bluuebunny/crossref_metadata_2025 huggingface dataset:
Prion protein NMR structure and species barrier for prion diseases
Authors: Billeter Martin , Riek Roland , Wider Gerhard , Hornemann Simone , Glockshuber Rudi , Wüthrich Kurt
Published: July 2002

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from
in vitro
and
in vivo
studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.
The solution structure of the N-terminal domain of α2-macroglobulin receptor-associated protein
Authors: Nielsen Peter R. , Ellgaard Lars , Etzerodt Michael , Thøgersen Hans C. , Poulsen Flemming M.
Published: July 2002

The three-dimensional structure of the N-terminal domain (residues 18–112) of α2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23–34, 39–65, and 73–88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix–loop–helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.
Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis
Authors: Jäger Stefan , Lewis James W. , Zvyaga Tatyana A. , Szundi Istvan , Sakmar Thomas P. , Kliger David S.
Published: July 2002

Rhodopsin is a prototypical G protein-coupled receptor that is activated by photoisomerization of its 11-
cis
-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin’s transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.
On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism
Authors: Lin Chao-Hsiung , Hoffman Timothy Z. , Wirsching Peter , Barbas Carlos F. , Janda Kim D. , Lerner Richard A.
Published: July 2002

Reactive immunization has emerged as a new tool for the study of biological catalysis. A powerful application resulted in catalytic antibodies that use an enamine mechanism akin to that used by the class I aldolases. With regard to the evolution of enzyme mechanisms, we investigated the utility of an enamine pathway for the allylic rearrangement exemplified by Δ5-3-ketosteroid isomerase (KSI; EC5.3.3.1). Our aldolase antibodies were found to catalyze the isomerization of both steroid model compounds and steroids. The kinetic and chemical studies showed that the antibodies afforded rate accelerations up to a factor of 104by means of an enamine mechanism in which imine formation was the rate-determining step. In light of our observations and the enzyme studies by other workers, we suggest that an enamine pathway could have been an early, viable KSI mechanism. Although this pathway is amenable to optimization for increased catalytic power, it appears that certain factors precluded its evolution in known KSI enzymes.
Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley
Authors: Koopmann Edda , Hahlbrock Klaus
Published: July 2002

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (
Petroselinum crispum
) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H.
Instrumental conditioning of fast (20- to 50-Hz) oscillations in corticothalamic networks
Authors: Amzica Florin , Neckelmann Dag , Steriade Mircea
Published: July 2002

Cats were instrumentally conditioned to generate grouped fast (20- to 50-Hz) oscillations in motor cortex (area 4). Over seven experimental sessions, there was a spatially selective increased generation of grouped fast oscillations in that electroencephalogram lead. This locally increased generation of fast oscillations in cortex was associated with a widespread increase in synchrony of fast oscillations in thalamocortical networks, as demonstrated by cross-correlations between intracortical, corticothalamic, and intrathalamic field potentials. A three-session extinction period abolished the local increase in generation of grouped fast oscillations and reset the thalamocortical synchrony of fast oscillations to control values. A subsequent series of seven sessions with instrumental conditioning of fast oscillations in visual cortex (area 17) reproduced the results from area 4, with a spatially selective increased generation of grouped fast oscillations in the criterion lead, associated with a widespread increase in thalamocortical synchrony of fast oscillations. In addition to their presence during the conditioning sessions, the changes in synchrony of fast oscillations were expressed during periods of quiet waking, rapid-eye-movement sleep, and nonrapid-eye-movement sleep recorded during the first hour after the end of the conditioning.
Encoded combinatorial chemistry: Synthesis and screening of a library of highly functionalized pyrrolidines
Authors: Maclean Derek , Schullek John R. , Murphy Martin M. , Ni Zhi-Jie , Gordon Eric M. , Gallop Mark A.
Published: July 2002

The application of a new encoding technology for drug discovery is described. A combinatorial library of mercaptoacyl pyrrolidines has been prepared on a beaded polymeric support. Each polymer bead carries one library constituent in association with an oligomeric “tag,” the structure of which is a record of the specific reagents from which that library member was prepared. After the ligands were solubilized, an array of such beads was screened for angiotensin-converting enzyme inhibitory activity, and the structures of active pyrrolidines were deduced by analysis of the associated tags at sub-picomole levels. Several extremely potent enzyme inhibitors were identified, many from multiple beads. The most potent inhibitor was found to have a
K
i
of 160 pM, ≈3-fold more active than captopril in the same assay. Direct comparison with iterative deconvolution shows that the encoded screening strategy is a much more efficient means for extracting information from such compound collections, producing more data on a larger number of active structures.
Transcriptional coactivator cAMP response element binding protein mediates induction of the human proliferating cell nuclear antigen promoter by the adenovirus E1A oncoprotein
Authors: Lee Benjamin H. , Mathews Michael B.
Published: July 2002

The proliferating cell nuclear antigen (PCNA), a crucial component of eukaryotic cell cycle and DNA replication complexes, is induced by the adenovirus E1A 243R oncoprotein through a cis-acting element termed the PERE (PCNA-E1A responsive element). The PERE contains a sequence homologous to an activating transcription factor (ATF) motif, and ATF-1 is a major component of PERE-protein complexes. We have identified a second PERE-binding protein, the cAMP response element binding protein (CREB) transcription factor, which forms heterodimers with ATF-1 at this site. CREB, but not ATF-1, is able to mediate transactivation of a minimal PCNA-chloramphenicol acetyltransferase reporter by E1A 243R. Further analysis revealed that the transcriptional coactivator, the CREB-binding protein (CBP), associates with PERE-related complexes, and that CBP is able to mediate a strong transactivation response to E1A 243R at the PCNA promoter. Experiments conducted with mutants in the E1A or CREB components support a model whereby E1A 243R transactivates the PCNA promoter via a CBP-CREB-PERE pathway. These findings delineate a paradigm by which E1A 243R can target and transactivate specific DNA promoter sequences.
X-ray emission from clusters and groups of galaxies
Authors: Mushotzky Richard
Published: July 2002

Recent major advances in x-ray imaging and spectroscopy of clusters
have allowed the determination of their mass and mass profile out to
≈1/2 the virial radius. In rich clusters, most of the baryonic mass
is in the gas phase, and the ratio of mass in gas/stars varies by a
factor of 2–4. The baryonic fractions vary by a factor of ≈3 from
cluster to cluster and almost always exceed 0.09
h
50
−[3/2]
and thus are in fundamental
conflict with the assumption of Ω = 1 and the results of big bang
nucleosynthesis. The derived Fe abundances are 0.2–0.45 solar, and the
abundances of O and Si for low redshift systems are 0.6–1.0 solar.
This distribution is consistent with an origin in pure type II
supernova. The amount of light and energy produced by these supernovae
is very large, indicating their importance in influencing the formation
of clusters and galaxies. The lack of evolution of Fe to a redshift of
z ≈ 0.4 argues for very early enrichment of the cluster gas.
Groups show a wide range of abundances, 0.1–0.5 solar. The results of
an x-ray survey indicate that the contribution of groups to the mass
density of the universe is likely to be larger than 0.1
h
50
−2
. Many of the very poor groups have large
x-ray halos and are filled with small galaxies whose velocity
dispersion is a good match to the x-ray temperatures.
Intestinal intraepithelial lymphocytes include precursors committed to the T cell receptor αβ lineage
Authors: Page Stephanie T. , Bogatzki Lisa Y. , Hamerman Jessica A. , Sweenie Claire H. , Hogarth Philip J. , Malissen Marie , Perlmutter Roger M. , Pullen Ann M.
Published: July 2002

The majority of T cells develop in the thymus and exhibit well characterized phenotypic changes associated with their maturation. Previous analysis of intestinal intraepithelial lymphocytes (IEL) from nude mice and a variety of experimentally manipulated models led to the view that at least a portion of these cells represent a distinct T cell population that matures extrathymically. The IEL that are postulated to mature within the intestine include both T cell receptor (TCR) αβ- and γδ-bearing subpopulations. They can be distinguished from conventional thymically derived T cells in that they express an unusual coreceptor, a CD8α homodimer. In addition, they can utilize the Fc receptor γ-chain in place of the CD3-associated ζ-chain for TCR signaling and their maturation depends on the interleukin 2 receptor β-chain. Moreover, TCRαβ+CD8αα+IEL are not subject to conventional thymic selection processes. To determine whether CD3−CD8αα+IEL represent precursors of T cells developing extrathymically, we examined IEL from knockout mice lacking the recombination activating gene-1 (rag-1), CD3ɛ, or both Lck and Fyn, in which thymic T cell development is arrested. CD3−CD8αα+CD16+IEL from all three mutant strains, as well as from nude mice, included cells that express pre-TCRα transcripts, a marker of T cell commitment. These IEL fromlck−/−fyn−/−animals exhibited TCR β-gene rearrangement. However, CD3−CD8αα+CD16+IEL from ɛ-deficient mice had not undergone Dβ-Jβ joining, despite normal rearrangement at the TCRβ locus in thymocytes from these animals. These results revealed another distinction between thymocytes and IEL, and suggested an unexpectedly early role for CD3ɛ in IEL maturation.
Identification and characterization of a single-stranded DNA-binding protein from the archaeon
Methanococcus jannaschii
Authors: Kelly Thomas J. , Simancek Pamela , Brush George S.
Published: July 2002

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria and eukarya. We report here the identification and characterization of the SSB of an archaeon,
Methanococcus jannaschii
. The
M. jannaschii
SSB (mjaSSB) has significant amino acid sequence similarity to the eukaryotic SSB, replication protein A (RPA), and contains four tandem repeats of the core single-stranded DNA (ssDNA) binding domain originally defined by structural studies of RPA. Homologous SSBs are encoded by the genomes of other archaeal species, including
Methanobacterium thermoautotrophicum
and
Archaeoglobus fulgidus
. The purified mjaSSB binds to ssDNA with high affinity and selectivity. The apparent association constant for binding to ssDNA is similar to that of RPA under comparable experimental conditions, and the affinity for ssDNA exceeds that for double-stranded DNA by at least two orders of magnitude. The binding site size for mjaSSB is ≈20 nucleotides. Given that RPA is related to mjaSSB at the sequence level and to
Escherichia coli
SSB at the structural level, we conclude that the SSBs of archaea, eukarya, and bacteria share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.
Localization of F plasmid SopB protein to positions near the poles ofEscherichia coli cells
Authors: Kim Sook-Kyung , Wang James C.
Published: July 2002

The subcellular localization of the SopB protein, which is encoded by theEscherichia coliF plasmid and is involved in the partition of the single-copy plasmid, was directly visualized through the expression of the protein fused to the jellyfish green fluorescent protein (GFP). The fusion protein, but not GFP itself, was found to localize to positions close but not at the poles of exponentially growing cells. Neither the presence of other F-encoded proteins nor the binding of SopB to its recognition sites within thesopClocus of F is required for this localization. Examination of derivatives of the fusion protein lacking various regions of SopB suggests that the signal for the cellular localization of SopB resides in a region close to its N terminus. It is plausible that the near polar localization of SopB may serve the function of keeping a segregated pair of F plasmids apart while the cell septum is being formed. The plausible relation between the specific location of SopB and its suppression ofsopC-linked genes when overexpressed is also discussed.
High affinity cross-reacting mAb generated by minimal mimicry: Implications for the pathogenesis of anti-nuclear autoantibodies and immunosuppression
Authors: Rothermel Annette L. , Altieri Dario C.
Published: July 2002

The antigen recognition of a profoundly immunosuppressive mAb, mAb 2E1,in vivowas investigated. In addition to the 62-kDa effector cell protease receptor 1, mAb 2E1 bound the 32-kDa T cell adhesion receptor E2 (CD99) and the 86-kDa p80 subunit of the nuclear antigen complex Ku. These molecules share no overall sequence similarity. Peptide mapping experiments identified the mAb 2E1 cross-reacting epitopes as the sequences66GSFSDADLAD75in E2 and571GGAHFSVSSLAEG583in p80 of Ku, sharing a minimal homology motif FSXXXLA, in which X is a nonconserved amino acid. Each of these peptides separately inhibited the binding of mAb 2E1 to E2, effector cell protease receptor 1, and p80 of Ku in a dose-dependent manner. Scatchard plot analysis of125I-labeled mAb 2E1 binding to peripheral blood mononuclear cells revealed a high-affinity interaction with a dissociation constant of 7 × 10−10M. An anti-E2 mAb bound the same epitope66GSFSDADLAD75recognized by mAb 2E1 but failed to react with p80 of Ku and was not immunosuppressive. These findings demonstrate that high-affinity cross-reacting mAbs can be generated by mimicry of a minimal surface on unrelated molecules. This model of minimal mimicry may determine the nuclear reactivity of certain autoantibodies to Ku and contribute to aberrant immunosuppressionin vivo.
Bivalency as a principle for proteasome inhibition
Authors: Loidl Günther , Groll Michael , Musiol Hans-Jürgen , Huber Robert , Moroder Luis
Published: July 2002

The proteasome, a multicatalytic protease, is known to degrade unfolded polypeptides with low specificity in substrate selection and cleavage pattern. This lack of well-defined substrate specificities makes the design of peptide-based highly selective inhibitors extremely difficult. However, the x-ray structure of the proteasome from
Saccharomyces cerevisiae
reveals a unique topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition. Structure-derived active site separation distances were exploited for the design of homo- and heterobivalent inhibitors based on peptide aldehyde head groups and polyoxyethylene as spacer element. Polyoxyethylene was chosen as a flexible, linear, and proteasome-resistant polymer to mimic unfolded polypeptide chains and thus to allow access to the proteolytic chamber. Spacer lengths were selected that satisfy the inter- and intra-ring distances for occupation of the active sites from the S subsites. X-ray analysis of the proteasome/bivalent inhibitor complexes confirmed independent recognition and binding of the inhibitory head groups. Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhibitors, result from the bivalent binding. The principle of multivalency, ubiquitous in nature, has been successfully applied in the past to enhance affinity and avidity of ligands in molecular recognition processes. The present study confirms its utility also for inhibition of multicatalytic protease complexes.
Selective interaction of the C2 domains of phospholipase C-β
1
and -β
2
with activated Gα
q
subunits: An alternative function for C2-signaling modules
Authors: Wang Tieli , Pentyala Srinivas , Elliott John T. , Dowal Louisa , Gupta Ekta , Rebecchi Mario J. , Scarlata Suzanne
Published: July 2002

Phospholipase C (PLC)-β
1
and PLC-β
2
are regulated by the G
q
family of heterotrimeric G proteins and contain C2 domains. These domains are Ca
2+
-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-β
1
and PLC-β
2
function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5′-[γ-thio]triphosphate (GTP[γS])-bound] Gα
q
subunits. The C2 domain of PLC-β
1
effectively suppressed the activation of the intact isozyme by Gα
q
(GTP[γS]), indicating that the C2-Gα
q
interaction may be physiologically relevant. C2 affinity for Gα
q
(GTP[γS]) was reduced when Gα
q
was deactivated to the GDP-bound state. Binding to activated Gα
i1
subunits or to Gβγ subunits was not detected. Also, Gα
q
(GTP[γS]) failed to associate with the C2 domain of PLC-δ, an isozyme that is not activated by Gα
q
. These results indicate that the C2 domains of PLC-β
1
and PLC-β
2
provide a surface to which Gα
q
subunits can dock, leading to activation of the native protein.
A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop
Authors: Slotboom Dirk Jan , Sobczak Iwona , Konings Wil N. , Lolkema Juke S.
Published: July 2002

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding sites for substrate and coupling ions. A serine-rich sequence motif in this part of the proteins is located in a putative intracellular loop. Cysteine-scanning mutagenesis was applied to this loop in the glutamate transporter GltT of
Bacillus stearothermophilus
. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, and E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with
N-
ethylmaleimide blocked the transport activity in six mutants (T267C, A268C, S269C, S270C, E271C, and T272C). Two milimolars
l
-glutamate effectively protected against the modification of the cysteines at position 269–271 from the periplasmic side of the membrane but was unable to protect cysteine modification from the cytoplasmic side of the membrane. The results indicate that the conserved serine-rich motif in the glutamate transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop is of crucial importance for the function of the glutamate transporter.
Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity
Authors: Pályi I. , Vincze B. , Lovas S. , Mező I. , Pató J. , Kálnay A. , Turi G. , Gaál D. , Mihalik R. , Péter I. , Teplán I. , Murphy R. F.
Published: July 2002

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(
N
-vinylpyrrolidone-
co
-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G
2
/M phase of the cell cycle after 24 h at lower doses and in the G
1
and G
2
phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.
Prehistoric birds from New Ireland, Papua New Guinea: Extinctions on a large Melanesian island
Authors: Steadman David W. , White J. Peter , Allen Jim
Published: July 2002

At least 50 species of birds are represented in 241 bird bones from five late Pleistocene and Holocene archaeological sites on New Ireland (Bismarck Archipelago, Papua New Guinea). The bones include only two of seabirds and none of migrant shorebirds or introduced species. Of the 50 species, at least 12 (petrel, hawk, megapode, quail, four rails, cockatoo, two owls, and crow) are not part of the current avifauna and have not been recorded previously from New Ireland. Larger samples of bones undoubtedly would indicate more extirpated species and refine the chronology of extinction. Humans have lived on New Ireland for
ca.
35,000 years, whereas most of the identified bones are 15,000 to 6,000 years old. It is suspected that most or all of New Ireland’s avian extinction was anthropogenic, but this suspicion remains undetermined. Our data show that significant prehistoric losses of birds, which are well documented on Pacific islands more remote than New Ireland, occurred also on large, high, mostly forested islands close to New Guinea.
Absence of interdomain contacts in the crystal structure of the RNA recognition motifs of Sex-lethal
Authors: Crowder Scott M. , Kanaar Roland , Rio Donald C. , Alber Tom
Published: July 2002

By binding specific RNA transcripts, the Sex-lethal protein (SXL) governs sexual differentiation and dosage compensation in
Drosophila melanogaster
.
To investigate the basis for RNA binding specificity, we determined the crystal structure of the tandem RNA recognition motifs (RRMs) of SXL. Both RRMs adopt the canonical RRM fold, and the 10-residue, interdomain linker shows significant disorder. In contrast to the previously determined structure of the two-RRM fragment of heterogeneous nuclear ribonucleoprotein Al, SXL displays no interdomain contacts between RRMs. These results suggest that the SXL RRMs are flexibly tethered in solution, and RNA binding restricts the orientation of RRMs. Therefore, the observed specificity for single-stranded, U-rich sequences does not arise from a predefined, rigid architecture of the isolated SXL RRMs.
Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy
Authors: Lang F. , Klingel K. , Wagner C. A. , Stegen C. , Wärntges S. , Friedrich B. , Lanzendörfer M. , Melzig J. , Moschen I. , Steuer S. , Waldegger S. , Sauter M. , Paulmichl M. , Gerke V. , Risler T. , Gamba G. , Capasso G. , Kandolf R. , Hebert S. C. , Massry S. G. , Bröer S.
Published: July 2002

Transforming growth factor β (TGF-β) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-β stimulates the expression of a distinct serine/threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-β (2 μg/liter), phorbol 12,13-didecanoate (1 μM), or the Ca
2+
ionophore ionomycin (1 μM) and blunted by high concentrations of nifedipine (10 and 100 μM).
In situ
hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in
Xenopus
oocytes expressing the renal epithelial Na
+
channel ENaC or the mouse thick ascending limb Na
+
,K
+
,2Cl
−
cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 μM) and chelerythrine (1 μM) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na
+
transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.
Abrupt climate change in the computer: Is it real?
Authors: Stocker Thomas F. , Marchal Olivier
Published: July 2002

Models suggest that dramatic changes in the ocean
circulation are responsible for abrupt climate changes during the last
ice age and may possibly alter the relative climate stability of the
last 10,000 years.
Psychiatry of the Elderly: A Consensus Statement
Authors: Silva J. A. Costa e , Wertheimer J.
Published: July 2002

This document is a consensus statement on Psychiatry of the Elderly jointly produced by WHO and the Geriatric Psychiatry Section of the World Psychiatric Association, with the collaboration of several pertinent NGOs and the participation of experts from countries in several WHO regions.
The Place and Role of Psychology in Cognitive Science: An International Survey
Authors: Denis Michel
Published: July 2002

This paper reports the results of an international survey on the place and role of psychology in cognitive science (CS). The respondents were psychological societies from 31 countries illustrating a variety of situations, from countries where psychology has been established as a science for a long time to countries where it has been established more recently and is still developing. The paper reviews topics covered by the CS approach, as well as research operations and initiatives taken in this domain. The analysis of responses reflects the belief that psychology, in general, has a central role in CS. This is based on epistemological considerations, as well as on the recognition of the methodological expertise of psychology for collecting and analyzing empirical data. Psychology is reported to have a unique integrative capacity and to take care to maintain the place of the human being at the centre of CS. Limitations and obstacles to the development of pluridisciplinary research on cognition are also reported, and information is provided on the training programmes in CS and the institutions that support research in this domain.
Development of Social Competences in the Primary School‐study of specific pedagogic practices
Authors: Morais Ana M. , Rocha Carmo
Published: July 2002

ABSTRACT This study intended to promote the development of socio‐affective competences in children in the first two years of primary school (age 6‐8). A model of pedagogic practice with particular characteristics in terms of the relations between subjects, discourses and spaces was implemented. The mediating influence of teacher's pedagogic practice, family's social class and gender was studied. The results showed that the pedagogic practice with general values of weak framing (i.e. where some control is given to the learner) but with strong framing in specific aspects was the practice which facilitated the development of the selected competences by all children.
The Hsp70 Homologue Lhs1p Is Involved in a Novel Function of the Yeast Endoplasmic Reticulum, Refolding and Stabilization of Heat-denatured Protein Aggregates
Authors: Saris Nina , Holkeri Heidi , Craven Rachel A. , Stirling Colin J. , Makarow Marja
Published: July 2002

Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50°C after preconditioning at 37°C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24°C. Here we show that refolding of the marker enzyme Hsp150Δ–β-lactamase, inactivated and aggregated by the 50°C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Δ–β-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Δ– β-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Δ–β-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50°C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24°C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.
Interactions of the Cytoplasmic Domain of P-Selectin with Clathrin-coated Pits Enhance Leukocyte Adhesion under Flow
Authors: Setiadi Hendra , Sedgewick Gerald , Erlandsen Stanley L. , McEver Rodger P.
Published: July 2002

Flowing leukocytes tether to and roll on P-selectin, a receptor on endothelial cells that is rapidly internalized in clathrin-coated pits. We asked whether the association of P-selectin with clathrin-coated pits contributes to its adhesive function. Under flow, rolling neutrophils accumulated efficiently on CHO cells expressing wild-type P-selectin or a P-selectin construct with a substitution in the cytoplasmic domain that caused even faster internalization than that of the wild-type protein. By contrast, far fewer rolling neutrophils accumulated on CHO cells expressing P-selectin constructs with a deletion or a substitution in the cytoplasmic domain that impaired internalization. Neutrophils rolled on the internalization-competent constructs with greater adhesive strength, slower velocity, and more uniform motion. Flowing neutrophils tethered equivalently to internalization-competent or internalization-defective P-selectin, but after tethering, they rolled further on internalization-competent P-selectin. Confocal microscopy demonstrated colocalization of α-adaptin, a component of clathrin-coated pits, with wild-type P-selectin, but not with P-selectin lacking the cytoplasmic domain. Treatment of CHO cells or endothelial cells with hypertonic medium reversibly impaired the clathrin-mediated internalization of P-selectin and its ability to support neutrophil rolling. Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits provide a novel mechanism to enhance leukocyte adhesion under flow.
Interaction among Gsk-3, Gbp, Axin, and APC in Xenopus Axis Specification
Authors: Farr Gist H. , Ferkey Denise M. , Yost Cynthia , Pierce Sarah B. , Weaver Carole , Kimelman David
Published: July 2002

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is β-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal–ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and β-catenin form a complex that promotes the GSK-3–mediated phosphorylation and subsequent degradation of β-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates β-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3–mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic β-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in β-catenin levels and establishment of the dorsal axis.
Developmentally Regulated Extinction of Ly-49 Receptor Expression Permits Maturation and Selection of NK1.1+ T Cells
Authors: Robson MacDonald H. , Lees Rosemary K. , Held Werner
Published: July 2002

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.
Transcription Factor SCL Is Required for c-kit Expression and c-Kit Function in Hemopoietic Cells
Authors: Krosl Gorazd , He Gang , Lefrancois Martin , Charron Frédéric , Roméo Paul-Henri , Jolicoeur Paul , Kirsch Ilan R. , Nemer Mona , Hoang Trang
Published: July 2002

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony–stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit–dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL– or dn-SCL– expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro–B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.
Metabolic Consequences of Adenosine Deaminase Deficiency in Mice Are Associated with Defects in Alveogenesis, Pulmonary Inflammation, and Airway Obstruction
Authors: Blackburn Michael R. , Volmer Jonathan B. , Thrasher Janci L. , Zhong Hongyan , Crosby Jeff R. , Lee James J. , Kellems Rodney E.
Published: July 2002

Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologically active purines adenosine and 2′-deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient mice included an accumulation of activated alveolar macrophages and eosinophils. These changes were accompanied by a pronounced enlargement of alveolar spaces and increases in mucus production in the bronchial airways. The alveolar enlargement was found to be due in part to abnormal alveogenesis. Lowering adenosine and 2′-deoxyadenosine levels using ADA enzyme therapy decreased the pulmonary eosinophilia and resolved many of the lung histopathologies. In addition, genetically restoring ADA to the forestomach of otherwise ADA-deficient mice prevented adenine metabolic disturbances as well as lung inflammation and damage. These data suggest that disturbances in purinergic signaling mediate the lung inflammation and damage seen in ADA-deficient mice.
Human Endothelial Cells Regulate Survival and Proliferation of Human Mast Cells
Authors: Mierke Claudia T. , Ballmaier Matthias , Werner Uwe , Manns Michael P. , Welte Karl , Bischoff Stephan C.
Published: July 2002

Mast cells (MCs) are immunoregulatory and inflammatory tissue cells preferentially located around blood vessels. Since endothelial cells have been suggested to regulate MC functions, we analyzed MC–endothelial cell interactions in vitro by performing coculture experiments with purified human intestinal MCs and human umbilical vein endothelial cells (HUVECs). We found that HUVECs provide signals allowing MCs to survive for at least 3 wk and to proliferate without addition of cytokines; otherwise all MCs died. HUVEC-dependent MC proliferation was more pronounced than that induced by stem cell factor (SCF), known to act as an MC growth factor both in vitro and in vivo. After coculture with HUVECs, most MCs were of the tryptase and chymase double-positive phenotype (MCTC). Transwell experiments suggested that the HUVECs' effects on MCs are not mediated by soluble factors. HUVEC-dependent MC adhesion and proliferation were inhibited by neutralizing antibodies directed against SCF and vascular cell adhesion molecule (VCAM)-1 expressed on HUVECs, and c-kit and very late antigen 4 (VLA-4) on MCs. The data suggest that two mechanisms (membrane-bound SCF/c-kit and VCAM-1/VLA-4) are involved in human MC–endothelial cell interactions. In conclusion, our study provides evidence that endothelial cells regulate MC survival and preferentially support human MCTC development.
Interferon γ Stabilizes the T Helper Cell Type 1 Phenotype
Authors: Zhang Yongkang , Apilado Ron , Coleman John , Ben-Sasson Shlomo , Tsang Sharon , Hu-Li Jane , Paul William E. , Huang Hua
Published: July 2002

T helper cell (Th)1-primed CD4 T cells from wild-type donors make little interleukin (IL)-4 when restimulated under Th2 conditions. However, such restimulation of Th1-primed cells from interferon (IFN)-γ2/− or IFN-γ receptor (IFN-γR)−/− mice resulted in substantial production of IL-4 and other Th2 cytokines. Adding IFN-γ to the priming culture markedly diminished the capacity of Th1-primed IFN-γ2/− cells to express IL-4. Even IFN-γ–producing cells from IFN-γR−/− mice could acquire IL-4–producing capacity. Thus, IFN-γ is not required for the development of IFN-γ–producing capacity, but it plays a critical role in suppressing the IL-4–producing potential of Th1 cells.
Genetic Influences on the End-Stage Effector Phase of Arthritis
Authors: Ji Hong , Gauguier Dominique , Ohmura Koichiro , Gonzalez Antonio , Duchatelle Veronique , Danoy Patrick , Garchon Henri-Jean , Degott Claude , Lathrop Mark , Benoist Christophe , Mathis Diane
Published: July 2002

K/B×N T cell receptor transgenic mice are a model of inflammatory arthritis, most similar to rheumatoid arthritis, that is critically dependent on both T and B lymphocytes. Transfer of serum, or just immunoglobulins, from arthritic K/B×N animals into healthy recipients provokes arthritis efficiently, rapidly, and with high penetrance. We have explored the genetic heterogeneity in the response to serum transfer, thereby focussing on the end-stage effector phase of arthritis, leap-frogging the initiating events. Inbred mouse strains showed clear variability in their responses. A few were entirely refractory to disease induction, and those which did develop disease exhibited a range of severities. F1 analyses suggested that in most cases susceptibility was controlled in a polygenic additive fashion. One responder/nonresponder pair (C57Bl/6 × NOD) was studied in detail via a genome scan of F2 mice; supplementary information was provided by the examination of knock-out and congenic strains. Two genomic regions that are major, additive determinants of the rapidity and severity of K/B×N serum-transferred arthritis were highlighted. Concerning the first region, on proximal chromosome (chr)2, candidate assignment to the complement gene C5 was confirmed by both strain segregation analysis and functional data. Concerning the second, on distal chr1, coinciding with the Sle1 locus implicated in susceptibility to lupus-like autoimmune disease, a contribution by the fcgr2 candidate gene was excluded. Two other regions, on chr12 and chr18 may also contribute to susceptibility to serum-transferred arthritis, albeit to a more limited degree. The contributions of these loci are additive, but gene dosage effects at the C5 locus are such that it largely dominates. The clarity of these results argues that our focus on the terminal effector phase of arthritis in the K/B×N model will bear fruit.
Diffeomorphisms of the circle and hyperbolic curvature
Authors: Singer David
Published: July 2002

The trace

T
f

Tf

of a smooth function

f
f

of a real or complex variable is defined and shown to be invariant under conjugation by Möbius transformations. We associate with a convex curve of class

C
2

C^2

in the unit disk with the Poincaré metric a diffeomorphism of the circle and show that the trace of the diffeomorphism is twice the reciprocal of the geodesic curvature of the curve. Then applying a theorem of Ghys on Schwarzian derivatives we give a new proof of the four-vertex theorem for closed convex curves in the hyperbolic plane.