bluuebunny/crossref_metadata_2025 huggingface dataset:
Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion
Authors: Strömberg Anette , Ryttsén Frida , Chiu Daniel T. , Davidson Max , Eriksson Peter S. , Wilson Clyde F. , Orwar Owe , Zare Richard N.
Published: July 2002

A method for cell–cell and cell–liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell–cell or cell–liposome pair was achieved by the application of a highly focused electric field through a pair of 5-μm o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell–liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (γ-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.
Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins
Authors: Lombardi Angela , Summa Christopher M. , Geremia Silvano , Randaccio Lucio , Pavone Vincenzo , DeGrado William F.
Published: July 2002

De novoprotein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit ofEscherichia coliribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.
Functional implications from crystal structures of the conserved
Bacillus subtilis
protein Maf with and without dUTP
Authors: Minasov George , Teplova Marianna , Stewart George C. , Koonin Eugene V. , Anderson Wayne F. , Egli Martin
Published: July 2002

Three-dimensional structures of functionally uncharacterized proteins may furnish insight into their functions. The potential benefits of three-dimensional structural information regarding such proteins are particularly obvious when the corresponding genes are conserved during evolution, implying an important function, and no functional classification can be inferred from their sequences. The
Bacillus subtilis
Maf protein is representative of a family of proteins that has homologs in many of the completely sequenced genomes from archaea, prokaryotes, and eukaryotes, but whose function is unknown. As an aid in exploring function, we determined the crystal structure of this protein at a resolution of 1.85 Å. The structure, in combination with multiple sequence alignment, reveals a putative active site. Phosphate ions present at this site and structural similarities between a portion of Maf and the anticodon-binding domains of several tRNA synthetases suggest that Maf may be a nucleic acid-binding protein. The crystal structure of a Maf-nucleoside triphosphate complex provides support for this hypothesis and hints at di- or oligonucleotides with either 5′- or 3′-terminal phosphate groups as ligands or substrates of Maf. A further clue comes from the observation that the structure of the Maf monomer bears similarity to that of the recently reported
Methanococcus jannaschii
Mj0226 protein. Just as for Maf, the structure of this predicted NTPase was determined as part of a structural genomics pilot project. The structural relation between Maf and Mj0226 was not apparent from sequence analysis approaches. These results emphasize the potential of structural genomics to reveal new unexpected connections between protein families previously considered unrelated.
Regulation of the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa
in vivo
by dopamine D
1
, dopamine D
2
, and adenosine A
2A
receptors
Authors: Svenningsson P. , Lindskog M. , Ledent C. , Parmentier M. , Greengard P. , Fredholm B. B. , Fisone G.
Published: July 2002

Dopamine D
1
, dopamine D
2
, and adenosine A
2A
receptors are highly expressed in striatal medium-sized spiny neurons. We have examined,
in vivo
, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D
2
receptor antagonist eticlopride (0.1–2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A
2A
receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A
2A
receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D
1
receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that,
in vivo
, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D
1
, D
2
, and A
2A
receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D
2
antagonists
in vivo
.
Phylogeny of seed plants based on all three genomic compartments: Extant gymnosperms are monophyletic and Gnetales' closest relatives are conifers
Authors: Bowe L. Michelle , Coat Gwénaële , dePamphilis Claude W.
Published: July 2002

Efforts to resolve Darwin's “abominable mystery”—the origin of angiosperms—have led to the conclusion that Gnetales and various fossil groups are sister to angiosperms, forming the “anthophytes.” Morphological homologies, however, are difficult to interpret, and molecular data have not provided clear resolution of relationships among major groups of seed plants. We introduce two sequence data sets from slowly evolving mitochondrial genes,
cox
1 and
atp
A, which unambiguously reject the anthophyte hypothesis, favoring instead a close relationship between Gnetales and conifers. Parsimony- and likelihood-based analyses of plastid
rbc
L and nuclear 18S rDNA alone and with
cox
1 and
atp
A also strongly support a gnetophyte–conifer grouping. Surprisingly, three of four genes (all but nuclear rDNA) and combined three-genome analyses also suggest or strongly support Gnetales as derived conifers, sister to Pinaceae. Analyses with outgroups screened to avoid long branches consistently identify all gymnosperms as a monophyletic sister group to angiosperms. Combined three- and four-gene rooted analyses resolve the branching order for the remaining major groups—cycads separate from other gymnosperms first, followed by
Ginkgo
and then (Gnetales + Pinaceae) sister to a monophyletic group with all other conifer families. The molecular phylogeny strongly conflicts with current interpretations of seed plant morphology, and implies that many similarities between gnetophytes and angiosperms, such as “flower-like” reproductive structures and double fertilization, were independently derived, whereas other characters could emerge as synapomorphies for an expanded conifer group including Gnetales. An initial angiosperm–gymnosperm split implies a long stem lineage preceding the explosive Mesozoic radiation of flowering plants and suggests that angiosperm origins and homologies should be sought among extinct seed plant groups.
Cognition and character traits as determinants of young children’s behaviour in traffic situations
Authors: Briem Valdimar , Bengtsson Hans
Published: July 2002

Young children, 3 to 6 years’ old, were observed in two situations: (1) a traffic model, where they used dolls to enact the movements of two children on the way to and from day care; and (2) as they crossed a lightly trafficked, minor road in a situation analogous to that in the model. Atotal of 131 children participated. All were tested in the model situation (a), both on understanding of safety and safety devices and on road-crossing behaviour. The latter was seen as a task consisting of three components (i) using a zebra crossing, (ii) stopping at the curb, and (iii) looking for cars. Asubgroup of 47 children was tested on three character traits, activity, distraction, and impulsivity. Another subgroup of 45 children participated in the roadside situation (b). The results show that although both age and understanding were important predictors of appropriate behaviour in both traffic situations, the behaviour components were differentially related to these factors. Of the character traits, impulsivity was found to be reliably related to traffic behaviour.
Timing of School-to-Work Transitions: A Developmental-Contextual Perspective
Authors: Reitzle Matthias , Vondracek Fred W. , Silbereisen Rainer K.
Published: July 2002

The critical role of the timing of person-context interactions, a central notion of developmental contextualism, is recognised as important in the conceptualisation of the school-to-work transition. As a result, an expanded framework for understanding this transition is employed in the present study. A stratified sample of young adults from former East and from West Germany participated in a 1991 national survey of youth. Retrospective data concerning life events during childhood and adolescence, and progress through school and occupational training, as well as extensive current information regarding attitudes, values, and occupational status, were analysed through the use of survival analyses. Findings revealed important differences in how individual differences and contextual factors contributed to the school-to-work transition in the contrasting environments of former East and West Germany.
Career Development of Academics: Cross-cultural and Lifespan Factors
Authors: Poole Millicent , Bornholt Laurel
Published: July 2002

This study takes a lifespan perspective on academic career development across several countries, in examining contributions to aspects of academic work in terms of gender, age groups, and years of experience in higher education. The study was based on the recent International Survey of the Academic Profession (Altbach, 1996). Findings suggest common themes regarding attitudes and activities within the gendered context of academic work that vary from one country to another and among working conditions, activities of teaching, research and service, issues of governance and management, and international dimensions of academic work. A general model is then described of activities and attitudes that constitute academic work. These findings are discussed in terms of strategies for career development that optimise the academic in a context.
Levels of soluble ICAM‐1 in premature and full‐term neonates with infection
Authors: Apostolou Melita , Dimitriou Helen , Kaleyias Joseph , Perdikogianni Chrissoula , Stiakaki Eftichia , Costalos Christos , Kalmanti Maria
Published: July 2002

Background: Infection in the neonatal period is an extremely serious condition and diagnosis is difficult. C‐reactive protein (CRP) is widely used as a marker of infection; however, its usefulness is limited in the early phase. The role of soluble intracellular adhesion molecule‐1 (sICAM‐1), an adhesion molecule, has been examined in recent studies as an early marker of neonatal infection with controversial results.

Aim: Assessment of sICAM‐1 concentrations and correlation with CRP, which is the currently used marker of infection, in order to use sICAM as an early diagnostic tool in neonates suspected for infection

Methods: Blood samples and blood cultures were obtained from two groups of pre‐term and full‐term neonates with clinical suspicion of infection prior to the initiation of antibiotics. The sICAM‐1 and CRP values were compared with the corresponding non‐infected ones
(n = 10 each).

Results: The sICAM‐1 levels were found increased in the group of both premature and term neonates with infection compared with the corresponding healthy ones (p < 0.0001). Prematurity combined with infection resulted in excessive increase of the levels of sICAM‐1 in comparison with full‐term infected newborns (p < 0.001). CRP values were normal in all samples except one in both full‐term and premature infected neonates on day 1 of clinically suspected infection. Serial detection of CRP values on days 2 and 4 of infection revealed a pattern according to which CRP values in premature neonates continued rising, while in the group of full terms these values, after rising on the second day, lowered on day 4.

Conclusions: Increased sICAM‐1 levels can be detected early in both full‐term and premature neonates with sepsis while CRP levels are within normal range at the sametime. Assessment of sICAM‐1 concentrationsmay be used as a diagnostic tool in neonates suspected for infection, resulting in earlier initiation of antibiotic therapy and therefore improving their outcome.
Cromoglycate and nedocromil enhanced the reactive oxygen species‐dependent suppressions with, but not without, dexamethasone in ischaemic and histamine paw oedema of mice
Authors: Oyanagui Y.
Published: July 2002

Anti‐inflammatory actions of two anti‐allergic drugs, alone or with dexamethasone (Dex) were examined in two models, because inflammation is claimed to be important for allergic events, especially for asthma. Cromoglycate and nedocromil were tested in ischaemic‐ and histamineinduced paw oedema models of mice. These antiallergic drugs (1–100 mg/kg, i.p.) failed to suppress these oedemata, but enhanced the suppressions by a low dose of dexamethasone (0.1 mg/kg, s.c.) at 3–8 h after Dex injection. The mode of effects by anti‐allergic drugs resembled that of a natural antioxidant (α‐tocopherol, β‐carotene etc.), and was different from that of an immunosuppressant like FK506. The enhancing potencies of the two anti‐allergic drugs were similar at 6 h after Dex in both oedemata, and were diminished by superoxide dismutase (SOD) or catalase (i.p.). Cycloheximide completely abolished suppressions. Nedocromil, but not cromoglycate, inhibits inflammatory events. Therefore, there are common unknown actions by which the two anti‐allergics enhance suppression by Dex. A possible mechanism of this action was supposed to enhance the superoxide and/or hydrogen peroxide‐dependent glucocorticoid receptor (GR) signalling in the target cells.
The immunoregulatory abilities of polymorphonuclear neutrophils in the course of multiple sclerosis
Authors: Ziąber J. , PaŚnik J. , Baj Z. , Pokoca L. , Chmielewski H. , Tchórzewski H.
Published: July 2002

The polymorphonuclear neutrophils (PMN) possess sufficient potential to affect both immune response and inflammation, however it has not been yet described in the course of multiple sclerosis (MS). We have studied binding of fluorescein isothiocyanate (FITC)‐ stained TNF‐α by PMN, the expression of CD11a, CD11b, and CD18 molecules of β 2‐integrines and the expression of CD10 (neutral endopeptidaseNEP) and of CD13 (aminopeptidase N; APN) antigens on PMN in three different groups of MS patients. The control group included neurological patients (OND) with noninflammatory diseases. The obtained results have proved that during MS exacerbation and in the course of chronic progressive MS, PMN reveal several forms of preactivation, including significantly higher stained‐TNF‐α binding, higher expression of CD11b and CD18, as well as CD10 and CD13 antigens, in comparison with MS remission or OND. We suggest that the increased expression of these molecules on PMN of MS patients in exacerbation of the disease and to a lower degree in the course of CP‐MS is a result of PMN priming, and directly prove the PMN involvement in the disease pathogenesis.
Alterations in excitatory amino acid‐mediated regulation of midbrain dopaminergic neurones induced by chronic psychostimulant administration and stress: relevance to behavioural sensitization and drug addiction
Authors: CLARK DAVID , OVERTON PAUL G.
Published: July 2002

Repeated, intermittent administration of the psychostimulants d‐amphetamine and cocaine, as well as other drugs of abuse, leads to an enduring augmentation of certain behavioural responses (e.g. locomotor activity) produced by these drugs. This behavioural sensitization has been the subject of considerable interest due to its potential relevance to drug addiction. Repeated administration of d‐amphetamine also leads to an enhancement in the ability of electrical stimulation of the prefrontal cortex to induce burst firing in midbrain dopaminergic (DA) neurones. This hyper‐responsiveness probably reflects a potentiation of transmission at excitatory amino acid (EAA)ergic synapses on DA neurones. In addition, we have previously reported that selective activation of mineralocorticoid receptors (MRs) by corticosterone leads to a potentiation of EAA‐induced burst firing in midbrain DA neurones, an effect antagonized by glucocorticoid receptor (GR) activation. In this review article, we propose a model describing how drugs of abuse and stress alter EAA function at the level of DA cells in the ventral tegmental area (VTA), which can result in a long‐lasting impact on behaviour. D‐amphetamine produces a transitory increase in EAA‐mediated transmission at the level of DA cells in the VTA, which triggers a more long‐lasting change in EAAergic function resembling hippocampal long‐term potentiation. Dopaminergic burst events are likely to be a critical link between enhanced EAAergic activity in afferents synapsing on DA neurones and plasticity at these synapses, by increasing calcium transport into the cell, which is known to be an important factor in synaptic plasticity. Selective MR occupation by corticosterone in the VTA facilitates the development of this plasticity. However, we hypothesize that during stress, GR‐occupation also activates EAAergic afferents to DA neurones in a manner similar to that following psychostimulants. Under these circumstances, GR‐occupation acts via circuitry external to the VTA, which may include the hippocampus. Thus, potentiation of EAAergic synapses on DA neurones in the VTA may represent a final common pathway by which two divserse means (psychostimulants and stress) achieve the same end (sensitization). Alterations in EAA‐mediated transmission at the level of DA cells not only plays a critical role in the induction of behavioural sensitization, but probably continues to produce abnormal DA cell responses in the drug‐free situation.
Metastatic Osteosarcoma: A Review of Current Issues in Systemic Treatment
Authors: Bramwell Vivien
Published: July 2002

Purpose. Original articles and abstracts published between January 1991 and January 1997
were selected according to specified criteria and reviewed to provide answers to five interesting questions about the
systemic treatment of metastatic osteosarcoma.

Results.

(1) In patients with metastatic disease at presentation, what is the outcome after intensive multi‐agent chemotherapy?

Historically, survival has been poor, but may be improving with the use of ifosfamide‐containing regimens.

(2) Can response to new agents be evaluated better in patients who have received no previous chemotherapy?

Based on limited data, this is probably true.

(3) Is the response to neo‐adjuvant chemotherapy, as determined by histopathology, similar for the primary tumor and
synchronous pulmonary metastases?

With intensive multi‐agent chemotherapy, good histological response rates are in the range 70–90% for both groups.

(4) What is the outcome, after intensive combined modality treatment with chemotherapy and surgery, in patients
relapsing with metastases after previous adjuvant chemotherapy, and what are the important prognostic factors?

Outcome is highly variable, but 5‐year survival ranges between 25 and 50% and a good outcome is more likely if
recurrent disease is limited to resectable lung metastases.

(5) Can a biological agent (L‐MTP‐PE) prolong the time to relapse in patients with resected metastatic osteosarcoma?

Preliminary data suggest that this is possible, but more studies are required.
The Geometric Osteotomy: Joint Preservation in Juxta‐Articular Surface Bone Neoplasms
Authors: Masterson Eric L. , Ferracini Riccardo , Davis Aileen M. , Wunder Jay S. , Bell Robert S.
Published: July 2002

Purpose. To present the oncologic and functional results of a consecutive series of patients treated by geometric
osteotomy and allograft reconstruction for juxta‐articular surface bone neoplasms.

Patients. Twelve consecutive patients (mean age 28 years) underwent excision of benign aggressive or malignant
juxta‐articular surface bone neoplasms. In each case, only part of the circumference of the bone was excised, and the joint
surface was preserved and kept in continuity with the remainder of the bone. The defects were filled with allograft or
autograff, and internally fixed as appropriate.

Methods. Patients were eligible for the study if they had a histologically proven, primary tumour of bone adjacent to a
joint such that the turnout could be completely excised with a partial cortical resection and preservation of the joint. The
database at the University Musculoskeletal Oncology Unit was used to identify all cases. Patient demographics and
oncologic results were recorded. Functional assessment was performed using the Musculoskeletal Tumor Society rating
scale and the Toronto Extremity Salvage Score.

Results. Nine tumours were about the knee and three were in the proximal humerus. Negative margins were achieved in
all cases. No patient had metastatic disease at a mean follow‐up of 56.5 months. There was one local recurrence and this
was managed by conversion to a Van Nes rotationplasty. Functional results were excellent in the proximal humeral cases
and in cases about the knee where the stabilizing ligaments were preserved. Cases with post‐operative knee instability were
less successful but none the less were well controlled with bracing.

Discussion. This technique offers an alternative to joint excision and prosthetic replacement in
a group of young patients.
Histologic Correlation With Magnetic Resonance Imaging for Benign and Malignant Lipomatous Masses
Authors: Rougraff Bruce T. , Durbin Mark , Lawerence Jackie , Buckwalter Kenneth
Published: July 2002

Purpose/results. We evaluated the diagnostic accuracy of magnetic resonance imaging (MRI) for 46 consecutive patients
with lipomatous soft tissue tumors prior to biopsy and resection. Twenty‐eight patients had benign lipomas and 18 had
liposarcomas. Clinical differences between thdse patients with benign disease and those with malignant lesions were
average age at the time of presentation (49 years for benign vs 62 years for malignant, p < 0.001) and average length of
symptoms prior to resection (64 months for benign versus 38 months for malignant, p = 0.01). MRI characteristics
associated with benign disease included: smaller tumor size (9.4 cm average greatest dimension for benign lesions vs
13.4 cm for malignant masses, p = 0.022); a mass with a uniformly homogeneous signal (p = 0.0003); a mass with
homogeneous high T1 and T2 signals and a low short‐time‐inversion‐recovery (STIR) signal comparable to normal fat
(p < 0.0001). This last signal pattern was not seen in malignant lesions (0/18) and was present in almost all benign lipomas
(25/28). The usual MRI descriptions of soft tissue masses such as infiltrating vs encapsulating, deep vs subcutaneous and
septated vs non‐septated were not helpful predictors of malignancy in this series. Needle biopsies of lipomatous masses
with heterogeneous signals on MRI resulted in inaccurate diagnoses due to sampling error in 5/9 patients.

Discussion. A carefully planned and performed MRI study of lipomatous masses can accurately predict a benign lipoma
whenever a homogeneous high T1 and T2, as well as a low STIR, signal is present. However, a mass with any other signal
characteristics must be biopsied carefully in order to make an accurate diagnosis.
Soft Tissue Edema Around Musculoskeletal Sarcomas at Magnetic Resonance Imaging
Authors: Panicek David M. , Schwartz Lawrence H.
Published: July 2002

The presence of soft tissue edema around a malignant musculoskeletal neoplasm can interfere with accurate local tumor
staging at magnetic resonance imaging. This article discusses and illustrates such edema, emphasizing means for avoiding
misinterpretation of edema and subsequent overstaging.
Inhibition of a Mitotic Motor Compromises the Formation of Dendrite-like Processes from Neuroblastoma Cells
Authors: Yu Wenqian , Sharp David J. , Kuriyama Ryoko , Mallik Prabhat , Baas Peter W.
Published: July 2002

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.
Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin
Authors: Delft Sanne van , Schumacher Christopher , Hage Willem , Verkleij Arie J. , Henegouwen Paul M.P. van Bergen en
Published: July 2002

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.
Genetic Analysis of Myoblast Fusion: blown fuse Is Required for Progression Beyond the Prefusion Complex
Authors: Doberstein Stephen K. , Fetter Richard D. , Mehta Anand Y. , Goodman Corey S.
Published: July 2002

The events of myoblast fusion in Drosophila are dissected here by combining genetic analysis with light and electron microscopy. We describe a new and essential intermediate step in the process, the formation of a prefusion complex consisting of “paired vesicles.” These pairs of vesicles from different cells align with each other across apposed plasma membranes. This prefusion complex resolves into dense membrane plaques between apposed cells; these cells then establish cytoplasmic continuity by fusion of small areas of plasma membrane followed by vesiculation of apposed membranes. Different steps in this process are specifically blocked by mutations in four genes required for myoblast fusion. One of these genes, blown fuse, encodes a novel cytoplasmic protein expressed in unfused myoblasts that is essential for progression beyond the prefusion complex stage.
Chromatin Degradation in Differentiating Fiber Cells of the Eye Lens
Authors: Bassnett Steven , Mataic Danijela
Published: July 2002

During development, the lens of the eye becomes transparent, in part because of the elimination of nuclei and other organelles from the central lens fiber cells by an apoptotic-like mechanism. Using confocal microscopy we showed that, at the border of the organelle-free zone (OFZ), fiber cell nuclei became suddenly irregular in shape, with marginalized chromatin. Subsequently, holes appeared in the nuclear envelope and underlying laminae, and the nuclei collapsed into condensed, spherical structures. Nuclear remnants, containing DNA, histones, lamin B2, and fragments of nuclear membrane, were detected deep in the OFZ. We used in situ electrophoresis to demonstrate that fragmented DNA was present only in cells bordering the OFZ. Confocal microscopy of terminal deoxynucleotidyl transferase (TdT)–labeled lens slices confirmed that DNA fragmentation was a relatively late event in fiber differentiation, occurring after the loss of the nuclear membrane. DNA fragments with 3′-OH or 3′-PO4 ends were not observed elsewhere in the lens under normal conditions, although they could be produced by pretreatment with DNase I or micrococcal nuclease, respectively. Dual labeling with TdT and an antibody against protein disulfide isomerase, an ER-resident protein, revealed a distinct spatial and temporal gap between the disappearance of ER and nuclear membranes and the onset of DNA degradation. Thus, fiber cell chromatin disassembly differs significantly from classical apoptosis, in both the sequence of events and the time course of the process. The fact that DNA degradation occurs only after the disappearance of mitochondrial, ER, and nuclear membranes suggests that damage to intracellular membranes may be an initiating event in nuclear breakdown.
The Mesodermal Expression of rolling stone (rost) Is Essential for Myoblast Fusion in Drosophila and Encodes a Potential Transmembrane Protein
Authors: Paululat Achim , Goubeaud Anette , Damm Christine , Knirr Stefan , Burchard Susanne , Renkawitz-Pohl Renate
Published: July 2002

In homozygous rolling stone embryos, the fusion of myoblasts to syncytial myotubes is diminished. Nevertheless, the visceral mesoderm, the heart mesoderm, and few somatic muscles are properly formed. Thus, we postulate a central role of rolling stone for the fusion process within the somatic mesoderm. We have cloned the rolling stone gene, and the deduced protein sequence is in accordance with a transmembrane protein, which agrees with the enrichment of Rost in the membrane fraction of Drosophila embryos. No homologous genes have been described so far. rolling stone is expressed in the embryonic nervous system and cells of the somatic mesoderm, most notable in muscle founder cells. To elucidate the function of rolling stone for myoblast fusion, we applied a knock-out strategy. The expression of an antisense rolling stone transcript specifically within the mesoderm of wild-type embryos results in fusion defects of myoblasts, proving that the rolling stone expression in the mesoderm is responsible for the rolling stone phenotype. We suggest that rolling stone is a member of a group of genes that are necessary for the fusion process during myogenesis.
Transportin-mediated Nuclear Import of Heterogeneous Nuclear RNP Proteins
Authors: Siomi Mikiko C. , Eder Paul S. , Kataoka Naoyuki , Wan Lili , Liu Qing , Dreyfuss Gideon
Published: July 2002

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase–M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1–A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.
Cell Cycle–regulated Expression of the Muscle Determination Factor Myf5 in Proliferating Myoblasts
Authors: Lindon Catherine , Montarras Didier , Pinset Christian
Published: July 2002

Myf5 is the earliest-known muscle-specific factor to be expressed in vivo and its expression is associated with determination of the myoblast lineage. In C2 cells, we show by immunocytolocalization that Myf5 disappears rapidly from cells in which the differentiation program has been initiated. In proliferating myoblasts, the levels of Myf5 and MyoD detected from cell to cell are very heterogeneous. We find that some of the heterogeneity of Myf5 expression arises from a posttranscriptional regulation of Myf5 by the cell cycle. Immunoblotting of extracts from synchronized cultures reveals that Myf5 undergoes periodic fluctuations during the cell cycle and is absent from cells blocked early in mitosis by use of nocodazole. The disappearance of Myf5 from mitotic cells involves proteolytic degradation of a phosphorylated form of Myf5 specific to this phase of the cell cycle. In contrast, MyoD levels are not depleted in mitotic C2 cells. The mitotic destruction of Myf5 is the first example of a transcription factor showing cell cycle–regulated degradation. These results may be significant in view of the possible role of Myf5 in maintaining the determination of proliferating cells and in timing the onset of differentiation.
Glycosylation Provides Both Stimulatory and Inhibitory Effects on Cell Surface and Soluble CD44 Binding to Hyaluronan
Authors: Skelton Timothy P. , Zeng Chunxun , Nocks Aaron , Stamenkovic Ivan
Published: July 2002

Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (Kd = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.
Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus
Authors: Llorente Alicia , Rapak Andrzej , Schmid Sandra L. , van Deurs Bo , Sandvig Kirsten
Published: July 2002

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.
Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src
Authors: Postma Friso R. , Hengeveld Trudi , Alblas Jacqueline , Giepmans Ben N.G. , Zondag Gerben C.M. , Jalink Kees , Moolenaar Wouter H.
Published: July 2002

Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication.
ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin
Authors: Haskins Julie , Gu Lijie , Wittchen Erika S. , Hibbard Jennifer , Stevenson Bruce R.
Published: July 2002

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.
Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes
Authors: Shibata A. , Wright M.V. , David S. , McKerracher L. , Braun P.E. , Kater S.B.
Published: July 2002

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.
Cartilage Fibrils of Mammals are Biochemically Heterogeneous: Differential Distribution of Decorin and Collagen IX
Authors: Hagg Rupert , Bruckner Peter , Hedbom Erik
Published: July 2002

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents—decorin and collagen IX—in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.