bluuebunny/crossref_metadata_2025 huggingface dataset:
The insect neuropeptide prothoracicotropic hormone is released with a daily rhythm: re-evaluation of its role in development.
Authors: Vafopoulou X , Steel C G
Published: July 2002

Prothoracicotropic hormone (PTTH) is the central cerebral neurohormone in insect development. Its release has been believed for decades to be confined to one (or two) critical moments early in each developmental stage at which time it triggers prolonged activation of the prothoracic glands to synthesize and release the steroid molting hormones (ecdysteroids), which elicit developmental responses in target tissues. We used an in vitro assay for PTTH released from excised brains of the bug Rhodnius prolixus and report that release of PTTH does occur at the expected time on day 6, but that this release is merely the first in a daily rhythm of release that continues throughout most of the 21 days of larval-adult development. This finding, together with reports of circadian control of ecdysteroid synthesis and titer throughout this time, raises significant challenges to several features of the current understanding of the hormonal control of insect development. New questions are raised concerning the function(s) of PTTH, its relationship with the prothoracic glands, and the significance of circadian rhythmicity throughout this endocrine axis. The significance of the reported observations derives from the set of entirely new questions they raise concerning the regulation of insect development.
Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells.
Authors: Ollert M W , David K , Schmitt C , Hauenschild A , Bredehorst R , Erttmann R , Vogel C W
Published: July 2002

Neuroblastoma (NB) is characterized by the second highest spontaneous regression of any human malignant disorder, a phenomenon that remains to be elucidated. In this study, a survey of 94 normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines in approximately one-third of the tested sera of both genders. Specific cell killing by these sera was in the range of 40% to 95%. Serum cytotoxicity was dependent on an intact classical pathway of complement. By several lines of evidence, IgM antibodies were identified as the cytotoxic factor in the sera. Further analyses revealed that a 260-kDa protein was recognized by natural IgM of cytotoxic sera in Western blots of NB cell extracts. The antigen was expressed on the surface of seven human NB cell lines but not on human melanoma or other control tumor cell lines derived from kidney, pancreas, colon, bone, skeletal muscle, lymphatic system, and bone marrow. Furthermore, no reactivity was observed with normal human fibroblasts, melanocytes, and epidermal keratinocytes. The antigen was expressed in vivo as detected by immunohistochemistry in both the tumor of a NB patient and NB tumors established in nude rats from human NB cell lines. Most interestingly, the IgM anti-NB antibody was absent from the sera of 11 human NB patients with active disease. The anti-NB IgM also could not be detected in tumor tissue obtained from a NB patient. Collectively, our data suggest the existence of a natural humoral immunological tumor defense mechanism, which could account for the in vivo phenomenon of spontaneous NB tumor regression.
CCAAT/enhancer binding protein ɛ is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing
Authors: Yamanaka Ryuya , Kim Gun-Do , Radomska Hanna S. , Lekstrom-Himes Julie , Smith Laura T. , Antonson Per , Tenen Daniel G. , Xanthopoulos Kleanthis G.
Published: July 2002

CCAAT/enhancer binding protein (C/EBP) ɛ is a recently cloned member of the C/EBP family of transcription factors and is expressed exclusively in cells of hematopoietic origin. The human C/EBPɛ gene is transcribed by two alternative promoters, Pα and Pβ. A combination of differential splicing and alternative use of promoters generates four mRNA isoforms, of 2.6 kb and 1.3–1.5 kb in size. These transcripts can encode three proteins of calculated molecular mass 32.2 kDa, 27.8 kDa, and 14.3 kDa. Accordingly, Western blots with antibodies specific for the DNA-binding domain, that is common to all forms, identify multiple proteins. C/EBPɛ mRNA was greatly induced during
in vitro
granulocytic differentiation of human primary CD34
+
cells. Retinoic acid treatment of HL60 promyelocytic leukemia cells for 24 hr induced C/EBPɛ mRNA levels by 4-fold, while prolonged treatment gradually reduced mRNA expression to pretreatment levels. Transient transfection experiments with expression vectors for two of the isoforms demonstrated that the 32.2-kDa protein is an activator of transcription of granulocyte colony-stimulating factor receptor promoter, while the 14.3-kDa protein is not. Thus, C/EBPɛ is regulated in a complex fashion and may play a role in the regulation of genes involved in myeloid differentiation.
Reported emissions of organic gases are not consistent with observations
Authors: Henry Ronald C. , Spiegelman Clifford H. , Collins John F. , Park EunSug
Published: July 2002

Regulatory agencies and photochemical models of ozone rely on self-reported industrial emission rates of organic gases. Incorrect self-reported emissions can severely impact on air quality models and regulatory decisions. We compared self-reported emissions of organic gases in Houston, Texas, to measurements at a receptor site near the Houston ship channel, a major petrochemical complex. We analyzed hourly observations of total nonmethane organic carbon and 54 hydrocarbon compounds from C-2 to C-9 for the period June through November, 1993. We were able to demonstrate severe inconsistencies between reported emissions and major sources as derived from the data using a multivariate receptor model. The composition and the location of the sources as deduced from the data are not consistent with the reported industrial emissions. On the other hand, our observationally based methods did correctly identify the location and composition of a relatively small nearby chemical plant. This paper provides strong empirical evidence that regulatory agencies and photochemical models are making predictions based on inaccurate industrial emissions.
The load dependence of kinesin’s mechanical cycle
Authors: Coppin Chris M. , Pierce Daniel W. , Hsu Long , Vale Ronald D.
Published: July 2002

Kinesin is a dimeric motor protein that transports organelles in a stepwise manner toward the plus-end of microtubules by converting the energy of ATP hydrolysis into mechanical work. External forces can influence the behavior of kinesin, and force-velocity curves have shown that the motor will slow down and eventually stall under opposing loads of ≈5 pN. Using an
in vitro
motility assay in conjunction with a high-resolution optical trapping microscope, we have examined the behavior of individual kinesin molecules under two previously unexplored loading regimes: super-stall loads (>5 pN) and forward (plus-end directed) loads. Whereas some theories of kinesin function predict a reversal of directionality under high loads, we found that kinesin does not walk backwards under loads of up to 13 pN, probably because of an irreversible transition in the mechanical cycle. We also found that this cycle can be significantly accelerated by forward loads under a wide range of ATP concentrations. Finally, we noted an increase in kinesin’s rate of dissociation from the microtubule with increasing load, which is consistent with a load dependent partitioning between two recently described kinetic pathways: a coordinated-head pathway (which leads to stepping) and an independent-head pathway (which is static).
Substrate-induced conformational change in a trimeric ornithine transcarbamoylase
Authors: Ha Ya , McCann Mark T. , Tuchman Mendel , Allewell Norma M.
Published: July 2002

The crystal structure of
Escherichia coli
ornithine transcarbamoylase (OTCase, EC
2.1.3.3
) complexed with the bisubstrate analog
N
-(phosphonacetyl)-
l
-ornithine (PALO) has been determined at 2.8-Å resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein–protein interactions, and conformational change. The structure was solved by molecular replacement with the
Pseudomonas aeruginosa
catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995)
Proc. Natl. Acad. Sci. USA
92, 10762–10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic
R
value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an
l
-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated
P. aeruginosa
catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in
E. coli
aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.
Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos
Authors: Schrank Bertold , Götz Rudolf , Gunnersen Jennifer M. , Ure Janice M. , Toyka Klaus V. , Smith Austin G. , Sendtner Michael
Published: July 2002

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (
SMN
) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the
SMN
gene product
in vivo
we generated
SMN
-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one
SMN
gene. Mice with homozygous
SMN
disruption display massive cell death during early embryonic development, indicating that the
SMN
gene product is necessary for cellular survival and function.
A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes
Authors: Kelly Edward J. , Sandgren Eric P. , Brinster Ralph L. , Palmiter Richard D.
Published: July 2002

Synthesis of mouse metallothionein (MT)-I and MT-II is transcriptionally induced by the synthetic glucocorticoid, dexamethasone (DEX) or both
in vivo
as well as in numerous cell lines. However, the location(s) of a glucocorticoid response element (GRE) has not been described. The observation that a marked
MT-I
gene, as well as heterologous genes, when placed in the context of 17 kb of flanking sequence from the MT locus, are inducible by DEX and lipopolysaccharide in transgenic mice renewed the search for the GRE. Analysis of a series of deletion constructs from this 17-kb region in cultured cells identified a single 455-bp region that conferred DEX induction on a reporter gene. This 455-bp region contains two GREs that bind to the glucocorticoid receptor as assessed by gel mobility shift. Deletion of this fragment from the 17-kb flanking region eliminates the DEX responsiveness of reporter genes. The two GREs, which are located ≈1 kb upstream of the
MT-II
gene and ≈7 kb upstream of the
MT-I
gene, are necessary for induction of both genes and can function independently of elements within the proximal promoter region of either gene.
Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome
Authors: Hughes Eric A. , Hammond Craig , Cresswell Peter
Published: July 2002

N
-acetyl-
l
-leucyl-
l
-leucyl-
l
-norleucinal (LLnL), which reversibly inhibits the proteasome in addition to other proteases, and a more specific irreversible inhibitor of the proteasome, lactacystin, were found to cause the accumulation of major histocompatibility complex (MHC) class I heavy chains in the cytosol of the β
2
-microglobulin-deficient cell line Daudi and the TAP-deficient cell line .174. These cell lines, which are severely impaired in their ability to fold MHC class I heavy chain, showed an accumulation of soluble class I heavy chains at different rates over a period of hours in the presence of LLnL. The accumulation of soluble class I heavy chains in the presence of either LLnL or lactacystin was easily revealed in Daudi and .174 but almost undetectable in a Daudi transfectant expressing β
2
-microglobulin and in 45.1, the wild-type parent of .174. The soluble class I heavy chain was also found to be devoid of its N-linked glycan and to be located in the cytosol. When the gene for ICP47, a herpes simplex virus protein that blocks the translocation of peptides into the endoplasmic reticulum, was transfected into 45.1, a similar accumulation of soluble MHC class I heavy chain was detectable. These data suggest that in cells where the MHC class I molecule is unable to assemble properly, the misfolded heavy chain is removed from the endoplasmic reticulum to the cytosol, deglycosylated, and degraded by the proteasome.
Avian vocalizations and phylogenetic signal
Authors: McCracken Kevin G. , Sheldon Frederick H.
Published: July 2002

The difficulty of separating genetic and ecological components of vocalizations has discouraged biologists from using vocal characters to reconstruct phylogenetic and ecological history. By considering the physics of vocalizations in terms of habitat structure, we predict which of five vocal characters of herons are most likely to be influenced by ecology and which by phylogeny, and test this prediction against a molecular-based phylogeny. The characters most subject to ecological convergence, and thus of least phylogenetic value, are first peak-energy frequency and frequency range, because sound penetration through vegetation depends largely on frequency. The most phylogenetically informative characters are number of syllables, syllable structure, and fundamental frequency, because these are more reflective of behavior and syringeal structure. Continued study of the physical principles that distinguish between potentially informative and convergent vocal characters and general patterns of homology in such characters should lead to wider use of vocalizations in the study of evolutionary history.
A cell surface receptor defined by a mAb mediates a unique type of cell death similar to oncosis
Authors: Zhang Chonghui , Xu Yuhui , Gu Jijie , Schlossman Stuart F.
Published: July 2002

Cell death is mediated by distinct pathways including apoptosis and oncosis in response to various death signals. To characterize molecules involved in cell death, a panel of mAbs was raised by immunizing mice with apoptotic cells. One of these antibodies, designated anti-Porimin (forpro-oncosisreceptorinducingmembraneinjury), was found to directly induce a unique type of cell death in Jurkat cells. Anti-Porimin defines a 110-kDa cell surface receptor on Jurkat cells. Functionally, anti-Porimin alone rapidly mediates pore formation on the plasma membrane and induces cell death without participation of complement. Both the cellular expression and functional characteristics of the Porimin antigen indicate that it is distinct from the CD95 (Fas/Apo-1) and other cell receptors known to induce apoptosis. Anti-Porimin-mediated cell death was preceded by cell aggregation, formation of plasma membrane pores, and the appearance of membrane blebs. More important, these cells show neither DNA fragmentation nor apoptotic bodies, but display lethal damage of the cell membrane. Cell death by anti-Porimin is distinct from complement-dependent cytolysis or complement-independent apoptosis but is similar to that described for oncosis, a form of cell death accompanied by the membrane damage followed by karyolysis. The induction of cell death by anti-Porimin may represent a unique cell surface receptor-mediated pathway of cell death in the human lymphoid system.
Localization of myosin-V in the centrosome
Authors: Espreafico Enilza M. , Coling Donald E. , Tsakraklides Vasiliki , Krogh Karin , Wolenski Joseph S. , Kalinec Gilda , Kachar Bechara
Published: July 2002

The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green fluorescent protein tag, but not in cells transfected with the vector encoding green fluorescent protein by itself.
A protein tyrosine phosphatase-like protein from baculovirus has RNA 5′-triphosphatase and diphosphatase activities
Authors: Takagi Toshimitsu , Taylor Gregory S. , Kusakabe Takahiro , Charbonneau Harry , Buratowski Stephen
Published: July 2002

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the
Caenorhabditis elegans
mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the
Autographa californica
nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5′-phosphatase. BVP sequentially removes γ and β phosphates from the 5′ end of triphosphate-terminated RNA, leaving a 5′-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA.
Transformation of hematopoietic cells by the Ski oncoprotein involves repression of retinoic acid receptor signaling
Authors: Dahl Richard , Kieslinger Matthias , Beug Hartmut , Hayman Michael J.
Published: July 2002

The Ski oncogene has dramatic effects on the differentiation of several different cell types. It induces the differentiation of quail embryo cells into myoblasts and arrests the differentiation of chicken hematopoietic cells. The mechanism that Ski uses to carry out these disparate biological activities is unknown. However, we were struck by the similarity of these effects to those of certain members of the nuclear hormone receptor family. Both Ski and the thyroid hormone receptor-derived oncogene v-ErbA can arrest the differentiation of avian erythroblasts, and v-Ski-transformed avian multipotent progenitor cells resemble murine hematopoietic cells that express a dominant-negative form of the retinoic acid receptor, RARα. In this paper, we have tested the hypothesis that v-Ski and its cellular homologue c-Ski exert their effects by interfering with nuclear hormone receptor-induced transcription. We demonstrate that Ski associates with the RAR complex and can repress transcription from a retinoic acid response element. The physiological significance of this finding is demonstrated by the ability of high concentrations of a RARα-specific ligand to abolish v-Ski-induced transformation of the multipotent progenitors. These results strongly suggest that the ability of Ski to alter cell differentiation is caused in part by the modulation of RAR signaling pathways.
Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins
Authors: Mead Paul E. , Zhou Yi , Lustig Kevin D. , Huber Tara L. , Kirschner Marc W. , Zon Leonard I.
Published: July 2002

We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in
Xenopus
. The Mix family of proteins are expressed during late blastula and gastrula stages of
Xenopus
development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation.
Cardiac reperfusion injury: Aging, lipid peroxidation, and mitochondrial dysfunction
Authors: Lucas David T. , Szweda Luke I.
Published: July 2002

Cardiac reperfusion and aging are associated with increased rates of mitochondrial free radical production. Mitochondria are therefore a likely site of reperfusion-induced oxidative damage, the severity of which may increase with age. 4-Hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, increases in concentration upon reperfusion of ischemic cardiac tissue, can react with and inactivate enzymes, and inhibits mitochondrial respiration
in vitro
. HNE modification of mitochondrial protein(s) might, therefore, be expected to occur during reperfusion and result in loss in mitochondrial function. In addition, this process may be more prevalent in aged animals. To begin to test this hypothesis, hearts from 8- and 24-month-old rats were perfused in Langendorff fashion and subjected to periods of ischemia and/or reperfusion. The rate of state 3 respiration of mitochondria isolated from hearts exposed to ischemia (25 min) was approximately 25% less than that of controls, independent of age. Reperfusion (40 min) caused a further decline in the rate of state 3 respiration in hearts isolated from 24- but not 8-month-old rats. Furthermore, HNE modification of mitochondrial protein (∼30 and 44 kDa) occurred only during reperfusion of hearts from 24-month-old rats. Thus, HNE-modified protein was present in only those mitochondria exhibiting reperfusion-induced declines in function. These studies therefore identify mitochondria as a subcellular target of reperfusion damage and a site of age-related increases in susceptibility to injury.
Negative correlation between male allocation and rate of self-fertilization in a hermaphroditic animal
Authors: Johnston Mark O. , Das Bijon , Hoeh Walter R.
Published: July 2002

Sex-allocation theory predicts that the evolution of increased rates of self-fertilization should be accompanied by decreased allocation to male reproduction (sperm production and broadcast). This prediction has found support in plants but has not previously been tested in animals, which, in contrast to biotically pollinated plants, are free of complications associated with incorporating the costs of attractive structures such as petals. Here we report rates of self-fertilization as well as proportional allocation to male reproductive tissues within populations of the simultaneous hermaphrodite
Utterbackia imbecillis
, a freshwater mussel. Individuals from populations with higher selfing rates devoted a lower proportion of reproductive tissue to sperm production (correlation = −0.99), in support of theory.
Comparison of the 5′ nuclease activities of
Taq
DNA polymerase and its isolated nuclease domain
Authors: Lyamichev Victor , Brow Mary Ann D. , Varvel Virgil E. , Dahlberg James E.
Published: July 2002

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5′ nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of
Thermus aquaticus
(TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5′ arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5′ nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3′ arm of the bifurcation; optimal stimulation requires overlap of the 3′ terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn
2+
ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.
JC virus DNA is present in the mucosa of the human colon and in colorectal cancers
Authors: Laghi Luigi , Randolph Ann E. , Chauhan D. P. , Marra Giancarlo , Major Eugene O. , Neel James V. , Boland C. Richard
Published: July 2002

JC virus (JCV) is a polyoma virus that commonly infects humans. We have found T antigen DNA sequences of JCV in the mucosa of normal human colons, colorectal cancers, colorectal cancer xenografts raised in nude mice, and in the human colon cancer cell line SW480. A larger number of viral copies is present in cancer cells than in non-neoplastic colon cells, and sequence microheterogeneity occurs within individual colonic mucosal specimens. The improved yield of detection after treatment with topoisomerase I suggests that the viral DNA is negatively supercoiled in the human tissues. These results indicate that JCV DNA can be found in colonic tissues, which raises the possibility that this virus may play a role in the chromosomal instability observed in colorectal carcinogenesis.
An important base triple anchors the substrate helix recognition surface within the
Tetrahymena
ribozyme active site
Authors: Szewczak Alexander A. , Ortoleva-Donnelly Lori , Zivarts Maris V. , Oyelere Adegboyega K. , Kazantsev Alexei V. , Strobel Scott A.
Published: July 2002

Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300⋅A97-U277), which anchors the substrate helix recognition surface within the
Tetrahymena
group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300⋅A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C
+
⋅G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme’s active site. The U300⋅A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998)
Science
282, 259–264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.
Measures of residue density in protein structures
Authors: Baud Franck , Karlin Samuel
Published: July 2002

A hierarchy of residue density assessments and packing properties in protein structures are contrasted, including a regular density, a variety of charge densities, a hydrophobic density, a polar density, and an aromatic density. These densities are investigated by alternative distance measures and also at the interface of multiunit structures. Amino acids are divided into nine structural categories according to three secondary structure states and three solvent accessibility levels. To take account of amino acid abundance differences across protein structures, we normalize the observed density by the expected density defining a density index. Solvent accessibility levels exert the predominant influence in determinations of the regular residue density. Explicitly, the regular density values vary approximately linearly with respect to solvent accessibility levels, the linearity parameters depending on the amino acid. The charge index reveals pronounced inequalities between lysine and arginine in their interactions with acidic residues. The aromatic density calculations in all structural categories parallel the regular density calculations, indicating that the aromatic residues are distributed as a random sample of all residues. Moreover, aromatic residues are found to be over-represented in the neighborhood of all amino acids. This result might be attributed to nucleation sites and protein stability being substantially associated with aromatic residues.
The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood–brain barrier disruption: Implications for multiple sclerosis treatment
Authors: Bourrié Bernard , Bribes Estelle , Esclangon Martine , Garcia Laurent , Marchand Jean , Thomas Corinne , Maffrand Jean-Pierre , Casellas Pierre
Published: July 2002

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood–brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor α and IFN-γ mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.
Variation in expression of the
Haemophilus influenzae
HMW adhesins: A prokaryotic system reminiscent of eukaryotes
Authors: Dawid Suzanne , Barenkamp Stephen J. , St. Geme Joseph W.
Published: July 2002

Expression of a number of eukaryotic genes is regulated by long stretches of tandem repeats located within the 5′ untranslated region of the particular gene. In this study, we describe a regulatory system in
Haemophilus influenzae
with striking similarities to those found in eukaryotes. We show that expression of the HMW1 and HMW2 adhesins varies based on the number of 7-bp tandem repeats in the
hmw1A
and
hmw2A
promoters. The repeats lie between two separate transcription initiation sites and exert a repressive effect, such that increases in repeat number result in step-wise decreases in levels of specific mRNA and protein production and vice versa. The range of expression of HMW1 and HMW2 varies between very weak and very strong, with a series of gradations in between. Variation in the number of repeats in the
hmw1A
and
hmw2A
promoters occurs in individual colonies passaged
in vitro
, in an animal model of infection, and during natural infection in humans. This system of regulation is unique in prokaryotes and likely enhances the pathogenicity of the organism by increasing adaptive potential.
Tight frames of
k
-plane ridgelets and the problem of representing objects that are smooth away from
d
-dimensional singularities in R

n
Authors: Donoho David L.
Published: July 2002

For each pair (
n
,
k
) with 1 ≤
k
<
n
, we construct a tight frame (ρ
λ
: λ ∈ Λ) for
L
2
(R

n

), which we call a frame of
k-plane ridgelets
. The intent is to efficiently represent functions that are smooth away from singularities along
k
-planes in R

n

. We also develop tools to help decide whether
k
-plane ridgelets provide the desired efficient representation. We first construct a wavelet-like tight frame on the

X-ray bundle χ
n,k

—the fiber bundle having the Grassman manifold

G
n,k

of
k
-planes in R

n

for base space, and for fibers the orthocomplements of those planes. This wavelet-like tight frame is the pushout to

χ
n,k

, via the smooth local coordinates of

G
n,k

, of an orthonormal basis of tensor Meyer wavelets on Euclidean space R

k
(
n
−
k
)

× R

n
−
k

. We then use the
X-ray isometry
[Solmon, D. C. (1976)
J. Math. Anal. Appl.
56, 61–83] to map this tight frame isometrically to a tight frame for
L
2
(R

n

)—the
k
-plane ridgelets. This construction makes analysis of a function
f
∈
L
2
(R

n

) by
k
-plane ridgelets identical to the analysis of the
k
-plane
X
-ray transform of
f
by an appropriate wavelet-like system for
χ

n,k

. As wavelets are typically effective at representing point singularities, it may be expected that these new systems will be effective at representing objects whose
k
-plane
X
-ray transform has a point singularity. Objects with discontinuities across hyperplanes are of this form, for
k
=
n
− 1.
Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins
Authors: Kobe Bostjan , Center Rob J. , Kemp Bruce E. , Poumbourios Pantelis
Published: July 2002

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Å resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.
Toward antibody-catalyzed hydrolysis of organophosphorus poisons
Authors: Vayron Philippe , Renard Pierre-Yves , Taran Frédéric , Créminon Christophe , Frobert Yveline , Grassi Jacques , Mioskowski Charles
Published: July 2002

We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-α-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-α-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M
−1
⋅min
−1
at pH 7.4 and 25°C, which corresponds to a catalytic proficiency of 14,400 M
−1
toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents.
Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes
Authors: Ellsworth Rachel E. , Jamison D. Curtis , Touchman Jeffrey W. , Chissoe Stephanie L. , Braden Maduro Valerie V. , Bouffard Gerard G. , Dietrich Nicole L. , Beckstrom-Sternberg Stephen M. , Iyer Leslie M. , Weintraub Lauren A. , Cotton Marc , Courtney Laura , Edwards Jennifer , Maupin Rachel , Ozersky Philip , Rohlfing Theresa , Wohldmann Patricia , Miner Tracie , Kemp Kimberley , Kramer Jason , Korf Ian , Pepin Kimberlie , Antonacci-Fulton Lucinda , Fulton Robert S. , Minx Patrick , Hillier LaDeana W. , Wilson Richard K. , Waterston Robert H. , Miller Webb , Green Eric D.
Published: July 2002

The identification of the cystic fibrosis transmembrane conductance regulator gene (
CFTR
) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the
CFTR
gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of
CFTR
expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human
CFTR
and mouse
Cftr
genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding ≈1.6 Mb and ≈358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the ≈189-kb and ≈152-kb segments containing the human
CFTR
and mouse
Cftr
genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the
CFTR
/
Cftr
genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near
CFTR
/
Cftr
, including one known gene (
WNT2
/
Wnt2
) and two previously unknown genes that immediately flank
CFTR
/
Cftr
.
The Holliday junction in an inverted repeat DNA sequence: Sequence effects on the structure of four-way junctions
Authors: Eichman Brandt F. , Vargason Jeffrey M. , Mooers Blaine H. M. , Ho P. Shing
Published: July 2002

Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2.1 Å. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson–Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4° as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3′-C⋅G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are stacked in the stable form of the junction.
Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus
Authors: Krumenacker Joshua S. , Hyder Salman M. , Murad Ferid
Published: July 2002

Previous reports that investigated the regulation of the
NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic
compounds have focused primarily on the levels of NO, NO-producing
enzymes, and cGMP in various tissues. In this study, we demonstrate
that 17β-estradiol (E2) regulates the α
1
and
β
1
subunits of the NO receptor, sGC, at the mRNA and
protein levels in rat uterus. Using real-time quantitative PCR, we
found that within 1 h of
in vivo
E2 administration
to rats, sGC mRNA levels begin to diminish. After 3 h, there is a
maximal diminution of sGC mRNA expression (sGC α
1
10%
and sGC β
1
33% of untreated). This effect was blocked by
the estrogen receptor antagonist, ICI 182,780, indicating that estrogen
receptor is required. The effect of E2 also was observed
in
vitro
with incubations of uterine tissue, indicating that the
response does not depend on the secondary release of other hormones or
factors from other tissues. Puromycin did not block the effect,
suggesting the effects occur because of preexisting factors in uterine
tissues and do not require new protein synthesis. Using immunoblot
analysis, we found that sGC protein levels also were reduced by E2 over
a similar time course as the sGC mRNA. We conclude that sGC plays a
vital role in the NO/sGC/cGMP regulatory pathway during conditions
of elevated estrogen levels in the rat uterus as a result of the
reduction of sGC expression.
Mother's and father's parental adjustment: Links to externalising behaviour problems in sons and daughters
Authors: Verlaan Pierrette , Schwartzman Alex E.
Published: July 2002

This study examined links between parents (189 mothers, 153 fathers) adjustment and children's externalising behaviour problems (97 girls; 92 boys) representative of the general population. Structural equation modelling was used to examine models that included “direct” and “indirect” pathways. Externalising behaviour problems in children were strongly related to parental adjustment difficulties. Maternal antisocial behaviour and marital hostility were linked to sons’ and daughters’ externalising behaviour problems via dysfunctional child-rearing practices. Maternal antisocial behaviour, however, was not fully mediated by dysfunctional parenting. For fathers, the pattern related to externalising problems in sons was as those specified for mothers. The pattern did not appear relevant for daughters. Results are discussed in terms of the importance of adjustment patterns with regard to the sex composition of the parent-child dyads.
Development through media use? A German study on the use of radio in adolescence
Authors: Boehnke Klaus , Münch Thomas , Hoffmann Dagmar
Published: July 2002

A relationship between the developmental aspirations of adolescents and the modes of their media use is suggested. The hypothesis is forwarded that adolescents’ use of the radio is largely determined by their wish to accelerate and enhance their own development. Radio is assumed to be used by adolescents to master age-specific developmental tasks. A model linking developmental aspirations and music involvement with radio use is tested in a structural equations approach. Data from a German youth study of 10- to 18-year-olds (N = 1011) is analysed. Findings underscore the conjecture that developmental aspirations in the form of subjective discrepancies between developmental goals and the current developmental standing with regard to selected developmental tasks and certain modes of radio use (socioemotional) are indeed related. Adolescent music involvement also emerged as a powerful predictor of socioemotional radio use.
Drawing as a verbally mediated activity: A study of relationships between verbal, motor, and visuospatial skills and drawing in children
Authors: Toomela Aaro
Published: July 2002

The present studies analysed drawing as a system rather than a unitary ability. The aim was to investigate whether and which language factors influence drawing performance and drawing development. In two studies of 2- to 11-year-old children drawing as a system of components was investigated. Tests were designed to measure motor output, imagery, memory, perception, and verbal abilities. The analysis of relationships between drawing measures and putative components of a drawing system revealed that all of the proposed components independently affected drawing development. Results of analyses of the data from younger and older children separately suggest that different components of a system are crucial for drawing development at different phases of development.
Uncertainty of Estimates of Cancer Risks Derived by Extrapolation from High to Low Doses and from Animals to Humans
Authors: Kodell Ralph L. , Gaylor David W.
Published: July 2002

The uncertainties associated with extrapolating model-based cancer risks from high to low doses and animal-based cancer risks to humans are examined. It is argued that low-dose linear extrapolation based on statistical confidence limits calculated from animal data is designed to account for data uncertainty, model-selection uncertainty, and model-fitting instability. The intent is to err on the side of safety, that is, overstating rather than understating the true risk. The tendency toward conservatism in predicting human cancer risks from animal data based on linear extrapolation is confirmed by a real-data analysis of the various sources of uncertainty involved in extrapolating from animals to humans. Along with the tendency toward conservatism, a high degree of overall uncertainty in the interspecies extrapolation process is demonstrated. It is concluded that human cancer risk estimates based on animal data may underestimate the true risk by a factor of 10 or may overestimate that risk by a factor of 1,000.
Standards for Soil Contamination: Results of a Federal/State Working Group
Authors: Bachmann Günther , von Borries Dietrich , König Wilhelm
Published: July 2002

The German Soil Protection Legislation (draft) proposes generic soil trigger levels and action levels to assess contaminated sites. This article presents results of a working group set up by the Bundl Länder-Arbeitsgemeinschaft “Bodenschutz” (LABO) and the Länder-Arbeitsgemeinschaft “Abfall” (LAGA). The report on key issues for assessing public health effects of contaminated land for 14 substances of prime importance also has been negotiated with the Arbeitsgemeinschaft der Leitenden Medizinalbeamten der Länder (AGLMB [health service]). The report introduces toxicological reference data indicating a virtually safe body dose and a factor deriving a hazardous body dose, which will be used to calculate soil trigger levels. Calculations are based on exposure estimates covering the inhalative andingestive routes of exposure. Results of the calculations are to be checked against soil background data.