bluuebunny/crossref_metadata_2025 huggingface dataset:
The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.
Authors: Jiang H , Su Z Z , Lin J J , Goldstein N I , Young C S , Fisher P B
Published: July 2002

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.
Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.
Authors: Wieland K , Zuurmond H M , Krasel C , Ijzerman A P , Lohse M J
Published: July 2002

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.
Accurate reconstruction of a known HIV-1 transmission history by phylogenetic tree analysis.
Authors: Leitner T , Escanilla D , Franzén C , Uhlén M , Albert J
Published: July 2002

Phylogenetic analyses are increasingly used in attempts to clarify transmission patterns of human immunodeficiency virus type 1 (HIV-1), but there is a continuing discussion about their validity because convergent evolution and transmission of minor HIV variants may obscure epidemiological patterns. Here we have studied a unique HIV-1 transmission cluster consisting of nine infected individuals, for whom the time and direction of each virus transmission was exactly known. Most of the transmissions occurred between 1981 and 1983, and a total of 13 blood samples were obtained approximately 2-12 years later. The p17 gag and env V3 regions of the HIV-1 genome were directly sequenced from uncultured lymphocytes. A true phylogenetic tree was constructed based on the knowledge about when the transmissions had occurred and when the samples were obtained. This complex, known HIV-1 transmission history was compared with reconstructed molecular trees, which were calculated from the DNA sequences by several commonly used phylogenetic inference methods [Fitch-Margoliash, neighbor-joining, minimum-evolution, maximum-likelihood, maximum-parsimony, unweighted pair group method using arithmetic averages (UPGMA), and a Fitch-Margoliash method assuming a molecular clock (KITSCH)]. A majority of the reconstructed trees were good estimates of the true phylogeny; 12 of 13 taxa were correctly positioned in the most accurate trees. The choice of gene fragment was found to be more important than the choice of phylogenetic method and substitution model. However, methods that are sensitive to unequal rates of change performed more poorly (such as UPGMA and KITSCH, which assume a constant molecular clock). The rapidly evolving V3 fragment gave better reconstructions than p17, but a combined data set of both p17 and V3 performed best. The accuracy of the phylogenetic methods justifies their use in HIV-1 research and argues against convergent evolution and selective transmission of certain virus variants.
Selective response of ternary complex factor Sap1a to different mitogen-activated protein kinase subgroups.
Authors: Strahl T , Gille H , Shaw P E
Published: July 2002

Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear. Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions.
YY1 transcriptional initiator: Protein interactions and association with a DNA site containing unpaired strands
Authors: Usheva Anny , Shenk Thomas
Published: July 2002

The Ying–Yang 1 protein (YY1) DNA-binding site functions as an
initiator element at which YY1, transcription factor IIB (TFIIB), and
RNA polymerase II sponsor basal transcription from a supercoiled DNA
template. We show that TFIIB binds to YY1, stabilizing its interaction
with DNA, and YY1 contacts the large subunit of polymerase II,
directing it to the initiation site. YY1 directs initiation from linear
DNA containing mismatched sequences within its binding site, leading us
to infer that supercoiling facilitates the separation of DNA strands
and to suggest that YY1 likely remains bound to the start site as DNA
strands separate during initiation. These results provide a mechanistic
basis for transcriptional initiation directed by YY1 in the absence of
the TATA box-binding protein.
Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons.
Authors: Lambolez B , Ropert N , Perrais D , Rossier J , Hestrin S
Published: July 2002

In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-III neocortical neurons, we find that 90% of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% of the GluR1-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluR1 mRNA (GluR1/GluR1-4 = 59%), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance of the flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-type-specific manner, without changing permeation properties.
Host regulation of lysogenic decision in bacteriophage λ: Transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA)
Authors: Kihara Akio , Akiyama Yoshinori , Ito Koreaki
Published: July 2002

The
cII
gene product of bacteriophage λ is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of
Escherichia coli
are crucial for these commitments of infecting λ genome. One of them,
hflA
encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII
in vivo
and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene,
ftsH
(
hflB
) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation.
In vitro
, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over λ lysogenic decision.
Role of intrinsic synaptic circuitry in collicular sensorimotor integration
Authors: Lee Psyche H. , Helms Matthew C. , Augustine George J. , Hall William C.
Published: July 2002

The superficial gray layer of the superior colliculus contains a map that represents the visual field, whereas the underlying intermediate gray layer contains a vector map of the saccades that shift the direction of gaze. These two maps are aligned so that a particular region of the visual field is represented directly above the neurons that orient the highest acuity area of the retina toward that region. Although it has been proposed that the transmission of information from the visuosensory to the motor map plays an important role in the generation of visually guided saccades, experiments have failed to demonstrate any functional linkage between the two layers. We examined synaptic transmission between these layers
in vitro
by stimulating the superficial layer while using whole-cell patch-clamp methods to measure the responses of intermediate layer neurons. Stimulation of superficial layer neurons evoked excitatory postsynaptic currents in premotor cells. This synaptic input was columnar in organization, indicating that the connections between the layers link corresponding regions of the visuosensory and motor maps. Excitatory postsynaptic currents were large enough to evoke action potentials and often occurred in clusters similar in duration to the bursts of action potentials that premotor cells use to command saccades. Our results indicate the presence of functional connections between the superficial and intermediate layers and show that such connections could play a significant role in the generation of visually guided saccades.
The activation process of the α
1B
-adrenergic receptor: Potential role of protonation and hydrophobicity of a highly conserved aspartate
Authors: Scheer Alexander , Fanelli Francesca , Costa Tommaso , De Benedetti Pier G. , Cotecchia Susanna
Published: July 2002

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the α
1B
-adrenergic receptor (α
1B
-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the α
1B
-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.
Molecular characterization of Br-cadherin, a developmentally regulated, brain-specific cadherin
Authors: Selig Sara , Lidov Hart G. W. , Bruno Sandra A. , Segal Michael M. , Kunkel Louis M.
Published: July 2002

Cadherins are a family of transmembrane proteins that play a crucial role in cell adhesion and in morphogenesis. Several of the cadherins are expressed in the nervous system, but none is neuron-specific. We characterize a new member of the cadherin family, Br-cadherin, which is present exclusively in the central nervous system. Although the Br-cadherin protein is confined to the central nervous system, its mRNA is present in several additional tissues, suggesting that there is posttranscriptional control of this gene’s expression. Within the central nervous system, Br-cadherin appears to be expressed specifically by neurons. In the mouse, its expression becomes detectable during the first postnatal week, which corresponds temporally to the onset of synaptogenesis and dendrite outgrowth in the brain. This pattern of expression is consistent with a role for Br-cadherin in neuronal development, perhaps specifically with synaptogenesis.
Vitamin K-dependent carboxylation of the carboxylase
Authors: Berkner Kathleen L. , Pudota B. Nirmala
Published: July 2002

Vitamin K-dependent (VKD) proteins require modification by the VKD-γ-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated
in vitro
with
14
CO
2
and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of
in vitro
-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the
in vitro
reaction. Carboxylase carboxylation also was detected in baculovirus(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated
in vivo
. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation
in vitro
suggest a possible role for carboxylase carboxylation in factor IX turnover, and
in vivo
studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.
Conserved water molecules contribute to the extensive network of interactions at the active site of protein kinase A
Authors: Shaltiel Shmuel , Cox Sarah , Taylor Susan S.
Published: July 2002

Protein kinases constitute a large family of regulatory enzymes, each with a distinct specificity to restrict its action to its physiological target(s) only. The catalytic (C) subunit of protein kinase A, regarded as a structural prototype for this family, is composed of a conserved core flanked by two nonconserved segments at the amino and carboxyl termini. Here we summarize evidence to show that (
i
) the active site consists of an extended network of interactions that weave together both domains of the core as well as both segments that flank the core; (
ii
) the opening and closing of the active site cleft, including the dynamic and coordinated movement of the carboxyl terminal tail, contributes directly to substrate recognition and catalysis; and (
iii
) in addition to peptide and ATP, the active site contains six structured water molecules that constitute a conserved structural element of the active site. One of these active-site conserved water molecules is locked into place by its interactions with the nucleotide, the peptide substrate/inhibitor, the small and large domains of the conserved core, and Tyr-330 from the carboxyl-terminal “tail.”
Altered thalamic response to levodopa in Parkinson’s patients with dopa-induced dyskinesias
Authors: Hershey Tamara , Black Kevin J. , Stambuk Mikula K. , Carl Juanita L. , McGee-Minnich Lori A. , Perlmutter Joel S.
Published: July 2002

Parkinson’s disease (PD) is a progressive neurologic condition characterized by tremor, slowness, stiffness, and unstable posture. Degeneration of dopamine-producing neurons in the substantia nigra causes PD. Treatment with levodopa, a precursor of dopamine, initially ameliorates the clinical manifestations of PD. However, chronic levodopa treatment can produce severe involuntary movements (so-called dopa-induced dyskinesias or DID), limiting treatment. Pallidotomy, placement of a surgical lesion in the internal segment of the globus pallidus, reduces DID. Because this result is inconsistent with current theories of both basal ganglia function and DID, it prompted us to investigate the brain’s response to levodopa. We measured regional cerebral blood flow response to levodopa with positron-emission tomography in 6 PD patients with DID, 10 chronically treated PD patients without DID, 17 dopa-naïve PD patients, and 11 normals. The dose of levodopa was chosen to produce clinical benefit without inducing DID. This strategy allowed us to examine the brain response to levodopa across groups without the confounding effect of differences in motor behavior. We found that the DID group had a significantly greater response in ventrolateral thalamus than the other groups. This was associated with decreased activity in primary motor cortex. These findings are consistent with increased inhibitory output from the internal segment of the globus pallidus to thalamus after levodopa administration. They provide a physiological explanation for the clinical efficacy of pallidotomy and new insights into the physiology of the basal ganglia.
RNA helicase A is essential for normal gastrulation
Authors: Lee Chee-Gun , Soares Vera da Costa , Newberger Carol , Manova Katia , Lacy Elizabeth , Hurwitz Jerard
Published: July 2002

RNA helicase A (RHA) is the human homologue of the
Drosophila
maleless protein, an essential factor for the development of male flies. Recently, it was shown that RHA cooperates with the cAMP-responsive element in mediating the cAMP-dependent transcriptional activation of a number of genes. Due to the participation of cAMP as a second messenger in a number of signaling pathways, we examined the function of RHA during mammalian embryogenesis. To examine the role(s) of RHA in mammalian development, RHA knockout mice were generated by homologous recombination. Homozygosity for the mutant
RHA
allele led to early embryonic lethality. Histological analysis, combined with terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) reactions of
RHA
-null embryos, revealed marked apoptotic cell death specifically in embryonic ectodermal cells during gastrulation. RNA
in situ
analyses of the expression of
HNF-3β
and
Brachyury
, two molecular markers for gastrulation, showed that
RHA
-null embryos at days 7.5 and 8.5 expressed both
HNF-3β
and
Brachyury
in a pattern similar to those of pre- and early streak stages of embryos, respectively. These observations indicate that
RHA
is necessary for early embryonic development and suggest the requirement of RHA for the survival and differentiation of embryonic ectoderm.
Tumorigenic conversion of immortal human keratinocytes through stromal cell activation
Authors: Skobe Mihaela , Fusenig Norbert E.
Published: July 2002

The stromal microenvironment plays a crucial role in tumor development and progression. One of the most potent activators of stromal cells is the platelet-derived growth factor (PDGF). To investigate the role of PDGF in epithelial tumor development we stably transfected immortal nontumorigenic human keratinocytes with the PDGF-B cDNA. Transfected HaCaT cells overexpressed PDGF-B but remained negative for the PDGF receptors α and β (mRNA). Thus, they did not exhibit autocrine growth stimulation
in vitro,
but proliferation of cocultured fibroblasts was enhanced and this effect was inhibited by a neutralizing antibody to PDGF-BB. After subcutaneous injection into nude mice the transfected cells maintained high PDGF expression and formed progressively enlarging, rapidly proliferating cysts, classified as benign tumors. During early tumor development (up to 2 months), PDGF-B transfectants induced marked mesenchymal cell proliferation and angiogenesis, yet this effect vanished at later stages (2–6 months) concomitantly with increased epithelial cell proliferation and enhanced tumor growth. These results demonstrate that an activated stromal environment can promote tumorigenic conversion of nontumorigenic keratinocytes by inducing sustained epithelial hyperproliferation. This effect is apparently caused by a dual action of PDGF-BB: (
i
) PDGF-BB can promote tumor growth by inducing angiogenesis and stroma formation, and (
ii
) PDGF-activated stromal cells maintain elevated keratinocyte proliferation via a paracrine mechanism. Thus, PDGF, a major factor activated in wound healing, may play an important role as an endogenous promoter in epithelial tumor formation.
Structure of a complex between a cap analogue and mRNA guanylyl transferase demonstrates the structural chemistry of RNA capping
Authors: Håkansson Kjell , Wigley Dale B.
Published: July 2002

Paramecium bursaria Chlorella
virus PBCV-1 mRNA guanylyl transferase (capping enzyme) has been complexed with an mRNA cap analogue G[5′]ppp[5′]G and crystallized. The crystals belong to space group C222
1
with unit cell dimensions a = 78.4 Å, b = 164.1 Å, c = 103.3 Å, and diffraction data to 3.1 Å has been collected by using synchrotron radiation. The structure has been solved by molecular replacement by using each of the two domains in the previously determined structure of the enzyme in complex with GTP. The conformation is open with respect to the active site cleft, and all contacts between enzyme and ligand are mediated by domain 1. One of the guanine bases is bound in the same pocket that is utilized by GTP. The conformation of the ligand positions the β phosphate and the active site lysine on opposite sides of the α phosphate. This geometry is optimal for nucleophilic substitution reactions and has previously been found for GTP in the closed conformational form of the capping enzyme, where the lysine can be guanylylated upon treatment with excess manganese(II) ions. The remainder of the cap analogue runs along the conserved active site Lys
82
Thr
83
Asp
84
Gly
85
Ile
86
Arg
87
motif, and the second guanine, corresponding to the 5′ RNA base, is stacked against the hydrophobic Ile
86
. The ligand displays approximate 2-fold symmetry with intramolecular hydrogen bonding between the 2′ and 3′ hydroxyls of the two ribose rings.
Antibacterial agents that inhibit two-component signal transduction systems
Authors: Barrett J. F. , Goldschmidt R. M. , Lawrence L. E. , Foleno B. , Chen R. , Demers J. P. , Johnson S. , Kanojia R. , Fernandez J. , Bernstein J. , Licata L. , Donetz A. , Huang S. , Hlasta D. J. , Macielag M. J. , Ohemeng K. , Frechette R. , Frosco M. B. , Klaubert D. H. , Whiteley J. M. , Wang L. , Hoch J. A.
Published: July 2002

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA∷Spo0F two-component signal transduction system
in vitro
. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase∷response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.
A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins
Authors: Xu Yao , Piston David W. , Johnson Carl Hirschie
Published: July 2002

We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.
Retention of the Bub3 checkpoint protein on lagging chromosomes
Authors: Martinez-Exposito Maria J. , Kaplan Kenneth B. , Copeland Jay , Sorger Peter K.
Published: July 2002

Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment. When unattached kinetochores are present, anaphase is delayed and the time available for chromosome-microtubule capture increases. Genes required for this delay first were identified in budding yeast (the
MAD
and
BUB
genes), but it is not yet known how the checkpoint senses unattached chromosomes or how it signals cell-cycle arrest. We report the isolation and analysis of a murine homologue of
BUB3
, a gene whose deletion abolishes mitotic checkpoint function in
Saccharomyces cerevisiae
. mBub3 belongs to a small gene family that has been highly conserved through evolution. By expressing recombinant proteins in insect cells, we show that mBub3, like yeast Bub3p, binds to Bub1 to form a complex with protein kinase activity. During prophase and prometaphase, preceding kinetochore-microtubule attachment, Bub3 localizes to kinetochores. High levels of mBub3 remain associated with lagging chromosomes but not with correctly aligned chromosomes during metaphase, consistent with a role for Bub3 in sensing microtubule attachment. Intriguingly, the number of lagging chromosomes with high Bub3 staining increases dramatically in cells treated with low (and pharmacologically relevant) concentrations of the chemotherapeutic taxol and the microtubule poison nocodazole.
Morphological innovation and developmental genetics
Authors: Marshall Charles R. , Orr H. Allen , Patel Nipam H.
Published: July 2002

How do the actions of individual genes contribute to the complex
morphologies of animals and plants? How widespread are these genes
taxonomically? How many genes are involved in the morphological
differences observed between species, and can we identify them? To what
extent can empirical data and theory be reconciled? We provide an
overview of some recent attempts to answer these questions, answers
that have taken us to the threshold of understanding the mechanistic
basis and evolutionary factors that underlie morphological innovation.
Morphological analysis of the mammalian postcranium: A developmental perspective
Authors: Lovejoy C. Owen , Cohn Martin J. , White Tim D.
Published: July 2002

The past two decades have greatly improved our knowledge of vertebrate skeletal morphogenesis. It is now clear that bony morphology lacks individual descriptive specification and instead results from an interplay between positional information assigned during early limb bud deployment and its “execution” by highly conserved cellular response programs of derived connective tissue cells (e.g., chondroblasts and osteoblasts). Selection must therefore act on positional information and its apportionment, rather than on more individuated aspects of presumptive adult morphology. We suggest a trait classification system that can help integrate these findings in both functional and phylogenetic examinations of fossil mammals and provide examples from the human fossil record.
Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains
Authors: Palma Eleonora , Fucile Sergio , Barabino Benedetta , Miledi Ricardo , Eusebi Fabrizio
Published: July 2002

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on
Xenopus
oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα
7
receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors.
Mapping regions containing binding residues within functional domains of
Plasmodium vivax
and
Plasmodium knowlesi
erythrocyte-binding proteins
Authors: Ranjan Akash , Chitnis Chetan E.
Published: July 2002

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas
Plasmodium vivax
and
Plasmodium knowlesi
use the Duffy blood group antigen,
Plasmodium falciparum
uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes.
P. knowlesi
uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the
P. vivax
and
P. knowlesi
Duffy-binding proteins,
P. knowlesi
β and γ proteins, which bind alternate receptors on rhesus erythrocytes, and
P. falciparum
erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within
P. vivax
and
P. knowlesi
β region II. Chimeric domains containing
P. vivax
region II sequences fused to
P. knowlesi
β region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of
P. vivax
region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of
P. knowlesi
β region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.
The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B
Authors: Bognár László Kozma , Hall Anthony , Ádám Éva , Thain Simon C. , Nagy Ferenc , Millar Andrew J.
Published: July 2002

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the
Arabidopsis
circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of
PHYB
∷
LUC
reporter genes containing the promoter and 5′ untranslated region of the tobacco
NtPHYB1
or
Arabidopsis
AtPHYB
genes fused to the luciferase (
LUC
) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of
PHYB
RNA retains this circadian regulation and use a PHYB∷Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.
Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-
O
-glycosylation codes
Authors: Shpak Elena , Leykam Joseph F. , Kieliszewski Marcia J.
Published: July 2002

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively
O
-glycosylated through the Hyp residues. The patterns of Hyp-
O
-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide
O
-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an
O
-glycosylation code of plants.
Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development
Authors: Kim James I. , Li Tiansen , Ho I.-Cheng , Grusby Michael J. , Glimcher Laurie H.
Published: July 2002

The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens.
A conserved amino acid sequence directing intracellular type III secretion by
Salmonella typhimurium
Authors: Miao Edward A. , Miller Samuel I.
Published: July 2002

Type III secretion systems (TTSS) are important virulence factors that Gram-negative bacteria use to translocate proteins into the cytoplasm of eukaryotic host cells. Salmonellae encode two virulence-associated TTSS. The
Salmonella
pathogenicity island 1 (SPI1)-encoded TTSS is active on contact with host cells, whereas the
Salmonella
pathogenicity island 2 (SPI2)-encoded TTSS is expressed after phagocytosis of bacteria by host cells. Previously, no consensus signal sequence for translocation has been identified among TTSS effector proteins. In this work, seven proteins, termed
Salmonella
-translocated effectors (STE), are described that contain conserved amino acid sequences that direct translocation by TTSS in
Salmonella typhimurium
. STE that are coordinately regulated with SPI2 gene expression contain translocation signals that are recognized by the SPI2 but not by the SPI1 TTSS. STE that are constitutively expressed contain signals that direct translocation through both SPI1 and SPI2 TTSS. Of the seven STE examined, SspH1 and SspH2 have been previously shown to be translocated and involved in virulence; SlrP and SifA were identified as virulence factors, but were not previously known to be associated with TTSS; and SseI, SseJ, and SifB were previously unidentified. Three STE genes (
sspH1
,
sspH2
, and
sseI
) are located within temperate bacteriophages, suggesting a common mechanism for the dissemination of more recently evolved STE.
Nucleic acid-triggered catalytic drug release
Authors: Ma Zhaochun , Taylor John-Stephen
Published: July 2002

We propose a concept for the rational design and synthesis of
highly selective chemotherapeutic agents that makes direct use of
genetic information about the disease state. The key idea is to use the
mRNA or DNA specific to the disease state to trigger the catalytic
release of a cytotoxic drug by promoting the association of a prodrug
with a catalyst capable of releasing the drug. We demonstrate the
feasibility of such an approach
in vitro
with a model
system that is based on the hydrolysis of
p
-nitrophenyl
esters by imidazole. In our model system, the catalytic component
consists of an imidazole group linked to the 5′ end of a 15-mer that is
complementary to the 5′ end of the triggering oligodeoxynucleotide. The
corresponding prodrug component consists of a
p
-nitrophenol ester linked to the 3′ end of an 8-mer
oligodeoxynucleotide that is complementary to the 3′ end of the
triggering sequence. We show that this system efficiently releases
p
-nitrophenol in the presence of all three components
and that the reaction is catalytic and undergoes multiple turnovers. We
also show that the complex between the catalytic component and the
triggering oligodeoxynucleotide behaves like an enzyme and follows
Michaelis–Menten kinetics, with a
K
M
of 22
μM and a
k
cat
of 0.018 min
−1
.
Most importantly, we show that catalytic release of
p
-nitrophenol is sensitive to the presence of a single
base-pair mismatch.
PCGEM1, a prostate-specific gene, is overexpressed in prostate cancer
Authors: Srikantan Vasantha , Zou Zhiqiang , Petrovics Gyorgy , Xu Linda , Augustus Meena , Davis Leland , Livezey Jeffrey R. , Connell Theresa , Sesterhenn Isabell A. , Yoshino Kiyoshi , Buzard Gregory S. , Mostofi F. K. , McLeod David G. , Moul Judd W. , Srivastava Shiv
Published: July 2002

A prostate-specific gene,PCGEM1, was identified by differential display analysis of paired normal and prostate cancer tissues. Multiple tissue Northern blot analysis revealed thatPCGEM1was expressed exclusively in human prostate tissue. Analysis ofPCGEM1expression in matched normal and primary tumor specimens revealed tumor-associated overexpression in 84% of patients with prostate cancer byin situhybridization assay and in 56% of patients by reverse transcription–PCR assay. Among various prostate cancer cell lines analyzed,PCGEM1expression was detected only in the androgen receptor-positive cell line LNCaP. Extensive DNA sequence analysis of thePCGEM1cDNA and genomic DNA revealed thatPCGEM1lacks protein-coding capacity and suggests that it may belong to an emerging class of noncoding RNAs, also called “riboregulators.” ThePCGEM1locus was mapped to chromosome 2q32. Taken together, the remarkable prostate-tissue specificity and androgen-dependent expression ofPCGEM1as well as its elevated expression in a significant percentage of tumor tissues suggest specific functions ofPCGEM1in the biology and tumorigenesis of the prostate gland.
Impaired hippocampal-dependent learning and functional abnormalities in the hippocampus in mice lacking serotonin
1A
receptors
Authors: Sarnyai Zoltán , Sibille Etienne L. , Pavlides Constantine , Fenster Robert J. , McEwen Bruce S. , Tóth Miklós
Published: July 2002

The hippocampus is a major limbic target of the brainstem
serotonergic neurons that modulate fear, anxiety, and learning through
postsynaptic serotonin
1A
receptors (5-HT
1A
receptors). Because chronic stress selectively down-regulates the
5-HT
1A
receptors in the hippocampus, we hypothesized that
mice lacking these receptors may exhibit abnormalities reminiscent of
symptoms of stress-related psychiatric disorders. In particular, a
hippocampal deficit in the 5-HT
1A
receptor could contribute
to the cognitive abnormalities often seen in these disorders. To test
whether a deficit in 5-HT
1A
receptors impairs
hippocampus-related functions, we studied hippocampal-dependent
learning and memory, synaptic plasticity in the hippocampus, and limbic
neuronal excitability in 5-HT
1A
-knockout (KO) mice.
5-HT
1A
-KO animals showed a deficit in hippocampal-dependent
learning and memory tests, such as the hidden platform (spatial)
version of the Morris water maze and the delayed version of the Y maze.
The performance of KO mice was not impaired in nonhippocampal memory
tasks such as the visible platform (nonspatial) version of the Morris
water maze, the immediate version of the Y maze, and the
spontaneous-alternation test of working memory. Furthermore,
paired-pulse facilitation in the dentate gyrus of the hippocampus was
impaired in 5-HT
1A
-KO mice. Finally, 5-HT
1A
-KO
mice, as compared with wild-type animals, displayed higher limbic
excitability manifested as lower seizure threshold and higher lethality
in response to kainic acid administration. These results demonstrate
that 5-HT
1A
receptors are required for maintaining normal
hippocampal functions and implicate a role for the 5-HT
1A
receptor in hippocampal-related symptoms, such as cognitive
disturbances, in stress-related disorders.
Expression profiling reveals fundamental biological differences in acute myeloid leukemia with isolated trisomy 8 and normal cytogenetics
Authors: Virtaneva Kimmo , Wright Fred A. , Tanner Stephan M. , Yuan Bo , Lemon William J. , Caligiuri Michael A. , Bloomfield Clara D. , de la Chapelle Albert , Krahe Ralf
Published: July 2002

Acute myeloid leukemia (AML) is a heterogeneous group of diseases. Normal cytogenetics (CN) constitutes the single largest group, while trisomy 8 (+8) as a sole abnormality is the most frequent trisomy. How trisomy contributes to tumorigenesis is unknown. We used oligonucleotide-based DNA microarrays to study global gene expression in AML+8 patients with +8 as the sole chromosomal abnormality and AML-CN patients. CD34+cells purified from normal bone marrow (BM) were also analyzed as a representative heterogeneous population of stem and progenitor cells. Expression patterns of AML patients were clearly distinct from those of CD34+cells of normal individuals. We show that AML+8 blasts overexpress genes on chromosome 8, estimated at 32% on average, suggesting gene-dosage effects underlying AML+8. Systematic analysis by cellular function indicated up-regulation of genes involved in cell adhesion in both groups of AML compared with CD34+blasts from normal individuals. Perhaps most interestingly, apoptosis-regulating genes were significantly down-regulated in AML+8 compared with AML-CN. We conclude that the clinical and cytogenetic heterogeneity of AML is due to fundamental biological differences.
Psychological Ethics Codes: A Comparison of Twenty‐four Countries
Authors: Leach Mark M. , Harbin J. Judd
Published: July 2002

Codes of ethics from 19 countries, representing a total of 24 countries, were compared to the United States using the American Psychological Association's (APA) Ethical Principles of Psychologists and Code of Conduct. Canada's code of ethics was most similar to the US/APA code and China's the most dissimilar. Ten individual standards may be approaching universal agreement, while eight others were unique to the APA ethical code. The relationship of ethical codes to cultural values, and future research suggestions, are discussed.