bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide
Authors: Boger Dale L. , Patterson Jean E. , Guan Xiaojun , Cravatt Benjamin F. , Lerner Richard A. , Gilula Norton B.
Published: July 2002

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16–24 carbons of which 16–18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Δ
9
cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve
in vivo
properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.
Mycobacterium bovisbacille Calmette–Guérin strains secreting listeriolysin ofListeria monocytogenes
Authors: Hess Jürgen , Miko Diana , Catic André , Lehmensiek Vera , Russell David G. , Kaufmann Stefan H. E.
Published: July 2002

Recombinant (r)Mycobacterium bovisstrains were constructed that secrete biologically active listeriolysin (Hly) fusion protein ofListeria monocytogenes. The r-BCG strains pAT261:Hly or pMV306:Hly expressed plasmid multicopies or chromosomal single copies of thehlygene, respectively. Human and murine macrophage-like cell lines were infected with r-BCG pAT261:Hly and pMV306:Hly strains. Interestingly, intracellular persistence of both r-BCG strains was reduced in macrophages as compared with the parental BCG strain. By immunogold labeling Hly was detected in membrane structures and within the phagosomal space of macrophages. In addition, Hly was localized within cytoplasmic vacuoles outside the mycobacteria-containing phagosome of host cells infected with r-BCG pAT261:Hly or r-BCG pMV306:Hly. Hly fusions consistently colocalized with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered. Although r-BCG pAT261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of cophagocytosed soluble protein as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly secreting r-BCG constructs are antituberculosis vaccine candidates.
Fish and mammals in the economy of an ancient Peruvian kingdom
Authors: Marcus Joyce , Sommer Jeffrey D. , Glew Christopher P.
Published: July 2002

Fish and mammal bones from the coastal site of Cerro Azul, Peru shed light on economic specialization just before the Inca conquest of A.D. 1470. The site devoted itself to procuring anchovies and sardines in quantity for shipment to agricultural communities. These small fish were dried, stored, and eventually transported inland via caravans of pack llamas. Cerro Azul itself did not raise llamas but obtainedcharqui(or dried meat) as well as occasional whole adult animals from the caravans. Guinea pigs were locally raised. Some 20 species of larger fish were caught by using nets; the more prestigious varieties of these show up mainly in residential compounds occupied by elite families.
IL-4 enhances proliferation and mediator release in mature human mast cells
Authors: Bischoff S. C. , Sellge G. , Lorentz A. , Sebald W. , Raab R. , Manns M. P.
Published: July 2002

Tissue mast cells (MC) are recognized as key effector cells of immediate-type allergic reactions releasing inflammatory mediators and cytokines on stimulation with antigen, but they also might be involved in IgE-independent inflammatory and tissue repair processes. The mechanism of human MC regulation in tissue is not fully understood. Here, we show that IL-4, in synergy with stem cell factor (SCF), regulates the function of purified human MC isolated from intestinal tissue. Whereas SCF induced only marginal proliferation of MC culturedin vitroup to 4 weeks, addition of IL-4 and SCF strongly increased the proliferation rate. Moreover, IL-4, which by itself had no visible effect on human MC, enhanced the release of histamine, leukotriene C4, and IL-5 in MC triggered by IgE receptor crosslinking. The IL-4 effects occurred in a dose-dependent fashion (ED50= 100 pg/ml) and could be totally blocked by a competitive IL-4 receptor antagonist. Our data indicate that IL-4 is an important regulator of human MC function and suggest that mature MC retain the capacity to proliferate in a particular tissue environment.
Protein prenylation in spinach chloroplasts
Authors: Parmryd Ingela , Andersson Bertil , Dallner Gustav
Published: July 2002

Protein prenylation in plants was studied by
in vivo
metabolic
3
H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach cotyledons, this posttranslational protein modification was found to be divided into two categories, one representing the conventional prenylation involving farnesyl and geranylgeranyl groups bound to cysteine residues via thioether linkages. This category revealed a similar pattern of prenylated proteins to that observed in mammalian cells and depends on nuclear gene expression. The other category was shown to represent a type of prenylation confined to chloroplasts. It depends on plastid gene expression and does not involve a thioether bond. The modifying isoprenoid could be released from the chloroplastic polypeptides by alkaline treatment and was identified as phytol upon GC-MS analysis. The phytol could readily be derived from all-
trans
-[
3
H]farnesol, which, like all-
trans
-[
3
H]geranylgeraniol, was taken up by the cotyledons, resulting in incorporation of radiolabel into proteins.
Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer
Authors: Chen Liaohai , McBranch Duncan W. , Wang Hsing-Lin , Helgeson Roger , Wudl Fred , Whitten David G.
Published: July 2002

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding “molecular excited states.” Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer
(−)
/quencher
(+)
], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.
In
Azotobacter vinelandii,
the E1 subunit of the pyruvate dehydrogenase complex binds
fpr
promoter region DNA and ferredoxin I
Authors: Regnström Karin , Sauge-Merle Sandrine , Chen Kaisheng , Burgess Barbara K.
Published: July 2002

In
Azotobacter vinelandii
, deletion of the
fdxA
gene that encodes a well characterized seven-iron ferredoxin (FdI) is known to lead to overexpression of the FdI redox partner, NADPH:ferredoxin reductase (FPR). Previous studies have established that this is an oxidative stress response in which the
fpr
gene is transcriptionally activated to the same extent in response to either addition of the superoxide propagator paraquat to the cells or to
fdxA
deletion. In both cases, the activation occurs through a specific DNA sequence located upstream of the
fpr
gene. Here, we report the identification of the
A. vinelandii
protein that binds specifically to the paraquat activatable
fpr
promoter region as the E1 subunit of the pyruvate dehydrogenase complex (PDHE1), a central enzyme in aerobic respiration. Sequence analysis shows that PDHE1, which was not previously suspected to be a DNA-binding protein, has a helix–turn–helix motif. The data presented here further show that FdI binds specifically to the DNA-bound PDHE1.
Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons
Authors: Morral Núria , O’Neal Wanda , Rice Karen , Leland Michele , Kaplan Johanne , Piedra Pedro A. , Zhou Heshan , Parks Robin J. , Velji Rizwan , Aguilar-Córdova Estuardo , Wadsworth Samuel , Graham Frank L. , Kochanek Stefan , Carey K. Dee , Beaudet Arthur L.
Published: July 2002

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α
1
-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes.
Progressive juvenile-onset punctate cataracts caused by mutation of the γD-crystallin gene
Authors: Stephan Dietrich A. , Gillanders Elizabeth , Vanderveen Deborah , Freas-Lutz Diana , Wistow Graeme , Baxevanis Andreas D. , Robbins Christiane M. , Van Auken Ann , Quesenberry Matthew I. , Bailey-Wilson Joan , Juo Suh-Hang Hank , Trent Jeffrey M. , Smith Lois , Brownstein Michael J.
Published: July 2002

Cataracts are a significant public health problem. Here, we describe the genetic alteration responsible for a progressive form of cataract, segregating as an autosomal dominant trait in a three-generation pedigree. Unlike most autosomal dominant cataracts, these are not clinically apparent at birth but are initially observed in the first year or two of life. The opacification evolves relatively slowly, generally necessitating removal of the lens in childhood or early adolescence. A genome-wide search in our kindred revealed linkage at 2q33–35 where the γ-crystallin gene cluster resides. A single base alteration resulting in an Arg- 14 → Cys (R14C) substitution in γD-crystallin was subsequently identified. Protein modeling suggests that the effect of this mutation is a subtle one, affecting the surface properties of the crystallin molecule rather than its tertiary structure, consistent with the fact that the patients’ lenses are normal at birth. This is the first gene defect shown to be responsible for a noncongenital progressive cataract, and studying the defective protein should teach us more about the mechanisms underlying cataract formation.
Melan-A/MART-151–73represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4+T cells
Authors: Zarour Hassane M. , Kirkwood John M. , Kierstead Lisa Salvucci , Herr Wolfgang , Brusic Vladimir , Slingluff Craig L. , Sidney John , Sette Alessandro , Storkus Walter J.
Published: July 2002

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8+cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4+T cells. The Melan-A/MART-151–73peptide was able to induce thein vitroexpansion of specific CD4+T cells derived from normal DR4+donors or from DR4+patients with melanoma when pulsed onto autologous dendritic cells. CD4+responder T cells specifically produced IFN-γ in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-151–73peptide and DR4+melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4+T cell immunoreactivity against the Melan-A/MART-151–73peptide typically coexisted with a high frequency of anti-Melan-A/MART-127–35reactive CD8+T cells in freshly isolated blood harvested from HLA-A2+/DR4+patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4+T cell responses against melanomain vivo.
Catalytic tempering: A method for sampling rough energy landscapes by Monte Carlo
Authors: Stolovitzky Gustavo , Berne B. J.
Published: July 2002

A new Monte Carlo algorithm is presented for the efficient sampling
of the Boltzmann distribution of configurations of systems with rough
energy landscapes. The method is based on the introduction of a
fictitious coordinate
y
so that the dimensionality of the
system is increased by one. This augmented system has a potential
surface and a temperature that is made to depend on the new coordinate
y
in such a way that for a small strip of the
y
space, called the “normal region,” the temperature is set equal
to the temperature desired and the potential is the original rough
energy potential. To enhance barrier crossing outside the “normal
region,” the energy barriers are reduced by truncation (with
preservation of the potential minima) and the temperature is made to
increase with |
y
|. The method, called catalytic
tempering or CAT, is found to greatly improve the rate of convergence
of Monte Carlo sampling in model systems and to eliminate the
quasi-ergodic behavior often found in the sampling of rough energy
landscapes.
Efficient studies of long-distance
Bmp5
gene regulation using bacterial artificial chromosomes
Authors: DiLeone Ralph J. , Marcus Gregory A. , Johnson Michelle D. , Kingsley David M.
Published: July 2002

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse
bone morphogenetic protein-5
(
Bmp5
) gene. Simple coinjection of large insert clones with
lacZ
reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the
Bmp5
gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the
Bmp5
locus, to test whether
Bmp5
control regions act similarly on endogenous and foreign promoters, and to show that
Bmp5
control elements are capable of rescuing phenotypic effects of a
Bmp5
deficiency. This rapid approach has identified new
Bmp5
control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.
Biodiversity of Costa Rican salamanders: Implications of high levels of genetic differentiation and phylogeographic structure for species formation
Authors: García-París Mario , Good David A. , Parra-Olea Gabriela , Wake David B.
Published: July 2002

Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenusBolitoglossa(Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genusBolitoglossain Costa Rica reveals strong phylogeographic structure within a single species,Bolitoglossa pesrubra.Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display “tropicality” in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.
The LPS‐induced neutrophil recruitment into rat air pouches is mediated by TNFα: likely macrophage origin
Authors: Arreto C-D. , Dumarey C. , Nahori M-A. , Vargaftig B. B.
Published: July 2002

The role of resident cells during the lipopolysaccharide (LPS)‐induced neutrophil recruitment into rat air pouches was investigated. In this model, LPS (Escherichia coli, O55: B5 strain; 2–2000 ng) induced a dose– and time‐dependent neutrophil recruitment accompanied by the generation of a tumour necrosis factor‐α (TNFα)‐like activity. Dexamethasone (0.05–5 mug) and cycloheximide (6 ng), injected 2 h before LPS into the pouches, inhibited the neutrophil recruitment and the generation of the TNFα‐like activity, while the H1‐receptor antagonist mepyramine (1 and 4 mg/kg, i.p., 0.5 h before LPS) and the PAF‐receptor antagonist WEB 2170 (0.05 and 1 mg/kg, i.p., 0.5 h before LPS) had no effect. Purified alveolar macrophages (AM) were used to replenish the pouches of cycloheximide‐treated recipient rats. AM provided by PBS‐treated animals led to the recovery of the LPS‐induced neutrophil recruitment and of the TNFα‐like formation contrasting with those from cycloheximide‐treated animals (1 mg/kg, i.p.). When delivered in situ, liposome‐encapsulated clodronate, a macrophage depletor, significantly impaired both the LPSinduced neutrophil recruitment and the TNFα‐like activity. An anti‐murine TNFα polyclonal antibody (0.5 h before LPS) was also effective. These results emphasize the pivotal role of macrophages for LPS‐induced neutrophil recruitment via the formation of TNFα.
Differential response to dexamethasone on the TXB2 release in guinea‐pig alveolar macrophages induced by zymosan and cytokines
Authors: Salgueiro M. E. , Conde M. , Seco A. J. , Méndez N. , Manso G.
Published: July 2002

Glucocorticosteroids reduce the production of inflammatory mediators but this effect may depend on the stimulus. We have compared the time course of the effect of dexamethasone on the thromboxane B2 (TXB2) release induced by cytokine stimulation and zymosan in guinea‐pig alveolar macrophages. Interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and opsonized zymosan (OZ), all stimulate TXB2 release. High concentrations of dexamethasone (1–10 μM) inhibit the TXB2 production induced by both cytokines and OZ, but the time course of this response is different. Four hours of incubation with dexamethasone reduce the basal
TXB2 release and that induced by IL‐1β and TNF‐α, but do not modify the TXB2 release induced by OZ. However, this stimulus was reduced after 24 h incubation. Our results suggest that the antiinflammatory activity of glucocorticosteroids shows some dependence on stimulus and, therefore, may have more than one mechanism involved.
Cocaine‐Induced Mood Disorder: Prevalence Rates and Psychiatric Symptoms in an Outpatient Cocaine‐Dependent Sample
Authors: Siqueland Lynne , Horn Amanda , Moras Karla , Woody George , Weiss Roger , Blaine Jack , Bishop Sarah , Barber Jacques , Thase Michael
Published: July 2002

This paper attempts to examine and compare prevalence rates and symptom patterns of DSM substance‐induced and other mood disorders. 243 cocaine‐dependent outpatients with cocaine‐induced mood disorder (CIMD), other mood disorders, or no mood disorder were compared on measures of psychiatric symptoms. The prevalence rate for CIMD was 12% at baseline. Introduction of the DSM‐IV diagnosis of CIMD did not substantially affect rates of the other depressive disorders. Patients with CIMD had symptom severity levels between those of patients with and without a mood disorder. These findings suggest some validity for the new DSM‐IV diagnosis of CIMD, but also suggest that it requires further specification and replication.
Amplification of the MYC Gene in Osteosarcoma Secondary to Paget′s Disease of Bone
Authors: Ueda Takafumi , Healey John H. , Huvos Andrew G. , Ladanyi Marc
Published: July 2002

Purpose. In a previous series of 25 human osteosarcoma samples studied for MYC gene amplification, we found
amplification in two cases (8%), including one arising in association with Paget′s disease (pagetic osteosarcoma). Based
on this observation, we further investigated the prevalence of MYC gene amplification in pagetic osteosarcomas.

Methods. MYC gene amplification was assessed by Southern blot analysis using frozen tissue samples in five cases of
pagetic osteosarcoma and 53 cases of primary (non‐pagetic) osteosarcoma. Amplification was considered present if the
MYC copy number was six or greater.

Results. Three out of five patients (60%) with pagetic osteosarcoma showed MYC gene amplification, whereas it was
present in only 5/53 patients (9.4%) with primary osteosarcoma. The incidence of MYC amplification in pagetic
osteosarcoma was thus significantly higher than that in primary osteosarcoma (p = 0.016).

Discussion. The finding that MYC gene amplification may be more common in pagetic than primary osteosarcoma
warrants further study and suggests pathogenetic differences between primary osteosarcomas and those arising in the
setting of Paget′s disease. Three of the four pagetic osteosarcomas from the present study were previously shown to be
immunoreactive for p53, suggesting that p53 mutation may also be a frequent genetic lesion in these tumors.
Detection of EWS/FLI‐1 by Immunostaining. An Adjunctive Tool in Diagnosis of Ewing′s Sarcoma and Primitive Neuroectodermal Tumour on Cytological Samples and Paraffin‐Embedded Archival Material
Authors: Nilsson Gunnar , Wang Min , Wejde Johan , Kreicbergs Andris , Larsson Olle
Published: July 2002

Purpose. Recently we showed that the 68‐kDa fusion protein derived
from the EWS/FLI1 hybrid gene can be specifically detected by Western blotting using
a polyclonal antibody to the C‐terminal of FLI1 on biopsy material from Ewing′s
sarcoma. The aim of this study was to investigate whether this antibody also could be
used for immunocytochemistry and immunohistochemistry in diagnosis of
Ewing′s sarcoma.

Methods. Immunostaining on paraffin‐embedded archival material,
fine‐needle aspirates and tumour touch imprints from Ewing′s sarcomas and primitive
neuroectodermal tumours (PNET) for detection of the fusion protein was performed.
Most cases were also analysed by Western blotting.Tumours of differential
diagnostic importance were also included.

Results. Eighty per cent (12/15 cases) of the Ewing tumours exhibited a positive
immunoreactivity for the FLI1 antibody. The signal was mainly localised in the nuclei
of the tumour cells, which seems reasonable since EWS/FLI1 is a transcription factor.
The signal was found to be specific since it did not appear when the blocking peptide
was added to the antibody solution.Moreover, two other types of small‐round cell tumours
(i.e. neuroblastoma and alveolar rhabdomyosarcoma) were negative as well as most
normal tissues.

Discussion. Immunostaining of histological and cytological specimens
with the FLI1 antibody can be of diagnostic relevance in Ewing tumours carrying
t(11;22).The absence of immunoreactivity in non‐Ewing cells is most likely due to a
low expression of the wild‐type FLI1 protein.
ERS-24, a Mammalian v-SNARE Implicated in Vesicle Traffic between the ER and the Golgi
Authors: Paek Inbok , Orci Lelio , Ravazzola Mariella , Erdjument-Bromage Hediye , Amherdt Mylene , Tempst Paul , Söllner Thomas H. , Rothman James E.
Published: July 2002

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.
N-Cadherin Extracellular Repeat 4 Mediates Epithelial to Mesenchymal Transition and Increased Motility
Authors: Kim Jae-Beom , Islam Shahidul , Kim Young J. , Prudoff Ryan S. , Sass Kristin M. , Wheelock Margaret J. , Johnson Keith R.
Published: July 2002

E- and N-cadherin are members of the classical cadherin family of proteins. E-cadherin plays an important role in maintaining the normal phenotype of epithelial cells. Previous studies from our laboratory and other laboratories have shown that inappropriate expression of N-cadherin by tumor cells derived from epithelial tissue results in conversion of the cell to a more fibroblast-like cell, with increased motility and invasion. Our present study was designed to determine which domains of N-cadherin make it different from E-cadherin, with respect to altering cellular behavior, such as which domains are responsible for the epithelial to mesenchymal transition and increased cell motility and invasion. To address this question, we constructed chimeric cadherins comprised of selected domains of E- and N-cadherin. The chimeras were transfected into epithelial cells to determine their effect on cell morphology and cellular behavior. We found that a 69–amino acid portion of EC-4 of N-cadherin was necessary and sufficient to promote both an epithelial to mesenchymal transition in squamous epithelial cells and increased cell motility. Here, we show that different cadherin family members promote different cellular behaviors. In addition, we identify a novel activity that can be ascribed to the extracellular domain of N-cadherin.
Mitotic Spindle Integrity and Kinetochore Function Linked by the Duo1p/Dam1p Complex
Authors: Cheeseman Iain M. , Enquist-Newman Maria , Müller-Reichert Thomas , Drubin David G. , Barnes Georjana
Published: July 2002

Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.
Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways
Authors: Puri Vishwajeet , Watanabe Rikio , Singh Raman Deep , Dominguez Michel , Brown Jennifer C. , Wheatley Christine L. , Marks David L. , Pagano Richard E.
Published: July 2002

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.
EGF-R signaling through Fyn kinase disrupts the function of integrin α6β4 at hemidesmosomes
Authors: Mariotti Agnese , Kedeshian Paul A. , Dans Michael , Curatola Anna Maria , Gagnoux-Palacios Laurent , Giancotti Filippo G.
Published: July 2002

We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin α6β4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the β4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of β4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with α6β4. By contrast, Src and Lck do not associate with α6β4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of β4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the β4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.
Roles of fission yeast tea1p in the localization of polarity factors and in organizing the microtubular cytoskeleton
Authors: Behrens Ralf , Nurse Paul
Published: July 2002

The cylindrical shape of the fission yeast cell is generated by linear polarized growth from its cell ends. Using immunofluorescence and live imaging microscopy, we have investigated the roles of the cell end marker tea1p in generating linear polarized growth. We found that tea1p is primarily transported on plus ends of microtubules from the vicinity of the nucleus to the cell ends, and that its movement near the nucleus is independent of the kinesin tea2p. Deletion analysis identified a coiled-coil domain in tea1p essential for its retention at cell ends, and demonstrated that tea1p exerts different functions dependent on its location. On the tips of microtubules, tea1p prevents the curling of microtubules around the cell ends, whereas it is required for maintaining linear cell growth and for retention of polarity factors such as the Dyrk kinase pom1p, the CLIP170-like tip1p, and tea2p at the cell ends. We propose that tea1p has roles in organizing the microtubule cytoskeleton on the tips of microtubules, and in the retention of factors at the cell ends necessary for the cell to grow in a straight line.
Induction of Airway Mucus Production By T Helper 2 (Th2) Cells: A Critical Role For Interleukin 4 In Cell Recruitment But Not Mucus Production
Authors: Cohn Lauren , Homer Robert J. , Marinov Anthony , Rankin John , Bottomly Kim
Published: July 2002

Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased mucus secretion is an important clinical symptom and contributes to airway obstruction in asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4–deficient mice were not recruited to the lung and did not induce mucus production. When this defect in homing was overcome by administration of TNF-α, IL-4 −/− Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.
Age-resolving Osteopetrosis: A Rat Model Implicating Microphthalmia and the Related Transcription Factor TFE3
Authors: Weilbaecher Katherine N. , Hershey Christine L. , Takemoto Clifford M. , Horstmann Martin A. , Hemesath Timothy J. , Tashjian Armen H. , Fisher David E.
Published: July 2002

Microphthalmia (Mi) is a basic helix-loop-helix–leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3′ half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.
Virulent Strains of Helicobacter pylori Demonstrate Delayed Phagocytosis and Stimulate Homotypic Phagosome Fusion in Macrophages
Authors: Allen Lee-Ann H. , Schlesinger Larry S. , Kang Byoung
Published: July 2002

Helicobacter pylori colonizes the gastric epithelium of ∼50% of the world's population and plays a causative role in the development of gastric and duodenal ulcers. H. pylori is phagocytosed by mononuclear phagocytes, but the internalized bacteria are not killed and the reasons for this host defense defect are unclear. We now show using immunofluorescence and electron microscopy that H. pylori employs an unusual mechanism to avoid phagocytic killing: delayed entry followed by homotypic phagosome fusion. Unopsonized type I H. pylori bound readily to macrophages and were internalized into actin-rich phagosomes after a lag of ∼4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next ∼2 h. The resulting “megasomes” contained multiple viable organisms and were stable for 24 h. Phagosome–phagosome fusion required bacterial protein synthesis and intact host microtubules, and both chloramphenicol and nocodazole increased killing of intracellular H. pylori. Type II strains of H. pylori are less virulent and lack the cag pathogenicity island. In contrast to type I strains, type II H. pylori were rapidly ingested and killed by macrophages and did not stimulate megasome formation. Collectively, our data suggest that megasome formation is an important feature of H. pylori pathogenesis.
Induction and Suppression of Collagen-Induced Arthritis Is Dependent on Distinct Fcγ Receptors
Authors: Kleinau Sandra , Martinsson Pernilla , Heyman Birgitta
Published: July 2002

Receptors for immunoglobulin (Ig)G (FcγRs) are important for the antibody-mediated effector functions of the immune system. FcγRI and FcγRIII trigger cell activation through a common γ chain, whereas FcγRII acts as a negative regulator of antibody production and immune complex–triggered activation. Here we describe the in vivo consequences of FcγR deficiency in a mouse model of human rheumatoid arthritis. FcRγ chain–deficient mice on arthritis-susceptible DBA/1 background were immunized with collagen for induction of collagen-induced arthritis. The DBA/1 mice lacking FcRγ chain were protected from collagen-induced arthritis in contrast to wild-type mice, although both groups produced similar levels of IgG anticollagen antibodies. In comparison, DBA/1 mice lacking FcγRII developed an augmented IgG anticollagen response and arthritis. These observations suggest a crucial role of FcγRI and FcγRIII in triggering autoimmune arthritis.
An Alternative Open Reading Frame of the Human Macrophage Colony-Stimulating Factor Gene Is Independently Translated and Codes for an Antigenic Peptide of 14 Amino Acids Recognized by Tumor-Infiltrating Cd8 T Lymphocytes
Authors: Probst-Kepper Michael , Stroobant Vincent , Kridel Robert , Gaugler Béatrice , Landry Claire , Brasseur Francis , Cosyns Jean-Pierre , Weynand Birgit , Boon Thierry , Van den Eynde Benoit J.
Published: July 2002

We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.
Induction of a Diverse T Cell Receptor γ/δ Repertoire by the Helix-Loop-Helix Proteins E2a and Heb in Nonlymphoid Cells
Authors: Ghosh Jimut Kanti , Romanow William J. , Murre Cornelis
Published: July 2002

During specific stages of thymocyte development, the T cell receptor (TCR) locus is assembled from variable (V), diversity (D), and joining (J) gene segments. Proper TCR γ and δ V(D)J rearrangement during thymocyte development requires the presence of the E2A proteins. Here we show that E2A and a closely related protein, HEB, in the presence of recombination activating gene (RAG)1 and RAG2, each have the ability to activate TCR γ and δ rearrangement in human kidney cells. The coding joints are diverse, contain nucleotide deletions, and occasionally show the presence of P nucleotides. Interestingly, only a subset of V, D, and J segments are targeted by the E2A and HEB proteins. Thus, E2A and HEB permit localized accessibility of the TCR γ and δ loci to the recombination machinery. These data indicate that a distinct but diverse TCR repertoire can be induced in nonlymphoid cells by the mere presence of the V(D)J recombinase and the transcriptional regulators, E2A and HEB.
CC Chemokine Receptor 7–dependent and –independent Pathways for Lymphocyte Homing
Authors: Henning Golo , Ohl Lars , Junt Tobias , Reiterer Phillip , Brinkmann Volker , Nakano Hideki , Hohenberger Werner , Lipp Martin , Förster Reinhold
Published: July 2002

Cognate interaction of chemokine receptor CCR7 on lymphocytes with its ligands CCL19 and CCL21 expressed on high endothelial venules (HEVs) is essential for effective migration of T and B cells across HEVs into secondary lymphoid organs. Plt mice, which lack expression of CCL19 and CCL21-ser, both ligands for CCR7 on HEVs, as well as CCR7-deficient mice, have a defective cell migration and reduced homing of lymphocytes. FTY720, a novel immunosuppressant, causes a reduction of lymphocytes in peripheral blood and tissues and their sequestration into lymphoid tissues. In this study we demonstrate that FTY720 rescues the homing defect in both CCR7−/− mice and plt mice. After FTY720 treatment, the number of CD4+ and CD8+ T cells as well as B cells in peripheral blood is reduced while pertussis toxin–sensitive homing into peripheral lymph nodes, mesenteric lymph node, and Peyer's patches is increased. Immunohistology demonstrates that FTY720 enables these cells to enter lymphoid tissue through HEVs. Thus, our data suggest an alternative G-αi-dependent, CCR7-CCL19/CCL21-independent mechanism for lymphocyte homing through HEVs which is strongly augmented in the presence of FTY720.
Properties of a Novel pH-dependent Ca2+ Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function
Authors: Santi Celia M. , Santos Teresa , Hernández-Cruz Arturo , Darszon Alberto
Published: July 2002

Rises of intracellular Ca2+ ([Ca2+]i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca2+]i and intracellular pH (pHi) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca2+ channels modulated by pHi in the origin of these Ca2+ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca2+]i on identified spermatogenic cells from mouse adult testes. In BCECF [(2′,7′)-bis(carboxymethyl)- (5,6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30–60 s) application of 25 mM NH4Cl increased pHi by ∼1.3 U from a resting pHi ∼6.65. A steady pHi plateau was maintained during NH4Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca2+]i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca2+ influx or intracellular Ca2+ release demonstrated that the majority of the Ca2+ rise results from plasma membrane Ca2+ influx, although a small component likely to result from intracellular Ca2+ release was occasionally observed. Ca2+ transients potentiated with repeated NH4Cl applications, gradually obliterating the initial [Ca2+]i drop. The pH-sensitive Ca2+ permeation pathway allows the passage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorganic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blocker nifedipine. The magnitude of these Ca2+ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca2+ channel also present in spermatogenic cells. We postulate that the Ca2+ permeation pathway regulated by pHi, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx required for initiating the acrosome reaction and perhaps other important sperm functions.