bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of insect-borne disease: An approach using transgenic symbiotic bacteria
Authors: Durvasula Ravi V. , Gumbs Andrew , Panackal Anil , Kruglov Oleg , Aksoy Serap , Merrifield R. Bruce , Richards Frank F. , Beard Charles B.
Published: July 2002

Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting insects may serve as a powerful approach to control certain arthropod-borne diseases. The endosymbiont of the Chagas disease vector,
Rhodnius prolixus
, has been transformed to express cecropin A, a peptide lethal to the parasite,
Trypanosoma cruzi
. In insects carrying the transformed bacteria, cecropin A expression results in elimination or reduction in number of
T. cruzi
. A method has been devised to spread the transgenic bacteria to populations of
R. prolixus
, in a manner that mimics their natural coprophagous route of symbiont acquisition.
Bacteria lacking a multidrug pump: A sensitive tool for drug discovery
Authors: Hsieh Pei-Chung , Siegel Scott A. , Rogers Bruce , Davis Deborah , Lewis Kim
Published: July 2002

Microorganisms express multidrug resistance pumps (MDRs) that can confound antibiotic discovery. We propose the use of mutants deficient in MDRs to overcome this problem. Sensitivity to quinolones and to amphipathic cations (norfloxacin, benzalkonium chloride, cetrimide, pentamidine, etc.) was increased 5- to 30-fold in a
Staphylococcus aureus
mutant with a disrupted chromosomal copy of the NorA MDR. NorA was required both for increased sensitivity to drugs in the presence of an MDR inhibitor and for increased rate of cation efflux. This requirement suggests that NorA is the major MDR protecting
S. aureus
from the antimicrobials studied. A 15- to 60-fold increase in sensitivity to antimicrobials also was observed in wild-type cells at an alkaline pH that favors accumulation of cations and weak bases. This effect was synergistic with a
norA
mutation, resulting in an increase up to 1,000-fold in sensitivity to antimicrobials. The usefulness of applying MDR mutants for natural product screening was demonstrated further by increased sensitivity of the
norA
−
strain to plant alkaloid antimicrobials, which might be natural MDR substrates.
In search of a mutational hotspot
Authors: Hatahet Zafer , Zhou Meixia , Reha-Krantz Linda J. , Morrical Scott W. , Wallace Susan S.
Published: July 2002

In vitro
selection was used to define sequence contexts that significantly enhanced the mutagenic potential of 7,8-dihydro-8-oxoguanine (8-oxoG). Contexts that simultaneously reduced the efficiency of 8-oxoG cleavage by formamidopyrimidine DNA
N
-glycosylase and increased the efficiency of misincorporating A opposite the lesion by DNA polymerase were isolated from a pool of 4
8
random octanucleotide sequences. Kinetic analysis showed that the combined effects of poor repair and high miscoding resulted in 10
2
- to 10
3
-fold increase in the mutagenic potential of 8-oxoG. Furthermore, the isolated sequence contexts correlated strongly with G → T transversion hotspots in spontaneous mutational spectra reported for the
Escherichia coli lacI
and human p53 and factor IX genes. We present an example directly linking the interplay between DNA repair and replication to a “high risk sequence” for base substitution.
A 1.3-Å resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation
Authors: Ippolito Joseph A. , Steitz Thomas A.
Published: July 2002

The crystal structure of an HIV-1 trans-activation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-Å resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the influence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T
in vivo
.
Indirect mutagenesis by oxidative DNA damage: Formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal
Authors: Dedon Peter C. , Plastaras John P. , Rouzer Carol A. , Marnett Lawrence J.
Published: July 2002

Oxidation of endogenous macromolecules can generate electrophiles capable of forming mutagenic adducts in DNA. The lipid peroxidation product malondialdehyde, for example, reacts with DNA to form M
1
G, the mutagenic pyrimidopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Among the products of oxidative DNA damage are base propenals. We hypothesized that these structural analogs of malondialdehyde would react with DNA to form M
1
G. Consistent with this hypothesis, we detected a dose-dependent increase in M
1
G in DNA treated with calicheamicin and bleomycin, oxidizing agents known to produce base propenal. The hypothesis was proven when we determined that 9-(3-oxoprop-1-enyl)adenine gives rise to the M
1
G adduct with greater efficiency than malondialdehyde itself. The reactivity of base propenals to form M
1
G and their presence in the target DNA suggest that base propenals derived from oxidative DNA damage may contribute to the mutagenic burden of a cell.
MLCK-A, an unconventional myosin light chain kinase from
Dictyostelium
, is activated by a cGMP-dependent pathway
Authors: Silveira Linda A. , Smith Janet L. , Tan John L. , Spudich James A.
Published: July 2002

Dictyostelium
myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca
2+
/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A’s activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when
Dictyostelium
cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh
Dictyostelium
lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.
TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins
Authors: Salzmann Michael , Pervushin Konstantin , Wider Gerhard , Senn Hans , Wüthrich Kurt
Published: July 2002

The NMR assignment of
13
C,
15
N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below ∼25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (
1
H
N
), the amide nitrogen (
15
N), and
13
C atoms, this size limit has been previously extended by additional labeling with deuterium (
2
H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([
15
N,
1
H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for
2
H/
13
C/
15
N-labeled proteins and approximately twofold sensitivity gain for
13
C/
15
N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [
15
N,
1
H]-TROSY-HNCA and [
15
N,
1
H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of
15
N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.
Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells
Authors: Hahn Gabriele , Jores Rita , Mocarski Edward S.
Published: July 2002

Hematopoietic cells and their progenitors play important roles in human cytomegalovirus latency and reactivation. Latent infection has been evaluated in defined populations of myeloid-lineage-committed progenitor cells coexpressing CD33 and CD15 or CD33 and CD14 along with the dendritic cell markers CD1a and CD10. These CD33+cell populations were found to support latency and expression of viral latency-associated transcripts and to undergo reactivation of productive viral replication when differentiated in the presence of human fibroblasts. Reactivation was also observed when myeloid cells were carried in the presence of fibroblast-conditioned medium or medium supplemented with certain cytokines (interferon γ, tumor necrosis factor α, interleukin 4, or granulocyte–macrophage colony-simulating factor), suggesting that cell differentiation pathways act as determinants of reactivation. More primitive CD34+hematopoietic cells were also found to be susceptible to viral infection and latency was maintained as these cells differentiated into CD33+-lineage-committed populations. Between 0.01% and 0.001% of CD33+CD14+or CD33+CD15+bone marrow mononuclear cells isolated from naturally infected individuals were found to express latent transcripts. Thus, cytomegalovirus is carried within a small percentage of myeloid and dendritic cell progenitors in the healthy seropositive host. Virus reactivation may be triggered by factors associated with the inflammatory response.
Intraprotein electron transfer between tyrosine and tryptophan in DNA photolyase from
Anacystis nidulans
Authors: Aubert Corinne , Mathis Paul , Eker André P. M. , Brettel Klaus
Published: July 2002

Light-induced electron transfer reactions leading to the fully reduced, catalytically competent state of the flavin adenine dinucleotide (FAD) cofactor have been studied by flash absorption spectroscopy in DNA photolyase from
Anacystis nidulans
. The protein, overproduced in
Escherichia coli
, was devoid of the antenna cofactor, and the FAD chromophore was present in the semireduced form, FADH
⋅
, which is inactive for DNA repair. We show that after selective excitation of FADH
⋅
by a 7-ns laser flash, fully reduced FAD (FADH
−
) is formed in less than 500 ns by electron abstraction from a tryptophan residue. Subsequently, a tyrosine residue is oxidized by the tryptophanyl radical with
t

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= 50 μs. The amino acid radicals were identified by their characteristic absorption spectra, with maxima at 520 nm for Trp
⋅
and 410 nm for TyrO
⋅
. The newly discovered electron transfer between tyrosine and tryptophan occurred for ≈40% of the tryptophanyl radicals, whereas 60% decayed by charge recombination with FADH
−
(
t

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= 1 ms). The tyrosyl radical can also recombine with FADH
−
but at a much slower rate (
t

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= 76 ms) than Trp
⋅
. In the presence of an external electron donor, however, TyrO
⋅
is rereduced efficiently in a bimolecular reaction that leaves FAD in the fully reduced state FADH
−
. These results show that electron transfer from tyrosine to Trp
⋅
is an essential step in the process leading to the active form of photolyase. They provide direct evidence that electron transfer between tyrosine and tryptophan occurs in a native biological reaction.
Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism
Authors: Galan Jean-Marc , Peter Matthias
Published: July 2002

Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction. Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitin-conjugating enzyme, Cdc34p. It is not understood how SCF complexes are regulated and how physiological conditions alter their levels. Here we show that three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin- and proteasome-dependent manner
in vivo
. Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism. Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels did not fluctuate through the cell cycle. Taken together, our results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle.
Lepidopteran DALP, and its mammalian ortholog HIC-5, function as negative regulators of muscle differentiation
Authors: Hu Yanhui , Cascone Pamela J. , Cheng Lihong , Sun Danhui , Nambu John R. , Schwartz Lawrence M.
Published: July 2002

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of
DALP
, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth
Manduca sexta
become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in
Drosophila
results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C
2
C
12
myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved “switches” to block muscle differentiation and induce death.
Rational design of nascent metalloenzymes
Authors: Benson David E. , Wisz Michael S. , Hellinga Homme W.
Published: July 2002

Understanding the early genesis of new enzymatic functions is one of the challenges in protein design, mechanistic enzymology, and molecular evolution. We have experimentally mimicked starting points in this process by introducing primitive iron and oxygen binding sites at various locations in thioredoxin, a small protein lacking metal centers, by using computational design. These rudimentary active sites show emerging enzymatic activities that select to varying degrees between different oxygen chemistries. Even within these nascent enzymes, mechanisms by which different reactions are controlled can be discerned. These involve both stabilizing and destabilizing interactions imposed on the metal center by the surrounding protein matrix.
T cell receptor interactions with class I heavy-chain influence T cell selection
Authors: Kuhns Scott T. , Tallquist Michelle D. , Johnson Aaron J. , Mendez-Fernandez Yanice , Pease Larry R.
Published: July 2002

The interaction of the T cell receptor (TCR) with peptide in the binding site of the major histocompatibility complex molecule provides the basis for T cell recognition during immune surveillance, repertoire development, and tolerance. Little is known about the extent to which repertoire selection is influenced directly by variation of the structure of the class I heavy chain. We find that the 2C TCR, normally positively selected in the context of the Kbmolecule, is minimally selected into the CD8 lineage in the absence of antigen-processing genes. This finding underscores the importance of peptides in determining the positive-selecting class I ligands in the thymus. In contrast, Kbm3, a variant class I molecule that normally exerts a negative selection pressure on 2C-bearing T cells, positively selects 2C transgenic T cells into the CD8 lineage in an antigen-processing gene-deficient environment. These findings indicate that structural changes in the heavy chain can have direct influence in T cell recognition, from which we conclude that the nature of TCR interaction with class I heavy chain influences the array of TCRs selected during development of the functional adult repertoire.
Introduction
Authors: Gustavsson Sven
Published: July 2002

Right at the start, one word in the title of this special topic issue requires clarification: the word “minority.” We will not try to define the term here, but it is only fair to say that some of the groups which are mentioned or discussed are not recognized as minorities or have a rather dubious legal position, as, for example, the Kashubs and Lemkos in Poland or Macedonians in Greece. As to the notion of linguistic minority: it is a term very often used but rarely defined. That it is often used has to do with the fact that most minorities in Europe speak a language or a dialect or variant which discriminates them from the majority. As a matter of fact, the nationalist traditions in Europe seem to take more or less for granted that a minority without a separate language is not a real minority. Language questions have been in the forefront of most nation-building projects or ethnic mobilizations in Europe, and so it has become more or less axiomatic that every nation or ethnic group should have its own language. As an example, the Bosnian language may be cited. The Bosnians—or rather the Bosniaks—insist on naming the language they use Bosnian instead of, as before, Serbo-Croatian or Croato-Serbian. They also insist on their right to standardize their language in their own fashion. Another good example is the Rusyn language, or rather—to be more precise—the Rusyn languages in the Carpathian area. As Rusyns, not Ukrainians, they are now trying to create their own standard language or languages on the basis of their own dialects. The Rusyns in Yugoslavia started this process earlier; consequently, the Rusyn language of Yugoslavia is now to be considered one of the standard Slavic languages.
The Position of the Slovenes in Austria: Recent Developments in Political (and other) Attitudes
Authors: Priestly Tom
Published: July 2002

The Slovene-speaking minority in Austria—when compared with many other linguistic minorities in Europe—is in an enviable position. Superficially, its minority rights are both constitutionally guaranteed and, for the most part, legally enforced; in the province of Carinthia/Kärnten/Koroška (the home of nearly all the minority; see Map 1) bilingual education is available in many communities at the primary level, and there is a thriving bilingual secondary school; Slovene is officially used in many offices and churches, and can be heard in many shops and on many street corners; there are two weekly newspapers. The picture below the surface is not quite as pleasant: there is anti-Slovene discrimination in several forms, and the pressure on minority members to Germanize themselves is strong; in particular, it must be emphasized that although the minority enjoys virtually full support from the federal government in Vienna, the provincial government in Carinthia has seldom been as favorably disposed. Still, most of the other minorities in Central and Eastern Europe can only dream of living in conditions like those of the Carinthian Slovenes.
Using Pupil Performance Data for Judging Schools and Teachers: Scope and limitations
Authors: Goldstein Harvey
Published: July 2002

Abstract The article reviews ways in which performance data are currently used within the England and Wales education system. It uses research evidence to critique many of these uses and presents some alternative proposals for a more rational approach.
The Influence of Personal Involvement on the Performance in a False Belief Task: A Structural Analysis
Authors: Battacchi Marco Walter , Celani Giorgio , Bertocchi Alberto
Published: July 2002

In this study, 200 children (age range 2;6-5;6 years) were tested by using a standard task aimed at assessing their understanding of other’s false belief. The children were then divided into two groups: those who passed and those who failed. Later, those children who had failed the first test, were put into different groups and tested again under different conditions. This was done in order to check the effect of representational and motivational variables related to personal involvement on performance in the second task. The results showed a tendency towards an improvement of performance in the second task, when the following two conditions were simultaneously satisfied: (1) children were retested with a familiar peer instead of puppets; and (2) children were actors who took part in the action instead of remaining external spectators of the scene. A tentative explanation of the findings on the basis of the representational complexity of the different false belief tasks was proposed.
Buprenorphine and carbamazepine as a treatment for detoxification of opiate addicts with multiple drug misuse: a pilot study
Authors: Schneider U. , Paetzold W. , Eronat V. , Huber T. J. , Seifert J. , Wiese B. , Emrich H. M.
Published: July 2002

The growing tendency of opioid addicts to misuse multiple other drugs leads to the investigation of new pharmacostrategies to prevent patients from suffering life‐threatening complications and minimize the withdrawal symptoms. The short‐term efficacy of a 10‐day low‐dose buprenorphine/19‐day carbamazepine regime (n = 15) to a 14‐day oxazepam/19‐day carbamazepine regime (n = 12) in an open‐labelled 21‐day inpatient detoxification treatment was compared. Twenty‐seven men and women dependent on opioids and misusing other drugs admitted to a detoxification unit were included in this protocol. Eighteen of 27 patients (67%) completed the study. Four non‐completers (27%) received buprenorphine/carbamazepine (four of 15) and five non‐completers (42%) were treated with oxazepam/carbamzepine (five of 12), but the difference in the dropout rate between the two treatment strategies was not significant.The buprenorphine/carbamazepine regime provided significantly more effective relief of withdrawal symptoms during the first week of treatment. No severe side effects occurred during treatment in both groups. The present study supports the hypothesis that buprenorphine/carbamazepine is more effective than oxazepam/carbamazepine in rapid opioid detoxification in patients with additional multiple drug misuse and both regimens were safe with no unexpected side effects.
Quality of Life in Children Following Treatment for a Malignant Primary Bone Tumour Around the Knee
Authors: Eiser Christine , Cool Paul , Grimer Robert J. , Carter Simon R. , Cotter Imogen M. , Ellis Ann J. , Kopel Sheryl
Published: July 2002

Purpose. We report on the quality of life following treatment for a malignant primary bone tumour around the knee in skeletally immature children.

Patients. Patients (n = 41; mean age = 18 years; range 8–28) had all experienced chemotherapy in a neo‐adjuvant setting, surgical excision of the tumour and endoprosthetic replacement.

Methods. Interviews were conducted separately with the child and mother and focused on mobility, body image and the impact of treatment on schooling, employment and plans for the future.

Results. Mobility in the group was variable. Only 12% reported that they could run with any confidence. The proportion who were able to swim (49%) or ride a bike (46%) was higher. All had experienced major disruption in schooling (mean absence following diagnosis = 12 months). Eight had repeated a school year and 41% patients reported that their schoolwork was affected. As a result of their experience, eight (six females and two males) chose health‐related employment. Concerns for the future were highest among males and those with manual jobs. Three patients were receiving psychiatric support, in relation to extreme concern about the risk of recurrence. All expressed satisfaction with treatment, and older patients believed that the prosthesis gave a better quality of life than amputation.

Discussion. Our data suggest that outcome following limb‐salvage surgery is variable. Education is disrupted. Even so, only two left school with no qualifications. Employment is most restricted among males with few qualifications who may benefit from sensitive vocational counselling.
Components of the Human SWI/SNF Complex Are Enriched in Active Chromatin and Are Associated with the Nuclear Matrix
Authors: Reyes Jose C. , Muchardt Christian , Yaniv Moshe
Published: July 2002

Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation. We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton. Immunofluorescence studies revealed a punctate nuclear labeling pattern that was excluded from the nucleoli and from regions of condensed chromatin. Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.
Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer
Authors: Kenworthy A.K. , Edidin M.
Published: July 2002

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.
The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP
Authors: Hellman Rachel , Vanhove Marc , Lejeune Annabelle , Stevens Fred J. , Hendershot Linda M.
Published: July 2002

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.
Human Ect2 Is an Exchange Factor for Rho Gtpases, Phosphorylated in G2/M Phases, and Involved in Cytokinesis
Authors: Tatsumoto Takashi , Xie Xiaozhen , Blumenthal Rayah , Okamoto Isamu , Miki Toru
Published: July 2002

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH2-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.
Requirements for CD1d Recognition by Human Invariant Vα24+ CD4−CD8− T Cells
Authors: Exley Mark , Garcia Jorge , Balk Steven P. , Porcelli Steven
Published: July 2002

A subset of human CD4−CD8− T cells that expresses an invariant Vα24-JαQ T cell receptor (TCR)-α chain, paired predominantly with Vβ11, has been identified. A series of these Vα24 Vβ11 clones were shown to have TCR-β CDR3 diversity and express the natural killer (NK) locus–encoded C-type lectins NKR-P1A, CD94, and CD69. However, in contrast to NK cells, they did not express killer inhibitory receptors, CD16, CD56, or CD57. All invariant Vα24+ clones recognized the MHC class I–like CD16 molecule and discriminated between CD1d and other closely related human CD1 proteins, indicating that recognition was TCR-mediated. Recognition was not dependent upon an endosomal targeting motif in the cytoplasmic tail of CD1d. Upon activation by anti-CD3 or CD1d, the clones produced both Th1 and Th2 cytokines. These results demonstrate that human invariant Vα24+ CD4−CD8− T cells, and presumably the homologous murine NK1+ T cell population, are CD1d reactive and functionally distinct from NK cells. The conservation of this cell population and of the CD1d ligand across species indicates an important immunological function.
Direct Visualization of Antigen-specific CD8+T Cells during the Primary Immune Response to Epstein-Barr Virus In Vivo
Authors: Callan M.F.C. , Tan L. , Annels N. , Ogg G.S. , Wilson J.D.K. , O'Callaghan C.A. , Steven N. , McMichael A.J. , Rickinson A.B.
Published: July 2002

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex–peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8+ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8+ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.
CD5 Expression Is Developmentally Regulated By T Cell Receptor (TCR) Signals and TCR Avidity
Authors: Azzam Hala S. , Grinberg Alex , Lui Kin , Shen Howard , Shores Elizabeth W. , Love Paul E.
Published: July 2002

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4−CD8− (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56lck. At the CD4+CD8+ (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR–major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4+ or CD8+ (single positive, SP) thymocytes is induced by engagement of the α/β-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR–MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.
Nonselective Suppression of Voltage-gated Currents by Odorants in the Newt Olfactory Receptor Cells
Authors: Kawai Fusao , Kurahashi Takashi , Kaneko Akimichi
Published: July 2002

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch–clamp technique. Under voltage clamp, membrane depolarization to voltages between −90 mV and +40 mV from a holding potential (Vh) of −100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na+ current: INa, T-type Ca2+ current: ICa,T, L-type Ca2+ current: ICa,L, delayed rectifier K+ current: IKv and Ca2+-activated K+ current: IK(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (INa), 0.15 mM (ICa,T), 0.14 mM (ICa,L), 1.7 mM (IKv), and 0.17 mM (IK(Ca)), and Hill coefficient was 1.4 (INa), 1.0 (ICa,T), 1.1 (ICa,L), 1.0 (IKv), and 1.1 (IK(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of INa was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were −53 mV at control, −66 mV at 0.01 mM, and −84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on INa in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.
Gating Induces a Conformational Change in the Outer Vestibule of Enac
Authors: Snyder Peter M. , Bucher Daniel B. , Olson Diane R.
Published: July 2002

The epithelial Na+ channel (ENaC) is comprised of three homologous subunits (α, β, and γ). The channel forms the pathway for Na+ absorption in the kidney, and mutations cause disorders of Na+ homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in α, β, and γENaC, respectively). Equivalent “DEG” mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na+ current was increased by mutagenesis or chemical modification of this residue and adjacent residues in α, β, and γENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in βENaC increased the open state probability from 0.12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na+ current (γN530K) was identified in a patient with renal disease.
Location of a Constriction in the Lumen of a Transmembrane Pore by Targeted Covalent Attachment of Polymer Molecules
Authors: Movileanu Liviu , Cheley Stephen , Howorka Stefan , Braha Orit , Bayley Hagan
Published: July 2002

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal α-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.
Ryanoid Modification of the Cardiac Muscle Ryanodine Receptor Channel Results in Relocation of the Tetraethylammonium Binding Site
Authors: Tanna Bhavna , Welch William , Ruest Luc , Sutko John L. , Williams Alan J.
Published: July 2002

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425–447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9α-hydroxyryanodine, 21-azido-9α-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9α-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.
The significance of an impact factor: implications for the publishing community
Authors: O'Neill Jill
Published: July 2002

The journal impact factor, as a metric developed in the mid‐1960s by Dr Eugene Garfield and Dr Irving Sher, represents the influence that an ‘average article’ published in a specific journal has on the scholarly discipline and audience that it serves. Originally intended to serve as an equalizer for use by the Institute for Scientific Information® (ISI®) in making comparative evaluations of large and small journals in a particular discipline, the impact factor now has numerous applications for publishers, librarians, and researchers. Ideally, the journal impact factor should be seen by publishers as a useful tool in gauging the effectiveness of their publication product in serving the needs of a particular scholarly community. The significance of a journal impact factor, its appropriate usage by the scholarly publishing community and its extension into the electronic environment are discussed.
On one problem of uniqueness of meromorphic functions concerning small functions
Authors: Yi Hong-Xun
Published: July 2002

In this paper, we show that if two non-constant meromorphic functions

f
f

and

g
g

satisfy

E
¯

(

a

j

,
k
,
f
)
=

E
¯

(

a

j

,
k
,
g
)

\overline {E}(a_{j},k,f)=\overline {E}(a_{j},k,g)

for

j
=
1
,
2
,
…
,
5

j=1,2,\dots ,5

, where

a

j

a_{j}

are five distinct small functions with respect to

f
f

and

g
g

, and

k
k

is a positive integer or

∞
\infty

with

k
≥
14

k\geq 14

, then

f
≡
g

f\equiv g

. As a special case this also answers the long-standing problem on uniqueness of meromorphic functions concerning small functions.
Convergence of difference schemes with high resolution for conservation laws
Authors: Chen Gui-Qiang , Liu Jian-Guo
Published: July 2002

We are concerned with the convergence of Lax-Wendroff type schemes with high resolution to the entropy solutions for conservation laws. These schemes include the original Lax-Wendroff scheme proposed by Lax and Wendroff in 1960 and its two step versions–the Richtmyer scheme and the MacCormack scheme. For the convex scalar conservation laws with algebraic growth flux functions, we prove the convergence of these schemes to the weak solutions satisfying appropriate entropy inequalities. The proof is based on detailed

L

p

L^{p}

estimates of the approximate solutions,

H

−
1

H^{-1}

compactness estimates of the corresponding entropy dissipation measures, and some compensated compactness frameworks. Then these techniques are generalized to study the convergence problem for the nonconvex scalar case and the hyperbolic systems of conservation laws.