bluuebunny/crossref_metadata_2025 huggingface dataset:
A scrapie-like unfolding intermediate of the prion protein domain PrP(121–231) induced by acidic pH
Authors: Hornemann Simone , Glockshuber Rudi
Published: July 2002

The infectious agent of transmissible spongiform encephalopathies is believed to consist of an oligomeric isoform, PrP
Sc
, of the monomeric cellular prion protein, PrP
C
. The conversion of PrP
C
to PrP
Sc
is characterized by a decrease in α-helical structure, an increase in β-sheet content, and the formation of PrP
Sc
amyloid. Whereas the N-terminal part of PrP
C
comprising residues 23–120 is flexibly disordered, its C-terminal part, PrP(121–231), forms a globular domain with three α-helices and a small β-sheet. Because the segment of residues 90–231 is protease-resistant in PrP
Sc
, it is most likely structured in the PrP
Sc
form. The conformational change of the segment containing residues 90–120 thus constitutes the minimal structural difference between PrP
C
and a PrP
Sc
monomer. To test whether PrP(121–231) is also capable to undergo conformational transitions, we analyzed its urea-dependent unfolding transitions at neutral and acidic pH. We identified an equilibrium unfolding intermediate of PrP(121–231) that is exclusively populated at acidic pH and shows spectral characteristics of a β-sheet protein. The intermediate is in rapid equilibrium with native PrP(121–231), significantly populated in the absence of urea at pH 4.0, and may have important implications for the presumed formation of PrP
Sc
during endocytosis.
Amyrel
, a paralogous gene of the amylase gene family in
Drosophila melanogaster
and the
Sophophora
subgenus
Authors: Da Lage Jean-Luc , Renard Emmanuelle , Chartois Frédérique , Lemeunier Françoise , Cariou Marie-Louise
Published: July 2002

We describe a gene from
Drosophila melanogaster
related to the alpha-amylase gene
Amy
. This gene, which exists as a single copy, was named
Amyrel.
It is strikingly divergent from
Amy
because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes.
Amyrel
has also been cloned in
Drosophila ananassae
,
Drosophila pseudoobscura
, and
Drosophila subobscura
and is likely to be present throughout the
Sophophora
subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between
Amyrel
genes from different species, which is not the case for
Amy
and which suggests that selection acts on these regions. In contrast to the
Amy
genes,
Amyrel
is transcribed in larvae of
D. melanogaster
but not in adults. However, the protein has not been detected yet.
Amyrel
evolves about twice as fast as
Amy
in the several species studied. We suggest that this gene could result from a duplication of
Amy
followed by accelerated and selected divergence toward a new adaptation.
α
1
-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent
Authors: Jean François , Stella Kori , Thomas Laurel , Liu Gseping , Xiang Yang , Reason Andrew J. , Thomas Gary
Published: July 2002

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α
1
-antitrypsin Portland (α
1
-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by
Pseudomonas
exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged α
1
-PDX (α
1
-PDX/hf) revealed the selectivity of the α
1
-PDX/hf reactive site loop for furin (
K
i
, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α
1
-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin’s active site, α
1
-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg
355
-Ile-Pro-Arg
358
-
↓
or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α
1
-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α
1
-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.
The N-terminal domain of PsaF: Precise recognition site for binding and fast electron transfer from cytochromec6and plastocyanin to photosystem I ofChlamydomonas reinhardtii
Authors: Hippler Michael , Drepper Friedel , Haehnel Wolfgang , Rochaix Jean-David
Published: July 2002

The PsaF-deficient mutant 3bF ofChlamydomonas reinhardtiiwas used to modify PsaF by nuclear transformation and site-directed mutagenesis. Four lysine residues in the N-terminal domain of PsaF, which have been postulated to form the positively charged face of a putative amphipathic α-helical structure were altered to K12P, K16Q, K23Q, and K30Q. The interactions between plastocyanin (pc) or cytochromec6(cytc6) and photosystem I (PSI) isolated from wild type and the different mutants were analyzed using crosslinking techniques and flash absorption spectroscopy. The K23Q change drastically affected crosslinking of pc to PSI and electron transfer from pc and cytc6to PSI. The corresponding second order rate constants for binding of pc and cytc6were reduced by a factor of 13 and 7, respectively. Smaller effects were observed for mutations K16Q and K30Q, whereas in K12P the binding was not changed relative to wild type. None of the mutations affected the half-life of the microsecond electron transfer performed within the intermolecular complex between the donors and PSI. The fact that these single amino acid changes within the N-terminal domain of PsaF have different effects on the electron transfer rate constants and dissociation constants for both electron donors suggests the existence of a rather precise recognition site for pc and cytc6that leads to the stabilization of the final electron transfer complex through electrostatic interactions.
Characterization of genes modulated during pheomelanogenesis using differential display
Authors: Furumura Minao , Sakai Chie , Potterf S. Brian , Vieira Wilfred D. , Barsh Gregory S. , Hearing Vincent J.
Published: July 2002

Molecular and biochemical mechanisms that modulate the production of eumelanin or pheomelanin pigments involve the opposing effects of two intercellular signaling molecules, α-melanocyte stimulating hormone (MSH) and agouti signal protein (ASP). ASP is an antagonist of MSH signaling through the melanocyte-specific MSH receptor, although its mechanism(s) of action is controversial. We previously have reported significant down-regulation of all known melanogenic genes during the eumelanin to pheomelanin switch in murine hair follicle melanocytes and in cultured melanocytes treated with recombinant ASP. To identify factors that might be involved in the switch to pheomelanogenesis, we screened ASP-treated melanocytes by using differential display and identified three up-regulated genes: a DNA replication control protein, a basic helix–loop–helix transcription factor, and a novel gene. We have simultaneously identified six down-regulated genes in ASP-treated melanocytes; two of those encode tyrosinase and TRP2, melanogenic genes known to be down-regulated during pheomelanogenesis, which provide good internal controls for this approach. These results suggest that there are complex mechanisms involved in the switch to pheomelanin production, and that these modulated genes might be involved in the pleiotropic changes seen in yellow mice, including the change in coat color.
Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes
Authors: Razi Nahid , Varki Ajit
Published: July 2002

CD22 is a B cell-restricted glycoprotein involved in signal transduction and modulation of cellular activation. It is also an I-type lectin (now designated Siglec-2), whose extracellular domain can specifically recognize α2–6-linked sialic acid (Sia) residues. This activity is postulated to mediate intercellular adhesion and/or to act as a coreceptor in antigen-induced B cell activation. However, studies with recombinant CD22 indicate that the lectin function can be inactivated by expression of α2–6-linked Sia residues on the same cell surface. To explore whether this masking phenomenon affects native CD22 on B cells, we first developed a probe to detect the lectin activity of recombinant CD22 expressed on Chinese hamster ovary cells (which have no endogenous α2–6-linked Sia residues). This probe is inactive against CD22-positive B lymphoma cells and Epstein–Barr virus-transformed lymphoblasts which express high levels of α2–6-linked Sia residues. Enzymatic desialylation unmasks the CD22 lectin activity, indicating that endogenous Sia residues block the CD22 lectin-binding site. Truncation of the side chains of cell surface Sia residues by mild periodate oxidation (known to abrogate Sia recognition by CD22) also had this unmasking effect, indicating that the effects of desialylation are not due to a loss of negative charge. Normal resting B cells from human peripheral blood gave similar findings. However, the lectin is partially unmasked duringin vitroactivation of these cells. Thus, the lectin activity of CD22 is restricted by endogenous sialylation in resting B cells and may be transiently unmasked duringin vivoactivation, perhaps to modulate intercellular or intracellular interactions at this critical stage in the humoral response.
Depletion of intracellular Ca
2+
stores, phosphorylation of eIF2α, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole
Authors: Aktas Huseyin , Flückiger Rudolf , Acosta Juan A. , Savage Joyce M. , Palakurthi Sangeetha S. , Halperin José A.
Published: July 2002

Regulation of translation initiation plays a critical role in the control of cell growth and division in eukaryotic cells. Translation of many growth regulatory proteins including cyclins depends critically on translation initiation factors because their mRNAs are translated inefficiently. We report that clotrimazole, a potent antiproliferative agent both
in vitro
and
in vivo
, inhibits cell growth by interfering with translation initiation. In particular, clotrimazole causes a sustained depletion of intracellular Ca
2+
stores, which results in activation of PKR, phosphorylation of eIF2α, and thereby in inhibition of protein synthesis at the level of translation initiation. Consequently, clotrimazole preferentially decreases the expression of the growth promoting proteins cyclin A, E and D1, resulting in inhibition of cyclin-dependent kinase activity and blockage of cell cycle in G
1
.
Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions
Authors: Greene Thomas W. , Hannah L. Curtis
Published: July 2002

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-
d
-glucose-1-phosphate adenylyltransferase, EC
2.7.7.27
), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP.
Escherichia coli
cells expressing wild type (WT)
Shrunken2 (Sh2)
, and
Brittle2 (Bt2)
exhibit a reduced capacity to produce glycogen when grown at 42°C. Mutagenesis of
Sh2
and coexpression with WT
Bt2
led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, S
h2hs33,
identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of
Sh2hs33
and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in
Sh2
can increase the stability of maize endosperm AGP through enhanced subunit interactions.
MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity
Authors: Sun Hui Bin , Zhu Yuan Xiao , Yin Tinggui , Sledge George , Yang Yu-Chung
Published: July 2002

Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene,mrg1[melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9,mrg1can be induced by other cytokines and biological stimuli, including IL-1α, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction ofmrg1by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reducedmrg1induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required formrg1induction, at least in response to IL-9. Transfection ofmrg1cDNA into TS1, an IL-9-dependent mouse T cell line, converted these cells to IL-9-independent growth through a nonautocrine mechanism. Overexpression ofmrg1in Rat1 cells resulted in loss of cell contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice, demonstrating thatmrg1is a transforming gene. MRG1 is a transcriptional activator and may represent a founding member of an additional family of transcription factors.
Altered regulation of platelet-derived growth factor receptor-α gene-transcription
in vitro
by spina bifida-associated mutant Pax1 proteins
Authors: Joosten Paul H. L. J. , Hol Frans A. , van Beersum Sylvia E. C. , Peters Heiko , Hamel Ben C. J. , Afink Gijs B. , van Zoelen Everardus J. J. , Mariman Edwin C. M.
Published: July 2002

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-α gene (
PDGFR
α). Mice heterozygous for the
PDGFR
α-mutation
Patch
, and at the same time homozygous for the
undulated
mutation in the
Pax1
gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRα and Pax1. Using the human
PDGFRα
promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the
PDGFRα
gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the
undulated
-mutation or the Gln → His mutation previously identified by us in the
PAX1
gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRα-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRα expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein–DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the
PDGFRα
gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRα expression may be causally related to NTDs.
Insulin-like growth factor I restores motor coordination in a rat model of cerebellar ataxia
Authors: Fernandez A. M. , de la Vega A. Gonzalez , Torres-Aleman I.
Published: July 2002

We tested the potential of insulin-like growth factor I (IGF-I) to induce functional recovery in an animal model of cerebellar ataxia because this motor impairment is accompanied in humans and rodents by distinct changes in several components of the IGF-I trophic system. Rats rendered ataxic by deafferentation of the cerebellar cortex with 3-acetylpyridine recovered motor function after IGF-I was administered, as determined by behavioral and electrophysiological tests. When treated with IGF-I, inferior olive neurons, the targets of the neurotoxin, were rescued to various degrees (from 92 to 27% of surviving neurons), depending on the time that treatment with IGF-I was initiated. Furthermore, full recovery was obtained regardless of the route by which the trophic factor was administered (intraventricular or subcutaneous) even in rats with severe neuronal loss. These results suggest that human ataxia could be treated with IGF-I by a simple procedure.
Functional and structural mapping of human cerebral cortex: Solutions are in the surfaces
Authors: Van Essen David C. , Drury Heather A. , Joshi Sarang , Miller Michael I.
Published: July 2002

The human cerebral cortex is notorious for the depth and
irregularity of its convolutions and for its variability from one
individual to the next. These complexities of cortical geography have
been a chronic impediment to studies of functional specialization in
the cortex. In this report, we discuss ways to compensate for the
convolutions by using a combination of strategies whose common
denominator involves explicit reconstructions of the cortical surface.
Surface-based visualization involves reconstructing cortical surfaces
and displaying them, along with associated experimental data, in
various complementary formats (including three-dimensional native
configurations, two-dimensional slices, extensively smoothed surfaces,
ellipsoidal representations, and cortical flat maps). Generating these
representations for the cortex of the Visible Man leads to a
surface-based atlas that has important advantages over conventional
stereotaxic atlases as a substrate for displaying and analyzing large
amounts of experimental data. We illustrate this by showing the
relationship between functionally specialized regions and
topographically organized areas in human visual cortex. Surface-based
warping allows data to be mapped from individual hemispheres to a
surface-based atlas while respecting surface topology, improving
registration of identifiable landmarks, and minimizing unwanted
distortions. Surface-based warping also can aid in comparisons between
species, which we illustrate by warping a macaque flat map to match the
shape of a human flat map. Collectively, these approaches will allow
more refined analyses of commonalities as well as individual
differences in the functional organization of primate cerebral cortex.
Inhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNA
Authors: Good Liam , Nielsen Peter E.
Published: July 2002

Peptide nucleic acid (PNA) is a DNA mimic that has shown considerable promise as a lead compound for developing gene therapeutic drugs. We report that PNAs targeted to functional and accessible sites in ribosomal RNA can inhibit translation in an
Escherichia coli
cell-free transcription/translation system, with 50% reductions caused by nanomolar PNA concentrations. The effect
in vitro
is quantitatively similar to that of the known translation inhibitor and antibiotic tetracycline. Also, the targeted PNAs inhibited bacterial growth on agar plates and in liquid culture. A strain of
E. coli
(AS19) that is more permeable to antibiotics was approximately 10-fold more sensitive to the active PNAs, suggesting that the effect on growth indeed was caused by PNAs that entered cells. Inhibition was not observed when using control PNAs of similar composition but with an unrelated or mismatched sequence. The results demonstrate that ribosomal RNA is a possible target for sequence-designed novel antibiotics based on DNA analogues or mimics.
Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes
Authors: Lavinsky Robert M. , Jepsen Kristen , Heinzel Thorsten , Torchia Joseph , Mullen Tina-Marie , Schiff Rachel , Del-Rio Alfonso Leon , Ricote Mercedes , Ngo Sally , Gemsch Joslin , Hilsenbeck Susan G. , Osborne C. Kent , Glass Christopher K. , Rosenfeld Michael G. , Rose David W.
Published: July 2002

Several lines of evidence indicate that the nuclear receptor corepressor (N-CoR) complex imposes ligand dependence on transcriptional activation by the retinoic acid receptor and mediates the inhibitory effects of estrogen receptor antagonists, such as tamoxifen, suppressing a constitutive N-terminal, Creb-binding protein/coactivator complex-dependent activation domain. Functional interactions between specific receptors and N-CoR or SMRT corepressor complexes are regulated, positively or negatively, by diverse signal transduction pathways. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in a mouse model system for human breast cancer. Our data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways.
The small GTP-binding protein Cdc42 is required for nerve growth factor withdrawal-induced neuronal death
Authors: Bazenet Chantal E. , Mota Mónica A. , Rubin Lee L.
Published: July 2002

An increase in the level of the c-Jun transcription factor and of its phosphorylation has previously been shown to be essential for nerve growth factor (NGF) withdrawal-induced apoptosis of rat sympathetic neurons (SCG). The Rho-like GTPases Cdc42 and Rac1 are involved in the regulation of a number of cellular processes, including activation of the c-Jun NH
2
-terminal kinase (JNK) pathway. Therefore, we have investigated the role of these GTPases in this process. Overexpression of activated Rac1 or Cdc42 in SCG neurons maintained in the presence of NGF induced apoptosis, whereas expression of dominant negative mutants of Cdc42 or Rac1 blocked apoptosis following NGF withdrawal. Cdc42 activation produced an increase in the level of c-Jun and of its phosphorylation. Furthermore, Cdc42-induced death was prevented by coexpressing the c-Jun dominant negative FLAGΔ169. Thus, Cdc42 appears to function as an initiator of neuronal cell death by activating a transcriptional pathway regulated by c-Jun.
A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1
Authors: Casasnovas José M. , Stehle Thilo , Liu Jin-huan , Wang Jia-huai , Springer Timothy A.
Published: July 2002

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding toPlasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.
Translocation of inserted foreign epitopes by a channelforming protein
Authors: Jakes Karen S. , Kienker Paul K. , Slatin Stephen L. , Finkelstein Alan
Published: July 2002

Certain bacterial protein toxins are able to insert themselves into, and at least partially across, lipid bilayer membranes in the absence of any auxiliary proteins, by using unknown mechanisms to overcome the high energy barrier presented by the hydrophobic bilayer core. We have previously shown that one such toxin, colicin Ia, translocates a large, hydrophilic part of itself completely across a lipid bilayer in conjunction with the formation of an ion-conducting channel. To address the question of whether the colicin can translocate any arbitrary amino acid sequence, we have altered the translocated segment by inserting, singly, two different foreign epitopes. Colicins containing either epitope retain significant bactericidal activity and form channels of normal conductance in planar bilayers. Furthermore, antibodies added on the side of the bilayer opposite that to which the colicin was added interact specifically with the corresponding epitopes, producing an inhibition of channel closing. Thus, the inserted epitopes are translocated along with the rest of the segment, suggesting that a surprisingly small part of colicin Ia, located elsewhere in the molecule, acts as a nonspecific protein translocator.
Src interacts with dynamin and synapsin in neuronal cells
Authors: Foster-Barber Audrey , Bishop J. Michael
Published: July 2002

The nonreceptor tyrosine kinase Src is expressed at a high level in cells that are specialized for regulated secretion, such as the neuron, and is concentrated on secretory vesicles or at the site of exocytosis. To investigate the possibility that Src may play a role in regulating membrane traffic, we searched for neuronal proteins that will interact with Src. The SH3 domain of Src, but not that of the splice variant N-Src, bound to three proteins from mouse synaptosomes or PC12 cells: dynamin, synapsin Ia, and synapsin Ib. Dynamin and the synapsins coprecipitated with Src from PC12 cell extracts, and they colocalized with a subset of Src in the PC12 cell by immunofluorescence. Neither dynamin nor the synapsins were phosphorylated by Src, suggesting that the interaction of these proteins serves to direct the kinase activity of Src toward other proteins in the vesicle population. In immunoprecipitates containing Src and dynamin, the clathrin adaptor protein α-adaptin was also found. The association of Src and synapsin suggests a role for Src in the life cycle of the synaptic vesicle. The identification of a complex containing Src, dynamin, and α-adaptin indicates that Src may play a more general role in membrane traffic as well.
Substrate sequestration by a proteolytically inactive Lon mutant
Authors: Van Melderen Laurence , Gottesman Susan
Published: July 2002

Lon protein of
Escherichia coli
is an ATP-dependent protease responsible for the rapid turnover of both abnormal and naturally unstable proteins, including SulA, a cell division inhibitor made after DNA damage, and RcsA, a positive regulator of transcription. Lon is a multimer of identical 94-kDa subunits, each containing a consensus ATPase motif and a serine active site. We found that overexpressing Lon, which is mutated for the serine active site (LonS679A) and is therefore devoid of proteolytic activity, unexpectedly led to complementation of the UV sensitivity and capsule overproduction of a
lon
deletion mutant. SulA was not degraded by LonS679A, but rather was completely protected by the Lon mutant from degradation by other cellular proteases. We interpret these results to mean that the mutant LonS679A binds but does not degrade Lon substrates, resulting in sequestration of the substrate proteins and interference with their activities, resulting in apparent complementation. Lon that carried a mutation in the consensus ATPase site, either with or without the active site serine, was no longer able to complement a Δ
lon
mutant. These
in vivo
results suggest that the pathway of degradation by Lon couples ATP-dependent unfolding with movement of the substrate into protected chambers within Lon, where it is held until degradation proceeds. In the absence of degradation the substrate remains sequestered. Comparison of our results with those from a number of other systems suggest that proteins related to the regulatory portions of energy-dependent proteases act as energy-dependent sequestration proteins.
Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisxand Lewisx
Authors: Mao Shenlan , Gao Changshou , Lo Chih-Hung L. , Wirsching Peter , Wong Chi-Huey , Janda Kim D.
Published: July 2002

mAbs against tumor-associated carbohydrate antigens have the potential to play a prominent role in cancer immunotherapy. However, it has not been possible to fully exploit the clinical utility of such antibodies primarily, because those of adequate affinity could be derived only from murine sources. To address this problem, we prepared a single-chain Fv (scFv) antibody library from the peripheral blood lymphocytes of 20 patients with various cancer diseases. Completely human high-affinity scFv antibodies were then selected by using synthetic sialyl Lewisxand LewisxBSA conjugates. These human scFv antibodies were specific for sialyl Lewisxand Lewisx, as demonstrated by ELISA, BIAcore, and flow cytometry binding to the cell surface of pancreatic adenocarcinoma cells. Nucleotide sequencing revealed that at least four unique scFv genes were obtained. TheKdvalues ranged from 1.1 to 6.2 × 10−7M that were comparable to the affinities of mAbs derived from the secondary immune response. These antibodies could be valuable reagents for probing the structure and function of carbohydrate antigens and in the treatment of human tumor diseases.
Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions
in vivo
Authors: Stoffel Ruedi , Ziegler Sandra , Ghilardi Nico , Ledermann Birgit , de Sauvage Frederic J. , Skoda Radek C.
Published: July 2002

The question of whether extracellular signals influence hematopoiesis by instructing stem cells to commit to a specific hematopoietic lineage (instructive model) or solely by permitting the survival and proliferation of predetermined progenitors (permissive model) has been controversial since the discovery of lineage-dominant hematopoietic cytokines. To study the potential role of cytokines and their receptors in hematopoietic cell fate decisions, we used homologous recombination to replace the thrombopoietin receptor gene (
mpl
) with a chimeric construct encoding the extracellular domain of mpl and the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This chimeric receptor binds thrombopoietin but signals through the G-CSFR intracellular domain. We found that, despite the absence of a functional mpl signaling domain, homozygous knock-in mice had a normal platelet count, indicating that
in vivo
the cytoplasmic domain of G-CSFR can functionally replace mpl signaling to support normal megakaryopoiesis and platelet formation. This finding is compatible with the permissive model, according to which cytokine receptors provide a nonspecific survival or proliferation signal, and argues against an instructive role of mpl or G-CSFR in hematopoietic cell fate decisions.
Three-dimensional structure of phospho
enol
pyruvate carboxylase: A proposed mechanism for allosteric inhibition
Authors: Kai Yasushi , Matsumura Hiroyoshi , Inoue Tsuyoshi , Terada Kazutoyo , Nagara Yoshitaka , Yoshinaga Takeo , Kihara Akio , Tsumura Kennji , Izui Katsura
Published: July 2002

The crystal structure of phospho
enol
pyruvate carboxylase (PEPC; EC
4.1.1.31
) has been determined by x-ray diffraction methods at 2.8-Å resolution by using
Escherichia coli
PEPC complexed with
l
-aspartate, an allosteric inhibitor of all known PEPCs. The four subunits are arranged in a “dimer-of-dimers” form with respect to subunit contact, resulting in an overall square arrangement. The contents of α-helices and β-strands are 65% and 5%, respectively. All of the eight β-strands, which are widely dispersed in the primary structure, participate in the formation of a single β-barrel. Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers. The location of the catalytic site is likely to be near the C-terminal side of the β-barrel. The binding site for
l
-aspartate is located about 20 Å away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding. The participation of Arg-587 is unexpected, because it is known to be catalytically essential. Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC,
l
-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site. There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure. The importance of this loop in catalytic activity was also shown. Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by
l
-aspartate.
Interaction of the poliovirus receptor with poliovirus
Authors: He Yongning , Bowman Valorie D. , Mueller Steffen , Bator Carol M. , Bella Jordi , Peng Xiaozhong , Baker Timothy S. , Wimmer Eckard , Kuhn Richard J. , Rossmann Michael G.
Published: July 2002

The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-Å resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus “canyon” and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.
Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator
Authors: Ding Huangen , Demple Bruce
Published: July 2002

Nitric oxide (NO) has diverse roles in intercellular communication and (at higher levels) in immune-mediated cell killing. NO reacts with many cellular targets, with cell-killing effects correlated to inactivation of key enzymes through nitrosylation of their iron-sulfur centers. SoxR protein, a redox-sensitive transcription activator dependent on the oxidation state of its binuclear iron-sulfur ([2Fe-2S]) centers, is also activated in
Escherichia coli
on exposure to macrophage-generated NO. We show here that SoxR activation by NO occurs through direct modification of the [2Fe-2S] centers to form protein-bound dinitrosyl-iron-dithiol adducts, which we have observed both in intact bacterial cells and in purified SoxR after NO treatment. Functional activation through nitrosylation of iron-sulfur centers contrasts with the inactivation typically caused by this modification. Purified, nitrosylated SoxR has transcriptional activity similar to that of oxidized SoxR and is relatively stable. In contrast, nitrosylated SoxR is short-lived in intact cells, indicative of mechanisms that actively dispose of nitrosylated iron-sulfur centers.
A novel function for serotonin-mediated short-term facilitation in
Aplysia
: Conversion of a transient, cell-wide homosynaptic Hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement
Authors: Bailey Craig H. , Giustetto Maurizio , Zhu Hiuxiang , Chen Mary , Kandel Eric R.
Published: July 2002

Studies of sensitization and classical conditioning of the
gill-withdrawal reflex in
Aplysia
have shown that the
synaptic connections between identified glutamatergic sensory neurons
and motor neurons can be enhanced in one of two ways: by a
heterosynaptic (modulatory input-dependent) mechanism that gives rise
with repetition to long-term facilitation and by a homosynaptic
(activity-dependent) mechanism that gives rise with repetition to a
facilitation that is partially blocked by 2-amino-5-phosphonovaleric
acid and by injection of
1,2-bis(2-aminophenoxy)ethane-
N,N,N′,N′
-tetraacetate
(BAPTA) into the postsynaptic cell and is similar to long-term
potentiation in the hippocampus. We here have examined how these two
forms of facilitation interact at the level of an individual synaptic
connection by using a culture preparation consisting of a single
bifurcated sensory neuron that forms independent synaptic contacts with
each of two spatially separated motor neurons. We find that the
homosynaptic facilitation produced by a train of action potentials is
cell wide and is evident at all of the terminals of the sensory neuron.
By contrast, the heterosynaptic facilitation mediated by the modulatory
transmitter serotonin (5-HT) can operate at the level of a single
synapse. Homosynaptic activation gives rise to only a transient
facilitation lasting a few hours, even when repeated in a spaced
manner. The heterosynaptic facilitation produced by a single pulse of
5-HT, applied to one terminal of the sensory neuron, also lasts only
minutes. However, when one or more homosynaptic trains of spike
activity are paired with even a single pulse of 5-HT applied to one of
the two branches of the sensory neuron, the combined actions lead to a
selective enhancement in synaptic strength only at the 5-HT-treated
branch that now lasts more than a day, and thus amplifies, by more than
20-fold, the duration of the individually produced homo- and
heterosynaptic facilitation. This form of synapse-specific facilitation
has unusual long-term properties. It does not require protein
synthesis, nor is it accompanied by synaptic growth.
Crystal structure of the cystine
C-S
lyase from
Synechocystis
: Stabilization of cysteine persulfide for FeS cluster biosynthesis
Authors: Clausen Tim , Kaiser Jens T. , Steegborn Clemens , Huber Robert , Kessler Dorothea
Published: July 2002

FeS clusters are versatile cofactors of a variety of proteins, but the mechanisms of their biosynthesis are still unknown. The cystine C-S lyase from
Synechocystis
has been identified as a participant in ferredoxin FeS cluster formation. Herein, we report on the crystal structure of the lyase and of a complex with the reaction products of cystine cleavage at 1.8- and 1.55-Å resolution, respectively. The sulfur-containing product was unequivocally identified as cysteine persulfide. The reactive persulfide group is fixed by a hydrogen bond to His-114 in the center of a hydrophobic pocket and is thereby shielded from the solvent. Binding and stabilization of the cysteine persulfide represent an alternative to the generation of a protein-bound persulfide by NifS-like proteins and point to the general importance of persulfidic compounds for FeS cluster assembly.
Conditional expression of a G
i
-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy
Authors: Redfern Charles H. , Degtyarev Michael Y. , Kwa Andrew T. , Salomonis Nathan , Cotte Nathalie , Nanevicz Tania , Fidelman Nick , Desai Kavin , Vranizan Karen , Lee Elena K. , Coward Peter , Shah Nila , Warrington Janet A. , Fishman Glenn I. , Bernstein Daniel , Baker Anthony J. , Conklin Bruce R.
Published: July 2002

Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G
i
-signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G
i
-coupled receptor (Ro1), we provide evidence that increased G
i
signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G
i
signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G
i
signaling in causing cardiac pathology.
Shared cognitive representations of perceptual and semantic structures of basic colors in Chinese and English
Authors: Moore Carmella C. , Romney A. Kimball , Hsia Ti-lien
Published: July 2002

Two perceptual structures (based on colors) and one semantic
structure (based on names) of eight basic chromatic colors were
obtained from three judged similarity tasks for 41 Mandarin
Chinese-speaking females in Taiwan and 35 English-speaking females in
the United States. Major findings include the following:
(
i
) there are robust and reliable individual differences
among individuals, (
ii
) the structures for Chinese and
English speakers are similar, and (
iii
) the semantic
structure of color names is similar to the perceptual structures of
colors. The results place strong constraints on theories relating to
individual differences, linguistic relativity, and the relation of
perceptual and semantic structures for colors.
Personal Adjustment to Acculturative Transitions: The Turkish Experience
Authors: Phalet Karen , Hagendoorn Louk
Published: July 2002

This research examines personal adjustment to acculturative transitions. Internal adjustment, or well‐being, and external adjustment, or effectiveness, are analyzed in two acculturation contexts: migration from rural to urban settings in Turkey, and international migration from Turkey to Belgium. Comparisons are made between low‐SES (socioeconomic status) and high‐SES youth in Istanbul, as well as between these youngsters and Turkish youth in Belgium. Adjustment is explained by collectivism and achievement values among acculturating persons, and by social inequality and cultural distance between acculturating and dominant cultural groups. Full causal models are used to test individual‐level and group‐level explanations of acculturative adjustment. It is found that Turkish youngsters with a collectivistic value orientation have fewer adjustment problems. Second, social inequality increases internal adjustment problems of low‐SES youth in Turkey, and cultural distance adds to external adjustment problems of Turkish immigrant youth in Belgium. Third, social inequality reinforces collectivism of low‐SES youth in Turkey, whereas in Belgium, collectivism is reinforced by cultural distance. Simultaneously, cultural distance in Belgium lowers achievement values of Turkish immigrant youth.
International Representation in the Psychological Literature
Authors: Bauserman Robert
Published: July 2002

The author searched PsycLit records to examine the representation of psychological research from outside the US and changes in international representation over time. American research dominates, but international representation has increased since 1975. Western Europe, Japan, Australia, and Israel in particular have become better represented. In contrast, developing nations have remained poorly represented for most of the time period examined. Relative national influence may accurately reflect the numbers of psychologists in general, and research psychologists in particular, in each nation. Psychologists in developing nations also focus on teaching, clinical, and social issues, and have few resources for research activities. International representation is likely to continue increasing.
History, Europe and the “National Idea”: The “Official” Narrative of National Identity in Ukraine
Authors: Wolczuk Kataryna
Published: July 2002

The demise of the Soviet Union entailed a reconfiguration of the political space and a reforging of collective identities within the boundaries of the new successor states. In the view of Anthony Smith, this was inevitable: “the rediscovery of the national self is not an academic matter, it is a pressing practical issue, vexed, and contentious, which spells life or death for the nationalist project of creating a nation.” Defining the national “self not only accomplishes a symbolic break with the previous political community but also sets out the parameters of statehood with regard to language and minority rights. However, even if the newness of the polity precipitates ”the definition, creation, and solidarification of a viable collective identity,“ this can be anything but straightforward. New states dwell on particularism, that is they look to ”local mores, established institutions, and the unities of common experience—to ‘tradition,’ ‘culture,’ ‘national character,’ or even ‘race’ for the roots of a new identity.“ And yet defining the national particularism may be fraught with inherent difficulties because, as Geertz observed, ”new states tend to be bundles of competing traditions gathered accidentally into concocted political frameworks rather than organically evolving civilizations.“
National Identity and Political Legitimacy in Turkmenistan*
Authors: Akbarzadeh Shahram
Published: July 2002

This paper traces political events and modes of generating legitimacy in Turkmenistan since the Soviet collapse. The emphasis here is on state policies and social movements that relate to “nation building” for their contribution to political legitimacy. The extent of nation-building success is not an immediate subject of inquiry, for this paper is not about public perception and bottom-up response to state policies, but the reverse. It is certain that state-sponsored proclamations and nationalist ideas espoused by the intelligentsia do not always find resonance among the national population at large. However, attention given to social movements in this paper may compensate for this shortcoming in a small way. It must be stated that social movements in Turkmenistan, and Central Asia, as a whole, have been top heavy. They were principally initiated and steered by the urbanized intelligentsia. The extent of mass involvement in such movements is suspect and hard to gauge.