bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription
Authors: Chen Dan , Fong Yick , Zhou Qiang
Published: July 2002

Tat stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human–rodent “hybrid” P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb–Tat–TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb–Tat interaction.
Effect of G protein heterotrimer composition on coupling of neurotransmitter receptors to N-type Ca
2+
channel modulation in sympathetic neurons
Authors: Jeong Seong-Woo , Ikeda Stephen R.
Published: July 2002

Voltage-dependent (VD) inhibition of N-type Ca
2+
channels is mediated primarily by neurotransmitter receptors that couple to pertussis toxin (PTX)-sensitive G proteins (such as G
o
and G
i
). To date, however, the composition of heterotrimeric complexes, i.e., specific Gαβγ combinations, capable of coupling receptors to N-type Ca
2+
channels has not been defined. We addressed this question by heterologously expressing identified Gαβγ combinations in PTX-treated rat sympathetic neurons and testing for reconstitution of agonist-mediated VD inhibition. The heterologously expressed Gα subunits were rendered PTX-insensitive by mutating the codon specifying the ADP ribosylation site. The following results were obtained from this approach. (
i
) Expression of Gα
oA
, Gα
oB
, and Gα
i2
(along with Gβ
1
γ
2
) reconstituted VD inhibition mediated by α
2
-adrenergic, adenosine, somatostatin, and prostaglandin E
2
receptors. Conversely, expression of Gα
i1
and Gα
i3
was ineffective at restoring coupling. (
ii
) Coupling efficiency, as determined from the magnitude of reconstituted Ca
2+
current inhibition, depended on both the receptor and Gα subtype. The following rank order of coupling efficiency was observed: Gα
oA
= Gα
oB
> Gα
i2
for α
2
-adrenergic receptor; Gα
i2
> Gα
oA
= Gα
oB
for adenosine and prostaglandin E
2
receptors; and Gα
oB
= Gα
i2
> Gα
oA
for the somatostatin receptor. (
iii
) In general, varying the Gβγ composition of Gα
oA
-containing heterotrimers had little effect on the coupling of α
2
-adrenergic receptors to the VD pathway. Taken together, these results suggest that multiple, diverse Gαβγ combinations are capable of coupling neurotransmitter receptors to VD inhibition of N-type Ca
2+
channels. Thus, if exquisite Gαβγ-coupling specificity exists
in situ
, it cannot arise solely from the inherent inability of other Gαβγ combinations to form functional signaling complexes.
In Vivo Function of a Differentiation Inhibitor, Id2
Authors: Yokota Yoshifumi , Mori Seiichi , Narumi Osamu , Kitajima Kazuhito
Published: July 2002

Cell differentiation is an essential process for the development of various cell types that constitute multicellular organisms. During development, the large family of factors bearing a helix‐loop‐helix (HLH) motif participates profoundly in this process and these factors serve as good experimental tools for investigating mechanisms underlying tissue‐specific differentiation. The HLH family includes both positive and negative regulators of cell differentiation: basic HLH (bHLH)‐type transcription factors and Id proteins, respectively. Following an exciting decade focusing on bHLH factors, advances achieved in studies of the inhibitory factors in the last couple of years have placed them in the front line of the research on differentiation and proliferation control. Here, we present and discuss recent results obtained using Id2 ‐deficient mice, which manifest intriguing phenotypes in various systems.
Low Density Membranes Are Associated with RNA-binding Proteins and Thylakoids in the Chloroplast of Chlamydomonas reinhardtii
Authors: Zerges William , Rochaix Jean-David
Published: July 2002

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thylakoids.
Transcription Factor Erg Variants and Functional Diversification of Chondrocytes during Limb Long Bone Development
Authors: Iwamoto Masahiro , Higuchi Yoshinobu , Koyama Eiki , Enomoto-Iwamoto Motomi , Kurisu Kojiro , Yeh Helena , Abrams William R. , Rosenbloom Joel , Pacifici Maurizio
Published: July 2002

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27–amino acid segment located ∼80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.
Negative Regulation of Ros Receptor Tyrosine Kinase Signaling
Authors: Keilhack Heike , Müller Marit , Böhmer Sylvia-Annette , Frank Carsten , Weidner K. Michael , Birchmeier Walter , Ligensa Tanja , Berndt Alexander , Kosmehl Hartwig , Günther Bernd , Müller Thomas , Birchmeier Carmen , Böhmer Frank D.
Published: July 2002

Male “viable motheaten” (mev) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of mev mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH2-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.
Related Leucine-based Cytoplasmic Targeting Signals in Invariant Chain and Major Histocompatibility Complex Class II Molecules Control Endocytic Presentation of Distinct Determinants in a Single Protein
Authors: Zhong Guangming , Romagnoli Paola , Germain Ronald N.
Published: July 2002

Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II β chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for Ii-dependent determinants. This demonstrates that related leucine-based trafficking signals in Ii and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.
A Transgenic Marker for Mouse B Lymphoid Precursors
Authors: Mårtensson Inga-Lill , Melchers Fritz , Winkler Thomas H.
Published: July 2002

Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5′ of the precursor (pre)–B cell–specific λ5 gene. All three strains express human CD25 in parallel to endogenous λ5 on pre–B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220+CD19−λ5+ precursor of the B220+CD19+λ5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19− precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre–BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5′ region of the λ5 gene into the mouse genome confers tissue and differentiation stage–specific expression of a transgene.
Adherence of Erythrocytes during Exflagellation of Plasmodium falciparum Microgametes Is Dependent on Erythrocyte Surface Sialic Acid and Glycophorins
Authors: Templeton Thomas J. , Keister David B. , Muratova Olga , Procter Jo Lynn , Kaslow David C.
Published: July 2002

Malaria male gametocytes within a newly ingested infected blood meal in the mosquito midgut emerge from erythrocytes and extrude approximately eight flagellar microgametes in a process termed exflagellation. In culture, and in blood removed from infected patients, emerging microgametes avidly adhere to neighboring uninfected and infected erythrocytes, as well as to emerged female macrogametes, creating “exflagellation centers”. The mechanism of erythrocyte adherence is not known nor has it been determined for what purpose microgametes may bind to erythrocytes. The proposition of a function underlying erythrocyte adherence is supported by the observation of species-specificity in adhesion: microgametes of the human malaria Plasmodium falciparum can bind human erythrocytes but not chicken erythrocytes, whereas avian host Plasmodium gallinaceum microgametes bind chicken but not human erythrocytes. In this study we developed a binding assay in which normal, enzyme-treated, variant or null erythrocytes are identified by a cell surface fluorescent label and assayed for adherence to exflagellating microgametes. Neuraminidase, trypsin or ficin treatment of human erythrocytes eliminated their ability to adhere to Plasmodium falciparum microgametes, suggesting a role of sialic acid and one or more glycophorins in the binding to a putative gamete receptor. Using nulls lacking glycophorin A [En(a−)], glycophorin B (S−s−U−) or a combination of glycophorin A and B (Mk/Mk) we showed that erythrocytes lacking glycophorin B retain the ability to bind but a lack of glycophorin A reduced adherence by exflagellating microgametes. We propose that either the sialic acid moiety of glycophorins, predominantly glycophorin A, or a more complex interaction involving the glycophorin peptide backbone, is the erythrocyte receptor for adhesion to microgametes.
Conformational Correction Mechanisms Aiding Antigen Recognition by a Humanized Antibody
Authors: Holmes Margaret A. , Buss Timothy N. , Foote Jefferson
Published: July 2002

The crystal structure of the complex between hen egg lysozyme and the Fv fragment of a humanized antilysozyme antibody was determined to 2.7-Å resolution. The structure of the antigen combining site in the complex is nearly identical to that of the complexed form of the parent mouse antibody, D1.3. In contrast, the combining sites of the unliganded mouse and humanized antilysozymes show moderate conformational differences. This disparity suggests that a conformational readjustment process linked to antigen binding reverses adverse conformations in the complementarity determining regions that had been introduced by engineering these segments next to human framework regions in the humanized antibody.
Molecular Recognition of Lipid Antigens by T Cell Receptors
Authors: Grant Ethan P. , Degano Massimo , Rosat Jean-Pierre , Stenger Steffen , Modlin Robert L. , Wilson Ian A. , Porcelli Steven A. , Brenner Michael B.
Published: July 2002

The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the α and β chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the MHC α helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral putative antigen-binding groove beneath the CD1 α helices. Here, we demonstrate that α/β TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences permit a model for recognition in which the TCR CDR3 loops containing charged residues project between the CD1 α helices, contacting the lipid antigen hydrophilic head moieties as well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.
The Immunoevasive Function Encoded by the Mouse Cytomegalovirus Gene m152 Protects the Virus against T Cell Control in Vivo
Authors: Krmpotic Astrid , Messerle Martin , Crnkovic-Mertens Irena , Polic Bojan , Jonjic Stipan , Koszinowski Ulrich H.
Published: July 2002

Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum–Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57–66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in β2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8+ T lymphocytes in vivo.
A Highly Selective Cc Chemokine Receptor (Ccr)8 Antagonist Encoded by the Poxvirus Molluscum Contagiosum
Authors: Lüttichau Hans R. , Stine Johnny , Boesen Thomas P. , Johnsen Anders H. , Chantry David , Gerstoft Jan , Schwartz Thue W.
Published: July 2002

The MC148 CC chemokine from the human poxvirus molluscum contagiosum (MCV) was probed in parallel with viral macrophage inflammatory protein (vMIP)-II encoded by human herpesvirus 8 (HHV8) in 16 classified human chemokine receptors. In competition binding using radiolabeled endogenous chemokines as well as radiolabeled MC148, MC148 bound with high affinity only to CCR8. In calcium mobilization assays, MC148 had no effect on its own on any of the chemokine receptors, but in a dose-dependent manner blocked the stimulatory effect of the endogenous I-309 chemokine on CCR8 without affecting chemokine-induced signaling of any other receptor. In contrast, vMIP-II acted as an antagonist on 10 of the 16 chemokine receptors, covering all four classes: XCR, CCR, CXCR, and CX3CR. In chemotaxis assays, MC148 specifically blocked the I-309–induced response but, for example, not stromal cell–derived factor 1α, monocyte chemoattractant protein 1, or interleukin 8–induced chemotaxis. We thus concluded that the two viruses choose two different ways to block the chemokine system: HHV8 encodes the broad-spectrum chemokine antagonist vMIP-II, whereas MCV encodes a highly selective CCR8 antagonist, MC148, conceivably to interfere with monocyte invasion and dendritic cell function. Because of its pharmacological selectivity, the MC148 protein could be a useful tool in the delineation of the role played by CCR8 and its endogenous ligand, I-309.
Human Malaria in Immunocompromised Mice
Authors: Badell Edgar , Oeuvray Claude , Moreno Alicia , Soe Soe , van Rooijen Nico , Bouzidi Ahmed , Druilhe Pierre
Published: July 2002

We have recently described that sustained Plasmodium falciparum growth could be obtained in immunodeficient mice. We now report the potential of this new mouse model by assaying the effect of the passive transfer of antibodies (Abs) which in humans have had a well-established effect.

Our results show that the total African adult hyperimmune immunoglobulin Gs (HI-IgGs) strongly reduce P. falciparum parasitemia similarly to that reported in humans, but only when mice are concomitantly reconstituted with human monocytes (HuMNs). In contrast, neither HI-IgGs nor HuMNs alone had any direct effect upon parasitemia. We assessed the in vivo effect of epitope-specific human Abs affinity-purified on peptides derived either from the ring erythrocyte surface antigen (RESA) or the merozoite surface protein 3 (MSP3). The inoculation of low concentrations of anti-synthetic peptide from MSP3, but not of anti-RESA Abs, consistently suppressed P. falciparum in the presence of HuMNs. Parasitemia decrease was stronger and faster than that observed using HI-IgGs and as fast as that induced by chloroquine. Our observations demonstrate that this mouse model is of great value to evaluate the protective effect of different Abs with distinct specificity in the same animal, a step hardly accessible and therefore never performed before in humans.
Control of Gammaherpesvirus Latency by Latent Antigen-Specific Cd8+ T Cells
Authors: Usherwood Edward J. , Roy Douglas J. , Ward Kim , Surman Sherri L. , Dutia Bernadette M. , Blackman Marcia A. , Stewart James P. , Woodland David L.
Published: July 2002

The contribution of the latent antigen-specific CD8+ T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8+ T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M291–99/Kd CD8+ T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial “burst” of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8+ T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.
A Naturally Processed Mitochondrial Self-Peptide in Complex with Thymic Mhc Molecules Functions as a Selecting Ligand for a Viral-Specific T Cell Receptor
Authors: Sasada Tetsuro , Ghendler Yoseph , Neveu John M. , Lane William S. , Reinherz Ellis L.
Published: July 2002

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4+CD8+ double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2Kb molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed Kb-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to Kb. The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68–75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO68–75). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2−/− transporter associated with antigen processing 1−/− fetal thymic organ culture, NUbO68–75 induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO68–75 to support differentiation of a second Kb-restricted TCR indicates that its inductive effects are not general.
Bimodal Control of a Ca2+-Activated Cl− Channel by Different Ca2+ Signals
Authors: Kuruma Akinori , Hartzell H. Criss
Published: July 2002

Ca2+-activated Cl− channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca2+ and voltage. Excised inside-out patches containing many Ca2+-activated Cl− channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl− channel that exhibited an anionic selectivity of I− > Br− > Cl− (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca2+]. However, depending on the amplitude of the Ca2+ signal, this channel exhibited qualitatively different behaviors. At [Ca2+] < 1 μM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current–voltage relationship was strongly outwardly rectifying. At higher [Ca2+], the currents did not rectify and were time independent. This difference in behavior at different [Ca2+] was explained by an apparent voltage-dependent Ca2+ sensitivity of the channel. At +120 mV, the EC50 for channel activation by Ca2+ was approximately fourfold less than at −120 mV (0.9 vs. 4 μM). Thus, at [Ca2+] < 1 μM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca2+ binding. The voltage-dependent Ca2+ sensitivity was explained by a kinetic gating scheme in which channel activation was Ca2+ dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca2+ concentration jumps were voltage sensitive, but that the activation time constants were Ca2+ sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca2+ concentrations adds a new dimension to Ca2+ signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca2+ signal.
Proof of the prime power conjecture for projective planes of order 𝑛 with abelian collineation groups of order 𝑛²
Authors: Blokhuis Aart , Jungnickel Dieter , Schmidt Bernhard
Published: July 2002

Let

G
G

be an abelian collineation group of order

n
2

n^2

of a projective plane of order

n
n

. We show that

n
n

must be a prime power, and that the

p
p

-rank of

G
G

is at least

b
+
1

b+1

if

n
=

p
b

n=p^b

for an odd prime

p
p

.
The cost of computing integers
Authors: de Melo W. , Svaiter B.
Published: July 2002

We analyse the growth rate of a number theoretic function related to the operational complexity of integers
Lyapunov functions and attractors in arbitrary metric spaces
Authors: Hurley Mike
Published: July 2002

We prove two theorems concerning Lyapunov functions on metric spaces. The new element in these theorems is the lack of a hypothesis of compactness or local compactness. The first theorem applies to a discrete dynamical system on any metric space; the result is that if

A
A

is an attractor for a continuous map

g
g

of a metric space

X
X

to itself, then there is a Lyapunov function for

A
A

. The second theorem applies only to separable metric spaces; the theorem is that there is a complete Lyapunov function for any continuously-generated discrete dynamical system on a separable metric space. (A complete Lyapunov function is a real-valued function that is constant on orbits in the chain recurrent set, is strictly decreasing along all other orbits, and separates different components of the chain recurrent set.)
A family of fifth-order Runge-Kutta pairs
Authors: Papakostas S. , Papageorgiou G.
Published: July 2002

The construction of a Runge-Kutta pair of order

5
(
4
)

5(4)

with the minimal number of stages requires the solution of a nonlinear system of

25
25

order conditions in

27
27

unknowns. We define a new family of pairs which includes pairs using

6
6

function evaluations per integration step as well as pairs which additionally use the first function evaluation from the next step. This is achieved by making use of Kutta’s simplifying assumption on the original system of the order conditions, i.e., that all the internal nodes of a method contributing to the estimation of the endpoint solution provide, at these nodes, cost-free second-order approximations to the true solution of any differential equation. In both cases the solution of the resulting system of nonlinear equations is completely classified and described in terms of five free parameters. Optimal Runge-Kutta pairs with respect to minimized truncation error coefficients, maximal phase-lag order and various stability characteristics are presented. These pairs were selected under the assumption that they are used in Local Extrapolation Mode (the propagated solution of a problem is the one provided by the fifth-order formula of the pair). Numerical results obtained by testing the new pairs over a standard set of test problems suggest a significant improvement in efficiency when using a specific pair of the new family with minimized truncation error coefficients, instead of some other existing pairs.
A fast algorithm to compute cubic fields
Authors: Belabas K.
Published: July 2002

We present a very fast algorithm to build up tables of cubic fields. Real cubic fields with discriminant up to

10

11

10^{11}

and complex cubic fields down to

−

10

11

-10^{11}

have been computed.
A space efficient algorithm for group structure computation
Authors: Teske Edlyn
Published: July 2002

We present a new algorithm for computing the structure of a finite abelian group, which has to store only a fixed, small number of group elements, independent of the group order. We estimate the computational complexity by counting the group operations such as multiplications and equality checks. Under some plausible assumptions, we prove that the expected run time is

O
(

n

)

O(\sqrt {n})

(with

n
n

denoting the group order), and we explicitly determine the

O
O

-constants. We implemented our algorithm for ideal class groups of imaginary quadratic orders and present experimental results.
Random matrix theory over finite fields
Authors: Fulman Jason
Published: July 2002

The first part of this paper surveys generating functions methods in the study of random matrices over finite fields, explaining how they arose from theoretical need. Then we describe a probabilistic picture of conjugacy classes of the finite classical groups. Connections are made with symmetric function theory, Markov chains, Rogers-Ramanujan type identities, potential theory, and various measures on partitions.
Down-Regulation of Protease-activated Receptor-1 Is Regulated by Sorting Nexin 1
Authors: Wang Yingjie , Zhou Yixing , Szabo Katalin , Haft Carol Renfrew , Trejo JoAnn
Published: July 2002

Degradation or “down-regulation” of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, is critical for termination of receptor signaling. Toward understanding the molecular mechanisms by which activated PAR1 is internalized, sorted to lysosomes, and degraded, we investigated whether PAR1 interacted with sorting nexin 1 (SNX1). SNX1 is a membrane-associated protein that functions in lysosomal sorting of the epidermal growth factor receptor. In vitro biochemical binding assays revealed a specific interaction between a glutathione S-transferase fusion of SNX1 and PAR1. In HeLa cells, activated PAR1 colocalized with endogenous SNX1 and coimmunoprecipitated SNX1. SNX1 contains a phox homology domain predicted to bind phosphatidylinositol-3-phosphate and a C-terminal coiled-coil region. To assess SNX1 function, we examined the effects of SNX1 deletion mutants on PAR1 trafficking. Neither the N terminus nor phox homology domain of SNX1 affected PAR1 trafficking. By contrast, overexpression of SNX1 C-terminal domain markedly inhibited agonist-induced degradation of PAR1, whereas internalization remained virtually intact. Immunofluorescence microscopy studies revealed substantial PAR1 accumulation in an early endosome antigen-1-positive compartment in agonist-treated cells expressing SNX1 C terminus. By contrast, lysosome-associated membrane protein-1 distribution was unperturbed. Together, these findings strongly suggest a role for SNX1 in sorting of PAR1 from early endosomes to lysosomes. Moreover, this study provides the first example of a protein involved in lysosomal sorting of a G protein-coupled receptor in mammalian cells.
Amphibole fibres in Chinese chrysotile asbestos
Authors: Tossavainen Antti , Kotilainen Mia , Takahashi Ken , Pan Guowei , Vanhala Esa
Published: July 2002

Ten chrysotile bulk samples originating from six Chinese chrysotile mines were studied for amphibole fibres. Five of the mines operate on ultramafic rocks whereas one exploits a dolomite-hosted deposit. The asbestos fibre content in lung tissue was examined from seven deceased workers of the Shenyang asbestos plant using these raw materials. The bulk samples were pretreated with acid/alkali-digestion, and thereafter, scanning and transmission electron microscopy, X-ray microanalysis, selected area electron diffraction and X-ray powder diffractometry were used to identify the minerals. Sample preparation of lung tissue involved drying and low-temperature ashing.

All of the bulk samples contained amphibole fibres as an impurity. The amphibole asbestos contents were between 0.002 and 0.310 w-%. Tremolite fibres were detected in every sample but anthophyllite fibres were present only in the sample originating from the dolomite-hosted deposit. In comparison, anthophyllite (71%), tremolite (9%) and chrysotile (10%) were the main fibre types in the lung tissue samples indicating faster pulmonary clearance of chrysotile fibres. The total levels ranged from 2.4 to 148.3 million fibres (over 1 μm in length) per gram of dry tissue, and they were consistent with heavy occupational exposure to asbestos.
Pathway analysis in metabolic databases via differential

metabolic display (DMD)
Authors: Küffner Robert , Zimmer Ralf , Lengauer Thomas
Published: July 2002

Motivation: A number of metabolic databases are available electronically, some with features for querying and visualizing metabolic pathways and regulatory networks. We present a unifying, systematic approach based on Petri nets for storing, displaying, comparing, searching and simulating such nets from a number of different sources.

Results: Information from each data source is extracted and compiled into a Petri net. Such Petri nets then allow to investigate the (differential) content in metabolic databases, to map and integrate genomic information and functional annotations, to compare sequence and metabolic databases with respect to their functional annotations, and to define, generate and search paths and pathways in nets. We present an algorithm to systematically generate all pathways satisfying additional constraints in such Petri nets. Finally, based on the set of valid pathways, so-called differential metabolic displays (DMDs) are introduced to exhibit specific differences between biological systems, i.e. different developmental states, disease states, or different organisms, on the level of paths and pathways. DMDs will be useful for target finding and function prediction, especially in the context of the interpretation of expression data.

Availability: Part of this work has been integrated into the software package ToPLign available for use over the WWW at http://cartan.gmd.de/ToPLign.html.

Contact: Robert.Kueffner@gmd.de; Ralf.Zimmer@gmd.de; Thomas.Lengauer@gmd.de

To whom correspondence should be addressed.
INCLUSive: INtegrated Clustering, Upstream sequence retrieval
and motif Sampling
Authors: Thijs Gert , Moreau Yves , De Smet Frank , Mathys Janick , Lescot Magali , Rombauts Stephane , Rouze Pierre , De Moor Bart , Marchal Kathleen
Published: July 2002

Summary: INCLUSive allows automatic multistep analysis of microarray data (clustering and motif finding). The clustering algorithm (adaptive quality-based clustering) groups together genes with highly similar expression profiles. The upstream sequences of the genes belonging to a cluster are automatically retrieved from GenBank and can be fed directly into Motif Sampler, a Gibbs sampling algorithm that retrieves statistically over-represented motifs in sets of sequences, in this case upstream regions of co-expressed genes.

Availability: For academic purposes at http://www.esat.kuleuven.ac.be/~dna/BioI/Software.html

Contact: Gert.Thijs@esat.kuleuven.ac.be

* To whom correspondence should be addressed. Email: Kathleen.Marchal@esat.kuleuven.ac.be.
Financial Market Variables do not Predict Real Activity: Further Evidence
Authors: Weber Christian E.
Published: July 2002

Previous studies argue that financial variables do not help forecast U.S. output growth. F statistics for excluding financial variables from output growth equations depend on the sample period and the inclusion of 1974:12 in the sample. Also, an autoregressive model of output growth often provides better forecasts than models with lagged financial variables included. I decompose output into permanent and cyclical components and ask whether financial variables help forecast either component in isolation. The paper‐bill spread does improve in‐sample forecasts of cyclical output, but no financial variable helps forecast either cyclical output or permanent output growth out of sample.
One RNA polymerase serving two genomes
Authors: Hedtke Boris , Börner Thomas , Weihe Andreas
Published: July 2002

The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage‐type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3). The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively. Here we show that the transit peptide of RpoT;2 possesses dual targeting properties. Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N‐terminus of RpoT;2 joined to green fluorescent protein to both organelles. Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes. RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage‐type polymerase.
Marketing Diversity: The Making of a Global Consumer Product—Hohner's Harmonicas, 1857–1930
Authors: Berghoff Hartmut
Published: July 2002

Unlike most nineteenth-century makers of traditional musical instruments, who did not aim at mass markets, German harmonica manufacturers were able to develop a large and worldwide market for their product. Located in regions outside Germany's industrial core districts, they epitomized successful specialty production. From the beginning, they relied on exports, especially to North America, a trade that was initially assisted by transatlantic emigrants who helped test market potential and tap into commercial networks. Hohner, the most successful harmonica manufacturer, created a well-known brand and extensively applied strategies of market segmentation and product differentiation from the 1890s onward, in part to satisfy customers' demand for myriad product variations. Sales channels and marketing methods also were far from homogeneous, being most effective when adapted to local conditions and cultures. For harmonica makers, diversity rather than uniformity was the key to global marketing success.
In-vitro antibacterial activity of levofloxacin against hospital isolates: a multicentre study
Authors: Soussy C.-J. , Cluzel M. , Ploy M.-C. , Kitzis M.-D. , Morel C. , Bryskier A. , Courvalin P.
Published: July 2002

The objective of this study was to evaluate the activity of the fluoroquinolone, levofloxacin, against hospital isolates of bacteria. MICs of levofloxacin were determined for 2154 strains by agar dilution. Breakpoints for susceptibility testing were calculated using the agar diffusion technique with 5 µ g discs. The activity of levofloxacin against nalidixic acid- and pefloxacin-susceptible Enterobacteriaceae ( n = 668) was higher (MIC 50/90 0.06–0.12 mg/L) than previously reported for ofloxacin. As seen with other fluoroquinolones, this activity was reduced against nalidixic acid-resistant and pefloxacin-intermediate and -resistant strains (MIC 1–8 mg/L). MICs for Pseudomonas aeruginosa ( n = 104) were between 0.12 and 128 mg/L. Levofloxacin had good activity against nalidixic acid- and pefloxacin-susceptible Acinetobacter baumannii ( n = 12; MIC 0.06–0.25 mg/L), but the activity was reduced against nalidixic acid- and pefloxacin-resistant strains ( n = 80; MIC 1–32 mg/L). Haemophilus influenzae ( n = 70), Haemophilus parainfluenzae ( n = 47) and Moraxella catarrhalis ( n = 64) were inhibited by low concentrations of levofloxacin (MICs 0.016–0.03 mg/L, 0.03–0.12 mg/L and 0.03–0.12 mg/L, respectively). Clostridium perfringens ( n = 23; MIC 0.25–1 mg/L) was more susceptible than Bacteroides fragilis ( n = 60; MIC 0.5–4 mg/L). Levofloxacin showed superior activity compared with ofloxacin against methicillin-susceptible staphylococci ( n = 107; MIC 0.03–0.5 mg/L); the resistant strains (MICs 2–32 mg/L) were usually also resistant to methicillin. Levofloxacin was less effective against enterococci ( n = 105; MIC 1–32 mg/L), but streptococci ( n = 192) and pneumococci ( n = 129), including 58 penicillin-non-susceptible strains, were inhibited by low concentrations (MICs 0.5–2 mg/L). According to the regression curve, zone diameters were usually 20–22 mm, 17–19 mm and 15–16 mm for MICs of 1, 2 and 4 mg/L, respectively. In conclusion, this study, performed on a large number of strains, confirms the superior antibacterial activity of levofloxacin compared with ofloxacin, especially against pathogens isolated from respiratory tract infections.
Marine biological controls on climate via the carbon and sulphur geochemical cycles
Authors: Watson Andrew J. , Liss Peter S.
Published: July 2002

We review aspects of the influence of the marine biota on climate, focusing particularly on their role in mediating surface temperatures via their influence on atmospheric carbon dioxide (CO2) and dimethyl sulphide (DMS) concentrations. Variation in natural CO2concentrations occurring over 103to 105years are set by oceanic processes, and in particular by conditions in the Southern Ocean, so it is to this region that we must look to understand the glacial–interglacial changes in CO2concentrations. It seems likely that marine productivity in the Southern Ocean is limited by a combination of restricted iron supply to the region and insufficient light.

Plankton–produced DMS is thought to influence climate by changing the numbers of cloud condensation nuclei available in remote regions; the efficiency of this mechanism is still unknown, but calculations suggest it may be a powerful influence on climate. It has a much shorter time–scale than the CO2effect, and as a consequence may well be a player on the ‘global change’ timescale. The direction of both the CO2and the DMS mechanisms is such that more marine productivity would lead to lower global temperatures, and we speculate that the overall effect of the marine biota today is to cool the planet by ca. 6°C as a result of these two mechanisms, with one–third of this figure being due to CO2effects and two–thirds due to DMS.

While the marine biota influence climate, climate also influences the marine biota, chiefly via changing atmospheric circulation. This in turn alters ocean circulation patterns, responsible for mixing up sub–surface nutrients, and also influences the transport of nutrients, such as iron, in atmospheric dust. A more vigorous atmospheric circulation would be expected to increase the productivity of the marine biota on both counts. Thus during glacial time, the colder and drier climate might be expected to stimulate greater marine productivity than occurs today. Since more production leads to greater cooling by reduction in CO2and increase in DMS, the marine biota–climate system appears to have been in positive feedback in the glacial–interglacial transition, with the changes in the climate system being reinforced by changes in the marine biota. In the context of anthropogenic change, we cannot at present say what sign the feedback on climate will have, because we have no clear idea whether circulation will become more or less vigorous in the future.
Reconstructing the origins of human hepatitis viruses
Authors: Simmonds P.
Published: July 2002

Infections with hepatitis B and C viruses (HBV, HCV) are widespread in human populations throughout the world, and are major causes of chronic liver disease and liver cancer. HBV, HCV and the related hepatitis G virus or GB virus C (referred to here as HGV/GBV–C) are capable of establishing persistent, frequently lifelong infections characterized by high levels of continuous replication. All three viruses show substantial genetic heterogeneity, which has allowed each to be classified into a number of distinct genotypes that have different geographical distributions and associations with different risk groups for infection. Information on their past transmission and epidemiology might be obtained by estimation of the time of divergence of the different genotypes of HCV, HBV and HGV/GBV–C using knowledge of their rates of sequence change. While information on the latter is limited to short observation periods and is therefore subject to considerable error and uncertainty, the relatively recent times of origin for genotype of each virus predicted by this method (HCV, 500–2000 years; HBV, 3000 years; HGV/GBV–C, 200 years) are quite incompatible with their epidemiological distributions in human populations. They also cannot easily be reconciled with the recent evidence for species–associated variants of HBV and HGV/GBV–C in a range of non–human primates. The apparent conservatism of viruses over long periods implied by their epidemiological distributions instead suggests that nucleotide sequence change may be subject to constraints peculiar to viruses with single–stranded genomes, or with overlapping reading frames that defy attempts to reconstruct evolution according to the principles of the ‘molecular clock’. Large population sizes and intense selection pressures that optimize fitness may be additional factors that set virus evolution apart from that of their hosts.