bluuebunny/crossref_metadata_2025 huggingface dataset:
X-ray crystal structure of an anti-Buckminsterfullerene antibody Fab fragment: Biomolecular recognition of C
60
Authors: Braden Bradford C. , Goldbaum Fernando A. , Chen Bi-Xing , Kirschner Austin N. , Wilson Stephen R. , Erlanger Bernard F.
Published: July 2002

We have prepared a monoclonal Buckminsterfullerene specific
antibody and report the sequences of its light and heavy chains. We
also show, by x-ray crystallographic analysis of the Fab fragment and
by model building, that the fullerene binding site is formed by the
interface of the antibody light and heavy chains. Shape-complementary
clustering of hydrophobic amino acids, several of which participate in
putative stacking interactions with fullerene, form the binding site.
Moreover, an induced fit mechanism appears to participate in the
fullerene binding process. Affinity of the antibody–fullerene complex
is 22 nM as measured by competitive binding. These findings should be
applicable not only to the use of antibodies to assay and direct
potential fullerene-based drug design but could also lead to new
methodologies for the production of fullerene derivatives and nanotubes
as well.
Statistical equilibrium predictions of jets and spots on Jupiter
Authors: Turkington Bruce , Majda Andrew , Haven Kyle , DiBattista Mark
Published: July 2002

An equilibrium statistical theory of coherent structures is applied to midlatitude bands in the northern and southern hemispheres of Jupiter. The theory imposes energy and circulation constraints on the large-scale motion and uses a prior distribution on potential vorticity fluctuations to parameterize the small-scale turbulent eddies. Nonlinearly stable coherent structures are computed by solving the constrained maximum entropy principle governing the equilibrium states of the statistical theory. The theoretical predictions are consistent with the observed large-scale features of the weather layer if and only if the prior distribution has anticyclonic skewness, meaning that intense anticyclones predominate at small scales. Then the computations show that anticyclonic vortices emerge at the latitudes of the Great Red Spot and the White Ovals in the southern band, whereas in the northern band no vortices form within the zonal jets. Recent observational data from the Galileo mission support the occurrence of intense small-scale anticyclonic forcing. The results suggest the possibility of using equilibrium statistical theory for inverse modeling of the small-scale characteristics of the Jovian atmosphere from observed features.
Comprehensive identification of conditionally essential genes in mycobacteria
Authors: Sassetti Christopher M. , Boyd Dana H. , Rubin Eric J.
Published: July 2002

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a
mariner-
based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of
Mycobacterium bovis
bacillus Calmette–Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.
The transition from quantity to quality: A neglected causal mechanism in accounting for social evolution
Authors: Carneiro Robert L.
Published: July 2002

Students of social evolution are concerned not only with the
general course it has followed, but also with the mechanisms that have
brought it about. One such mechanism comes into play when the
quantitative increase in some entity, usually population, reaching a
certain threshold, gives rise to a qualitative change in the structure
of a society. This mechanism, first recognized by Hegel, was seized on
by Marx and Engels. However, neither they nor their current followers
among anthropologists have made much use of it in attempting to explain
social evolution. But as this paper attempts to show, in those few
instances when the mechanism has been invoked, it has heightened our
understanding of the process of social evolution. And, it is argued, if
the mechanism were more widely applied, further understanding of the
course of evolution could be expected to result.
The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance
Authors: Temiakov Dmitry , Mentesana Pamela E. , Ma Kaiyu , Mustaev Arkady , Borukhov Sergei , McAllister William T.
Published: July 2002

During the early stages of transcription, T7 RNA polymerase forms
an unstable initiation complex that synthesizes and releases
transcripts 2–8 nt in length before disengaging from the promoter and
isomerizing to a stable elongation complex. In this study, we used
RNA⋅protein and RNA⋅DNA crosslinking methods to probe the
location of newly synthesized RNA in halted elongation complexes. The
results indicate that the RNA in an elongation complex remains in an
RNA⋅DNA hybrid for about 8 nt from the site of nucleotide addition
and emerges to the surface of the enzyme about 12 nt from the addition
site. Strikingly, as the transcript leaves its hybrid with the
template, the crosslinks it forms with the RNA polymerase involve a
portion of a hairpin loop (the specificity loop) that makes specific
contacts with the binding region of the promoter during initiation.
This observation suggests that the specificity loop may have a dual
role in transcription, binding first to the promoter and subsequently
interacting with the RNA product. It seems likely that association of
the nascent RNA with the specificity loop facilitates disengagement
from the promoter and is an important part of the process that leads to
a stable elongation complex.
Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON
Authors: Jaffrey Samie R. , Benfenati Fabio , Snowman Adele M. , Czernik Andrew J. , Snyder Solomon H.
Published: July 2002

The specificity of the reactions of nitric oxide (NO) with its neuronal targets is determined in part by the precise localizations of neuronal NO synthase (nNOS) within the cell. The targeting of nNOS is mediated by adapter proteins that interact with its PDZ domain. Here, we show that the nNOS adapter protein, CAPON, interacts with synapsins I, II, and III through an N-terminal phosphotyrosine-binding domain interaction, which leads to a ternary complex comprising nNOS, CAPON, and synapsin I. The significance of this ternary complex is demonstrated by changes in subcellular localization of nNOS in mice harboring genomic deletions of both
synapsin I
and
synapsin II
. These results suggest a mechanism for specific actions of NO at presynaptic sites.
Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein.
Authors: Leopardi R , Roizman B
Published: July 2002

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.
A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.
Authors: Campos-Caro A , Sala S , Ballesta J J , Vicente-Agulló F , Criado M , Sala F
Published: July 2002

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspartate residue therein, Asp-266 in the alpha7 subunit, was identified by site-directed mutagenesis as crucial, since mutants at this position exhibited very poor functional responses to three different nicotinic agonists. We have extended this investigation to another neuronal nicotinic receptor (alpha3/beta4), and found that a homologous residue in the beta4 subunit, Asp-268, played a similar role in coupling. These findings are consistent with a hypothesis that the aspartate residue in the M2-M3 loop, which is conserved in all homomer-forming alpha-type subunits and all neuronal beta-type subunits that combine to form functional receptors, is a major determinant of information transmission from binding site to channel gate in all neuronal nicotinic receptors.
Radioimmunotherapy with a 64Cu-labeled monoclonal antibody: a comparison with 67Cu.
Authors: Connett J M , Anderson C J , Guo L W , Schwarz S W , Zinn K R , Rogers B E , Siegel B A , Philpott G W , Welch M J
Published: July 2002

67Cu (t1/2 = 62 h) has demonstrated potential as a radionuclide for radioimmunotherapy, but limited availability severely restricts its widespread use. 64Cu (t1/2 = 12.8 h) has been shown to have comparable effectiveness in vitro and in vivo. The present study was undertaken to examine the therapeutic potential of 64Cu- and 67Cu-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradeca ne-N, N',N",N"'-tetraacetic acid (BAT)-2-iminothiolane (2IT)-1A3 (1A3 is a mouse anti-human colorectal cancer mAb) for treatment of GW39 human colon carcinoma carried in hamster thighs. Hamsters were injected with 64Cu- or 67Cu-BAT-2IT-1A3 or Cu-labeled nonspecific IgG (MOPC) or saline. Hamsters were killed 6-7 months after therapy or when tumors were > or = 10 g. Of the hamsters with small tumors (mean weight 0.43 +/- 0.25 g), 87.5% were disease-free 7 months after treatment with 2 mCi (1 Ci = 37 GBq) of 64Cu-BAT-2IT-1A3 or 0.4 MCi of 67Cu-BAT-2IT-1A3. The mean tumor doses at these activities of 64Cu- and 67Cu-BAT-2IT-1A3 were 586 and 1269 rad (1 rad = 0.01 Gy), respectively. In contrast, 76% of hamsters treated with 2 mCi of 64Cu-BAT-2IT-MOPC or 0.4 mCi of 67Cu-BAT-2IT-MOPC had to be killed before 6 months because of tumor regrowth. When hamsters with larger tumors (mean weight 0.66 +/- 0.11 g) were treated with 64Cu- or 67Cu-BAT-2IT-1A3, survival was extended compared with controls, but only one animal remained tumor-free to 6 months. These results demonstrate that 64Cu- and 67Cu-BAT-2IT-1A3 given in a single administered dose can eradicate small tumors without significant host toxicity, but additional strategies to deliver higher tumor doses will be needed for larger tumors.
Dopamine inhibits mammalian photoreceptor Na+,K+-ATPase activity via a selective effect on the alpha3 isozyme.
Authors: Shulman L M , Fox D A
Published: July 2002

The rat retina contains dopaminergic interplexiform cells that send processes to the outer plexiform layer where dopamine is released in a light-dependent manner. We report herein that physiologically relevant concentrations of dopamine inhibited ouabain-sensitive photoreceptor oxygen consumption in dark- and light-adapted rat retinas and inhibited Na+,K+-ATPase specific activity (EC 3.6.1.37) in a rat rod outer-inner segment preparation. Experiments with the selective D1 agonist fenoldopam or D2 agonist quinpirole and experiments with dopamine plus either the D1 antagonist SCH23390 or D2/D4 antagonist clozapine showed that the inhibition of oxygen consumption and enzyme activity were mediated by D2/D4-like receptors. The amphetamine-induced release of dopamine, monitored by the inhibition of oxygen consumption, was blocked by L-2-amino-4-phosphonobutyric acid and kynurenic acid. Pharmacological and biochemical experiments determined that the IC50 values of ouabain for the alpha1-low and alpha3-high ouabain affinity isozymes of photoreceptor Na+,K+-ATPase were approximately 10(-5) and approximately 10(-7) M, respectively, and that the D2/D4-like mediated inhibition of Na+,K+-ATPase was exclusively selective for the alpha3 isozyme. The dopamine-mediated inhibition of alpha3 first occurred at 5 nM, was maximal at 100 microM (-47%), had an IC50 value of 382 +/- 23 nM, and exhibited negative cooperativity (Hill coefficient, 0.27). Prior homogenization of the rod outer-inner segment completely prevented the long-lasting inhibition, suggesting that the effect was coupled to a second messenger. Although the physiological significance of our findings to photoreceptor function is unknown, we hypothesize that these results may have relevance for the temporal tuning properties of rods.
Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.
Authors: Hunyady L , Zhang M , Jagadeesh G , Bor M , Balla T , Catt K J
Published: July 2002

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.
Intracellular pH in adipocytes: effects of free fatty acid diffusion across the plasma membrane, lipolytic agonists, and insulin.
Authors: Civelek V N , Hamilton J A , Tornheim K , Kelly K L , Corkey B E
Published: July 2002

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.
Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells.
Authors: Miano J M , Firulli A B , Olson E N , Hara P , Giachelli C M , Schwartz S M
Published: July 2002

Smooth muscle cell plasticity is considered a prerequisite for atherosclerosis and restenosis following angioplasty and bypass surgery. Identification of transcription factors that specify one smooth muscle cell phenotype over another therefore may be of major importance in understanding the molecular basis of these vascular disorders. Homeobox genes exemplify one class of transcription factors that could govern smooth muscle cell phenotypic diversity. Accordingly, we screened adult and fetal human smooth muscle cell cDNA libraries with a degenerate oligonucleotide corresponding to a highly conserved region of the homeodomain with the idea that homeobox genes, if present, would display a smooth muscle cell phenotype-dependent pattern of expression. No homeobox genes were detected in the adult human smooth muscle cell library; however, five nonparalogous homeobox genes were uncovered from the fetal library (HoxA5, HoxA11, HoxB1, HoxB7, and HoxC9). Northern blotting of adult and fetal tissues revealed low and restricted expression of all five homeobox genes. No significant differences in transcripts of HoxA5, HoxA11, and HoxB1 were detected between adult or fetal human smooth muscle cells in culture. HoxB7 and HoxC9, however, showed preferential mRNA expression in fetal human smooth muscle cells that appeared to correlate with the age of the donor. This phenotype-dependent expression of homeobox genes was also noted in rat pup versus adult smooth muscle cells. While similar differences in gene expression have been reported between subsets of smooth muscle cells from rat vessels of different-aged animals or clones of rat smooth muscle, our findings represent a demonstration of a transcription factor distinguishing two human smooth muscle cell phenotypes.
Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.
Authors: Sandvig K , Garred O , van Deurs B
Published: July 2002

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.
Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase.
Authors: Pear J R , Kawagoe Y , Schreckengost W E , Delmer D P , Stalker D M
Published: July 2002

In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of cellulose, the major cell wall polymer of plants. We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are homologs of the bacterial celA genes that encode the catalytic subunit of cellulose synthase. Three regions in the deduced amino acid sequences of the plant celA gene products are conserved with respect to the proteins encoded by bacterial celA genes. Within these conserved regions, there are four highly conserved subdomains previously suggested to be critical for catalysis and/or binding of the substrate UDP-glucose (UDP-Glc). An overexpressed DNA segment of the cotton celA1 gene encodes a polypeptide fragment that spans these domains and binds UDP-Glc, while a similar fragment having one of these domains deleted does not. The plant celA genes show little homology at the N- and C-terminal regions and also contain two internal insertions of sequence, one conserved and one hypervariable, that are not found in the bacterial gene sequences. Cotton celA1 and celA2 genes are expressed at high levels during active secondary wall cellulose synthesis in developing cotton fibers. Genomic Southern blot analyses in cotton demonstrate that celA forms a small gene family.
Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists
Authors: McGall Glenn , Labadie Jeff , Brock Phil , Wallraff Greg , Nguyen Tiffany , Hinsberg William
Published: July 2002

High-density arrays of oligonucleotide probes are proving to be
powerful new tools for large-scale DNA and RNA sequence analysis. A
method for constructing these arrays, using light-directed DNA
synthesis with photoactivatable monomers, can currently achieve
densities on the order of 10
6
sequences/cm
2
.
One of the challenges facing this technology is to further increase the
volume, complexity, and density of sequence information encoded in
these arrays. Here we demonstrate a new approach for synthesizing DNA
probe arrays that combines standard solid-phase oligonucleotide
synthesis with polymeric photoresist films serving as the
photoimageable component. This opens the way to exploiting
high-resolution imaging materials and processes from the
microelectronics industry for the fabrication of DNA probe arrays with
substantially higher densities than are currently available.
Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex.
Authors: Tang H , Sun X , Reinberg D , Ebright R H
Published: July 2002

We have used alanine scanning to analyze protein-protein interactions by human TATA-element binding protein (TBP) within the transcription preinitiation complex. The results indicate that TBP interacts with RNA polymerase II and general transcription factors IIA, IIB, and IIF within the functional transcription preinitiation complex and define the determinants of TBP for each of these interactions. The results permit construction of a model for the structure of the preinitiation complex.
Cue recognition and cue elaboration in learning from examples.
Authors: Zhu X , Lee Y , Simon H A , Zhu D
Published: July 2002

This paper describes the processes used by students to learn from worked-out examples and by working through problems. Evidence is derived from protocols of students learning secondary school mathematics and physics. The students acquired knowledge from the examples in the form of productions (condition-->action): first discovering conditions under which the actions are appropriate and then elaborating the conditions to enhance efficiency. Students devoted most of their attention to the condition side of the productions. Subsequently, they generalized the productions for broader application and acquired specialized productions for special problem classes.
Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells.
Authors: Moffett J , Kratz E , Florkiewicz R , Stachowiak M K
Published: July 2002

Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density-dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density-dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low-density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp -650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells.
Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.
Authors: Moskovitz J , Jenkins N A , Gilbert D J , Copeland N G , Jursky F , Weissbach H , Brot N
Published: July 2002

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.
Homophilic adhesion mediated by the neural cell adhesion molecule involves multiple immunoglobulin domains.
Authors: Ranheim T S , Edelman G M , Cunningham B A
Published: July 2002

The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig I-V) and the two fibronectin type III repeats (FnIII 1-2) in the extracellular region of N-CAM are involved in this process have led to ambiguous results. We have generated soluble recombinant proteins corresponding to each of the individual immunoglobulin domains and the combined FnIII 1-2 and prepared polyclonal antibodies specific for each. The purified proteins and antibodies were used in aggregation experiments with fluorescent microspheres and chicken embryo brain cells to determine possible contributions of each domain to homophilic adhesion. The recombinant domains were tested for their ability to bind to purified native N-CAM, to bind to each other, and to inhibit the aggregation of N-CAM on microspheres and the aggregation of neuronal cells. Each of the immunoglobulin domains bound to N-CAM, and in solution all of the immunoglobulin domains inhibited the aggregation of N-CAM-coated microspheres. Soluble Ig II, Ig III, and Ig IV inhibited neuronal aggregation; antibodies against whole N-CAM, the Ig III domain, and the Ig I domain all strongly inhibited neuronal aggregation, as well as the aggregation of N-CAM-coated microspheres. Of all the domains, the third immunoglobulin domain alone demonstrated the ability to self-aggregate, whereas Ig I bound to Ig V and Ig II bound to Ig IV. The combined FnIII 1-2 exhibited a slight ability to self-aggregate but did not bind to any of the immunoglobulin-like domains. These results suggest that N-CAM-N-CAM binding involves all five immunoglobulin domains and prompt the hypothesis that in homophilic cell-cell binding mediated by N-CAM these domains may interact pairwise in an antiparallel orientation.
Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53.
Authors: Yap N , Yu C L , Cheng S Y
Published: July 2002

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these effects are unclear. One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects. In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53. p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction. We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain. As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner. In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1. In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements. These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells.
The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F
1
-ATPase
Authors: Hingorani Manju M. , Washington M. Todd , Moore Kristen C. , Patel Smita S.
Published: July 2002

Bacteriophage T7 DNA helicase is a ring-shaped hexamer that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. Of the six potential nucleotide binding sites on the hexamer, we have found that three are noncatalytic sites and three are catalytic sites. The noncatalytic sites bind nucleotides with a high affinity, but dTTPs bound to these sites do not dissociate or hydrolyze through many dTTPase turnovers at the catalytic sites. The catalytic sites show strong cooperativity which leads to sequential binding and hydrolysis of dTTP. The elucidated dTTPase mechanism of the catalytic sites of T7 helicase is remarkably similar to the binding change mechanism of the ATP synthase. Based on the similarity, a general mechanism for hexameric helicases is proposed. In this mechanism, an F
1
-ATPase-like rotational movement around the single-stranded DNA, which is bound through the central hole of the hexamer, is proposed to lead to unidirectional translocation along single-stranded DNA and duplex DNA unwinding.
Synergy between adjacent zinc fingers in sequence-specific DNA recognition
Authors: Isalan Mark , Choo Yen , Klug Aaron
Published: July 2002

Zif268-like zinc fingers are generally regarded as independent DNA-binding modules that each specify three base pairs in adjacent, but discrete, subsites. However, crystallographic evidence suggests that a contact also can occur from the second helical position of one finger to the subsite of the preceding finger. Here we show for the three-finger DNA-binding domain of the protein Zif268, and a panel of variants, that deleting the putative contact from finger 3 can affect the binding specificity for the 5′ base in the adjoining triplet, which forms part of the binding site of finger 2. This finding demonstrates that Zif268-like zinc fingers can specify overlapping 4-bp subsites, and that sequence specificity at the boundary between subsites arises from synergy between adjacent fingers. This has important implications for the design and selection of zinc fingers with novel DNA binding specificities.
Suppression of insulin-like growth factor type I receptor by a triple-helix strategy inhibits IGF-I transcription and tumorigenic potential of rat C6 glioblastoma cells
Authors: Rininsland Frauke , Johnson Thomas R. , Chernicky Cheryl L. , Schulze Ekkehard , Burfeind Peter , Ilan Judith , Ilan Joseph
Published: July 2002

Homopurine (AG) and homopyrimidine (CT) oligodeoxyribonucleotides predicted to form triple-helical (triplex) structures have been shown to specifically suppress gene expression when supplied to cultured cells. Here we present evidence that homopurine RNA (effector) sequences designed to form a triplex with a homopurine⋅homopyrimidine sequence 3′ to the termination codon of the insulin-like growth factor type I receptor (IGF-IR) structural gene can efficiently suppress IGF-IR gene transcription. Transfection vectors were constructed to drive transcription of either AG or CT variant triplex-forming strands. To increase the probability of obtaining stable transfectants with adequate expression of effector sequences, these were designed to be transcribed together with cDNA sequences conferring neomycin resistance as a fusion transcript. Rat C6 glioblastoma cells transfected with the AG variant showed dramatic reduction of IGF-IR transcripts compared with untransfected cells. The AG transfectants also exhibited marked down-regulation of the IGF-I, and an enhanced accumulation of serine protease inhibitor nexin-I mRNA. Similar changes in gene expression were observed following transfection of C6 cells with constructs transcribing antisense RNA to IGF-IR transcripts, but were not observed in C6 cells transfected with either the CT triplex variant or with vector lacking triplex-forming sequences. Moreover, C6 cells transfected with AG triplex variant displayed a dramatic inhibition of tumor growth when injected into nude mice. The results suggest that a triple-helix strategy can be used to inhibit transcription elongation of the IGF-IR gene, and emphasize the efficacy of triplex-mediated gene inhibition in an animal model.
The α-bungarotoxin binding site on the nicotinic acetylcholine receptor: Analysis using a phage–epitope library
Authors: Balass Moshe , Katchalski-Katzir Ephraim , Fuchs Sara
Published: July 2002

The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. α-Neurotoxins, in particular α-bungarotoxin (α-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with α-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to α-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the α-subunit of theTorpedomuscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind α-BTX. The library-derived peptide and the corresponding peptides containing residues 187–199 of theTorpedoAcChoR α-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human α7; ERFYECCKEPYPD) that binds α-BTX, inhibit the binding of α-BTX to the intactTorpedoAcChoR with IC50values of 10−6M. A synthetic peptide from a neuronal AcChoR that does not bind α-BTX (e.g., human α2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the α-BTX-binding protein (α7)—i.e., Lys in α2and Tyr in α7—does not inhibit the binding of α-BTX toTorpedoAcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to α-BTX.
Essential role of a kinesin-like protein in
Arabidopsis
trichome morphogenesis
Authors: Oppenheimer David G. , Pollock Mary A. , Vacik Joshua , Szymanski Daniel B. , Ericson Brad , Feldmann Ken , Marks M. David
Published: July 2002

Little is known about how cell shape is controlled. We are using the morphogenesis of trichomes (plant hairs) on the plant
Arabidopsis thaliana
as a model to study how cell shape is controlled. Wild-type
Arabidopsis
trichomes are large, single epidermal cells with a stalk and three or four branches, whereas in
zwichel
(
zwi
) mutants the trichomes have a shortened stalk and only two branches. To further understand the role of the
ZWI
gene in trichome morphogenesis we have cloned the wild-type
ZWICHEL
(
ZWI
) gene by T-DNA tagging, and report here that it encodes a member of the kinesin superfamily of microtubule motor proteins. Kinesin proteins transport diverse cellular materials in a directional manner along microtubules. Kinesin-like proteins are characterized by a highly conserved “head” region that comprises the motor domain, and a nonconserved “tail” region that is thought to participate in recognition and binding of the appropriate cargo.
Mast cell tumor necrosis factor α production is regulated by MEK kinases
Authors: Ishizuka Tamotsu , Terada Naohiro , Gerwins Pär , Hamelmann Eckard , Oshiba Akihiro , Fanger Gary R. , Johnson Gary L. , Gelfand Erwin W.
Published: July 2002

Mast cells synthesize and secrete specific cytokines and chemokines which play an important role in allergic inflammation. Aggregation of the high-affinity Fc receptor (FcɛRI) for immunoglobulin E (IgE) in MC/9 mouse mast cells stimulates the synthesis and secretion of tumor necrosis factor α (TNF-α). FcɛRI aggregation activates several sequential protein kinase pathways, leading to increased activity of extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and the p38 mitogen-activated protein (MAP) kinase. Inhibition of ERKs with the compound PD 098059 had little effect on FcɛRI-stimulated TNF-α production. Aggregation of FcɛRI stimulated MEK kinase 1 (MEKK1) activity, which activates JNK kinase (JNKK), the kinase that phosphorylates and activates JNKs. Expression of activated MEKK1 (ΔMEKK1) in MC/9 cells strongly stimulated JNK activity but only weakly stimulated p38 activity, and it induced a large activation of TNF-α promoter-regulated luciferase gene expression. Inhibitory mutant JNK2 expressed in MC/9 cells significantly blunted FcɛRI stimulation of TNF-α promoter-driven luciferase expression. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, diminished FcɛRI-mediated TNF-α synthesis, significantly blunted JNK activation and TNF-α promoter-driven luciferase expression, and only weakly inhibited p38 kinase activation. Inhibition of NFκB activation resulting from ΔMEKK1 expression or FcɛRI stimulation did not affect TNF-α promoter-driven luciferase expression. Our findings define a MEKK-regulated JNK pathway activated by FcɛRI that regulates TNF-α production in mast cells.
Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: Tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines
Authors: Nielsen Mette , Kaltoft Keld , Nordahl Mette , Röpke Carsten , Geisler Carsten , Mustelin Tomas , Dobson Pauline , Svejgaard Arne , Ødum Niels
Published: July 2002

Mycosis fungoides (MF) is a low-grade cutaneous T cell lymphoma of unknown etiology. In this report, the Jak/Stat (Janus kinase/signal transducer and activator of transcription) signaling pathway was investigated in tumor cell lines established from skin biopsy specimens from a patient with MF. Jaks link cytokine receptors to Stats, and abnormal Jak/Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3
sm
, was found to be constitutively activated, i.e., (
i
) Stat3
sm
was constitutively phosphorylated on tyrosine residues, and tyrosine phosphorylation was not enhanced by growth factor stimulation; (
ii
) band shift assays and immunoprecipitations of DNA/Stat complexes showed constitutive DNA-binding properties of Stat3
sm
; and (
iii
) Stat3
sm
was constitutively associated with Jak3. The abnormal activation of Stat3
sm
was highly specific. Thus, neither the fast migrating isoform of Stat3 (Stat3
fm
) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3
sm
and inhibited spontaneous as well as interleukin 2-induced growth of MF tumor cells. In conclusion, we have provided evidence for an abnormal Jak/Stat signaling and growth regulation in tumor cells obtained from affected skin of an MF patient.
The regulators of G protein signaling (RGS) domains of RGS4, RGS10, and GAIP retain GTPase activating protein activity
in vitro
Authors: Popov Serguei , Yu Kan , Kozasa Tohru , Wilkie Thomas M.
Published: July 2002

Regulators of G protein signaling (RGS) proteins accelerate GTP
hydrolysis by G
i
but not by G
s
class
α-subunits. All RGS proteins share a conserved 120-amino acid
sequence termed the RGS domain. We have demonstrated that the RGS
domains of RGS4, RGS10, and GAIP retain GTPase accelerating activity
with the G
i
class substrates G
iα1,
G
oα
, and G
zα
in vitro
. No
regulatory activity of the RGS domains was detected for
G
sα
. Short deletions within the RGS domain of RGS4
destroyed GTPase activating protein activity and G
iα1
substrate binding. Comparable protein–protein interactions between
G
iα1
–GDP–AlF
4
−
and the RGS domain or
full-length RGS4 were detected using surface plasmon resonance.
Mitosis-promoting factor-mediated suppression of a cloned delayed rectifier potassium channel expressed in
Xenopus
oocytes
Authors: Brüggemann Andrea , Stühmer Walter , Pardo Luis A.
Published: July 2002

The cell cycle is the crucial process that leads to mitosis in all cell types. The dramatic redirectioning of many cellular processes during the cycle is known to involve ion channels, either changing their level of expression or their voltage dependence, as in the case of inward rectifiers. Here we describe the specific inhibition of heterologously expressed ionic channels at the onset of maturation in
Xenopus
oocytes. In cells expressing rat
eag
(R-
eag
) potassium channels, maturation induces a dramatic reduction in the current amplitude, which is almost complete in most cases. The key molecule in oocyte maturation, the mitosis-promoting factor (a complex of cyclin B and p34

cdc2

), is able to induce similar changes when injected into the oocytes.
Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules
Authors: Nelson Christopher A. , Vidavsky Ilan , Viner Nicholas J. , Gross Michael L. , Unanue Emil R.
Published: July 2002

Major histocompatibility complex (MHC) class II molecules bind antigenic peptides for display to T lymphocytes. Although the enzymes involved remain to be identified, it is commonly believed that class II associated peptides are released from intact antigens through a series of proteolytic steps carried out inside antigen presenting cells. We have examined the effect of amino acid substitutions on proteolytic processing of the model antigen hen-egg lysozyme (HEL). Altered HEL molecules, engineered by site-directed mutagenesis of a HEL cDNA, were expressed as separate stable transfectants in a B cell lymphoma line. Each transfectant processed a different mutant HEL protein for presentation on MHC class II. We purified the resulting class II-associated peptides and analyzed them by mass spectrometry. Our results strongly support the hypothesis that antigen processing continues after peptide binding to the MHC class II molecule and are most consistent with a scenario in which long peptides first bind to MHC class II and are then trimmed by exopeptidase.
A homolog of the mammalian GTPase Rab2 is present in
Arabidopsis
and is expressed predominantly in pollen grains and seedlings
Authors: Moore Ian , Diefenthal Thomas , Zarsky Viktor , Schell Jeff , Palme Klaus
Published: July 2002

Vesicle traffic between the endoplasmic reticulum and the Golgi apparatus in mammals requires the small GTP-binding protein Rab2, but
Saccharomyces cerevisiae
appears not to have a Rab2 homolog. Here it is shown that the higher plant,
Arabidopsis thaliana
, contains a gene,
At-RAB2
, whose predicted product shares 79% identity with human Rab2 protein. Transgenic plants containing fusions between β-glucuronidase and sequences upstream of
At-RAB2
demonstrated histochemical staining predominantly in maturing pollen and rapidly growing organs of germinating seedlings. β-glucuronidase activity in pollen is first detectable at microspore mitosis and increases thereafter. In this respect, the promoter of
At-RAB2
behaves like those of class II pollen-specific genes, whose products are often required after germination for pollen tube growth. Seedling germination and pollen tube growth are notable for their unusually high rates of cell wall and membrane biosynthesis. These results are consistent with a role for
At-RAB2
in secretory activity.
Ca
2+
-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca
2+
mobilization
Authors: Patel Sandip , Morris Stephen A. , Adkins Charles E. , O’Beirne Gerry , Taylor Colin W.
Published: July 2002

The interactions between calmodulin, inositol 1,4,5-trisphosphate (Ins
P
3
), and pure cerebellar Ins
P
3
receptors were characterized by using a scintillation proximity assay. In the absence of Ca
2+
,
125
I-labeled calmodulin reversibly bound to multiple sites on Ins
P
3
receptors and Ca
2+
increased the binding by 190% ± 10%; the half-maximal effect occurred when the Ca
2+
concentration was 184 ± 14 nM. In the absence of Ca
2+
, calmodulin caused a reversible, concentration-dependent (IC
50
= 3.1 ± 0.2 μM) inhibition of [
3
H]Ins
P
3
binding by decreasing the affinity of the receptor for Ins
P
3
. This effect was similar at all Ca
2+
concentrations, indicating that the site through which calmodulin inhibits Ins
P
3
binding has similar affinities for calmodulin and Ca
2+
-calmodulin. Calmodulin (10 μM) inhibited the Ca
2+
release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of Ins
P
3
. Tonic inhibition of Ins
P
3
receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to Ins
P
3
and limit spontaneous activation of Ins
P
3
receptors in the dendritic spines. Inhibition of Ins
P
3
receptors by calmodulin at all cytosolic Ca
2+
concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca
2+
, suggests that calmodulin may also allow both feedback control of Ins
P
3
receptors and integration of inputs from other signaling pathways.