bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR
Authors: Middleton David A. , Rankin Saffron , Esmann Mikael , Watts Anthony
Published: July 2002

Several biologically active derivatives of the cardiotonic steroid
ouabain have been made containing NMR isotopes (
13
C,
2
H, and
19
F) in the rhamnose sugar and steroid
moieties, and examined at the digitalis receptor site of renal
Na
+
/K
+
-ATPase by a combination of solid-state
NMR methods. Deuterium NMR spectra of
2
H-labeled inhibitors
revealed that the sugar group was only loosely associated with the
binding site, whereas the steroid group was more constrained, probably
because of hydrogen bonding to residues around the
K
+
-channel region. Crosspolarization magic-angle spinning
NMR showed that chemical shifts of inhibitors
13
C-labeled
in the sugar group moved downfield by 0.5 ppm after binding to the
digitalis site, suggesting that the sugar was close to aromatic side
groups. A
19
F,
13
C- rotational-echo
double-resonance NMR strategy was used to determine the structure of an
inhibitor in the digitalis receptor site, and it showed that the
ouabain derivatives adopt a conformation in which the sugar extends out
of the plane of the steroid ring system. The combined structural and
dynamic information favors a model for inhibition in which the ouabain
analogues lie across the surface of the
Na
+
/K
+
-ATPase α-subunit with the sugar
group facing away from the surface of the membrane but free to move
into contact with one or more aromatic residues.
The transcription factors c-myb and GATA-2 act independently in the regulation of normal hematopoiesis.
Authors: Melotti P , Calabretta B
Published: July 2002

The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.
Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma.
Authors: Bennicelli J L , Edwards R H , Barr F G
Published: July 2002

The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.
Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.
Authors: Bénitah J P , Tomaselli G F , Marban E
Published: July 2002

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.
Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.
Authors: McNicholas C M , Guggino W B , Schwiebert E M , Hebert S C , Giebisch G , Egan M E
Published: July 2002

We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels.
NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.
Authors: Zhao Q , Abeygunawardana C , Talalay P , Mildvan A S
Published: July 2002

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.
The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture.
Authors: Scheidler S , Fredericks W J , Rauscher F J , Barr F G , Vogt P K
Published: July 2002

Pediatric alveolar rhabdomyosarcoma is characterized by a chromosomal translocation that fuses parts of the PAX3 and FKHR genes. PAX3 codes for a transcriptional regulator that controls developmental programs, and FKHR codes for a forkhead-winged helix protein, also a likely transcription factor. The PAX3-FKHR fusion product retains the DNA binding domains of the PAX3 protein and the putative activator domain of the FKHR protein. The PAX3-FKHR protein has been shown to function as a transcriptional activator. Using the RCAS retroviral vector, we have introduced the PAX3-FKHR gene into chicken embryo fibroblasts. Expression of the PAX3-FKHR protein in these cells leads to transformation: the cells become enlarged, grow tightly packed and in multiple layers, and acquire the ability for anchorage-independent growth. This cellular transformation in vitro will facilitate studies on the mechanism of PAX3-FKHR-induced oncogenesis.
Behavioral responses of Escherichia coli to changes in redox potential.
Authors: Bespalov V A , Zhulin I B , Taylor B L
Published: July 2002

Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential. In a spatial redox gradient of benzoquinone/benzoquinol, E. coli cells migrated to form a sharply defined band. Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band. This behavioral response was named redox taxis. Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force. The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector. The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis. A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis. A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein. A similar mechanism has been proposed for aerotaxis. Redox taxis may play an important role in the distribution of bacterial species in natural environments.
Candida albicans
strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development
Authors: Köhler Julia R. , Fink Gerald R.
Published: July 2002

The
Candida albicans
genes,
CST20
and
HST7
, were cloned by their ability to suppress the
mating defects of
Saccharomyces cerevisiae
mutants in
the
ste20
and
ste7
genes, which code for
elements of the mating mitogen-activated protein (MAP) kinase pathway.
These
Candida
genes are both structural and functional
homologs of the cognate
Saccharomyces
genes. The pattern
of suppression in
Saccharomyces
is related to their
presumptive position in the MAP kinase cascade. Null alleles of these
genes were constructed in
Candida
. The
Candida
homozygous null mutants are defective in hyphal
formation on some media, but are still induced to form hyphae by serum,
showing that serum induction of hyphae is independent of the MAP kinase
cascade. The
Candida
heterozygotes
CST20
/
cst20
and
HST7
/
hst7
are also defective in hyphal
formation. This lack of dominance of the wild-type allele suggests that
gene dosage is important in
Candida
.
New perspectives on forbidden symmetries, quasicrystals, and Penrose tilings
Authors: Steinhardt Paul J.
Published: July 2002

Quasicrystals are solids with quasiperiodic atomic structures and
symmetries forbidden to ordinary periodic crystals—e.g., 5-fold
symmetry axes. A powerful model for understanding their structure and
properties has been the two-dimensional Penrose tiling. Recently
discovered properties of Penrose tilings suggest a simple picture of
the structure of quasicrystals and shed new light on why they form. The
results show that quasicrystals can be constructed from a single
repeating cluster of atoms and that the rigid matching rules of Penrose
tilings can be replaced by more physically plausible cluster
energetics. The new concepts make the conditions for forming
quasicrystals appear to be closely related to the conditions for
forming periodic crystals.
Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.
Authors: Kuang Y , Wu Y , Smrcka A , Jiang H , Wu D
Published: July 2002

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.
Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target
Authors: Giovannangeli Carine , Diviacco Silvia , Labrousse Valérie , Gryaznov Sergei , Charneau Pierre , Helene Claude
Published: July 2002

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide–psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence byDraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.
Interaction of the S phase regulator Cdc18 with cyclin-dependent kinase in fission yeast
Authors: Brown Grant W. , Jallepalli Prasad V. , Huneycutt Brenda J. , Kelly Thomas J.
Published: July 2002

The fission yeast gene
cdc18
+
is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G
1
/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase
in vivo
. We have expressed Cdc18 as a glutathione
S
-transferase fusion in fission yeast and demonstrated that the fusion protein is functional
in vivo
. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the
cdc2
+
gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G
1
/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.
Production of secretory immunoglobulin A by a single mammalian cell
Authors: Chintalacharuvu Koteswara R. , Morrison Sherie L.
Published: July 2002

Secretory IgA (sIgA) plays a critical role in providing protection against infection at the mucosal surfaces. Normally, sIgA is the product of two different cell types with heavy, light, and J chains produced by the plasma cells, whereas secretory component (SC), a cleavage product of the polymeric immunoglobulin receptor (pIgR), is added during the transit of dimeric IgA through the epithelial cell layer. In the current study, by introducing a gene for the processed form of SC into a cell line that produces dimeric IgA, we have succeeded in creating a single cell that is able to produce and secrete covalently joined sIgA. To our knowledge, this is the first time it has been possible to efficiently produce large quantities of sIgA of defined specificity in mammalian cells. The sIgA made using this approach has great potential as an immunotherapeutic.
An extended DNA structure through deoxyribose-base stacking induced by RecA protein
Authors: Nishinaka Taro , Ito Yutaka , Yokoyama Shigeyuki , Shibata Takehiko
Published: July 2002

The family of proteins that are homologous to RecA protein of
Escherichia coli
is essential to homologous genetic recombination in various organisms including viruses, bacteria, lower eukaryotes, and mammals. In the presence of ATP (or ATPγS), these proteins form helical filaments containing single-stranded DNA at the center. The single-stranded DNA bound to RecA protein is extended 1.5 times relative to B-form DNA with the same sequence, and the extension is critical to pairing with homologous double-stranded DNA. This pairing reaction, called homologous pairing, is a key reaction in homologous recombination. In this NMR study, we determined a three-dimensional structure of the single-stranded DNA bound to RecA protein. The DNA structure contains novel deoxyribose-base stacking in which the 2′-methylene moiety of each deoxyribose is placed above the base of the following residue, instead of normal stacking of adjacent bases. As a result of this deoxyribose-base stacking, bases of the single-stranded DNA are spaced out nearly 5 Å. Thus, this novel structure well explains the axial extension of DNA in the RecA-filaments relative to B-form DNA and leads to a possible interpretation of the role of this extension in homologous pairing.
Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host
Authors: Casu Rosanne , Eisemann Craig , Pearson Roger , Riding George , East Iain , Donaldson Alan , Cadogan Lee , Tellam Ross
Published: July 2002

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly
Lucilia cuprina
feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar
L. cuprina
larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of
L. cuprina
larvae that may have general application to the control of other insect pests.
Vitamin A in serum is a survival factor for fibroblasts
Authors: Chen Yanqiu , Derguini Fadila , Buck Jochen
Published: July 2002

Murine 3T3 cells arrest in a quiescent, nondividing state when transferred into medium containing little or no serum. Within the first day after transfer, fibroblasts can be activated to proliferate by platelet-derived growth factor (PDGF) alone; cells starved longer than 1 day, however, are activated only by serum. We demonstrate that endogenous vitamin A (retinol) or retinol supplied by serum prevents cell death and that retinol, in combination with PDGF, can fully replace serum in activating cells starved longer than 1 day. The physiological retinol derivative 14-hydroxy-4,14-
retro
-retinol, but not retinoic acid, can replace retinol in rescuing or activating 3T3 cells. Anhydroretinol, another physiological retinol metabolite that acts as a competitive antagonist of retinol, blocks cell activation by serum, indicating that retinol is a necessary component of serum. It previously has been proposed that activation of 3T3 cells requires two factors in serum, an activation factor shown to be PDGF and an unidentified survival factor. We report that retinol is the survival factor in serum.
Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: A possible mechanistic basis for its antitumor action
Authors: Panda Dulal , Miller Herbert P. , Islam Khalid , Wilson Leslie
Published: July 2002

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (
K
d
, ≈30 μM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.
Maintenance of a somatotopic cortical map in the face of diminishing thalamocortical inputs
Authors: Jones E. G. , Manger P. R. , Woods T. M.
Published: July 2002

This study addresses the extent of divergence in the ascending somatosensory pathways of primates. Divergence of inputs from a particular body part at each successive synaptic step in these pathways results in a potential magnification of the representation of that body part in the somatosensory cortex, so that the representation can be expanded when peripheral input from other parts is lost, as in nerve lesions or amputations. Lesions of increasing size were placed in the representation of a finger in the ventral posterior thalamic nucleus (VPL) of macaque monkeys. After a survival period of 1–5 weeks, area 3b of the somatosensory cortex ipsilateral to the lesion was mapped physiologically, and the extent of the representation of the affected and adjacent fingers was determined. Lesions affecting less than 30% of the thalamic VPL nucleus were without effect upon the cortical representation of the finger whose thalamic representation was at the center of the lesion. Lesions affecting about 35% of the VPL nucleus resulted in a shrinkage of the cortical representation of the finger whose thalamic representation was lesioned, with concomitant expansion of the representations of adjacent fingers. Beyond 35–40%, the whole cortical representation of the hand became silent. These results suggest that divergence of brainstem and thalamocortical projections, although normally not expressed, are sufficiently great to maintain a representation after a major loss of inputs from the periphery. This is likely to be one mechanism of representational plasticity in the cerebral cortex.
Identification and characterization of a human protein kinase related to budding yeast Cdc7p
Authors: Jiang Wei , Hunter Tony
Published: July 2002

The Cdc7p protein kinase is essential for the G
1
/S transition and initiation of DNA replication during the cell division cycle in
Saccharomyces cerevisiae
. Cdc7p appears to be an evolutionarily conserved protein, since a homolog Hsk1 has been isolated from
Schizosaccharomyces pombe
. Here, we report the isolation of a human cDNA, HsCdc7, whose product is closely related in sequence to Cdc7p and Hsk1. The HsCdc7 cDNA encodes a protein of 574 amino acids with predicted size of 64 kDa. HsCdc7 contains the conserved subdomains common to all protein-serine/threonine kinases and three “kinase inserts” that are characteristic of Cdc7p and Hsk1. Immune complexes of HsCdc7 from cell lysates were able to phosphorylate histone H1
in vitro
. Indirect immunofluorescence staining demonstrated that HsCdc7 protein was predominantly localized in the nucleus. Although the expression levels of HsCdc7 appeared to be constant throughout the cell cycle, the protein kinase activity of HsCdc7 increased during S phase of the cell cycle at approximately the same time as that of Cdk2. These results, together with the functions of Cdc7p in yeast, suggest that HsCdc7 may phosphorylate critical substrate(s) that regulate the G
1
/S phase transition and/or DNA replication in mammalian cells.
Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2
in vivo
Authors: Gadbois Ellen L. , Chao David M. , Reese Joseph C. , Green Michael R. , Young Richard A.
Published: July 2002

Activation of eukaryotic class II gene expression involves the formation of a transcription initiation complex that includes RNA polymerase II, general transcription factors, and SRB components of the holoenzyme. Negative regulators of transcription have been described, but it is not clear whether any are general repressors of class II genes
in vivo
. We reasoned that defects in truly global negative regulators should compensate for deficiencies in SRB4 because SRB4 plays a positive role in holoenzyme function. Genetic experiments reveal that this is indeed the case: a defect in the yeast homologue of the human negative regulator NC2 (Dr1·DRAP1) suppresses a mutation in SRB4. Global defects in mRNA synthesis caused by the defective yeast holoenzyme are alleviated by the NC2 suppressing mutation
in vivo
, indicating that yeast NC2 is a global negative regulator of class II transcription. These results imply that relief from repression at class II promoters is a general feature of gene activation
in vivo
.
Centaurea corymbosa, a cliff-dwelling species tottering on the brink of extinction: A demographic and genetic study
Authors: Colas Bruno , Olivieri Isabelle , Riba Miquel
Published: July 2002

Centaurea corymbosa(Asteraceae) is endemic to a small area (≤3 km2), and <500 individuals reproduce in any given year. Nevertheless, enzyme polymorphism was found within and among the six local extant populations, the most distant at 2.3 km. Levels of gene flow among populations and seed and pollen dispersal data indicated very low dispersal capacity. Rarity of long distance dispersal events coupled with traits such as prolonged juvenile period, monocarpy, and self-incompatibility precludes the establishment of new populations and thus the evolution toward colonization ability through increased dispersal rate, polycarpy, or self-compatibility. The species thus appears to be trapped on an evolutionary dead-end toward extinction, even though, from a preliminary introduction experiment, we conclude that several nearby unoccupied sites would be suitable for the species.
Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments
Authors: Sugi Haruo , Akimoto Tsuyoshi , Sutoh Kazuo , Chaen Shigeru , Oishi Noboru , Suzuki Suechika
Published: July 2002

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M⋅ADP⋅P
i
) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP → M⋅ADP⋅P
i
. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.
Localization of guanylate cyclase-activating protein 2 in mammalian retinas
Authors: Otto-Bruc Annie , Fariss Robert N. , Haeseleer Françoise , Huang Jing , Surgucheva Janina BuczyŁko*, Irina , Baehr Wolfgang , Milam Ann H. , Palczewski Krzysztof
Published: July 2002

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca
2+
, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used
in situ
hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca
2+
-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.
The Weyl character formula, the half-spin representations, and equal rank subgroups
Authors: Gross Benedict , Kostant Bertram , Ramond Pierre , Sternberg Shlomo
Published: July 2002

Let
B
be a reductive Lie subalgebra of a semi-simple Lie algebra
F
of the same rank both over the complex numbers. To each finite dimensional irreducible representation
V
λ
of
F
we assign a multiplet of irreducible representations of
B
with m elements in each multiplet, where m is the index of the Weyl group of B in the Weyl group of
F
. We obtain a generalization of the Weyl character formula; our formula gives the character of
V
λ
as a quotient whose numerator is an alternating sum of the characters in the multiplet associated to
V
λ
and whose denominator is an alternating sum of the characters of the multiplet associated to the trivial representation of
F
.
Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease
Authors: Geng Liping , Solimena Michele , Flavell Richard A. , Sherwin Robert S. , Hayday Adrian C.
Published: July 2002

Glutamic acid decarboxylase (GAD)65 is a pancreatic β cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.
Site-specific gene delivery
in vivo
through engineered Sendai viral envelopes
Authors: Ramani Komal , Hassan Quamarul , Venkaiah Betapudi , Hasnain Seyed E. , Sarkar Debi P.
Published: July 2002

Inspite of several stimulating developments in gene therapy, the formulation of a targeted gene delivery “vector” is still far from ideal. We have demonstrated the potential of reconstituted Sendai viral envelopes containing only the fusion glycoprotein (F-virosomes) in targeted delivery of reporter genes to liver cells of BALB/c mouse
in vivo.
The membrane fusion-mediated high efficiency of gene transfer to liver cells was ascertained following a critical evaluation of the level of the DNA, mRNA, and relevant proteins. Furthermore, the involvement of viral glycoprotein both as a unique natural ligand and as a membrane fusogen could lead to preferential transfection of parenchymal cell types of liver. The integration of transgenes in the mouse chromosomal DNA and its stable expression up to 4 mo after single i.v. administration of this gene carrier has bolstered its efficiency and novelty. Moreover, the F-virosomes did not elicit significant humoral immune response against the fusion protein in the injected animal. The findings reported here open up the possibility for considering “F-virosomes” as a promising “vehicle” for site-specific DNA delivery in gene therapy.
Enhanced transcription factor access to arrays of histone H3/H4 tetramer⋅DNA complexes
in vitro
: Implications for replication and transcription
Authors: Tse Christin , Fletcher Terace M. , Hansen Jeffrey C.
Published: July 2002

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer⋅5S rDNA complexes have been assembled
in vitro
from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA, the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions where nucleosomal arrays are extensively folded. We further show that the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays.
Single prenyl-binding site on protein prenyl transferases
Authors: Desnoyers Luc , Seabra Miguel C.
Published: July 2002

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal C
AAX
motif (C is Cys,
A
is aliphatic, and
X
is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as
XX
CC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of C
AAX
prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the C
AAX
prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.
Rump white
inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of
Kit
expression
Authors: Hough R. Barry , Lengeling Andreas , Bedian Vahe , Lo Cecilia , Bucan Maja
Published: July 2002

The mouse
rump white
(
Rw
) mutation causes a pigmentation defect in heterozygotes and embryonic lethality in homozygotes. At embryonic day (E) 7.5,
Rw
/
Rw
embryos are retarded in growth, fail to complete neurulation and die around E 9.5. The
Rw
mutation is associated with a chromosomal inversion spanning 30 cM of the proximal portion of mouse chromosome 5. The
Rw
embryonic lethality is complemented by the

W
19H

deletion, which spans the distal boundary of the
Rw
inversion, suggesting that the
Rw
lethality is not caused by the disruption of a gene at the distal end of the inversion. Here, we report the molecular characterization of sequences disrupted by both inversion breakpoints. These studies indicate that the distal breakpoint of the inversion is associated with ectopic
Kit
expression and therefore may be responsible for the dominant pigmentation defect in
Rw
/+ mice; whereas the recessive lethality of
Rw
is probably due to the disruption of the gene encoding dipeptidyl aminopeptidase-like protein 6,
Dpp6
[Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J. & Shimasaki, S. (1992)
Proc. Natl. Acad. Sci. USA
89, 197–201] located at the proximal inversion breakpoint.
Modulation of intracellular transport by transported proteins: Insight from regulation of COPI-mediated transport
Authors: Aoe Tomohiko , Lee Agnes J. , van Donselaar Elly , Peters Peter J. , Hsu Victor W.
Published: July 2002

Intracellular transport is best understood for how proteins are shuttled among different compartments of the secretory pathway by membrane-bound transport carriers. However, it remains unclear whether regulation of this transport is modulated by the transported (cargo) proteins in the lumen of transport pathways. In the early secretory pathways that connect the endoplasmic reticulum (ER) and the Golgi complex, the small GTPase ADP-ribosylation factor 1 (ARF1) recruits a cytosolic coat protein complex named COPI onto membranes as a key step in the formation of transport vesicles. Transport of newly synthesized proteins that leave the ER includes a class of cargo proteins with a sequence motif of KDEL. When these KDEL proteins leave the ER to reach the Golgi complex, they are recognized by their receptor and transported retrograde in COPI-coated vesicles back to the ER. We now demonstrate that stimulation of the KDEL receptor by a KDEL protein enhances an interaction between the KDEL receptor and a GTPase-activating protein for ARF1. As a result, more cytosolic GTPase-activating protein is recruited to membranes to inactivate ARF1. Thus, the KDEL proteins are examples of luminal cargo proteins that regulate transport by activating their receptor. Most likely, this regulation affects retrograde transport from the Golgi complex to the ER, as activated KDEL receptor appears to reside only in retrograde COPI-coated vesicles.
De novo
insertion of an intron into the mammalian sex determining gene,
SRY
Authors: O’Neill Rachel J. Waugh , Brennan Francine E. , Delbridge Margaret L. , Crozier Ross H. , Graves Jennifer A. Marshall
Published: July 2002

Two theories have been proposed to explain the evolution of introns within eukaryotic genes. The introns early theory, or “exon theory of genes,” proposes that introns are ancient and that recombination within introns provided new exon structure, and thus new genes. The introns late theory, or “insertional theory of introns,” proposes that ancient genes existed as uninterrupted exons and that introns have been introduced during the course of evolution. There is still controversy as to how intron–exon structure evolved and whether the majority of introns are ancient or novel. Although there is extensive evidence in support of the introns early theory, phylogenetic comparisons of several genes indicate recent gain and loss of introns within these genes. However, no example has been shown of a protein coding gene, intronless in its ancestral form, which has acquired an intron in a derived form. The mammalian sex determining gene,
SRY
, is intronless in all mammals studied to date, as is the gene from which it recently evolved. However, we report here comparisons of genomic and cDNA sequences that now provide evidence of a
de novo
insertion of an intron into the
SRY
gene of dasyurid marsupials. This recently (approximately 45 million years ago) inserted sequence is not homologous with known transposable elements. Our data demonstrate that introns may be inserted as spliced units within a developmentally crucial gene without disrupting its function.
An X chromosome gene regulates hematopoietic stem cell kinetics
Authors: Abkowitz Janis L. , Taboada Monica , Shelton Grady H. , Catlin Sandra N. , Guttorp Peter , Kiklevich J. Veronika
Published: July 2002

Females are natural mosaics for X chromosome-linked genes. As X chromosome inactivation occurs randomly, the ratio of parental phenotypes among blood cells is approximately 1:1. Recently, however, ratios of greater than 3:1 have been observed in 38–56% of women over age 60. This could result from a depletion of hematopoietic stem cells (HSCs) with aging (and the maintenance of hematopoiesis by a few residual clones) or from myelodysplasia (the dominance of a neoplastic clone). Each possibility has major implications for chemotherapy and for transplantation in elderly patients. We report similar findings in longitudinal studies of female Safari cats and demonstrate that the excessive skewing that develops with aging results from a third mechanism that has no pathologic consequence, hemizygous selection. We show that there is a competitive advantage for all HSCs with a specific X chromosome phenotype and, thus, demonstrate that an X chromosome gene (or genes) regulates HSC replication, differentiation, and/or survival.
Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B
Authors: Zeman Steven M. , Depew Kristopher M. , Danishefsky Samuel J. , Crothers Donald M.
Published: July 2002

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (φ) and intrinsic association constant (
K
a
). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand–DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin γ
1
I
. The unwinding angle for CRB calculated by this method is −5.3 ± 0.5°. Its
K
a
value is 0.20 × 10
6
M
−1
. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.