bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding β-hairpin formation
Authors: Dinner Aaron R. , Lazaridis Themis , Karplus Martin
Published: July 2002

The kinetics of formation of protein structural motifs (e.g., α-helices and β-hairpins) can provide information about the early events in protein folding. A recent study has used fluorescence measurements to monitor the folding thermodynamics and kinetics of a 16-residue β-hairpin. In the present paper, we obtain the free energy surface and conformations involved in the folding of an atomistic model for the β-hairpin from multicanonical Monte Carlo simulations. The results suggest that folding proceeds by a collapse that is downhill in free energy, followed by rearrangement to form a structure with part of the hydrophobic cluster; the hairpin hydrogen bonds propagate outwards in both directions from the partial cluster. Such a folding mechanism differs from the published interpretation of the experimental results, which is based on a helix–coil-type phenomenological model.
Failure of spinal cord oligodendrocyte development in mice lacking neuregulin
Authors: Vartanian Timothy , Fischbach Gerald , Miller Robert
Published: July 2002

Oligodendrocytes develop from a subpopulation of precursor cells within the ventral ventricular zone of the spinal cord. The molecular cues that direct this spatially and temporally restricted event seem to originate in part from structures ventral to and within the spinal cord. Here, we present evidence that the family of ligands termed neuregulins are necessary for the normal generation of mouse spinal cord oligodendrocytes. Oligodendrocytes mature in spinal cord explants from wild-type mice and mice heterozygotic for a null mutation in the neuregulin gene (NRG +/−) in a temporal sequence of developmental events that replicates that observed
in vivo
. However, in spinal cord explants derived from mice lacking neuregulin (NRG −/−), oligodendrocytes fail to develop. Addition of recombinant neuregulin to spinal cord explants from NRG −/− mice rescues oligodendrocyte development. In wild-type spinal cord explants, inhibitors of neuregulin mimic the inhibition of oligodendrocyte development that occurs in NRG −/− explants. In embryonic mouse spinal cord, neuregulins are present in motor neurons and the ventral ventricular zone where they likely exert their influence on early oligodendrocyte precursor cells.
Persistence of lymphocytic choriomeningitis virus at very low levels in immune mice
Authors: Ciurea Adrian , Klenerman Paul , Hunziker Lukas , Horvath Edit , Odermatt Bernhard , Ochsenbein Adrian F. , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Lymphocytic choriomeningitis virus (LCMV), strain WE, is a non-cytopathic RNA virus that is highly adapted to its natural host, the mouse. Acute infection of adult mice leads to generalized virus spread, followed by cytotoxic T lymphocyte-mediated virus clearance below the detection levels of conventional assays within 2–3 weeks. Indirect evidence had suggested that virus or viral antigen might persist in the immune mouse. Here we demonstrate LCMV-WE persistence at low levels after infection with 102or 106plaque-forming units, shown as viral genome, viral antigen, and replicative virus using sensitivein vitroandin vivoassays. The finding that LCMV-WE persists in the face of apparently intact immune responses resembles the situation in some viral (hepatitis B and C, HIV) and bacterial (tuberculosis, leprosy) infections in humans; the results are relevant to the understanding not only of other murine and human persistent viral infections but also of protective immunological memory by “infection immunity.”
Accumulation of specific RNAs encoding transcriptional factors and stress response proteins against a background of severe depletion of cellular RNAs in cells infected with herpes simplex virus 1
Authors: Khodarev Nikolai N. , Advani Sunil J. , Gupta Nalin , Roizman Bernard , Weichselbaum Ralph R.
Published: July 2002

Herpes simplex virus 1 encodes several functions to preclude the shutoff of host response to infection, including degradation of mRNA immediately after infection. To determine whether any cellular mRNAs accumulate in infected cells against a background of severe loss of host RNA, we hybridized cDNAs derived from three different cell lines infected with wild type and a mutant virus to a DNA array containing probes for 588 human genes representing different functional groups. The results were that (
i
) infected cells accumulated at levels above those of mock-infected cells, a small number of transcripts representing transcriptional factors that could regulate gene expression both positively and negatively, and one stress response protein (GADD45), (
ii
) the amount and nature of the accumulated transcripts showed limited variability depending on the cell and virus, and (
iii
) at least some of the proteins encoded by the accumulated transcripts could benefit either the virus or the host.
The small ribosomal subunit from
Thermus thermophilus
at 4.5 Å resolution: Pattern fittings and the identification of a functional site
Authors: Tocilj Ante , Schlünzen Frank , Janell Daniela , Glühmann Marco , Hansen Harly A. S. , Harms Jörg , Bashan Anat , Bartels Heike , Agmon Ilana , Franceschi Francois , Yonath Ada
Published: July 2002

The electron density map of the small ribosomal subunit from
Thermus thermophilus
, constructed at 4.5 Å resolution, shows the recognizable morphology of this particle, as well as structural features that were interpreted as ribosomal RNA and proteins. Unbiased assignments, carried out by quantitative covalent binding of heavy atom compounds at predetermined sites, led to the localization of the surface of the ribosomal protein S13 at a position compatible with previous assignments, whereas the surface of S11 was localized at a distance of about twice its diameter from the site suggested for its center by neutron scattering. Proteins S5 and S7, whose structures have been determined crystallographically, were visually placed in the map with no alterations in their conformations. Regions suitable to host the fold of protein S15 were detected in several positions, all at a significant distance from the location of this protein in the neutron scattering map. Targeting the 16S RNA region, where mRNA docks to allow the formation of the initiation complex by a mercurated mRNA analog, led to the characterization of its vicinity.
A common pharmacophore for cytotoxic natural products that stabilize microtubules
Authors: Ojima Iwao , Chakravarty Subrata , Inoue Tadashi , Lin Songnian , He Lifeng , Horwitz Susan Band , Kuduk Scott D. , Danishefsky Samuel J.
Published: July 2002

Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew,
Taxus brevifolia
, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel—i.e., the stabilization of microtubules leading to mitotic arrest—is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [
3
H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure–activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity.
RXP 407, a phosphinic peptide, is a potent inhibitor of angiotensin I converting enzyme able to differentiate between its two active sites
Authors: Dive Vincent , Cotton Joël , Yiotakis Athanasios , Michaud Annie , Vassiliou Stamatia , Jiracek Jiri , Vazeux Gilles , Chauvet Marie-Thérèse , Cuniasse Philippe , Corvol Pierre
Published: July 2002

The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-Lysyl-Proline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L)Pheψ(PO2-CH2)(L)Ala-Ala-NH2, called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (Ki= 12 nM), RXP 407, which is metabolically stablein vivo, may lead to a new generation of ACE inhibitors able to blockin vivoonly a subset of the different functions regulated by ACE.
Somatic polyploidization and cellular proliferation drive body size evolution in nematodes
Authors: Flemming Anthony J. , Shen Zai-Zhong , Cunha Ana , Emmons Scott W. , Leroi Armand M.
Published: July 2002

Most of the hypodermis of a rhabditid nematode such as
Caenorhabditis elegans
is a single syncytium. The size of this syncytium (as measured by body size) has evolved repeatedly in the rhabditid nematodes. Two cellular mechanisms are important in the evolution of body size: changes in the numbers of cells that fuse with the syncytium, and the extent of its acellular growth. Thus nematodes differ from mammals and other invertebrates in which body size evolution is caused by changes in cell number alone. The evolution of acellular syncytial growth in nematodes is also associated with changes in the ploidy of hypodermal nuclei. These nuclei are polyploid as a consequence of iterative rounds of endoreduplication, and this endocycle has evolved repeatedly. The association between acellular growth and endoreduplication is also seen in
C. elegans
mutations that interrupt transforming growth factor-β signaling and that result in dwarfism and deficiencies in hypodermal ploidy. The transforming growth factor-β pathway is a candidate for being involved in nematode body size evolution.
Recent common ancestry of human Y chromosomes: Evidence from DNA sequence data
Authors: Thomson Russell , Pritchard Jonathan K. , Shen Peidong , Oefner Peter J. , Feldman Marcus W.
Published: July 2002

We consider a data set of DNA sequence variation at three Y
chromosome genes (SMCY, DBY, and DFFRY) in a worldwide sample of human
Y chromosomes. Between 53 and 70 chromosomes were fully screened for
sequence variation at each locus by using the method of denaturing
high-performance liquid chromatography. The sum of the lengths of the
three genes is 64,120 bp. We have used these data to study the
ancestral genealogy of human Y chromosomes. In particular, we focused
on estimating the expected time to the most recent common ancestor and
the expected ages of certain mutations with interesting geographic
distributions. Although the geographic structure of the inferred
haplotype tree is reminiscent of that obtained for other loci
(the root is in Africa, and most of the oldest non-African lineages are
Asian), the expected time to the most recent common ancestor is
remarkably short, on the order of 50,000 years. Thus, although previous
studies have noted that Y chromosome variation shows extreme geographic
structure, we estimate that the spread of Y chromosomes out of Africa
is much more recent than previously was thought. We also show that our
data indicate substantial population growth in the effective number of
human Y chromosomes.
Identification of the MATa mating-type locus of
Cryptococcus neoformans
reveals a serotype A MATa strain thought to have been extinct
Authors: Lengeler Klaus B. , Wang Ping , Cox Gary M. , Perfect John R. , Heitman Joseph
Published: July 2002

Cryptococcus neoformans
is an opportunistic fungal
pathogen with a defined sexual cycle involving mating between haploid
MATa and MATα cells. Here we describe the isolation of part of the
MATa mating-type locus encoding a Ste20 kinase homolog, Ste20a. We show
that the
STE20a
gene cosegregates with the MATa mating
type in genetic crosses, maps within the mating-type locus on a 1.8-Mb
chromosome, and is allelic with the MATα locus. We identify the first
MATa isolate of the most common pathogenic variety of
C.
neoformans
(serotype A, variety
grubii
) which
had been thought to be extinct. This serotype A MATa strain is sterile,
fails to produce mating pheromone, and is less virulent than a serotype
A MATα strain in an animal model. Our studies illustrate an
association of mating type with virulence and suggest that, like
Candida albicans
, pathogenic isolates of
C.
neoformans
may be largely asexual.
The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons
Authors: Rowe-Magnus Dean A. , Guerout Anne-Marie , Ploncard Pascaline , Dychinco Broderick , Davies Julian , Mazel Didier
Published: July 2002

Integrons are genetic elements that acquire and exchange exogenous
DNA, known as gene cassettes, by a site-specific recombination
mechanism. Characterized gene cassettes consist of a target
recombination sequence (
attC
site) usually associated
with a single open reading frame coding for an antibiotic resistance
determinant. The affiliation of multiresistant integrons (MRIs), which
contain various combinations of antibiotic resistance gene cassettes,
with transferable elements underlies the rapid evolution of multidrug
resistance among diverse Gram-negative bacteria. Yet the origin of MRIs
remains unknown. Recently, a chromosomal super-integron (SI) harboring
hundreds of cassettes was identified in the
Vibrio
cholerae
genome. Here, we demonstrate that the activity of its
associated integrase is identical to that of the MRI integrase, IntI1.
We have also identified equivalent integron superstructures in nine
distinct genera throughout the γ-proteobacterial radiation.
Phylogenetic analysis revealed that the evolutionary history of the
system paralleled that of the radiation, indicating that integrons are
ancient structures. The
attC
sites of the 63
antibiotic-resistance gene cassettes identified thus far in MRIs are
highly variable. Strikingly, one-fifth of these were virtually
identical to the highly related yet species-specific
attC
sites of the SIs described here. Furthermore,
antimicrobial resistance homologues were identified among the thousands
of genes entrapped by these SIs. Because the gene cassettes of SIs are
substrates for MRIs, these data identify SIs as the source of
contemporary MRIs and their cassettes. However, our demonstration of
the metabolic functions, beyond antibiotic resistance and virulence, of
three distinct SI gene cassettes indicates that integrons function as a
general gene-capture system for bacterial innovation.
Paget′s Osteosarcoma — no Cure in Sight
Authors: Shaylor Phillip J. , Peake David , Grimer Robert J. , Carter Simon R. , Tillman Roger M. , Spooner David
Published: July 2002

Purpose Paget′ s osteosarcoma has a fearful reputation with a quoted
survival of at best 5% at 5 years.We therefore reviewed our experience of 26 patients treated
over the last 25 years using modern staging and limb salvage techniques to see if there had
been any improvement in survival.

Subjects: We identified 26 patients on the Royal Orthopaedic Hospital Oncological
database with a diagnosis of sarcoma secondary to Paget′s disease.

Results: The survival rate was 53% at 1 year, 25% at 2 years and no
patient survived for 5 years.The median survival was 21 months for those treated with
curative intent and 7 months for those treated palliatively. Four of the five patients treated
with limb‐sparing surgery developed local recurrence between 5 and 12 months, the fifth
died at 14 months.There was no difference in survival between amputation and limb
salvage.

Discussion: The development of sarcomatous change in Paget′s
disease is well recognised. It represents an important segment of primary bone tumours in
patients over 40 years of age.The prognosis is appalling. Indeed only 15 of 368 cases (4%)
from a number of historical series have survived more than 5 years. Our results are similarly
disappointing with no survivors at 5 years despite modern methods of management of bone
tumours.While there is no difference in local recurrence rates or survival between limb
reconstruction and limb ablation the poor prognosis for both means that neither can be
recommended at present. Sarcomatous change in Pagetoid bone should therefore be
regarded as a different disease to primary osteosarcoma. It remains an incurable disease
with a poor prognosis.
Age‐Related Changes of Liver Antioxidant Enzymes and 8‐Hydroxy‐2‐Deoxyguanosine During Fetal?Neonate Transition and Early Rat Development
Authors: MuÑiz Pilar , JosÉ Maria , Barchino Garcia , Iradi Antonio , Mahiques Eduardo , Marco Vicente
Published: July 2002

We have studied the pro‐antioxidant status of the rat liver on the last day of gestation and at 1, 15, and 30 days of extrauterine life. Representative variables, such as activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase and concentrations of reduced glutathione and 8‐hydroxy‐2‐deoxyguanosine, were determined in liver to assess the degree of birth‐associated oxidative stress during the fetal‐neonatal transition and early development of the rat. Percentages by which liver Cu/ZnSOD activity increased over the basal value of the fetal liver were 54%, 95%, and 127% at neonatal days 1, 15, and 30, respectively. There was a lack of induction in the development profile of MnSOD. Catalase activity was clearly and progressively induced with time from the fetal state up to the neonatal age of 1 month. Glutathione peroxidase activity and glutathione content showed a tendency to decline during the first day after birth, though they increased to significantly higher values on days 15 and 30. However, the amount of rat liver 8‐hydroxy‐2‐deoxyguanosine did not increase. These results suggest that the induced antioxidant activities may be responsible for maintaining DNA stability during the perinatal development of the rat liver.
Passive Sorting in Maturing Granules of AtT-20 Cells: The Entry and Exit of Salivary Amylase and Proline-rich Protein
Authors: Castle Anna M. , Huang Amy Y. , Castle J. David
Published: July 2002

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093– 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711– 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.
The O-glycosylated Stalk Domain Is Required for Apical Sorting of Neurotrophin Receptors in Polarized MDCK Cells
Authors: Yeaman Charles , Gall Annick H. Le , Baldwin Anne N. , Monlauzeur Laure , Bivic Andre Le , Rodriguez-Boulan Enrique
Published: July 2002

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75NTR) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75NTR causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75NTR does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75NTR, providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75NTR is not required for apical sorting of either transmembrane or secreted forms.
A Requirement for Cyclin D3–Cyclin-dependent Kinase (cdk)-4 Assembly in the Cyclic Adenosine Monophosphate–dependent Proliferation of Thyrocytes
Authors: Depoortere Fabienne , Van Keymeulen Alexandra , Lukas Jiri , Costagliola Sabine , Bartkova Jirina , Dumont Jacques E. , Bartek Jiri , Roger Pierre P. , Dremier Sarah
Published: July 2002

In different systems, cyclic adenosine monophosphate (cAMP) either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (TSH). The cAMP-dependent mitogenic pathway is unique as it is independent of mitogen-activated protein kinase activation and differs from growth factor–dependent pathways at the level of the expression of several protooncogenes/transcription factors. This study examined the involvement of D-type G1 cyclins and their associated cyclin-dependent kinase (cdk4) in the cAMP-dependent G1 phase progression of dog thyroid cells. Unlike epidermal growth factor (EGF)+serum and other cAMP-independent mitogens, TSH did not induce the accumulation of cyclins D1 and D2 and partially inhibited the basal expression of the most abundant cyclin D3. However, TSH stimulation enhanced the nuclear detection of cyclin D3. This effect correlated with G1 and S phase progression. It was found to reflect both the unmasking of an epitope of cyclin D3 close to its domain of interaction with cdk4, and the nuclear translocation of cyclin D3. TSH and EGF+serum also induced a previously undescribed nuclear translocation of cdk4, the assembly of precipitable cyclin D3–cdk4 complexes, and the Rb kinase activity of these complexes. Previously, cdk4 activity was found to be required in the cAMP-dependent mitogenic pathway of dog thyrocytes, as in growth factor pathways. Here, microinjections of a cyclin D3 antibody showed that cyclin D3 is essential in the TSH/ cAMP-dependent mitogenesis, but not in the pathway of growth factors that induce cyclins D1 and D2. The present study (a) provides the first example in a normal cell of a stimulation of G1 phase progression occurring independently of an enhanced accumulation of cyclins D, (b) identifies the activation of cyclin D3 and cdk4 through their enhanced assembly and/or nuclear translocation, as first convergence steps of the parallel cAMP-dependent and growth factor mitogenic pathways, and (c) strongly suggests that this new mechanism is essential in the cAMP-dependent mitogenesis, which provides the first direct demonstration of the requirement for cyclin D3 in a G1 phase progression.
Characterization of the Human Homologue of the Yeast Spc98p and Its Association with γ-Tubulin
Authors: Tassin Anne-Marie , Celati Claude , Moudjou Mohammed , Bornens Michel
Published: July 2002

A trimeric complex formed by Tub4p, the budding yeast γ-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5′ and 3′ cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3′ end. The deduced protein possesses 22% identity and 45% similarity with the yeast homologue. We further report that the human Spc98p, like γ-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both γ-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and γ-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction.
Myosin Heavy Chains IIa and IId Are Functionally Distinct in the Mouse
Authors: Sartorius Carol A. , Lu Brian D. , Acakpo-Satchivi Leslie , Jacobsen Renee P. , Byrnes William C. , Leinwand Leslie A.
Published: July 2002

Myosin in adult murine skeletal muscle is composed primarily of three adult fast myosin heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa– containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.
Layilin, A Novel Talin-binding Transmembrane Protein Homologous with C-type Lectins, is Localized in Membrane Ruffles
Authors: Borowsky Mark L. , Hynes Richard O.
Published: July 2002

Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten–amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. We have identified a short region within talin's amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase.
kakapo, a Gene Required for Adhesion Between and Within Cell Layers in Drosophila, Encodes a Large Cytoskeletal Linker Protein Related to Plectin and Dystrophin
Authors: Gregory Stephen L. , Brown Nicholas H.
Published: July 2002

Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000–amino acid region, and contains within this region an α-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest–specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.
Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p
Authors: Marelli Marcello , Aitchison John D. , Wozniak Richard W.
Published: July 2002

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.
Analysis of the Roles of 14-3-3 in the Platelet Glycoprotein Ib-IX–Mediated Activation of Integrin αIIbβ3 Using a Reconstituted Mammalian Cell Expression Model
Authors: Gu Minyi , Xi Xiaodong , Englund Graham D. , Berndt Michael C. , Du Xiaoping
Published: July 2002

We have reconstituted the platelet glycoprotein (GP) Ib-IX–mediated activation of the integrin αIIbβ3 in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing αIIbβ3 adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin αIIbβ3 and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against αIIbβ3. vWF binding to GPIb-IX also activates soluble fibrinogen binding to αIIbβ3 indicating that GPIb-IX mediates a cellular signal leading to αIIbβ3 activation. Deletion of the 14-3-3–binding site in GPIbα inhibited GPIb-IX–mediated fibrinogen binding to αIIbβ3 and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbα induced an endogenous integrin-dependent cell spreading on vWF without requiring αIIbβ3, but inhibited vWF-induced fibrinogen binding to αIIbβ3. Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX–mediated activation of αIIbβ3 requires 14-3-3 interaction with GPIbα.
Adenomatous Polyposis Coli (APC) Protein Moves along Microtubules and Concentrates at Their Growing Ends in Epithelial Cells
Authors: Mimori-Kiyosue Yuko , Shiina Nobuyuki , Tsukita Shoichiro
Published: July 2002

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.
Dynamin:Gtp Controls the Formation of Constricted Coated Pits, the Rate Limiting Step in Clathrin-Mediated Endocytosis
Authors: Sever Sanja , Damke Hanna , Schmid Sandra L.
Published: July 2002

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481–486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.
Flagellar Radial Spoke Protein 3 Is an a-Kinase Anchoring Protein (Akap)
Authors: Gaillard Anne Roush , Diener Dennis R. , Rosenbaum Joel L. , Sale Winfield S.
Published: July 2002

Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144–180. Amino acids 161–180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.
Tyrosine-Phosphorylated Extracellular Signal–Regulated Kinase Associates with the Golgi Complex during G2/M Phase of the Cell Cycle
Authors: Cha Hyukjin , Shapiro Paul
Published: July 2002

Phosphorylation of the extracellular signal–regulated kinases (ERKs) on tyrosine and threonine residues within the TEY tripeptide motif induces ERK activation and targeting of substrates. Although it is recognized that phosphorylation of both residues is required for ERK activation, it is not known if a single phosphorylation of either residue regulates physiological functions. In light of recent evidence indicating that ERK proteins regulate substrate function in the absence of ERK enzymatic activity, we have begun to examine functional roles for partially phosphorylated forms of ERK. Using phosphorylation site–specific ERK antibodies and immunofluorescence, we demonstrate that ERK phosphorylated on the tyrosine residue (pY ERK) within the TEY activation sequence is found constitutively in the nucleus, and localizes to the Golgi complex of cells that are in late G2 or early mitosis of the cell cycle. As cells progress through metaphase and anaphase, pY ERK localization to Golgi vesicles is most evident around the mitotic spindle poles. During telophase, pY ERK associates with newly formed Golgi vesicles but is not found on there after cytokinesis and entry into G1. Increased ERK phosphorylation causes punctate distribution of several Golgi proteins, indicating disruption of the Golgi structure. This observation is reversible by overexpression of a tyrosine phosphorylation–defective ERK mutant, but not by a kinase-inactive ERK2 mutant that is tyrosine phosphorylated. These data provide the first evidence that pY ERK and not ERK kinase activity regulates Golgi structure and may be involved in mitotic Golgi fragmentation and reformation.
Critical Points of Tumor Necrosis Factor Action in Central Nervous System Autoimmune Inflammation Defined by Gene Targeting
Authors: Körner Heinrich , Riminton D. Sean , Strickland Deborah H. , Lemckert Frances A. , Pollard John D. , Sedgwick Jonathon D.
Published: July 2002

Tumor necrosis factor (TNF)–dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35–55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF−/− mice. However, in the TNF−/− mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF−/− and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF−/− mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF−/− mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.
Dendritic Cells Induce Autoimmune Diabetes and Maintain Disease via De Novo Formation of Local Lymphoid Tissue
Authors: Ludewig Burkhard , Odermatt Bernhard , Landmann Salome , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Activation of autoreactive T cells can lead to autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). The initiation and maintenance of IDDM by dendritic cells (DC), the most potent professional antigen-presenting cells, were investigated in transgenic mice expressing the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) under the control of the rat insulin promoter (RIP-GP mice). We show that after adoptive transfer of DC constitutively expressing the immunodominant cytotoxic T lymphocyte (CTL) epitope of the LCMV-GP, RIP-GP mice developed autoimmune diabetes. Kinetic and functional studies of DC-activated CTL revealed that development of IDDM was dependent on dose and timing of antigenic stimulation. Strikingly, repeated CTL activation by DC led to severe destructive mononuclear infiltration of the pancreatic islets but also to de novo formation of islet-associated organized lymphoid structures in the pancreatic parenchyma. In addition, repetitive DC immunization induced IDDM with lymphoid neogenesis also in perforin-deficient RIP-GP mice, illustrating that CD8+ T cell–dependent inflammatory mechanisms independent of perforin could induce IDDM. Thus, DC presenting self-antigens not only are potent inducers of autoreactive T cells, but also help to maintain a peripheral immune response locally; therefore, the induction of autoimmunity against previously ignored autoantigens represents a potential hazard, particularly in DC-based antitumor therapies.
An Essential Role for Tumor Necrosis Factor in Natural Killer Cell–mediated Tumor Rejection in the Peritoneum
Authors: Smyth Mark J. , Kelly Janice M. , Baxter Alan G. , Körner Heinrich , Sedgwick Jonathon D.
Published: July 2002

Natural killer (NK) cells are thought to provide the first line of defence against tumors, particularly major histocompatibility complex (MHC) class I− variants. We have confirmed in C57BL/6 (B6) mice lacking perforin that peritoneal growth of MHC class I− RMA-S tumor cells in unprimed mice is controlled by perforin-dependent cytotoxicity mediated by CD3− NK1.1+ cells. Furthermore, we demonstrate that B6 mice lacking tumor necrosis factor (TNF) are also significantly defective in their rejection of RMA-S, despite the fact that RMA-S is insensitive to TNF in vitro and that spleen NK cells from B6 and TNF-deficient mice are equally lytic towards RMA-S. NK cell recruitment into the peritoneum was abrogated in TNF-deficient mice challenged with RMA-S or RM-1, a B6 MHC class I− prostate carcinoma, compared with B6 or perforin-deficient mice. The reduced NK cell migration to the peritoneum of TNF-deficient mice correlated with the defective NK cell response to tumor in these mice. By contrast, a lack of TNF did not affect peptide-specific cytotoxic T lymphocyte–mediated rejection of tumor from the peritoneum of preimmunized mice. Overall, these data show that NK cells delivering perforin are the major effectors of class I− tumor rejection in the peritoneum, and that TNF is specifically critical for their recruitment to the peritoneum.
Neutrophil-specific Granule Deficiency Results from a Novel Mutation with Loss of Function of the Transcription Factor CCAAT/Enhancer Binding Protein ε
Authors: Lekstrom-Himes Julie A. , Dorman Susan E. , Kopar Piroska , Holland Steven M. , Gallin John I.
Published: July 2002

Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)ε, a member of the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a five-basepair deletion in the second exon of the C/EBPε locus. The predicted frame shift results in a truncation of the 32-kD major C/EBPε isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBPε deficiency, define SGD as a defect in myelopoiesis and establish the requirement for C/EBPε for the promyelocyte–myelocyte transition in myeloid differentiation.
Migration Kinetics and Final Destination of  Type 1 and Type 2 CD8 Effector Cells Predict Protection against Pulmonary Virus Infection
Authors: Cerwenka Adelheid , Morgan Tammy M. , Harmsen Allen G. , Dutton Richard W.
Published: July 2002

The requirements for CD8 T cells to provide protection against a localized virus infection in models of adoptive immunotherapy are not well defined. Here we investigated the protective value of defined in vitro–generated hemagglutinin (HA) peptide-specific primary CD8 T cell effectors from the clone 4 T cell receptor transgenic mice, secreting type 1 or type 2 cytokines, against pulmonary infection with whole influenza virus. Cytotoxic T lymphocytes producing type 1 and type 2 cytokine (Tc1 and Tc2) populations were equally cytolytic, but Tc1 effectors and not Tc2 effectors reduced the pulmonary virus titer early during infection. Host recovery mediated by Tc1 effectors was found to be independent of interferon γ production. Tc2 effectors entered the lung with delayed kinetics as compared with Tc1 effectors, and after lung entry Tc2 effector cells did not localize near the infected airway epithelium as did Tc1 effectors but were found within clusters of inflammatory cells distant from the epithelium. We also show that the expression of several chemokine receptors was selectively regulated in the Tc1 and Tc2 subsets. Thus, the protective value of a CD8 cell population against pulmonary influenza virus infection is strongly correlated with its ability to exert its effector potential at the site of virus infection.