bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of inducible cyclooxygenase 2 gene transcription by aspirin and sodium salicylate
Authors: Xu Xiao-Ming , Sansores-Garcia Leticia , Chen Xian-Ming , Matijevic-Aleksic Nevenka , Du Min , Wu Kenneth K.
Published: July 2002

The pharmacological action of salicylate cannot be explained by its inhibition of cyclooxygenase (COX) activity. In this report, the effects of aspirin and sodium salicylate on COX-2 expressions in human umbilical vein endothelial cells and foreskin fibroblasts were evaluated. Aspirin and sodium salicylate at therapeutic concentrations equipotently blocked COX-2 mRNA and protein levels induced by interleukin-1β and phorbol 12-myristate 13-acetate. The suppressing effect was more pronounced in cultured cells deprived of fetal bovine serum for 24 h, suggesting that it may be cell cycle related. Salicylate inhibited nascent COX-2 transcript synthesis but had no effect on COX-2 mRNA stability. It inhibited COX-2 promoter activity in a concentration-dependent manner. In mice pretreated with aspirin (10 and 30 mg/kg), followed by challenge with lipopolysaccharide, COX-2 mRNA expression in peritoneal macrophages was markedly suppressed. These findings suggest that salicylate exerts its antiinflammatory action in part by suppressing COX-2 induction, thereby reducing the synthesis of proinflammatory prostaglandins.
DNA polymerase active site is highly mutable: Evolutionary consequences
Authors: Patel Premal H. , Loeb Lawrence A.
Published: July 2002

DNA polymerases contain active sites that are structurally superimposable and highly conserved in sequence. To assess the significance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of
Thermus aquaticus
DNA polymerase I. After selection, by using genetic complementation, we obtained a library of approximately 8,000 active mutant DNA polymerases, of which 350 were sequenced and analyzed. This is the largest collection of physiologically active polymerase mutants. We find that all residues of motif A, except one (Asp-610), are mutable while preserving wild-type activity. A wide variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative substitutions predominate at regions that stabilize tertiary structures. Several mutants exhibit unique properties, including DNA polymerase activity higher than the wild-type enzyme or the ability to incorporate ribonucleotide analogs. Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. The high mutability of the polymerase active site
in vivo
and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis. The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.
The complete unique long sequence and the overall genomic organization of the GA strain of Marek's disease virus
Authors: Lee Lucy F. , Wu Ping , Sui Dexin , Ren Delin , Kamil Jeremy , Kung Hsing Jien , Witter Richard L.
Published: July 2002

We have determined the DNA sequence of the unique long (UL) region and the repeat long (RL) region in the genome of serotype 1 GA strain of Marek's disease virus (MDV), a member of the α-herpesvirus family. With this information, the complete nucleotide sequence of GA-MDV is now known. The entire GA-MDV genome is predicted to be about 174 kbp in size, with an organization of TRL-UL-IRL-IRS-US-TRS, typical of a α-herpesvirus. The UL sequence contains 113,508 bp and has a base composition of 41.7% G + C. A total of 67 ORFs were identified completely within the UL region, among which 55 are homologous to genes encoded by herpes simplex virus-1. Twelve of them are unique with presently unknown functions. The sequence of RL reported here together with those published earlier reveal the major structural features of the RL. Virtually all of the ORFs encoded by RL are specific to serotype I of MDV. These ORFs are likely to contribute to some of the unique biological properties of MDV. Among the proteins encoded by MDV-specific ORFs are Meq, a jun/fos family of transcriptional factor implicated in transformation and latency, virus-encoded interleukin-8, a CXC chemokine, and pp38 and pp24, two phosphoproteins with undefined functions. There is also a putative lipase gene (LORF2) that has homologies in HPRS-24 (serotype II) strain of MDV and in various avian adenoviruses. An additional unique feature of MDV is the presence of long terminal repeat remnant sequences of avian retrovirus reticuloendotheliosis virus. These remnant sequences are derived from the U3-enhancer region through ancestral insertions by reticuloendotheliosis virus proviruses.
Tumor selective G
2
/M cell cycle arrest and apoptosis of epithelial and hematological malignancies by BBL22, a benzazepine
Authors: Xia Wenle , Spector Sydney , Hardy Lys , Zhao Sumin , Saluk Alan , Alemane Lourdes , Spector Neil L.
Published: July 2002

Two distinct benzodiazepine binding sites have been identified, (
i
) a central site restricted to brain and (
ii
) a ubiquitously expressed mitochondrial binding site, the so-called peripheral-type benzodiazepine receptor (PBR). In this paper, we show that a benzazepine referred to as BBL22 (2-amino 9-chloro-7-(2-fluorophenyl)-5H-pyrimidol[5,4-
d
][2]benzazepine), which is classified as a PBR ligand based on structure, induces arrest in G
2
/M phase of the cell cycle in human tumor cell lines of both epithelial and hematopoietic cellular origin. After G
2
/M arrest, several tumor types, notably prostate and certain breast cancer lines exhibited significant apoptosis. Ideally, cancer therapies should selectively target tumor cells while sparing normal cell counterparts. BBL22 exhibited such selectivity, as it did not affect the growth and survival of nonmalignant breast and prostate epithelial lines. Moreover, BBL22 demonstrated structural requirements for this selective antitumor activity as 11 structurally related PBR ligands, including high-affinity ligands Ro5–4864 and PK11195, failed to induce tumor cell growth arrest or apoptosis. The
in vivo
antitumor activity of BBL22 was examined in a human xenograft model of androgen-independent prostate cancer where BBL22 significantly reduced the growth of PC3 prostate tumors without eliciting overt toxicity. Identification of BBL22 represents a tumor selective therapeutic strategy for a variety of human tumors.
Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism
Authors: Sasaki Masayuki , Gonzalez-Zulueta Mirella , Huang Hui , Herring William J. , Ahn Sohyun , Ginty David D. , Dawson Valina L. , Dawson Ted M.
Published: July 2002

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca
2+
entry through voltage-sensitive Ca
2+
channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca
2+
. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca
2+
response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca
2+
-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.
Information theory, atoms in molecules, and molecular similarity
Authors: Nalewajski Roman F. , Parr Robert G.
Published: July 2002

Using information theory, it is argued that from among possible definitions of what an atom is when it is in a molecule, a particular one merits special attention. Namely, it is the atom defined by the “stockholders partitioning” of a molecule invented by Hirshfeld [(1977)
Theor. Chim. Acta
44, 129]. The theoretical tool used is the minimum entropy deficiency principle (minimum missing information principle) of Kullback and Liebler [(1951)
Ann. Math. Stat.
22, 79]. A corresponding analysis is given of the problem of assessing similarity between molecules or pieces of molecules.
Molecular basis for CD40 signaling mediated by TRAF3
Authors: Ni Chao-Zhou , Welsh Kate , Leo Eugen , Chiou Chu-kuan , Wu Hao , Reed John C. , Ely Kathryn R.
Published: July 2002

Tumor necrosis factor receptors (TNFR) are single transmembrane-spanning glycoproteins that bind cytokines and trigger multiple signal transduction pathways. Many of these TNFRs rely on interactions with TRAF proteins that bind to the intracellular domain of the receptors. CD40 is a member of the TNFR family that binds to several different TRAF proteins. We have determined the crystal structure of a 20-residue fragment from the cytoplasmic domain of CD40 in complex with the TRAF domain of TRAF3. The CD40 fragment binds as a hairpin loop across the surface of the TRAF domain. Residues shown by mutagenesis and deletion analysis to be critical for TRAF3 binding are involved either in direct contact with TRAF3 or in intramolecular interactions that stabilize the hairpin. Comparison of the interactions of CD40 with TRAF3 vs. TRAF2 suggests that CD40 may assume different conformations when bound to different TRAF family members. Thismolecular adaptationmay influence binding affinity and specific cellular triggers.
Heterochromatic deposition of centromeric histone H3-like proteins
Authors: Henikoff Steven , Ahmad Kami , Platero J. Suso , van Steensel Bas
Published: July 2002

Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. The functional significance of this centromere–heterochromatin relationship, if any, is unknown. One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins. We describe a
Drosophila
H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres. When the
cid
upstream region drives expression of H3 and H2B histone–green fluorescent protein fusion genes in
Drosophila
cells, euchromatin-specific deposition results. Remarkably, when the
cid
upstream region drives expression of yeast, worm, and human centromeric histone–green fluorescent protein fusion proteins, localization is preferentially within
Drosophila
pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, the heterochromatic state itself may help localize centromeric components.
Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin
Authors: Kandori Hideki , Yamazaki Yoichi , Shichida Yoshinori , Raap Jan , Lugtenburg Johan , Belenky Marina , Herzfeld Judith
Published: July 2002

Unidirectional proton transport in
bacteriorhodopsin is enforced by the switching
machinery of the active site. Threonine 89 is located in this region,
with its O—H group forming a hydrogen bond with Asp-85, the acceptor
for proton transfer from the Schiff base of the retinal chromophore.
Previous IR spectroscopy of [3-
18
O]threonine-labeled
bacteriorhodopsin showed that the hydrogen bond of
the O—D group of Thr-89 in D
2
O is strengthened in the K
photocycle intermediate. Here, we show that the strength and
orientation of this hydrogen bond remains unchanged in the L
intermediate and through the M intermediate. Furthermore, a strong
interaction between Asp-85 and the O—H (O—D) group of Thr-89 in M
is indicated by a shift in the C⩵O stretching vibration of the former
because of
18
O substitution in the latter. Thus, the strong
hydrogen bond between Asp-85 and Thr-89 in K persists through M,
contrary to structural models based on x-ray crystallography of the
photocycle intermediates. We propose that, upon photoisomerization of
the chromophore, Thr-89 forms a tight, persistent complex with one of
the side-chain oxygens of Asp-85 and is thereby precluded from
participating in the switching process. On the other hand, the loss of
hydrogen bonding at the other oxygen of Asp-85 in M may be related to
the switching event.
Evaluating the Role of the Sunshine Coast University (USC) in the Regional Economy
Authors: Allison Janelle , Keane Julie
Published: July 2002

The research findings discussed in this paper present ideas from the second stage of a project to evaluate the role of higher education institutions (HEIs) in regional economies. Using data developed through a learning region framework, the research schematically represents HEI linkages to the region . Possible linkage scenarios for each of six dimensions associated with embeddedness are presented. The findings suggest that this qualitative approach moves us closer to better capturing the complexity and role of HEI in regional development.
Critical thinking and the academic vocational divide
Authors: Halliday John
Published: July 2002

There is considerable interest in unifying post‐compulsory education and training systems through the common teaching of core or key skills. Critical thinking appears to be a very attractive example of such a skill but this article argues against the idea that critical thinking is a core skill that should be widely taught to bridge what is termed ‘the academic vocational divide’. Some of the attractions of this idea are acknowledged but it is argued that the very meanings of critical, academic and vocational depend upon the context and purpose of thinking and it is misleading to suggest that thinking is any kind of a skill. On the basis of this argument and an empirical study it is concluded that when the idea informs curricular policy and practice, far from bridging an academic vocational divide, the idea will actually widen it. If this conclusion is correct for what appears to be a most plausible candidate for a core or key skill, then policies designed to bridge an academic vocational divide that are based on the core or key skills idea should be abandoned.
Statistical Issues in Large Animal Toxicology
Authors: Thakur Ajit K.
Published: July 2002

Large animal toxicology studies generally contain small numbers of animals, 3–5/sex/group. Traditionally, people do not take the multivariate and multifactor aspects of the design into consideration while analyzing the parameters of interest of such studies. As a consequence, many investigators reach inappropriate and sometimes incorrect statistical conclusions from their analyses. The main purpose of this article is to show how, given the typically minimal sample sizes, one can reach more meaningful conclusions from such studies if one takes into consideration the factorial and multivariate nature of the design. The secondary purpose is to point out the need of careful examination of model specification and use of some popular statistical software. Examples are given to demonstrate the points.
Anorectic Toxicity of Dih Ydroartemisinin, Artemether, and Arteether in Rats Following Multiple Intramuscular Doses
Authors: Li Qigui , Brewer Thomas G. , Peggins James O.
Published: July 2002

During studies of arteether (AE), artemether (AM), and dihydroartemisinin (DQHS) neurotoxicity, the effect of 7 daily intramuscular doses (25, 50, and 100 mg/ kg/d) of those antimalarial drugs on gastrointestinal function was investigated in rats. A modified Nichols' method was used to measure daily food and water consumption. To estimate gastric transit, the total length amaranth (administered 40 minutes prior to sacrifice) dye traveled through small intestine were measured, and to determine gastric retention, the tied-off stomach pouch was removed and the contents weighed 24 hours after the last dose or when a rat became moribund or died. AM and AE dose solutions were prepared using sesame oil, whereas 50% dimethylacetamide (DMAC) sesame oil was used for DQHS. The results showed that after dosing with 50 mg/ kg for 7 days, 50% inhibition of food consumption (ID50) occurred at 1.9 days for DQHS, 3.9 days for AM, and 4.1 days for AE. Similar data were observed for water intake. After 100 mg/kg dosing, the ID50 s for food and water consumption decreased to 2.8-2.9 days for AM and 3.1-3.7 days for AE. Food consumption and body weights were decreased following all three treatments, and rats exhibited neurologic symptoms at 25-100 mg/kg dose of DQHS and 50-100 mg/kg dose of AM and AE. In addition, the results constituted a 53% and 82% inhibition of gastric transit for AM and AE, respectively, at 25 mg/kg animals compared to control, and 100% inhibition was found in high doses (50 and 100 mg/kg) for all the three drugs. The gastric retention ratio (controls equal 1.0) was 26.0 for DQHS, 5.8 with AE, and 2.3 for AM rats following 50 mg/kg dosing. When the 100 mg/kg dose was administered, the gastric retention ratio doubled for AE (11.6) and AM (4.3). The consumption data indicated that DQHS was about 2-3 times more toxic to the anorexia than AM and AE at 25 and 50 mg/kg/day dose levels. Significant differences in gastric emptying and gastric transit activities between AE and AM were observed. Data demonstrated that after multiple intramuscular doses of DQHS, AM, or AE in rats, food consumption and gastric emptying were decreased, gastric transit was inhibited, as reflected in a significant body weight reduction and death. Since an exhibition of the anorectic symptoms of AM and AE was at a lower dose than the neurologic signs in rats, the anorexia could be an early portent or prediction of the neurotoxicity in animals or humans.
Pragmatic Comprehension In Secondary School‐Aged Students With Specific Developmental Language Disorder
Authors: Rinaldi Wendy
Published: July 2002

This study explores the hypothesis that there may be particular difficulties for secondary school students with specific developmental language disorder (SDLD) in understanding contextual, pragmatic meaning. Sixty‐four SDLD students aged 11+ to 14+ years are compared with chronological‐age‐matched and language‐age‐matched non‐impaired students. New procedures are used to examine comprehension of two types of ambiguity where the context determines speaker intention: inconsistent messages of emotion and multiple meanings in context. These types of ambiguity are evident in a range of communicative intent, e.g. sarcasm, idiomatic expression, deceit and humour. Preliminary study into adolescent language suggests that at this age there is a growing expectation for students to understand these kinds of communication, both in the classroom and socially. The study finds that the SDLD students were less able than both comparison groups to use context to understand implied meanings. Nonimpaired children were also more able to rule out literal interpretations when they did not know the non‐literal meaning. These findings were statistically significant. The implications for research and practice are discussed, including those of diagnostic assessment, in the light of the literature survey revealing that many currently available do not assess pragmatic meaning comprehension. There is a challenge to the view that disorders in the semantic and pragmatic domains necessarily co‐occur, as suggested by the diagnostic category semanticpragmatic disorder.
The Yeast CDC37 Gene Interacts with MPS1 and Is Required for Proper Execution of Spindle Pole Body Duplication
Authors: Schutz Amy R. , Giddings Thomas H. , Steiner Estelle , Winey Mark
Published: July 2002

The MPS1 gene from Saccharomyces cerevisiae encodes an essential protein kinase required for spindle pole body (SPB) duplication and for the mitotic spindle assembly checkpoint. Cells with the mps1-1 mutation fail early in SPB duplication and proceed through monopolar mitosis with lethal consequences. We identified CDC37 as a multicopy suppressor of mps1-1 temperature-sensitive growth. Suppression is allele specific, and synthetic lethal interactions occur between mps1 and cdc37 alleles. We examined the cdc37-1 phenotype for defects related to the SPB cycle. The cdc37-1 temperature-sensitive allele causes unbudded, G1 arrest at Start (Reed, S.I. 1980. Genetics. 95: 561–577). Reciprocal shifts demonstrate that cdc37-1 arrest is interdependent with α-factor arrest but is not a normal Start arrest. Although the cells are responsive to α-factor at the arrest, SPB duplication is uncoupled from other aspects of G1 progression and proceeds past the satellite-bearing SPB stage normally seen at Start. Electron microscopy reveals side-by-side SPBs at cdc37-1 arrest. The outer plaque of one SPB is missing or reduced, while the other is normal. Using the mps2-1 mutation to distinguish between the SPBs, we find that the outer plaque defect is specific to the new SPB. This phenotype may arise in part from reduced Mps1p function: although Mps1p protein levels are unaffected by the cdc37-1 mutation, kinase activity is markedly reduced. These data demonstrate a requirement for CDC37 in SPB duplication and suggest a role for this gene in G1 control. CDC37 may provide a chaperone function that promotes the activity of protein kinases.
Dominant-Interfering Hsc70 Mutants Disrupt Multiple Stages of the Clathrin-Coated Vesicle Cycle in Vivo
Authors: Newmyer Sherri L. , Schmid Sandra L.
Published: July 2002

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.
Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase–deficient mice and primary cells despite increased chromosomal instability
Authors: Samper Enrique , Goytisolo Fermín A. , Murcia Josiane Ménissier-de , González-Suárez Eva , Cigudosa Juan C. , de Murcia Gilbert , Blasco María A.
Published: July 2002

Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1–deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1–deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. Nat. Genet. 23:76–80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1−/− littermate mice or PARP-1−/− primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1−/− cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1−/− primary cells is accordant with the absence of severe proliferative defects in PARP-1−/− mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1−/− primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.
High resolution mapping of mast cell membranes reveals primary and secondary domains of FcϵRI and LAT
Authors: Wilson Bridget S. , Pfeiffer Janet R. , Surviladze Zurab , Gaudet Elizabeth A. , Oliver Janet M.
Published: July 2002

In mast cells, cross-linking the high-affinity IgE receptor (FcϵRI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting FcϵRI colocalize loosely with Lyn, whereas cross-linked FcϵRI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of FcϵRI β is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCγ isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCγ1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCγ2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCγ2, Gab2, and a portion of p85 colocalize with FcϵRI β in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCγ1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, FcϵRI cross-linking increases PI3-kinase activity in anti-LAT, anti-FcεRIβ, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around FcεRIβ and from secondary domains, including one organized around LAT.
Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR
Authors: Lalli Giovanna , Schiavo Giampietro
Published: July 2002

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.
Nf1 Regulates Hematopoietic Progenitor Cell Growth and Ras Signaling in Response to Multiple Cytokines
Authors: Zhang You-Yan , Vik Terry A. , Ryder John W. , Srour Edward F. , Jacks Tyler , Shannon Kevin , Wade Clapp D.
Published: July 2002

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1−/− hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1−/− progenitors. Nf1−/− fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1−/− progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.
Wegener's Granulomatosis: Anti–proteinase 3 Antibodies Are Potent Inductors of Human Endothelial Cell Signaling and Leakage Response
Authors: Sibelius Ulf , Hattar Katja , Schenkel Angelika , Noll Thomas , Csernok Elena , Gross Wolfgang Ludwig , Mayet Werner-Johannes , Piper Hans-Michael , Seeger Werner , Grimminger Friedrich
Published: July 2002

Anti–neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor α induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng–2.5 μg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab)2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen–antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis–related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3–induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG.
Yersinia enterocolitica Impairs Activation of Transcription Factor NF-κB: Involvement in the Induction of Programmed Cell Death and in the Suppression of the Macrophage Tumor Necrosis Factor α Production
Authors: Ruckdeschel Klaus , Harb Suzanne , Roggenkamp Andreas , Hornef Mathias , Zumbihl Robert , Köhler Stephan , Heesemann Jürgen , Rouot Bruno
Published: July 2002

In this study, we investigated the activity of transcription factor NF-κB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-κB signal, Y. enterocolitica inhibited NF-κB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IκB-α and IκB-β observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-κB and to suppress the tumor necrosis factor α (TNF-α) production as well as to trigger macrophage apoptosis. When NF-κB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-α secretion. Y. enterocolitica also impaired the activity of NF-κB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-α could induce HeLa cell apoptosis alone, TNF-α provoked apoptosis when activation of NF-κB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-κB, which inhibits TNF-α release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.
Herpes Simplex Virus Type 1 Infection of Activated Cytotoxic T Cells
Authors: Raftery Martin J. , Behrens Christian K. , Müller Anke , Krammer Peter H. , Walczak Henning , Schönrich Günther
Published: July 2002

Herpes simplex virus type 1 (HSV1), a large DNA-containing virus, is endemic in all human populations investigated. After infection of mucocutaneous surfaces, HSV1 establishes a latent infection in nerve cells. Recently, it was demonstrated that HSV1 can also infect activated T lymphocytes. However, the consequences of T cell infection for viral pathogenesis and immunity are unknown. We have observed that in contrast to the situation in human fibroblasts, in human T cell lines antigen presentation by major histocompatibility complex class I molecules is not blocked after HSV1 infection. Moreover, HSV1 infection of T cells results in rapid elimination of antiviral T cells by fratricide. To dissect the underlying molecular events, we used a transgenic mouse model of HSV1 infection to demonstrate that CD95 (Apo-1, Fas)-triggered apoptosis is essential for HSV1-induced fratricide, whereas tumor necrosis factor (TNF) also contributes to this phenomenon but to a lesser extent. By contrast, neither TRAIL (TNF-related apoptosis-inducing ligand) nor perforin were involved. Finally, we defined two mechanisms associated with HSV1-associated fratricide of antiviral T cells: (a) T cell receptor–mediated upregulation of CD95 ligand and (b) a viral “competence-to-die” signal that renders activated T lymphocytes susceptible to CD95 signaling. We propose that induction of fratricide is an important immune evasion mechanism of HSV1, helping the virus to persist in the host organism throughout its lifetime.
Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line
Authors: Schromm Andra B. , Lien Egil , Henneke Philipp , Chow Jesse C. , Yoshimura Atsutoshi , Heine Holger , Latz Eicke , Monks Brian G. , Schwartz David A. , Miyake Kensuke , Golenbock Douglas T.
Published: July 2002

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.
DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences.
Authors: Lascaris R F , Mager W H , Planta R J
Published: July 2002

MOTIVATION: High-level transcriptional activation of most ribosomal protein (rp) genes in Saccharomyces cerevisiae is promoted by the global DNA-binding factor Rap1p. The creation of the complete database of yeast rp gene promoter sequences enabled us to develop a computational selection strategy aimed at acquiring detailed information concerning the DNA-binding specificity of Rap1p. RESULTS: Rap1p sites in rp gene promoters are often found in duplicate, exhibiting strong preferences in both spacing and orientation. Using these preferences, a weight matrix was selected that represents the in vivo binding requirements of Rap1p. The resulting matrix renders the identification of functional Rap1p binding sites more accurate and allowed us to re-evaluate previous in vitro data. Tandemly arranged Rap1p binding sites appear to be typical for rp gene promoters and differ in preferred spacing from sites occurring in (sub)telomeric repeats. The preferred spacing that is found in duplicate Rap1p binding sites of rp gene promoters is restricted to a small window between 15 and 26 bp. This is proposed to reflect the borders within which binding co-operativity operates. The data presented clearly illustrate that computational selection strategies provide information that reaches beyond experimental data. AVAILABILITY: The rp database is available at the url: http://www. chem.vu.nl/BMB/Database.html.
Improved performance in protein secondary structure prediction by inhomogeneous score combination.
Authors: Guermeur Y , Geourjon C , Gallinari P , Del√©age G
Published: July 2002

MOTIVATION: In many fields of pattern recognition, combination has proved efficient to increase the generalization performance of individual prediction methods. Numerous systems have been developed for protein secondary structure prediction, based on different principles. Finding better ensemble methods for this task may thus become crucial. Furthermore, efforts need to be made to help the biologist in the post-processing of the outputs. RESULTS: An ensemble method has been designed to post-process the outputs of discriminant models, in order to obtain an improvement in prediction accuracy while generating class posterior probability estimates. Experimental results establish that it can increase the recognition rate of protein secondary structure prediction methods that provide inhomogeneous scores, even though their individual prediction successes are largely different. This combination thus constitutes a help for the biologist, who can use it confidently on top of any set of prediction methods. Moreover, the resulting estimates can be used in various ways, for instance to determine which areas in the sequence are predicted with a given level of reliability. AVAILABILITY: The prediction is freely available over the Internet on the Network Protein Sequence Analysis (NPS@) WWW server at http://pbil.ibcp.fr/NPSA/npsa_server.ht ml. The source code of the combiner can be obtained on request for academic use.
HAT activity is essential for CBP‐1‐dependent transcription and differentiation in Caenorhabditis elegans
Authors: Victor Martin , Bei Yanxia , Gay Frédérique , Calvo Dominica , Mello Craig , Shi Yang
Published: July 2002

The p300/CBP family of transcriptional coactivators possesses multiple functional domains, including a histone acetyltransferase (HAT) and several activation domains. A number of models have been proposed to account for their roles in transcriptional activation, including interactions with basal transcription machinery and chromatin remodeling. However, individual contributions of these domains to transcriptional activation and their significance in living organisms remain unclear. We addressed the importance of the HAT activity of CBP‐1, the worm ortholog of p300/CBP, in Caenorhabditis elegans with three different and complementary approaches. These include allele‐specific RNA‐mediated interference (RNAi), genetic rescue and the use of a specific chemical inhibitor of the p300/CBP HAT. Our findings demonstrate that HAT activity is of primary importance for CBP‐1 to regulate transcription and to promote differentiation during C. elegans embryogenesis.
An intact NEDD8 pathway is required for Cullin‐dependent ubiquitylation in mammalian cells
Authors: Ohh Michael , Kim William Y , Moslehi Javid J , Chen Yuzhi , Chau Vincent , Read Margaret A , Kaelin William G
Published: July 2002

Skp1‐Cdc53/Cul1‐F‐box (SCF) complexes constitute a class of E3 ubiquitin ligases. Recently, a multiprotein complex containing pVHL, elongin C and Cul2 (VEC) was shown to structurally and functionally resemble SCF complexes. Cdc53 and the Cullins can become covalently linked to the ubiquitin‐like molecule Rub1/NEDD8. Inhibition of neddylation inhibits SCF function in vitro and in yeast and plants. Here we show that ongoing neddylation is likewise required for VEC function in vitro and for the degradation of SCF and VEC targets in mammalian cells. Thus, neddylation regulates the activity of two specific subclasses of mammalian ubiquitin ligases.
RWJ-270201 (BCX-1812): a novel neuraminidase inhibitor for influenza
Authors: Young Diane , Fowler Cynthia , Bush Karen
Published: July 2002

The influenza virus neuraminidase (NA) is important in the pathogenesis of infection and, thus, is an attractive target for agents used in the treatment and prophylaxis of influenza. This article describes preclinical and early clinical data related to RWJ–270201 (BCX–1812), a novel, orally active NA inhibitor that was rationally designed for having potent and selective activity against influenza A and B viruses. RWJ–270201 is a unique NA inhibitor with a cyclopentane ring structure and high selectivity for the influenza NA. RWJ–270201 has efficacy comparable to or better than earlier NA inhibitors against a wide range of influenza A and B isolates, including recently emerged and avian strains, both
in vitro
and in a lethal murine model of influenza. Based on the high selectivity and efficacy of RWJ–270201 against both type A and B influenza strains in preclinical studies as well as murine pharmacodynamic studies supporting the potential for once–daily administration, clinical trials were initiated in order to determine the tolerability and antiviral activity of RWJ–270201 in humans. To date, clinical studies have indicated that RWJ–270201 is well tolerated and has antiviral activity in human experimental influenza models when administered orally once daily.
Zanamivir: from drug design to the clinic
Authors: Elliott Michael
Published: July 2002

The development of the neuraminidase inhibitors has revolutionized the management options for influenza. Zanamivir was the first such inhibitor to be approved for the treatment of influenza in humans. It is delivered by inhalation to the respiratory tract, which is the site of viral replication, in order to ensure immediate antiviral activity. Early treatment with zanamivir in clinical trials rapidly reduced the severity and duration of influenza symptoms and associated complications. Furthermore, chemoprophylaxis with zanamivir was shown to be effective in the prevention of influenza illness. To date, there is no evidence for the emergence of clinically significant zanamivir–resistant isolates. In conclusion, zanamivir offers a useful complementary strategy to vaccination in the effective management of influenza.
Segmentation of carotid artery in ultrasound images: Method development and evaluation technique
Authors: Mao Fei , Gill Jeremy , Downey Donal , Fenster Aaron
Published: July 2002

Segmentation of carotid artery lumen in two‐dimensional and three‐dimensional ultrasonography is an important step in computerized evaluation of arterial disease severity and in finding vulnerable atherosclerotic plaques susceptible to rupture causing stroke. Because of the complexity of anatomical structures, noise as well as the requirement of accurate segmentation, interactions are necessary between observers and the computer segmentation process. In this paper a segmentation process is described based on the deformable model method with only one seed point to guide the initialization of the deformable model for each lumen cross section. With one seed, the initial contour of the deformable model is generated using the entropy map of the original image and mathematical morphology operations. The deformable model is driven to fit the lumen contour by an internal force and an external force that are calculated, respectively, with geometrical properties of deformed contour and with the image gray level features. The evaluation methodology using distance‐based and area‐based metrics is introduced in this paper. A contour probability distribution (CPD) method for calculating distance‐based metrics is introduced. The CPD is obtained by generating contours of the lumen using a set of possible seed locations. The mean contour can be compared to a manual outlined contour to provide accuracy metrics. The variance computed from the CPD can provide metrics of local and global variability. These metrics provide a complete performance evaluation of an interactive segmentation algorithm and a means for comparing different algorithm settings.