bluuebunny/crossref_metadata_2025 huggingface dataset:
SKIP is an indispensable factor for
Caenorhabditis elegans
development
Authors: Kostrouchova Marta , Housa Daniel , Kostrouch Zdenek , Saudek Vladimir , Rall Joseph Edward
Published: July 2002

SKI-binding protein (SKIP) is a transcription cofactor present in all eukaryotes. Here we show that SKIP is a unique protein that is required for
Caenorhabditis elegans
viability and development. Expression of
CeSKIP
(skp-1) assayed by RT-PCR and by GFP fluorescence in transgenic lines starts in embryos and continues to adulthood. Loss of
CeSKIP
activity by RNA-mediated inhibition results in early embryonic arrest similar to that seen following inhibition of RNA polymerase II. RNA polymerase II phosphorylation appears normal early in
CeSKIP
RNA-mediated inhibition treated embryos although the expression of several embryonic GFP reporter genes is severely restricted or absent. Our data suggest that CeSKIP is an essential component of many RNA polymerase II transcription complexes and is indispensable for
C. elegans
development.
Topological determinants of protein folding
Authors: Dokholyan Nikolay V. , Li Lewyn , Ding Feng , Shakhnovich Eugene I.
Published: July 2002

The folding of many small proteins is kinetically a two-state process that represents overcoming the major free-energy barrier. A kinetic characteristic of a conformation, its probability to descend to the native state domain in the amount of time that represents a small fraction of total folding time, has been introduced to determine to which side of the free-energy barrier a conformation belongs. However, which features make a protein conformation on the folding pathway become committed to rapidly descending to the native state has been a mystery. Using two small, well characterized proteins, CI2 and C-Src SH3, we show how topological properties of protein conformations determine their kinetic ability to fold. We use a macroscopic measure of the protein contact network topology, the average
graph connectivity
, by constructing graphs that are based on the geometry of protein conformations. We find that the average connectivity is higher for conformations with a high folding probability than for those with a high probability to unfold. Other macroscopic measures of protein structural and energetic properties such as radius of gyration, rms distance, solvent-accessible surface area, contact order, and potential energy fail to serve as predictors of the probability of a given conformation to fold.
Employing
Escherichia coli
to functionally express, purify, and characterize a human transporter
Authors: Quick Matthias , Wright Ernest M.
Published: July 2002

Large-scale purification of recombinant human membrane proteins represents a rate-limiting step toward the understanding of their role in health and disease. There are only four mammalian membrane proteins of known structure, and these were isolated from natural sources (see
http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html
). In addition, genetic diseases of membrane proteins are frequently caused by trafficking defects, and it is enigmatic whether these mutants are functional. Here, we report the employment of
Escherichia coli
for the functional expression, purification, and reconstitution of a human membrane protein, the human Na
+
/glucose cotransporter (hSGLT1). The use of an
E. coli
mutant defective in the outer membrane protease OmpT, incubation temperatures below 20°C, and transcriptional regulation from the
lac
promoter/operator are crucial to reduce proteolytic degradation. Purification of a recombinant hSGLT1 through affinity chromatography yields about 1 mg of purified recombinant hSGLT1 per 3 liters of cultured bacterial cells. Kinetic analysis of hSGLT1 in proteoliposomes reveals that a purified recombinant transporter, which is missorted in eukaryotic cells, retains full catalytic activity. These results indicate the power of bacteria to manufacture and isolate human membrane proteins implicated in genetic diseases.
Identification of gene function by cyclical packaging rescue of retroviral cDNA libraries
Authors: Bhattacharya Deepta , Logue Eric C. , Bakkour Sonia , DeGregori James , Sha William C.
Published: July 2002

Genes regulating responses in mammalian cells are often difficult to identify by functional cloning strategies limited to a single round of selection. Here we describe a strategy, cyclical packaging rescue (CPR), which allows rapid recovery and retransmission of retroviral cDNA libraries. CPR can be used not only with immortalized cell lines such as fibroblasts and Jurkat T cells, but also with primary B lymphocytes, which can be maintained only in short-term cultures. CPR allows for multiple rounds of selection and enrichment to identify cDNAs regulating responses in mammalian cells. Using CPR, five cDNAs were functionally cloned, which conferred protection against tumor necrosis factor α (TNFα)-induced apoptosis in RelA−/−fibroblasts. Three of the genes, RelA, cellular FLICE-like inhibitory protein (c-FLIP), and a dominant-negative mutant of TNF receptor 1 arising through CPR afforded strong protection against apoptosis. Two of the genes identified, Dbs and Fas-associated death domain protein (FADD), previously identified as a proapoptotic molecule, afforded partial protection against TNFα-induced apoptosis. These results suggest that CPR is a versatile method that permits functional identification of both wild-type and dominant-negative gene products that regulate cellular responses.
Two immediate-early pathogen-responsive members of the
AtCMPG
gene family in
Arabidopsis thaliana
and the W-box-containing elicitor-response element of
AtCMPG1
Authors: Heise Andreas , Lippok Bernadette , Kirsch Christoph , Hahlbrock Klaus
Published: July 2002

The
Arabidopsis thaliana
genome contains at least 50 predicted
AtCMPG
genes. The encoded protein family is defined by a common domain possessing four strictly conserved amino acid residues [Cys, Met, Pro, and Gly (CMPG)] that designate the family. Two members,
AtCMPG1
and
AtCMPG2
, with high sequence similarity to the previously described, immediate-early pathogen-responsive
PcCMPG1
gene from
Petroselinum crispum
were selected for analysis of their expression modes and defense-related promoter elements. Among the most striking similarities with
PcCMPG1
were immediate-early transcriptional activation on infection or treatment with a pathogen-derived elicitor and the functional importance of a W-box-containing
AtCMPG1
promoter element. Remarkably, this strongly pathogen/elicitor-responsive element, F, did not respond to wounding, in contrast to the
AtCMPG1
promoter itself. Comparative analysis, both within the
A. thaliana
genome and across species, provided further insight into the large structural diversity of W-box-containing elements. Possible roles of
At
CMPG proteins in regulatory processes are discussed with reference to a large variety of family members, partly with assigned functions, from plants as well as animals.
MHC class I-dependent Vγ4+pulmonary T cells regulate αβ T cell-independent airway responsiveness
Authors: Lahn Michael , Kanehiro Arihiko , Takeda Katsuyuki , Terry Jennifer , Hahn Youn-Soo , Aydintug M. Kemal , Konowal Anatole , Ikuta Koichi , O'Brien Rebecca L. , Gelfand Erwin W. , Born Willi K.
Published: July 2002

Mice exposed to aerosolized ovalbumin (OVA) develop increased airway responsiveness when deficient in γδ T cells. This finding suggests that γδ T cells function as negative regulators. The regulatory influence of γδ T cells is evident after OVA-sensitization and -challenge, and after OVA-challenge alone, but not in untreated mice. With aerosolized Abs to target pulmonary T cells, we now demonstrate that negative regulation of airway responsiveness is mediated by a small subpopulation of pulmonary γδ T cells. These cells express Vγ4 and depend in their function on the presence of IFN-γ and MHC class I. Moreover, their effect can be demonstrated in the absence of αβ T cells. This novel type of negative regulation seems to precede the development of the adaptive, antigen-specific allergic response.
Cocaine sensitization and reward are under the influence of circadian genes and rhythm
Authors: Abarca Carolina , Albrecht Urs , Spanagel Rainer
Published: July 2002

Investigations using the fruit fly
Drosophila melanogaster
have shown that the circadian clock gene
period
(
Per
) can influence behavioral responses to cocaine. Here we show that the mouse homologues of the
Drosophila Per
gene,
mPer1
and
mPer2
, modulate cocaine sensitization and reward, two phenomena extensively studied in humans and animals because of their importance for drug abuse. In response to an acute cocaine injection
mPer1
and
mPer2
mutant mice as well as wild-type mice exhibited an approximately 5-fold increase in activity compared with saline control levels, showing that there is no initial difference in sensitivity to acute cocaine administration in
Per
mutants. After repeated cocaine injections wild-type mice exhibited a sensitized behavioral response that was absent in
mPer1
knockout mice. In contrast,
mPer2
mutant mice exhibited a hypersensitized response to cocaine. Conditioned place preference experiments revealed similar behavioral reactions:
mPer1
knockout mice showed a complete lack of cocaine reward whereas
mPer2
mutants showed a strong cocaine-induced place preference. In another set of experiments, we tested C57/BL6J mice at different Zeitgeber times and found that cocaine-induced behavioral sensitization and place preference are under the control of the circadian clock. In conclusion, we demonstrate that processes involved in cocaine addiction depend on the circadian rhythm and are modulated in an opposing manner by
mPer1
and
mPer2
genes.
Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages
Authors: Kizaki Takako , Suzuki Kenji , Hitomi Yoshiaki , Taniguchi Naoyuki , Saitoh Daizoh , Watanabe Kenji , Onoé Kazunori , Day Noorbibi K. , Good Robert A. , Ohno Hideki
Published: July 2002

The expression of uncoupling protein 2 (UCP2) was reduced in macrophages after stimulation with lipopolysaccharide (LPS). The physiological consequence and the regulatory mechanisms of the UCP2 down-regulation by LPS were investigated in a macrophage cell line, RAW264 cells. UCP2 overexpression in RAW264 cells transfected with eukaryotic expression vector containingucp2cDNA markedly reduced the production of intracellular reactive oxygen species. Furthermore, in the UCP2 transfectant, nitric oxide (NO) synthesis, inducible NO synthase (NOS II) protein, NOS II mRNA, and NOS II promoter activity were definitely decreased after LPS stimulation compared with those in parental RAW264 or RAW264 cells transfected with the vector alone. Reporter assays suggested that an enhancer element was located in the region of intron 2 of the UCP2 gene and that the UCP2 expression was down-regulated not by the 7.3-kb promoter region but by the 5′ region of the UCP2 gene containing two introns. Deletion of intron 2 resulted in the low transcriptional activities and abolishment of the LPS-associated negative regulation. In addition, the mRNA expression of transfected UCP2 was suppressed in RAW264 cells transfected with expression vector containing UCP2 genomic DNA, but was markedly increased in cells transfected with the vector containing UCP2 intronless cDNA. These findings suggest that the LPS-stimulated signals suppress UCP2 expression by interrupting the function of intronic enhancer, leading to an up-regulation of intracellular reactive oxygen species, which activate the signal transduction cascade of NOS II expression, probably to ensure rapid and sufficient cellular responses to a microbial attack.
Specificity and affinity motifs for Grb2 SH2-ligand interactions
Authors: Kessels Helmut W. H. G. , Ward Alister C. , Schumacher Ton N. M.
Published: July 2002

Protein–protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains. However, in many biological systems specificity of interaction may be of equal or greater importance than affinity. To address this issue we have developed a peptide library screening technology that can be used to directly define ligands for protein domains based on both affinity and specificity of interaction. We demonstrate the value of this approach by the selection of peptide ligands that are either highly specific for the Grb2 Src homology 2 (SH2) domain or that are cross-reactive between a group of related SH2 domains. Examination of previously identified physiological ligands for the Grb2 SH2 domain suggests that for these ligands regulation of the specificity of interaction may be an important factor for
in vivo
ligand selection.
Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from
Mycobacterium leprae
Authors: Reeve Ian , Hummell David , Nelson Nathan , Voss John
Published: July 2002

It has long been recognized that the pathogenicity of a broad range of intracellular parasites depends on the availability of transition metal ions, especially iron. Nramp1 (natural resistance-associated macrophage protein 1), a proton-coupled divalent metal ion transporter, has been identified as a controlling factor in the resistance or susceptibility to infection with a diverse range of intracellular pathogens such as
Toxoplasma
,
Salmonella
,
Mycobacterium
, and
Leishmania
. The role of divalent metal ion transport is even more compelling given the existence of Nramp homologs in several intracellular parasites, such as mycobacteria. We have confirmed the functional homology of the Nramp homologue from
Mycobacterium
leprae
by using a yeast complementation assay for divalent cation uptake. To facilitate a concerted biochemical and structural analysis of this important class of transporters, the
M. leprae
Nramp was expressed in
Escherichia coli
. Dual affinity tags were engineered at the N and C termini to allow for isolation of full-length protein at >95% purity. Site-directed spin labeling of Cys-299 reveals a flexible hinge-like domain. A weak dipolar interaction is detected between the nitroxide and paramagnetic transition ions, indicating this position is approximately 19 Å from the nearest high affinity binding site.
Positional cloning of the murine flavivirus resistance gene
Authors: Perelygin Andrey A. , Scherbik Svetlana V. , Zhulin Igor B. , Stockman Bronislava M. , Li Yan , Brinton Margo A.
Published: July 2002

Inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genusFlavivirus, which includes human pathogens such as yellow fever, Dengue, and West Nile virus. A single gene, designatedFlv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. A positional cloning strategy was used to identify 22 genes from theFlvgene interval including 10 members of the 2′-5′-oligoadenylate synthetase gene family. One 2′-5′-oligoadenylate synthetase gene,Oas1b, was identified asFlvby correlation between genotype and phenotype in nine mouse strains. Susceptible mouse strains produce a protein lacking 30% of the C-terminal sequence as compared with the resistant counterpart because of the presence of a premature stop codon. TheOas1bgene differs from all the other murineOasgenes by a unique four-amino acid deletion in the P-loop located within the conserved RNA binding domain. Expression of the resistant allele ofOas1bin susceptible embryo fibroblasts resulted in partial inhibition of the replication of a flavivirus but not of an alpha togavirus.
Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma
Authors: Gorio Alfredo , Gokmen Necati , Erbayraktar Serhat , Yilmaz Osman , Madaschi Laura , Cichetti Cinzia , Di Giulio Anna Maria , Vardar Enver , Cerami Anthony , Brines Michael
Published: July 2002

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneursym clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.
Direct detection of potential selenium delivery proteins by using an
Escherichia coli
strain unable to incorporate selenium from selenite into proteins
Authors: Lacourciere Gerard M. , Levine Rodney L. , Stadtman Thressa C.
Published: July 2002

Selenium can be metabolized for protein synthesis by two major pathways
in vivo
. In a specific pathway it can be inserted into polypeptide chains as the amino acid selenocysteine, as directed by the UGA codon. Alternatively, selenium can be substituted for sulfur to generate the free amino acids selenocysteine and selenomethionine, and these are incorporated nonspecifically into proteins in place of cysteine and methionine, respectively. A mutant strain of
Escherichia coli
was constructed that is deficient in utilization of inorganic selenium for both specific and nonspecific pathways of selenoprotein synthesis. Disruption of the
cysK
gene prevented synthesis of free cysteine and selenocysteine from inorganic S and Se precursors. Inactivation of the
selD
gene prevented synthesis of selenophosphate, the reactive selenium donor, required for the specific incorporation pathway. As expected, the double mutant strain, RL165ΔselD, when grown anaerobically in LB + glucose medium containing
75
SeO
3
2−
, failed to synthesize selenium-dependent formate dehydrogenase H and seleno-tRNAs. However, it incorporated 24% as much selenium as the wild-type strain. Selenium in the deficient strain was bound to five different proteins. A 39-kDa species was identified as glyceraldehyde-3-phosphate dehydrogenase. It is possible that selenium was bound as a perselenide derivative to the reactive cysteine residue of this enzyme. A 28-kDa protein identified as deoxyribose phosphate aldolase also contained bound selenium. These
75
Se-labeled proteins may have alternate roles as selenium delivery proteins.
Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (
DGAT1
) with variation at a quantitative trait locus for milk fat content
Authors: Winter Andreas , Krämer Wolfgang , Werner Fabian A. O. , Kollers Sonja , Kata Srinivas , Durstewitz Gregor , Buitkamp Johannes , Womack James E. , Thaller Georg , Fries Ruedi
Published: July 2002

DGAT1
encodes diacylglycerol
O
-acyltransferase (EC
2.3.1.20
), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of
DGAT1
are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed
DGAT1
close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of
DGAT1
from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein–Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (
Qq
) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely
qq
at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof.
Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down-regulation
Authors: Bao Jianjun , Oishi Kazuhiko , Yamada Tomohisa , Liu Liqun , Nakamura Akio , Uchida Masaatsu K. , Kohama Kazuhiro
Published: July 2002

GbaSM-4 cells, smooth muscle cells derived from brain basilar artery, which express both 210-kDa long and 130-kDa short isoforms of myosin light chain kinase (MLCK), were infected with an adenovirus vector carrying a 1.4-kb catalytic portion of MLCK–cDNA in an antisense orientation. Western blot analysis showed that the expression of short MLCK was depressed without affecting long MLCK expression. The contraction of the down-regulated cells was measured by the cell-populated collagen-fiber method. The tension development after stimulation with norepinephrine or
A23187
was depressed. The additional infection of the down-regulated cells with the adenovirus construct containing the same insert in a sense direction rescued not only the short MLCK expression but also contraction, confirming the physiological role of short MLCK in the contraction. To examine the role of long MLCK in the residual contraction persisting in the short MLCK-deficient cells, long MLCK was further down-regulated by increasing the multiplicity of infection of the antisense construct. The additional down-regulation of long MLCK expression, however, did not alter the residual contraction, ruling out the involvement of long MLCK in the contractile activity. Further, in the cells where short MLCK was down-regulated specifically, the extent of phosphorylation of 20-kDa myosin light chain (MLC20) after the agonist stimulation was not affected. This finding suggests that there are additional factors to MLC20 phosphorylation that contribute to regulate smooth muscle contraction.
Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responsesexvivo
Authors: Whiteside Theresa L. , Gambotto Andrea , Albers Andreas , Stanson Joanna , Cohen Edward P.
Published: July 2002

This article describes a DNA-based vaccination strategy evaluatedex vivowith human cells. The vaccine was prepared by transferring tumor-derived genomic DNA to PCI-13 cells, a highly immunogenic tumor cell line (“recipient cell”), which had been genetically modified to secrete IL-2 (PCI-13/IL-2). PCI-13 cells expressed class I MHC determinants (HLA-A2) shared with the tumor from which the DNA was obtained as well as allogeneic determinants. DNA from a gp100+melanoma cell line was transduced into gp100−PCI-13/IL-2 cells (PCI-13/IL-2/DNA). A T cell line specific for the gp100 epitope responded to PCI-13/IL-2/DNA cells by IFN-γ-secretion measured in enzyme-linked immunospot assays. The T cell line also recognized the gp100 epitope presented by dendritic cells that ingested PCI-13/IL-2/DNA cells, which had been induced by UVB irradiation to undergo apoptosis. After up-take and processing of apoptotic PCI-13/IL-2/DNA cells, the dendritic cells primed normal peripheral blood lymphocytes to generate effector T cells specific for the tumor donating the DNA. The results indicate that tumor epitopes encoded in such DNA are expressed in recipient cells and can induce tumor-specific T cells. The findings support translation of this vaccination strategy to a phase I trial in patients with cancer.
Maternal transmission of
P
element transposase activity in
Drosophila melanogaster
depends on the last
P
intron
Authors: Simmons Michael J. , Haley Kevin J. , Thompson Sarah J.
Published: July 2002

Maternal transmission of RNAs or proteins through the egg cytoplasm plays an important role in eukaryotic development. We show that the transposase activity encoded by the
P
transposable element of
Drosophila melanogaster
is transmitted through the oocytes of females heterozygous for this element even when these oocytes do not carry the element itself. However, this maternal transmission is abolished when the last of three introns is removed from the
P
element. These facts imply that maternal transmission of transposase activity involves the RNA transcribed from the
P
element rather than the polypeptide it encodes, and that to be transmitted maternally, this RNA must possess the last intron. Examination of the intron's sequence reveals that it contains a motif of nine nucleotides that has been implicated in the maternal transmission of developmentally significant RNAs. This same intron limits expression of the P transposase to the germ line of
Drosophila
. Thus, the last
P
intron has two important biological functions.
The relationship between cortical activation and perception investigated with invisible stimuli
Authors: Moutoussis K. , Zeki S.
Published: July 2002

The aim of this work was to study the relationship between cortical activity and visual perception. To do so, we developed a psychophysical technique that is able to dissociate the visual percept from the visual stimulus and thus distinguish brain activity reflecting the perceptual state from that reflecting other stages of stimulus processing. We used dichoptic color fusion to make identical monocular stimuli of opposite color contrast “disappear” at the binocular level and thus become “invisible” as far as conscious visual perception is concerned. By imaging brain activity in subjects during a discrimination task between face and house stimuli presented in this way, we found that house-specific and face-specific brain areas are always activated in a stimulus-specific way regardless of whether the stimuli are perceived. Absolute levels of cortical activation, however, were lower with invisible stimulation compared with visible stimulation. We conclude that there is no terminal “perceptual” area in the visual brain, but that the brain regions involved in processing a visual stimulus are also involved in its perception, the difference between the two being dictated by a higher level of activity in the specific brain region when the stimulus is perceived.
Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis
Authors: Chai Yaohui , Shao Jianqiang , Miller Victor M. , Williams Aislinn , Paulson Henry L.
Published: July 2002

Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent protein-tagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.
Individual adjustment of sperm expenditure accords with sperm competition theory
Authors: Pilastro Andrea , Scaggiante Marta , Rasotto Maria B.
Published: July 2002

Sperm competition theory predicts that males should strategically allocate their sperm reserves according to the level of sperm competition, defined as the probability that the sperm of two males compete for fertilizing a given set of ova. Substantial evidence from numerous animal taxa suggests that, at the individual level, sperm expenditure increases when the risk of sperm competition is greater. In contrast, according to the “intensity model” of sperm competition [Parker, G. A., Ball, M. A., Stockley, P. & Gage, M. J. G. (1996)
Proc. R. Soc. London Ser. B
263, 1291–1297], when more than two ejaculates compete during a given mating event, sperm expenditure should decrease as the number of competing males increases. Empirical evidence supporting this prediction, however, is still lacking. Here we measured sperm expenditure in two gobiid fishes, the grass (
Zosterisessor ophiocephalus
) and black goby (
Gobius niger
), in which up to six sneakers can congregate around the nest of territorial males and release their sperm when females spawn. We show that, in accordance with theory, sneaker males of both species release fewer sperm as the number of competitors increases.
A 76-kb duplicon maps close to the BCR gene on chromosome 22 and the ABL gene on chromosome 9: Possible involvement in the genesis of the Philadelphia chromosome translocation
Authors: Saglio Giuseppe , Storlazzi Clelia T. , Giugliano Emilia , Surace Cecilia , Anelli Luisa , Rege-Cambrin Giovanna , Zagaria Antonella , Velasco Antonio Jimenez , Heiniger Anabel , Scaravaglio Patrizia , Gomez Antonio Torres , Gomez Josè Roman , Archidiacono Nicoletta , Banfi Sandro , Rocchi Mariano
Published: July 2002

A patient with a typical form of chronic myeloid leukemia was found to carry a large deletion on the derivative chromosome 9q+ and an unusual BCR-ABL transcript characterized by the insertion, between BCR exon 14 and ABL exon 2, of 126 bp derived from a region located on chromosome 9, 1.4 Mb 5′ to ABL. This sequence was contained in the bacterial artificial chromosome RP11-65J3, which in fluorescence
in situ
hybridization experiments on normal metaphases was found to detect, in addition to the predicted clear signal at 9q34, a faint but distinct signal at 22q11.2, where the BCR gene is located, suggesting the presence of a large region of homology between the two chromosomal regions. Indeed,
blast
analysis of the RP11-65J3 sequence against the entire human genome revealed the presence of a stretch of homology, about 76 kb long, located approximately 150 kb 3′ to the BCR gene, and containing the 126-bp insertion sequence. Evolutionary studies using fluorescence
in situ
hybridization identified the region as a duplicon, which transposed from the region orthologous to human 9q34 to chromosome 22 after the divergence of orangutan from the human-chimpanzee-gorilla common ancestor about 14 million years ago. Recent sequence analyses have disclosed an unpredicted extensive segmental duplication of our genome, and the impact of duplicons in triggering genomic disorders is becoming more and more apparent. The discovery of a large duplicon relatively close to the ABL and BCR genes and the finding that the 126-bp insertion is very close to the duplicon at 9q34 open the question of the possible involvement of the duplicon in the formation of the Philadelphia chromosome translocation.
Multiple centrosomes arise from tetraploidy checkpoint failure and mitotic centrosome clusters in p53 and RB pocket protein-compromised cells
Authors: Borel Franck , Lohez Olivier D. , Lacroix Françoise B. , Margolis Robert L.
Published: July 2002

A high degree of aneuploidy characterizes the majority of human tumors. Aneuploid status can arise through mitotic or cleavage failure coupled with failure of tetraploid G
1
checkpoint control, or through deregulation of centrosome number, thus altering the number of mitotic spindle poles. p53 and the RB pocket proteins are important to the control of G
1
progression, and p53 has previously been suggested as important to the control of centrosome duplication. We demonstrate here that neither suppression of p53 nor of the RB pocket protein family directly generates altered centrosome numbers in any of several mammalian primary cell lines. Instead, amplification of centrosome number occurs in two steps. The first step is failure to arrest at a G
1
tetraploidy checkpoint after failure to segregate the genome in mitosis, and the second step is clustering of centrosomes at a single spindle pole in subsequent tetraploid or aneuploid mitosis. The trigger for these events is mitotic or cleavage failure that is independent of p53 or RB status. Finally, we find that mouse embryo fibroblasts spontaneously enter tetraploid G
1
, explaining the previous demonstration of centrosome amplification by p53 abrogation alone in these cells.
Designed to penetrate: Time-resolved interaction of single antibiotic molecules with bacterial pores
Authors: Nestorovich Ekaterina M. , Danelon Christophe , Winterhalter Mathias , Bezrukov Sergey M.
Published: July 2002

Membrane permeability barriers are among the factors contributing to the intrinsic resistance of bacteria to antibiotics. We have been able to resolve single ampicillin molecules moving through a channel of the general bacterial porin, OmpF (outer membrane protein F), believed to be the principal pathway for the β-lactam antibiotics. With ion channel reconstitution and high-resolution conductance recording, we find that ampicillin and several other efficient penicillins and cephalosporins strongly interact with the residues of the constriction zone of the OmpF channel. Therefore, we hypothesize that, in analogy to substrate-specific channels that evolved to bind certain metabolite molecules, antibiotics have “evolved” to be channel-specific. Molecular modeling suggests that the charge distribution of the ampicillin molecule complements the charge distribution at the narrowest part of the bacterial porin. Interaction of these charges creates a region of attraction inside the channel that facilitates drug translocation through the constriction zone and results in higher permeability rates.
Endo-β-
N
-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol
Authors: Suzuki Tadashi , Yano Keiichi , Sugimoto Seiji , Kitajima Ken , Lennarz William J. , Inoue Sadako , Inoue Yasuo , Emori Yasufumi
Published: July 2002

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-β-
N
-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in
Drosophila melanogaster, Caenorhabditis elegans
, and
Arabidopsis thaliana,
indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.
The outer membrane localization of the
Neisseria gonorrhoeae
MsrA/B is involved in survival against reactive oxygen species
Authors: Skaar Eric P. , Tobiason Deborah M. , Quick J. , Judd Ralph C. , Weissbach Herbert , Etienne Frantzy , Brot Nathan , Seifert H. Steven
Published: July 2002

The PilB protein of
Neisseria gonorrhoeae
has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine sulfoxide reductase family of enzymes, specifically MsrA and MsrB from
Escherichia coli
. MsrA and MsrB in
E. coli
are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of
Neisseria gonorrhoeae
is not involved in pilus expression. Additionally, we show that wild-type
N. gonorrhoeae
produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage.
Effects of cerebral ischemia in mice deficient in
Persephin
Authors: Tomac Andreas C. , Agulnick Alan D. , Haughey Norman , Chang Chen-Fu , Zhang Yajun , Bäckman Cristina , Morales Marisela , Mattson Mark P. , Wang Yun , Westphal Heiner , Hoffer Barry J.
Published: July 2002

Persephin
(Pspn), a recently cloned member of the transforming growth factor-β superfamily (TGF-β) and glial cell line-derived neurotrophic factor (GDNF) subfamily, is distributed throughout the nervous system at extremely low levels and is thought to function as a survival factor for midbrain dopaminergic and spinal motor neurons
in vivo
. Here, we report that mice lacking Pspn by homologous recombination show normal development and behavior, but are hypersensitive to cerebral ischemia. A 300% increase in infarction volume was observed after middle cerebral artery occlusion. We find that glutamate-induced Ca
2+
influx, thought to be a major component of ischemic neuronal cell death, can be regulated directly by the
Persephin
protein (PSP) and that PSP can reduce hypoxia/reperfusion cell death
in vitro
. Neuronal cell death can be prevented or markedly attenuated by administration of recombinant human PSP
in vivo
before ischemia in both mouse and rat models. Taken together, these data indicate that PSP is a potent modulator of excitotoxicity in the central nervous system with pronounced neuroprotective activity. Our findings support the view that PSP signaling can exert an important control function in the context of stroke and glutamate-mediated neurotoxicity, and also suggest that future therapeutic approaches may involve this novel trophic protein.
A postsynaptic transient K
+
current modulated by arachidonic acid regulates synaptic integration and threshold for LTP induction in hippocampal pyramidal cells
Authors: Ramakers Geert M. J. , Storm Johan F.
Published: July 2002

Voltage-gated ion channels in the dendrites and somata of central neurons can modulate the impact of synaptic inputs. One of the ionic currents contributing to such modulation is the fast inactivating A-type potassium current (
I
A
). We have investigated the role of
I
A
in synaptic integration in rat CA1 pyramidal cells by using arachidonic acid (AA) and heteropodatoxin-3 (HpTX3), a selective blocker of the Kv4 channels underlying much of the somatodendritic
I
A
. AA and HpTX3 each reduced
I
A
by 60–70% (measured at the soma) and strongly enhanced the amplitude and summation of excitatory postsynaptic responses, thus facilitating action potential discharges. HpTX3 also reduced the threshold for induction of long-term potentiation. We conclude that the postsynaptic
I
A
is activated during synaptic depolarizations and effectively regulates the somatodendritic integration of high-frequency trains of synaptic input. AA, which can be released by such input, enhances synaptic efficacy by suppressing
I
A
, which could play an important role in frequency-dependent synaptic plasticity in the hippocampus.
A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors
Authors: Andersson Patrik , McGuire Jacqueline , Rubio Carlos , Gradin Katarina , Whitelaw Murray L. , Pettersson Sven , Hanberg Annika , Poellinger Lorenz
Published: July 2002

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.
Working artefacts: ethnomethods of the prototype
Authors: Suchman Lucy , Trigg Randall , Blomberg Jeanette
Published: July 2002

This paper follows recent science studies in theorizing information technologies as socio‐material configurations, aligned into more and less durable forms. The study of how new technologies emerge shifts, on this view, from a focus on invention to an interest in ongoing practices of assembly, demonstration, and performance. This view is developed in relation to the case of the ‘prototype’, an exploratory technology designed to effect alignment between the multiple interests and working practices of technology research and development, and sites of technologies‐in‐use. In so far as it is successful, the prototype works as an exemplary artefact that is at once intelligibly familiar to the actors involved, and recognizably new.
Roundtable: The Progress of Heterodox Economics
Authors: Coats A. W. Bob , Backhouse Roger E. , Dow Sheila C. , Fusfeld Daniel R. , Goodwin Craufurd D. , Rutherford Malcolm
Published: July 2002

The central theme of this session is the changing relationship between “orthodox” (i.e., mainstream, neoclassical) and “heterodox” economics, especially in the USA, during the past two or three decades. Economics is such a large and heterogeneous discipline that it cannot be characterized both briefly and accurately. Alongside the growth of formalization and mathematization, and the high degree of uniformity in the undergraduate and graduate curricula and in the leading textbooks, there are also within the subject a number of dissenting or deviant doctrinal schools, rival methodological approaches, and innovative developments designed to remedy its defects and/or overcome its limitations. Moreover, many of the outspoken criticisms of the status quo, proposed remedies, and innovations, originate with or are endorsed by prominent economists with impeccable professional credentials. Indeed, in some cases their contributions threaten the discipline's foundations and can, therefore, be considered a species of “orthodox subversion.”
Understanding Institutional Economics: 1918–1929
Authors: Rutherford Malcolm
Published: July 2002

All attempts to define American institutionalism, whether in terms of a set of key methodological or theoretical principles or in terms of the contributions of the three generally accepted “founding” figures of Thorstein Veblen, Wesley Mitchell, and John R. Commons, have run into a problem with the apparent disparities within the movement. In terms of the three “founders” there are obvious and quite dramatic differences between the methodologies and theoretical directions of the three men. Veblen is associated with an evolutionary approach, a key distinction between pecuniary institutions and technological or industrial requirements, and a biting critique of orthodox theory and business practices; Mitchell with quantitative methods and detailed research on business cycles, an approach he established at the National Bureau of Economic Research (NBER); Commons with documentary histories, work on labor issues and public utility regulation, and an analytical scheme emphasizing the evolution of legal institutions and processes of dispute resolution. The same problem shows up with more explicit types of definition that often seem to capture only some parts or aspects of the movement and not others, or are so broad as to lack much specific content. Institutionalism easily appears as incoherent, as little more than a set of individual research programs with nothing in common other than a questioning of more orthodox theory and method. Thus, Mark Blaug has stated that institutionalism “was never more than a tenuous inclination to dissent from orthodox economics” (Blaug 1978, p. 712), and George Stigler has claimed that institutionalism had “no positive agenda of research,” “no set of problems or new methods,” nothing but “a stance of hostility to the standard theoretical tradition” (quoted in Kitch 1983, p. 170). This view still finds wide currency— for example Oliver Williamson has recently argued that “unable or unwilling to offer a rival research agenda, the older institutional economics was given over to methodological objections to orthodoxy” (Williamson 1998b, p. 24; see also 1998a).
Perspectives on Allyn Young in Theories of Endogenous Growth
Authors: Sandilands Roger J.
Published: July 2002

A central message of Allyn Abbott Young's (1928) seminal paper on increasing returns and economic progress was the seeming tautology that “the division of labor depends in large part upon the division of labor.” From this he deduced that a major source of growth is growth itself. In this fundamental sense growth is endogenous and cumulative. He spoke of the “togetherness” of economic phenomena and doubted that the apparatus of supply and demand and marginal productivity theory could be integrated to give the social picture or explain why growth tends to be self-sustaining rather than self-exhausting.