virus–infected Macaques
Authors: Jin Xia , Bauer Daniel E. , Tuttleton Sarah E. , Lewin Sharon , Gettie Agegnehu , Blanchard James , Irwin Craig E. , Safrit Jeffrey T. , Mittler John , Weinberger Leor , Kostrikis Leondios G. , Zhang Linqi , Perelson Alan S. , Ho David D.
Published: July 2002

To determine the role of CD8+ T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8+ T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo.
A Human Histocompatibility Leukocyte Antigen (HLA)-G–specific Receptor Expressed on All Natural Killer Cells
Authors: Rajagopalan Sumati , Long Eric O.
Published: July 2002

Human natural killer (NK) cells express several killer cell immunoglobulin (Ig)-like receptors (KIRs) that inhibit their cytotoxicity upon recognition of human histocompatibility leukocyte antigen (HLA) class I molecules on target cells. Additional members of the KIR family, including some that deliver activation signals, have unknown ligand specificity and function. One such KIR, denoted KIR2DL4, is structurally divergent from other KIRs in the configuration of its two extracellular Ig domains and of its transmembrane and cytoplasmic domains. Here we show that recombinant soluble KIR2DL4 binds to cells expressing HLA-G but not to cells expressing other HLA class I molecules. Unlike other HLA class I–specific KIRs, which are clonally distributed on NK cells, KIR2DL4 is expressed at the surface of all NK cells. Furthermore, functional transfer of KIR2DL4 into the cell line NK-92 resulted in inhibition of lysis of target cells that express HLA-G, but not target cells that express other class I molecules including HLA-E. Therefore, given that HLA-G expression is restricted to fetal trophoblast cells, KIR2DL4 may provide important signals to maternal NK decidual cells that interact with trophoblast cells at the maternal–fetal interface during pregnancy.
Enhanced Hematopoiesis by Hematopoietic Progenitor Cells Lacking Intracellular Adaptor Protein, Lnk
Authors: Takaki Satoshi , Morita Hatsue , Tezuka Yoshinari , Takatsu Kiyoshi
Published: July 2002

Hematopoietic stem cells (HSCs) give rise to variety of hematopoietic cells via pluripotential progenitors and lineage-committed progenitors and are responsible for blood production throughout adult life. Amplification of HSCs or progenitors represents a potentially powerful approach to the treatment of various blood disorders and to applying gene therapy by bone marrow transplantation. Lnk is an adaptor protein regulating the production of B cells. Here we show that Lnk is also expressed in hematopoietic progenitors in bone marrow, and that in the absence of Lnk, the number and the hematopoietic ability of progenitors are significantly increased. Augmented growth signals through c-Kit partly contributed to the enhanced hematopoiesis by lnk−/− cells. Lnk was phosphorylated by and associated with c-Kit, and selectively inhibited c-Kit–mediated proliferation by attenuating phosphorylation of Gab2 and activation of mitogen-activated protein kinase cascade. These observations indicate that Lnk plays critical roles in the expansion and function of early hematopoietic progenitors, and provide useful clues for the amplification of hematopoietic progenitor cells.
Sodium Channel Activation Gating Is Affected by Substitutions of Voltage Sensor Positive Charges in All Four Domains
Authors: Kontis Kris J. , Rounaghi Amir , Goldin Alan L.
Published: July 2002

The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel α subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III–IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910–10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.
A Conserved Arginine Residue in the Pore Region of an Inward Rectifier K Channel (IRK1) as an External Barrier for Cationic Blockers
Authors: Sabirov Ravshan Z. , Tominaga Tomoko , Miwa Akiko , Okada Yasunobu , Oiki Shigetoshi
Published: July 2002

The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg148, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2.1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K+ > Rb+ > NH4+ >> Na+) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg2+ and Ca2+, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg2+ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs+ and Ba2+, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg2+ or Ca2+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg148 is located between the external blocking site for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in the IRK1 channel. It is concluded that Arg148 serves as a barrier to cationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.