bluuebunny/crossref_metadata_2025 huggingface dataset:
Further evidence for the presence of c=38.2 Å phase in a Bi-Ca-Sr-Cu-O superconductor
Authors: Ramesh R. , Hetherington C. J. D. , Thomas G. , Green S. M. , Jiang C. , Rudee M. L. , Luo H. L.
Published: July 2002

Results of high-resolution electron microscopy, convergent beam electron microscopy, and x-ray microanalysis on the high Tc phase are reported. Local inhomogeneities in the structure and composition have been observed along the c direction. Both c=30.56 Å (8ap) and c=38.2 Å (10ap) have been observed inside the same grain. The regions with the larger c parameter correspond to regions with higher Ca and Cu concentration suggesting that the range of transition temperatures observed (85 to 115 K) is due to the coexistence of such regions of c=38.2 Å and c=30.56 Å.
Visualization of electrical domains in semi-insulating GaAs:Cr and potential use for variable grating mode operation
Authors: Rajbenbach H. , Verdiell J. M. , Huignard J. P.
Published: July 2002

The results of an experimental optical technique for imaging the electrical domain repartition in semi-insulating GaAs:Cr are reported. The technique is based on the use of the crystal as the active component of a transverse electro-optic two-dimensional light modulator. Under dc applied voltage, the electrical domains are traveling from the cathode to the anode at a velocity that increases with the applied voltage and with the incident illumination (v≂10–100 mm/s). Results for ac applied voltages are also presented. In particular, the observation of stationary and periodically distributed high-field domains in GaAs:Cr is reported for sawtooth applied voltages (1 kV, 50–250 Hz). These high-field domains induce a phase structure whose period is shown to be electrically controllable. This is the first reported demonstration of the possibility of a variable grating mode operation in semiconductors.
Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response
Authors: Russo Vincenzo , Tanzarella Silvia , Dalerba Piero , Rigatti Donata , Rovere Patrizia , Villa Antonello , Bordignon Claudio , Traversari Catia
Published: July 2002

In an attempt to transduce monocyte-derived dendritic cells (DCs) with a retroviral vector coding for an intracytoplasmic tumor antigen (TAA), we were confronted by the evident dissociation between the ability of the treated DCs to induce a TAA-specific response, and the presence of integrated vector proviral DNA. The TAA, i.e., MAGE-3, was acquired by DCs and presented to immune effectors, thanks to the property of DCs to uptake the apoptotic bodies released by the irradiated vector-producing cells. Indeed, we observed that upon irradiation vector-producing cells underwent apoptotic cell death, monitored by annexin V and propidium iodide staining, and were phagocytosed by DCs. Lymphocytes obtained from a patient affected by a MAGE-3+melanoma, were stimulatedin vitrowith autologous DCs previously exposed to irradiated MAGE-3-expressing cells. This procedure led to the induction of MAGE-3-specific cytotoxic effectors, directed against a yet unknown MAGE-3 epitope presented by HLA-A*B5201 molecules. These data demonstrate that DCs can present engulfed human TAAs, thus providing strategies for cancer vaccination.
Unidirectional rotary motion in a liquid crystalline environment: Color tuning by a molecular motor
Authors: van Delden Richard A. , Koumura Nagatoshi , Harada Noboyuki , Feringa Ben L.
Published: July 2002

Life could not exist without motion induced by a variety of molecular motors. The construction of artificial motors by chemical synthesis, which can power motions that lead to macroscopic detectable effects in a system, is a major endeavor in contemporary science. To move toward this goal, a host–guest system, composed of a nematic liquid crystal film doped with a chiral light-driven molecular motor, is assembled. Irradiation of the film results in unidirectional rotary motion of the molecular motor, which induces a motion of the mesogenic molecules leading to a molecular reorganization and, as a consequence, a change in the color of the film. In this way, by control of the rotary motion at the molecular level, color tuning over the entire visible spectrum is achieved. These findings demonstrate that a molecular motor can exert a visually observable macroscopic change in a material.
Presence of mouse mammary tumor virus specifically alters the body odor of mice
Authors: Yamazaki Kunio , Boyse Edward A. , Bard Judith , Curran Maryanne , Kim David , Ross Susan R. , Beauchamp Gary K.
Published: July 2002

It has long been recognized that various genetic and metabolic human disorders alter body odor, which is not surprising because they may alter body chemistry. Thus, it has been suggested that some human diseases may be diagnosed by odor alone. In that regard, the mouse mammary tumor virus (MMTV) and its tumors of mice, which may have human counterparts, are of special interest because of the need for basic research possible only in inbred and genetically defined animals. Accordingly, we now show that the mouse MMTV, whether obtained environmentally or genetically transmitted, alters the body odor of mice in both males and females, and regardless of the presence or absence of tumors. These observations, together with the prospect of artificial human odor discrimination, may aid in the search for early human diagnostics.
On the mitogenic properties of Rap1b: cAMP-induced G
1
/S entry requires activated and phosphorylated Rap1b
Authors: Ribeiro-Neto Fernando , Urbani Julie , Lemee Nicolas , Lou Liguang , Altschuler Daniel L.
Published: July 2002

We have shown that the small GTPase Rap1b, a protein known to antagonize the mitogenic and transforming activity of Ras, is endowed with both mitogenic and tumorigenic properties. Rap1b can be activated by cAMP, an intracellular message known to either stimulate or inhibit cell proliferation. The oncogenic property of Rap1b was revealed in a model system in which cAMP stimulates cell proliferation and was linked to Rap's ability to promote S phase entry. We have now tested the significance of the mitogenic action of Rap1b in a physiologically relevant model, the differentiated thyroid follicular cells, a system that requires thyroid-stimulating hormone (TSH), acting via cAMP, to mediate a full mitogenic response. Here we report that cAMP-dependent hormonal stimulation of DNA synthesis requires Rap1b in a manner dependent on its phosphorylation by protein kinase A.
Mechanism of calcium/calmodulin inhibition of rod cyclic nucleotide-gated channels
Authors: Trudeau Matthew C. , Zagotta William N.
Published: July 2002

Rod cyclic nucleotide-gated (CNG) channels are heterotetramers comprised of both CNGA1 and CNGB1 subunits. Calcium/calmodulin (Ca
2+
/CaM) binds to a site in the N-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1/CNGB1 channels. Here, we show that polypeptides derived from an N-terminal region of CNGB1 form a specific interaction with polypeptides derived from a C-terminal region of CNGA1 that is distal to the cyclic nucleotide-binding domain. Deletion of the Ca
2+
/CaM-binding site from the N-terminal region of CNGB1 eliminated both Ca
2+
/CaM modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca
2+
/CaM. These results suggest that Ca
2+
/CaM-dependent inhibition of rod channels is caused by the direct binding of Ca
2+
/CaM to a site in the N-terminal region in CNGB1, which disrupts the interaction between this region and a distal C-terminal region of CNGA1. The mechanism underlying Ca
2+
/CaM modulation of rod channels is distinct from that in olfactory (CNGA2) CNG channels.
Fibroblast growth factors: An epigenetic mechanism of broad spectrum resistance to anticancer drugs
Authors: Song SaeHeum , Wientjes M. Guillaume , Gan Yuebo , Au Jessie L.-S.
Published: July 2002

Based on the observation that removal of tumors from metastatic organs reversed their chemoresistance, we hypothesized that chemoresistance is induced by extracellular factors in tumor-bearing organs. By comparing chemosensitivity and proteins in different tumors (primary vs. metastases) and different culture systems (tumor fragment histocultures vs. monolayer cultures derived from the same tumor), we found elevated levels of acidic (aFGF) and basic (bFGF) fibroblast growth factors in the conditioned medium (CM) of solid and metastatic tumors. These CM induced broad spectrum resistance to drugs with diverse structures and action mechanisms (paclitaxel, doxorubicin, 5-fluorouracil). Inhibition of bFGF by mAb and its removal by immunoprecipitation resulted in complete reversal of the CM-induced chemoresistance, whereas inhibition/removal of aFGF resulted in partial reversal. Using CM that had been depleted of aFGF and/or bFGF and subsequently reconstituted with respective human recombinant proteins, we found that bFGF but not aFGF induced chemoresistance whereas aFGF amplified the bFGF effect. aFGF and bFGF fully accounted for the CM effect, indicating these proteins as the underlying mechanism of the chemoresistance. The FGF-induced resistance was not due to reduced intracellular drug accumulation or altered cell proliferation. We further showed that an inhibitor of aFGF/bFGF (suramin) enhanced the
in vitro
and
in vivo
activity of chemotherapy, resulting in shrinkage and eradication of well established human lung metastases in mice without enhancing toxicity. These results indicate elevated levels of extracellular aFGF/bFGF as an epigenetic mechanism by which cancer cells elude cytotoxic insult by chemotherapy, and provide a basis for designing new treatment strategies.
Xenopus
Bcl-X
L
selectively protects Rohon-Beard neurons from metamorphic degeneration
Authors: Coen Laurent , du Pasquier David , Le Mevel Sébastien , Brown Spencer , Tata Jamshed , Mazabraud André , Demeneix Barbara A.
Published: July 2002

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-X
L
plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the
Xenopus
Bcl-X
L
homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in
Xenopus
tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11
in vivo
as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.
The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase
Authors: Lau Edmond Y. , Bruice Thomas C.
Published: July 2002

A molecular dynamics study was performed to compare the differences
in the active-site dynamics of the wild-type and W137F mutant enzymes
of 4-chlorobenzoyl-CoA dehalogenase. Only in the wild-type simulation
are conformations formed between the catalytic Asp-145 and
4-chlorobenzoyl-CoA, which resemble the
ab initio
calculated gas-phase transition-state geometry. In the W137F
simulation, the hydrogen bond formed between His-90 and Asp-145
persisted throughout the simulation, causing the carboxylate of Asp-145
to be distant from the benzoyl ring of 4-chlorobenzoyl-CoA. In both
simulations, water molecules were able to diffuse into the active site
of the enzymes. The trajectories provide insight into the routes that
water may use to get into position for the hydrolysis portion of the
dehalogenation reaction. In both simulations, the water molecule
entering the active site forms a hydrogen bond with Asp-145.
Mapping the parameters of prion-induced neuropathology
Authors: Stumpf Michael P. H. , Krakauer David C.
Published: July 2002

We present a theoretical framework that enables us to dissect out
the parametric dependencies of the pathogenesis of prion diseases. We
are able to determine the influence of both host-dependent factors
(connectivity, cell density, protein synthesis rate, and cell death)
and strain-dependent factors (cell tropism, virulence, and replication
rate). We use a model based on a linked system of differential
equations on a lattice to explore how the regional distribution of
central nervous system pathology in Creutzfeldt-Jakob disease,
Gerstmann-Sträussler-Scheinker syndrome, and fatal familial
insomnia relates to each of these factors. The model then is used to
make qualitative predictions about the pathology for two possible
hypothetical triggers of neuronal loss in prion diseases. Pathological
progression in overexpressing mouse models has been shown to depend on
the site of initial infection. The model allows us to compare the
pathologies resulting from different inoculation routes.
Haploinsufficiency of the
Pten
tumor suppressor gene promotes prostate cancer progression
Authors: Kwabi-Addo Bernard , Giri Dipak , Schmidt Karen , Podsypanina Katrina , Parsons Ramon , Greenberg Norman , Ittmann Michael
Published: July 2002

The
PTEN
gene encodes a lipid phosphatase that
negatively regulates the phosphatidylinositol 3-kinase pathway and is
inactivated in a wide variety of malignant neoplasms. High rates of
loss of heterozygosity are observed at the 10q23.3 region containing
the human
PTEN
gene in prostate cancer and other human
malignancies, but the demonstrated rate of biallelic inactivation of
the
PTEN
gene by mutation or homozygous deletion is
significantly lower than the rate of loss of heterozygosity. The
transgenic adenocarcinoma of mouse prostate model is a well
characterized animal model of prostate cancer. Analysis of prostate
cancer progression in transgenic adenocarcinoma of mouse prostate mice
bred to
Pten
+/−
heterozygous mice, coupled
with analysis of the
Pten
gene and protein in the
resulting tumors, reveals that haploinsufficiency of the
Pten
gene promotes the progression of prostate cancer in
this model system. This observation provides a potential explanation
for the discordance in rates of loss of heterozygosity at 10q23 and
biallelic
PTEN
inactivation observed in prostate cancer
and many human malignancies.
Selective repression of transcriptional activators by Pbx1 does not require the homeodomain.
Authors: Lu Q , Kamps M P
Published: July 2002

PBX1 is a homeobox-containing gene identified as the chromosome 1 participant of the t(1;19) chromosomal translocation of childhood pre-B-cell acute lymphoblastic leukemia. This translocation produces a fusion gene encoding the chimeric oncoprotein E2A-Pbx1, which can induce both acute myeloid and T-lymphoid leukemia in mice. The binding of Pbx1 to DNA is weak; however, both Pbx1 and E2A-Pbx1 exhibit tight binding to specific DNA motifs in conjunction with certain other homeodomain proteins, and E2A-Pbx1 activates transcription through these motifs, whereas Pbx1 does not. In this report, we investigate potential transcriptional functions of Pbx1, using transient expression assays. While no segments of Pbx1 activated transcription, an internal domain of Pbx1 repressed transcription induced by the activation domain of Sp1, but not by the activation domains of VP16 or p53. This Pbx1 domain, which lies upstream of the homeodomain and is highly conserved among Pbx proteins, is thus predicted to bind a specific transcription factor. Surprisingly, the repression activity of Pbx1 did not require homeodomain-dependent DNA binding. Thus, Pbx1 may be able to alter gene transcription by both DNA-binding-dependent and DNA-binding-independent mechanisms.
Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.
Authors: Ichikawa S , Sakiyama H , Suzuki G , Hidari K I , Hirabayashi Y
Published: July 2002

We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.
Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.
Authors: Wizemann T M , Moskovitz J , Pearce B J , Cundell D , Arvidson C G , So M , Weissbach H , Brot N , Masure H R
Published: July 2002

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.
Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.
Authors: Marbaix E , Kokorine I , Moulin P , Donnez J , Eeckhout Y , Courtoy P J
Published: July 2002

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation.
On the etiology of Crohn disease.
Authors: Mishina D , Katsel P , Brown S T , Gilberts E C , Greenstein R J
Published: July 2002

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.
Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.
Authors: Liégeois N J , Horner J W , DePinho R A
Published: July 2002

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.
Going mobile: microtubule motors and chromosome segregation.
Authors: Barton N R , Goldstein L S
Published: July 2002

Proper chromosome segregation in eukaryotes depends upon the mitotic and meiotic spindles, which assemble at the time of cell division and then disassemble upon its completion. These spindles are composed in large part of microtubules, which either generate force by controlled polymerization and depolymerization or transduce force generated by molecular microtubule motors. In this review, we discuss recent insights into chromosome segregation mechanisms gained from the analyses of force generation during meiosis and mitosis. These analyses have demonstrated that members of the kinesin superfamily and the dynein family are essential in all organisms for proper chromosome and spindle behavior. It is also apparent that forces generated by microtubule polymerization and depolymerization are capable of generating forces sufficient for chromosome movement in vitro; whether they do so in vivo is as yet unclear. An important realization that has emerged is that some spindle activities can be accomplished by more than one motor so that functional redundancy is evident. In addition, some meiotic or mitotic movements apparently occur through the cooperative action of independent semiredundant processes. Finally, the molecular characterization of kinesin-related proteins has revealed that variations both in primary sequence and in associations with other proteins can produce motor complexes that may use a variety of mechanisms to transduce force in association with microtubules. Much remains to be learned about the regulation of these activities and the coordination of opposing and cooperative events involved in chromosome segregation; this set of problems represents one of the most important future frontiers of research.
Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase.
Authors: Alex L A , Borkovich K A , Simon M I
Published: July 2002

Two-component signal transduction systems are most often found in prokaryotic organisms where they are responsible for mediating the cellular responses to many environmental stimuli. These systems are composed of an autophosphorylating histidine kinase and a response regulator. We have found evidence for the existence of two-component histidine kinases in the eukaryotic filamentous fungus Neurospora crassa based on screening with degenerate primers to conserved regions of these signaling proteins. Subsequent cloning and sequencing of one member of this newly discovered group, nik-1+, shows that the predicted protein sequence shares homology with both the kinase and response regulator modules of two-component signaling proteins. In addition, the N-terminal region of the protein has a novel repeating 90-amino acid motif. Deletion of the nik-1+ gene in N. crassa results in an organism that displays aberrant hyphal structure, which is enhanced under conditions of high osmostress. Increased osmotic pressure during growth on solid medium leads to restricted colonial growth, loss of aerial hyphae formation, and no subsequent conidiophore development. This finding may have implications for mechanisms of fungal colonization and pathogenicity.
Proof for a nonproteinaceous calcium-selective channel in
Escherichia coli
by total synthesis from (
R
)-3-hydroxybutanoic acid and inorganic polyphosphate
Authors: Das Sudipto , Lengweiler Urs D. , Seebach Dieter , Reusch Rosetta N.
Published: July 2002

Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La
3+
) from channels isolated from
Escherichia coli
. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.
Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif
Authors: Balakrishnan R. , Ramasubbu N. , Varughese K. I. , Parthasarathy R.
Published: July 2002

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.
Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in
Escherichia coli
Authors: Fujita Satoshi , Koguma Tetsuhiko , Ohkawa Jun , Mori Kazuyuki , Kohda Takeo , Kise Hideo , Nishikawa Satoshi , Iwakura Masahiro , Taira Kazunari
Published: July 2002

For use of ribozymes
in vivo
, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg
2+
ions, etc.). As a first step toward this goal, we developed a new screening system for detection
in vivo
of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript
in vivo
, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (
i
) mainly those cells that harbored active ribozymes survived in the presence of TMP and (
ii
) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system
in vivo
. If the background “noise” can be removed completely in the future, the selection system might usefully complement existing selection systems
in vitro
.
Decreased blood pressure response in mice deficient of the α
1b
-adrenergic receptor
Authors: Cavalli Antonella , Lattion Anne-Laure , Hummler Edith , Nenniger Monique , Pedrazzini Thierry , Aubert Jean-François , Michel Martin C. , Yang Ming , Lembo Giuseppe , Vecchione Carmine , Mostardini Marina , Schmidt Andrea , Beermann Friedrich , Cotecchia Susanna
Published: July 2002

To investigate the functional role of different α
1
-adrenergic receptor (α
1
-AR) subtypes
in vivo
, we have applied a gene targeting approach to create a mouse model lacking the α
1b
-AR (α
1b
−/−). Reverse transcription–PCR and ligand binding studies were combined to elucidate the expression of the α
1
-AR subtypes in various tissues of α
1b
+/+ and −/− mice. Total α
1
-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the α
1b
−/− as compared with +/+ mice. Because of the large decrease of α
1
-AR in the heart and the loss of the α
1b
-AR mRNA in the aorta of the α
1b
−/− mice, the
in vivo
blood pressure and
in vitro
aorta contractile responses to α
1
-agonists were investigated in α
1b
+/+ and −/− mice. Our findings provide strong evidence that the α
1b
-AR is a mediator of the blood pressure and the aorta contractile responses induced by α
1
agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in α
1b
−/− as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in α
1b
−/− mice. The α
1b
-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different α
1
-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.
Estimating the probability of initiated cell death before tumor induction
Authors: Boucher Kenneth M. , Yakovlev Andrej Y.
Published: July 2002

The effects of cell toxicity are known to be inherent in carcinogenesis induced by radiation or chemical carcinogens. The event of cell death precludes tumor induction from occurring. A long standing problem is to estimate the proportion of initiated cells that die before tumor induction. No experimental techniques are currently available for directly gauging the rate of cell death over extended periods of time. The obstacle can be surmounted by newly developed theoretical methods of carcinogenesis modeling. In this paper, we apply such methods to published data on multiple lung tumors in mice receiving different schedules of urethane. Bioassays of this type play an important role in testing environmental chemicals for carcinogenic activity. Our estimates for urethane-induced carcinogenesis show that, unexpectedly, many initiated cells die early in the course of tumor promotion. We present numerical estimates for the probability of initiated cell death for different schedules (and doses) of urethane administration.
Phosphatidylinositol 3-kinase-γ activates Bruton’s tyrosine kinase in concert with Src family kinases
Authors: Li Zuomei , Wahl Matthew I. , Eguinoa Alicia , Stephens Leonard R. , Hawkins Phillip T. , Witte Owen N.
Published: July 2002

Bruton’s tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bindin vitroto several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-γ. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-γ and Src family kinases.
Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase
Authors: Tucker Chandra L. , Hurley James H. , Miller Taunya R. , Hurley James B.
Published: July 2002

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.
Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1α (SDF-1α) and SDF-1β are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage
Authors: Shioda Tatsuo , Kato Hiroyuki , Ohnishi Yukano , Tashiro Kei , Ikegawa Masaya , Nakayama Emi E. , Hu Huiling , Kato Atsushi , Sakai Yuko , Liu Huanliang , Honjo Tasuku , Nomoto Akio , Iwamoto Aikichi , Morimoto Chikao , Nagai Yoshiyuki
Published: July 2002

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1α (SDF-1α) and 1β (SDF-1β), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34+progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1α confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1α and SDF-1β likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4+cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1α and SDF-1β are involved.
Structural factors controlling ligand binding to myoglobin: A kinetic hole-burning study
Authors: Ormos Pál , Száraz Sándor , Cupane Antonio , Nienhaus G. Ulrich
Published: July 2002

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CO-bound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as “kinetic hole burning.” The study of these correlations gives direct insights into the structure–function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates influence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is
not
the iron displacement from the mean heme plane.
Interleukin-2 induces β2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)
Authors: Brockdorff Johannes , Kanner Steven B. , Nielsen Mette , Borregaard Niels , Geisler Carsten , Svejgaard Arne , Ødum Niels
Published: July 2002

β2integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of β2integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4+T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in β2integrin (CD18)-positive but not in β2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in β2-integrin-positive T cells. Likewise, a β2integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in β2-integrin-positive but not in β2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in β2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125FAK. In conclusion, our data indicate that IL-2 induces β2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.
Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse
Authors: Mohand-Said Saddek , Deudon-Combe Alain , Hicks David , Simonutti Manuel , Forster Valérie , Fintz Anne-Claire , Léveillard Thierry , Dreyfus Henri , Sahel José-Alain
Published: July 2002

The role of cellular interactions in the mechanism of secondary cone photoreceptor degeneration in inherited retinal degenerations in which the mutation specifically affects rod photoreceptors was studied. We developed an organ culture model of whole retinas from 5-week-old mice carrying the retinal degeneration mutation, which at this age contain few remaining rods and numerous surviving cones cocultured with primary cultures of mixed cells from postnatal day 8 normal-sighted mice (C57BL/6) retinas or retinal explants from normal (C57BL/6) or dystrophic (C3H/He) 5-week-old mice. After 7 days, the numbers of residual cone photoreceptors were quantified after specific peanut lectin or anti-arrestin antibody labeling by using an unbiased stereological approach. Examination of organ cultured retinas revealed significantly greater numbers of surviving cones (15–20%) if cultured in the presence of retinas containing normal rods as compared with controls or cocultures with rod-deprived retinas. These data indicate the existence of a diffusible trophic factor released from retinas containing rod cells and acting on retinas in which only cones are present. Because cones are responsible for high acuity and color vision, such data could have important implications not only for eventual therapeutic approaches to human retinal degenerations but also to define interactions between retinal photoreceptor types.
Folding
in vivo
of a newly translated yeast cytosolic enzyme is mediated by the SSA class of cytosolic yeast Hsp70 proteins
Authors: Kim Suwon , Schilke Brenda , Craig Elizabeth A. , Horwich Arthur L.
Published: July 2002

The nature of chaperone action in the eukaryotic cytosol that assists newly translated cytosolic proteins to reach the native state has remained poorly defined. Actin, tubulin, and G
α
transducin are assisted by the cytosolic chaperonin, CCT, but many other proteins, for example, ornithine transcarbamoylase (OTC), a cytosolic homotrimeric enzyme of yeast, do not require CCT action. Here, we observe that yeast cytosolic OTC is assisted to its native state by the SSA class of yeast cytosolic Hsp70 proteins.
In vitro
, refolding of OTC diluted from denaturant was assisted by crude yeast cytosol and ATP and found to be directed by SSA1/2.
In vivo
, when OTC was induced in a temperature-sensitive SSA-deficient strain, it exhibited reduced specific activity, and nonnative subunits were detected in the soluble fraction. These findings indicate that,
in vivo
, the Hsp70 system assists in folding at least some newly translated cytosolic enzymes, most likely functioning in a posttranslational manner.
A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway
Authors: Lange B. Markus , Wildung Mark R. , McCaskill David , Croteau Rodney
Published: July 2002

Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on
in vivo
feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (
Mentha × piperita
) and heterologous expression in
Escherichia coli
of 1-deoxy-
d
-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.
The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-Å resolution
Authors: Liu Yanshun , Hart P. John , Schlunegger Michael P. , Eisenberg David
Published: July 2002

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-Å resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1–15) of each subunit is swapped into the major domain (residues 23–124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of ∼160°. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the “closed interface” but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the “open interface.” The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.