bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of presenilin 1 is associated with enhanced β-catenin signaling and skin tumorigenesis
Authors: Xia Xuefeng , Qian Su , Soriano Salvador , Wu Ying , Fletcher Anthony M. , Wang Xiao-Jing , Koo Edward H. , Wu Xiangwei , Zheng Hui
Published: July 2002

Presenilin 1 (PS1) is required for the proteolytic processing of Notch and the β-amyloid precursor protein (APP), molecules that play pivotal roles in cell-fate determination during development and Alzheimer's disease pathogenesis, respectively. In addition, PS1 interacts with β-catenin and promotes its turnover through independent mechanisms. Consistent with this activity, we report here that PS1 is important in controlling epidermal cell proliferation
in vivo
. PS1 knockout mice that are rescued through neuronal expression of human
PS1
transgene develop spontaneous skin cancers. PS1-null keratinocytes exhibit higher cytosolic β-catenin and β-catenin/lymphoid enhancer factor-1/T cell factor (β-catenin/LEF)-mediated signaling. This effect can be reversed by reintroducing wild-type PS1, but not a PS1 mutant active in Notch processing but defective in β-catenin binding. Nuclear β-catenin protein can be detected in tumors. Elevated β-catenin/LEF signaling is correlated with activation of its downstream target cyclin D1 and accelerated entry from G
1
into S phase of the cell cycle. This report demonstrates a function of PS1 in adult tissues, and our analysis suggests that deregulation of β-catenin pathway contributes to the skin tumor phenotype.
Particle-hole symmetry in the antiferromagnetic state of the cuprates
Authors: Wang Yayu , Ong N. P.
Published: July 2002

In the layered cuprate perovskites, the occurrence of
high-temperature superconductivity seems deeply related to the unusual
nature of the hole excitations. The limiting case of a very small
number of holes diffusing in the antiferromagnetic (AF) background may
provide important insights into this problem. We have investigated the
transport properties in a series of crystals of
YBa
2
Cu
3
O
y
, and found that the
temperature dependencies of the Hall coefficient
R
H
and thermopower
S
change
abruptly as soon as the AF phase boundary is crossed. In the AF state
at low temperatures
T
, both
R
H
and
S
are unexpectedly
suppressed to nearly zero over a broad interval of
T
. We
argue that this suppression arises from near-exact symmetry in the
particle-hole currents. From the trends in
R
H
and
S
, we infer that the
symmetry is increasingly robust as the hole density
x
becomes very small (
x
≃ 0.01). We discuss
implications for electronic properties both within the AF state and
outside.
Water magnetic relaxation dispersion in biological systems: The contribution of proton exchange and implications for the noninvasive detection of cartilage degradation
Authors: Duvvuri Umamaheswar , Goldberg Ari D. , Kranz James K. , Hoang Linh , Reddy Ravinder , Wehrli Felix W. , Wand A. Joshua , Englander S. W. , Leigh John S.
Published: July 2002

Magnetic relaxation has been used extensively to study and characterize biological tissues. In particular, spin-lattice relaxation in the rotating frame (
T
1ρ
) of water in protein solutions has been demonstrated to be sensitive to macromolecular weight and composition. However, the nature of the contribution from low frequency processes to water relaxation remains unclear. We have examined this problem by studying the water
T
1ρ
dispersion in peptide solutions (
14
N- and
15
N-labeled), glycosaminoglycan solutions, and samples of bovine articular cartilage before and after proteoglycan degradation. We find in model systems and tissue that hydrogen exchange from NH and OH groups to water dominates the low frequency water
T
1ρ
dispersion, in the context of the model used to interpret the relaxation data. Further, low frequency dispersion changes are correlated with loss of proteoglycan from the extra-cellular matrix of articular cartilage. This finding has significance for the noninvasive detection of matrix degradation.
A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization
Authors: Chowdhury Kajal , Tabor Stanley , Richardson Charles C.
Published: July 2002

The three-dimensional structure of bacteriophage T7 DNA polymerase
reveals the presence of a loop of 4 aa (residues 401–404) within the
DNA-binding groove; this loop is not present in other members of the
DNA polymerase I family. A genetically altered T7 DNA polymerase, T7
polΔ401–404, lacking these residues, has been characterized
biochemically. The polymerase activity of T7 polΔ401–404 on primed
M13 single-stranded DNA template is one-third of the wild-type enzyme
and has a 3′-to-5′ exonuclease activity indistinguishable from that of
wild-type T7 DNA polymerase. T7 polΔ401–404 polymerizes nucleotides
processively on a primed M13 single-stranded DNA template. T7 DNA
polymerase cannot initiate
de novo
DNA synthesis; it
requires tetraribonucleotides synthesized by the primase activity of
the T7 gene 4 protein to serve as primers. T7 primase-dependent DNA
synthesis on single-stranded DNA is 3- to 6-fold less with T7
polΔ401–404 compared with the wild-type enzyme. Furthermore, the
altered polymerase is defective (10-fold) in its ability to use
preformed tetraribonucleotides to initiate DNA synthesis in the
presence of gene 4 protein. The location of the loop places it in
precisely the position to interact with the tetraribonucleotide primer
and, presumably, with the T7 gene 4 primase. Gene 4 protein also
provides helicase activity for the replication of duplex DNA. T7
polΔ401–404 and T7 gene 4 protein catalyze strand-displacement DNA
synthesis at nearly the same rate as does wild-type polymerase and T7
gene 4 protein, suggesting that the coupling of helicase and polymerase
activities is unaffected.
Structural basis for autoantibody recognition of phosphatidylserine-β2 glycoprotein I and apoptotic cells
Authors: Cocca Brian A. , Seal Samarendra N. , D'Agnillo Paolo , Mueller Yvonne M. , Katsikis Peter D. , Rauch Joyce , Weigert Martin , Radic Marko Z.
Published: July 2002

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) inEscherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by β2 glycoprotein I (β2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-β2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.
Activation by GDNF of a transcriptional program repressing neurite growth in dorsal root ganglia
Authors: Linnarsson Sten , Mikaels Åsa , Baudet Christel , Ernfors Patrik
Published: July 2002

Glial cell line-derived neurotrophic factor (GDNF) promotes
the survival of postnatal—but not embryonic—mouse dorsal root
ganglion cells
in vitro
, despite the fact that its
receptors are expressed at both ages. To understand this difference, we
have performed an oligonucleotide microarray experiment. We found that
several hundred genes were regulated between embryonic and postnatal
stages, and that several important classes of genes were differentially
regulated by GDNF treatment, including genes related to translation and
to phenotypic specification and maturation. Interestingly, a set of
genes related to cell adhesion, cytoskeleton and cellular morphology
were consistently down-regulated by GDNF, suggesting a previously
uncharacterized role for GDNF in repressing neurite growth
and/or branching. This nuclear program initiated by GDNF was
functionally confirmed in cultures of embryonic wild-type neurons
sustained with nerve growth factor and in bax
−/−
neurons that survive in the absence of trophic support.
Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase.
Authors: Drennan C L , Matthews R G , Rosenblatt D S , Ledley F D , Fenton W A , Ludwig M L
Published: July 2002

Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.
Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.
Authors: Lakso M , Pichel J G , Gorman J R , Sauer B , Okamoto Y , Lee E , Alt F W , Westphal H
Published: July 2002

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.
Association of yeast SIN1 with the tetratrico peptide repeats of CDC23.
Authors: Shpungin S , Liberzon A , Bangio H , Yona E , Katcoff D J
Published: July 2002

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo. Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SIN1. Furthermore, using recombinant molecules in vitro, we show that the N terminal of SIN1 is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats. Earlier experiments identified the C-terminal domain of SIN1 to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. Taken together with the results presented here, we suggest that SIN1 is a chromatin protein having at least a dual function: The N terminal of SIN1 interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SIN1 binds proteins involved in transcriptional regulation.
Loss of sequences 3' to the testis-determining gene, SRY, including the Y pseudoautosomal boundary associated with partial testicular determination.
Authors: McElreavey K , Vilain E , Barbaux S , Fuqua J S , Fechner P Y , Souleyreau N , Doco-Fenzy M , Gabriel R , Quereux C , Fellous M , Berkovitz G D
Published: July 2002

The condition termed 46,XY complete gonadal dysgenesis is characterized by a completely female phenotype and streak gonads. In contrast, subjects with 46,XY partial gonadal dysgenesis and those with embryonic testicular regression sequence usually present ambiguous genitalia and a mix of Müllerian and Wolffian structures. In 46,XY partial gonadal dysgenesis gonadal histology shows evidence of incomplete testis determination. In 46,XY embryonic testicular regression sequence there is lack of gonadal tissue on both sides. Various lines of evidence suggest that embryonic testicular regression sequence is a variant form of 46,XY gonadal dysgenesis. The sex-determining region Y chromosome gene (SRY) encodes sequences for the testis-determining factor. To date germ-line mutations in SRY have been reported in approximately 20% of subjects with 46,XY complete gonadal dysgenesis. However, no germ-line mutations of SRY have been reported in subjects with the partial forms. We studied 20 subjects who presented either 46,XY partial gonadal dysgenesis or 46,XY embryonic testicular regression sequence. We examined the SRY gene and the minimum region of Y-specific DNA known to confer a male phenotype. The SRY-open reading frame (ORF) was normal in all subjects. However a de novo interstitial deletion 3' to the SRY-ORF was found in one subject. Although it is possible that the deletion was unrelated to the subject's phenotype, we propose that the deletion was responsible for the abnormal gonadal development by diminishing expression of SRY. We suggest that the deletion resulted either in the loss of sequences necessary for normal SRY expression or in a position effect that altered SRY expression. This case provides further evidence that deletions of the Y chromosome outside the SRY-ORF can result in either complete or incomplete sex reversal.
Information processing in the human brain: magnetoencephalographic approach.
Authors: Lounasmaa O V , Hämäläinen M , Hari R , Salmelin R
Published: July 2002

Rapid progress in effective methods to image brain functions has revolutionized neuroscience. It is now possible to study noninvasively in humans neural processes that were previously only accessible in experimental animals and in brain-injured patients. In this endeavor, positron emission tomography has been the leader, but the superconducting quantum interference device-based magnetoencephalography (MEG) is gaining a firm role, too. With the advent of instruments covering the whole scalp, MEG, typically with 5-mm spatial and 1-ms temporal resolution, allows neuroscientists to track cortical functions accurately in time and space. We present five representative examples of recent MEG studies in our laboratory that demonstrate the usefulness of whole-head magnetoencephalography in investigations of spatiotemporal dynamics of cortical signal processing.
Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors.
Authors: Molkentin J D , Olson E N
Published: July 2002

Members of the MyoD family of muscle-specific basic helix-loop-helix (bHLH) proteins function within a genetic pathway to control skeletal muscle development. Mutational analyses of these factors suggested that their DNA binding domains mediated interaction with a coregulator required for activation of muscle-specific transcription. Members of the myocyte enhancer binding factor 2 (MEF2) family of MADS-box proteins are expressed at high levels in muscle and neural cells and at lower levels in several other cell types. MEF2 factors are unable to activate muscle gene expression alone, but they potentiate the transcriptional activity of myogenic bHLH proteins. This potentiation appears to be mediated by direct interactions between the DNA binding domains of these different types of transcription factors. Biochemical and genetic evidence suggests that MEF2 factors are the coregulators for myogenic bHLH proteins. The presence of MEF2 and cell-specific bHLH proteins in other cell types raises the possibility that these proteins may also cooperate to regulate other programs of cell-specific gene expression. We present a model to account for such cooperative interactions.
The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.
Authors: Belanger A E , Besra G S , Ford M E , Mikusová K , Belisle J T , Brennan P J , Inamine J M
Published: July 2002

The antimycobacterial compound ethambutol [Emb; dextro-2,2'-(ethylenediimino)-di-1-butanol] is used to treat tuberculosis as well as disseminated infections caused by Mycobacterium avium. The critical target for Emb lies in the pathway for the biosynthesis of cell wall arabinogalactan, but the molecular mechanisms for drug action and resistance are unknown. The cellular target for Emb was sought using drug resistance, via target overexpression by a plasmid vector, as a selection tool. This strategy led to the cloning of the M. avium emb region which rendered the otherwise susceptible Mycobacterium smegmatis host resistant to Emb. This region contains three complete open reading frames (ORFs), embR, embA, and embB. The translationally coupled embA and embB genes are necessary and sufficient for an Emb-resistant phenotype which depends on gene copy number, and their putative novel membrane proteins are homologous to each other. The predicted protein encoded by embR, which is related to known transcriptional activators from Streptomyces, is expendable for the phenotypic expression of Emb resistance, but an intact divergent promoter region between embR and embAB is required. An Emb-sensitive cell-free assay for arabinan biosynthesis shows that overexpression of embAB is associated with high-level Emb-resistant arabinosyl transferase activity, and that embR appears to modulate the in vitro level of this activity. These data suggest that embAB encode the drug target of Emb, the arabinosyl transferase responsible for the polymerization of arabinose into the arabinan of arabinogalactan, and that overproduction of this Emb-sensitive target leads to Emb resistance.
Fork-like DNA templates support bypass replication of lesions that block DNA synthesis on single-stranded templates
Authors: Hoffmann Jean-Sébastien , Pillaire Marie-Jeanne , Lesca Claire , Burnouf Dominique , Fuchs Robert P. P. , Defais Martine , Villani Giuseppe
Published: July 2002

DNA replication is an asymmetric process involving concurrent DNA
synthesis on leading and lagging strands. Leading strand synthesis
proceeds concomitantly with fork opening, whereas synthesis of the
lagging strand essentially takes place on a single-stranded template.
The effect of this duality on DNA damage processing by the cellular
replication machinery was tested using eukaryotic cell extracts and
model DNA substrates containing site-specific DNA adducts formed by the
anticancer drug cisplatin or by the carcinogen
N
-2-acetylaminofluorene. Bypass of both lesions was
observed only with fork-like substrates, whereas complete inhibition of
DNA synthesis occurred on damaged single-stranded DNA substrates. These
results suggest a role for additional accessory factors that permit DNA
polymerases to bypass lesions when present in fork-like DNA.
Astrophysical symmetries
Authors: Trimble Virginia
Published: July 2002

Astrophysical objects, ranging from meteorites to the entire
universe, can be classified into about a dozen characteristic
morphologies, at least as seen by a blurry eye. Some patterns exist
over an enormously wide range of distance scales, apparently as a
result of similar underlying physics. Bipolar ejection from protostars,
binary systems, and active galaxies is perhaps the clearest example.
The oral presentation included about 130 astronomical images which
cannot be reproduced here.
Detection of a Yb
3+
binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups
Authors: Roselli Cecile , Boussac Alain , Mattioli Tony A. , Griffiths Jennifer A. , El-Sayed Mostafa A.
Published: July 2002

Near infrared Yb
3+
vibronic sideband spectroscopy was
used to characterize specific lanthanide binding sites in
bacteriorhodopsin (bR) and retinal free
bacteriorhodopsin (bO). The VSB spectra for
deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are
identical. Application of a two-dimensional anti-correlation technique
suggests that only a single Yb
3+
site is observed. The
Yb
3+
binding site in bO is observed to consist of
PO
2
−
groups and carboxylic acid groups, both of which
are bound in a bidentate manner. An additional contribution most likely
arising from a phenolic group is also observed. This implies that the
ligands for the observed single binding site are the lipid head groups
and amino acid residues. The vibronic sidebands of Yb
3+
in
deionized bR regenerated at a ratio of 2:1 ion to bR are essentially
identical to those in bO. The other high-affinity binding site is thus
either not evident or its fluorescence is quenched. A discussion is
given on the difference in binding of Ca
2+
(or
Mg
2+
) and lanthanides in phospholipid membrane proteins.
A metric map of humans: 23,500 loci in 850 bands
Authors: Collins A. , Frezal J. , Teague J. , Morton N. E.
Published: July 2002

High-resolution maps integrated with the enhanced location data
base software (
ldb+
) give improved estimates of genetic
parameters and reveal characteristics of cytogenetic bands. Chiasma
interference is intermediate between Kosambi and Carter–Falconer
levels, as in
Drosophila
and the mouse. The autosomal
genetic map is 2832 and 4348 centimorgans in males and females,
respectively. Telomeric T-bands are strikingly associated with male
recombination and gene density. Position and centromeric
heterochromatin have large effects, but nontelomeric R-bands are not
significantly different from G-bands. Several possible reasons are
discussed. These regularities validate the maps, despite their high
resolution and inevitable local errors. No other approach has been
demonstrated to integrate such a large number of loci, which are
increasing at about 45% per year. The maps and the data and software
from which they are constructed are available through the Internet
(
http://cedar.genetics.soton.ac.uk/public_html
). Successive
versions of this location data base may also be accessed on CD-ROM.
Multiplex selection technique (MuST): an approach to clone transcription factor binding sites.
Authors: Nallur G N , Prakash K , Weissman S M
Published: July 2002

We have used a multiplex selection approach to construct a library of DNA-protein interaction sites recognized by many of the DNA-binding proteins present in a cell type. An estimated minimum of two-thirds of the binding sites present in a library prepared from activated Jurkat T cells represent authentic transcription factor binding sites. We used the library for isolation of "optimal" binding site probes that facilitated cloning of a factor and to identify binding activities induced within 2 hr of activation of Jurkat cells. Since a large fraction of the oligonucleotides obtained appear to represent "optimal" binding sites for sequence-specific DNA-binding proteins, it is feasible to construct a catalog of consensus binding sites for DNA-binding proteins in a given cell type. Qualitative and quantitative comparisons of the catalogs of binding site sequences from various cell types could provide valuable insights into the process of differentiation acting at the level of transcriptional control.
Mutational properties of the primary aflatoxin B1-DNA adduct.
Authors: Bailey E A , Iyer R S , Stone M P , Harris T M , Essigmann J M
Published: July 2002

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.
Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.
Authors: Denu J M , Lohse D L , Vijayalakshmi J , Saper M A , Dixon J E
Published: July 2002

Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed.
Vascular endothelial growth factor B, a novel growth factor for endothelial cells.
Authors: Olofsson B , Pajusola K , Kaipainen A , von Euler G , Joukov V , Saksela O , Orpana A , Pettersson R F , Alitalo K , Eriksson U
Published: July 2002

We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle.
The 5' coding region of Paramecium surface antigen genes controls mutually exclusive transcription.
Authors: Leeck C L , Forney J D
Published: July 2002

Paramecium tetraurelia stock 51 can express at least 11 different types of surface antigens, yet only a single type is expressed on the surface of an individual cell at any one time. The differential expression of stock 51 type A and B surface antigen genes (51A and 51B) is regulated at the level of transcription. Previously, we reported that nucleotide sequences upstream of position -26 (relative to the start of translation) in the 51A and 51B surface antigen genes are necessary for transcriptional activity but are not sufficient to direct differential transcriptional control. In this report we demonstrate that at least some of the critical elements necessary for differential transcription of the 51A and 51B genes lie within the 5' coding region. A hybrid gene that contains 51B upstream sequences (-475 to +1) attached to the ATG start codon of 51A is not cotranscribed with the 51B gene. In contrast, further substitution with 51B sequences (-1647 to +885) allows the chimeric gene to be coexpressed with 51B. A different hybrid gene containing a substitution of 51B sequence from -26 to +885 in the 51A gene is also coexpressed with 51B, revealing that the critical elements within the coding region of 51B do not require 51B upstream sequences for their effect. Coinjection of the 51A gene with the chimeric gene that contains 51B up to +885 showed that the same sequences that allow coexpression with 51B prevent cotranscription with 51A. Together, these results demonstrate that a region downstream of the transcriptional start site between nucleotide positions +1 and +885 (relative to translational start) is necessary to control differential transcriptional activity.
The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication.
Authors: Manna A C , Pai K S , Bussiere D E , White S W , Bastia D
Published: July 2002

The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence. The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function. However, it provides little information as to the surface of the protein involved in dimer-dimer interaction. Using site-directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction. Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein. The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs. This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so. Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains.
Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.
Authors: Jovanovic J N , Benfenati F , Siow Y L , Sihra T S , Sanghera J S , Pelech S L , Greengard P , Czernik A J
Published: July 2002

The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain-derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G-actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation.
Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.
Authors: Kaludov N K , Bowman T L , Sikorski E M , Hurt M M
Published: July 2002

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.
Collective electrodynamics I
Authors: Mead Carver A.
Published: July 2002

Standard results of electromagnetic theory are derived from the direct interaction of macroscopic quantum systems; the only assumptions used are the Einstein–deBroglie relations, the discrete nature of charge, the Green’s function for the vector potential, and the continuity of the wave function. No reference is needed to Maxwell’s equations or to traditional quantum formalism. Correspondence limits based on classical mechanics are shown to be inappropriate.
Intracellular structures of normal and aberrant
Plasmodium falciparum
malaria parasites imaged by soft x-ray microscopy
Authors: Magowan Cathleen , Brown John T. , Liang Joy , Heck John , Coppel Ross L. , Mohandas Narla , Meyer-Ilse Werner
Published: July 2002

Soft x-ray microscopy is a novel approach for investigation of intracellular organisms and subcellular structures with high spatial resolution. We used x-ray microscopy to investigate structural development of
Plasmodium falciparum
malaria parasites in normal and genetically abnormal erythrocytes and in infected erythrocytes treated with cysteine protease inhibitors. Investigations in normal red blood cells enabled us to recognize anomalies in parasite structures resulting from growth under unfavorable conditions. X-ray microscopy facilitated detection of newly elaborated structures in the cytosol of fixed, unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. In cysteine protease inhibitor-treated, infected erythrocytes, high concentrations of material were detected in abnormal digestive vacuoles and aggregated at the parasite plasma membrane. We have demonstrated that an abnormal host erythrocyte skeleton affects structural development of parasites and that this aberrant development can be detected in the following generation when parasites from protein 4.1-deficient red blood cells infect normal erythrocytes. This work extends our current understanding of the relationship between the host erythrocyte membrane and the intraerythrocytic malaria parasite by demonstrating for the first time that constituents of the erythrocyte membrane play a role in normal parasite structural development.
Activation of RNA polymerase II by topologically linked DNA-tracking proteins
Authors: Ouhammouch Mohamed , Sayre Michael H. , Kadonaga James T. , Geiduschek E. Peter
Published: July 2002

Almost all proteins mediating transcriptional activation from promoter-distal sites attach themselves, directly or indirectly, to specific DNA sequence elements. Nevertheless, a single instance of activation by a prokaryotic topologically linked DNA-tracking protein has also been demonstrated. The scope of the latter class of transcriptional activators is broadened in this work. Heterologous fusion proteins linking the transcriptional activation domain of herpes simplex virus VP16 protein to the sliding clamp protein β of the
Escherichia coli
DNA polymerase III holoenzyme are shown to function as topologically DNA-linked activators of yeast and
Drosophila
RNA polymerase II. The β:VP16 fusion proteins must be loaded onto DNA by the clamp-loading
E. coli
γ complex to be transcriptionally active, but they do not occupy fixed sites on the DNA. The DNA-loading sites of these activators have all the properties of enhancers: they can be inverted and their locations relative to the transcriptional start site are freely adjustable.
Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family
Authors: Szczepanowska Joanna , Zhang Xiaolong , Herring Christopher J. , Qin Jun , Korn Edward D. , Brzeska Hanna
Published: July 2002

Myosin I heavy chain kinase from
Acanthamoeba castellanii
is activated
in vitro
by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation
in vitro
. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.
Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca
2+
channel α
1
subunit is responsible for channel inhibition by G protein-coupled receptors
Authors: Qin Ning , Platano Daniela , Olcese Riccardo , Stefani Enrico , Birnbaumer Lutz
Published: July 2002

Several classes of voltage-gated Ca
2+
channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca
2+
channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α
1
subunits that form non-L-type Ca
2+
channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the
ca.
600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca
2+
channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α
1E
of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α
1C
did not abolish inhibition by G proteins, but the exchange of the α
1E
C terminus with that of α
1C
did. This and properties of α
1E
C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca
2+
channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α
1
-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α
1
function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.
Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53
Authors: Wu Yu , Huang Hua , Miner Zoe , Kulesz-Martin Molly
Published: July 2002

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21

WAF1/Cip-1/Sdi

and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21

WAF1/Cip-1/Sdi

protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.
Chymase cleavage of stem cell factor yields a bioactive, soluble product
Authors: Longley B. Jack , Tyrrell Lynda , Ma Yongsheng , Williams David A. , Halaban Ruth , Langley Keith , Lu Hsieng S. , Schechter Norman M.
Published: July 2002

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.
p
-adic
L
functions and trivial zeroes
Authors: Perrin-Riou Bernadette
Published: July 2002

The following is adapted from the notes for the lecture. It
announces results and conjectures about values of the
p
-adic
L
function of the symmetric square of an
elliptic curve.
A histone deacetylase inhibitor potentiates retinoid receptor action in embryonal carcinoma cells
Authors: Minucci Saverio , Horn Valerie , Bhattacharyya Nisan , Russanova Valya , Ogryzko Vasily V. , Gabriele Lucia , Howard Bruce H. , Ozato Keiko
Published: July 2002

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, we studied the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. We show that coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhanced transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA required the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. Furthermore, TSA led to promoter activation by an RXR-selective ligand that was otherwise inactive in transcription. In addition, TSA enhanced transcription from a minimum basal promoter, independently of the RA-responsive element. Finally, we show that TSA alone or in combination with RA increases
in vivo
endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase presented elsewhere.