bluuebunny/crossref_metadata_2025 huggingface dataset:
The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development
Authors: Yang Huang-Tian , Tweedie David , Wang Su , Guia Antonio , Vinogradova Tatiana , Bogdanov Konstantin , Allen Paul D. , Stern Michael D. , Lakatta Edward G. , Boheler Kenneth R.
Published: July 2002

In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca
2+
release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca
2+
release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.
Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts
Authors: Zhang Wen-Hui , Svarovskaia Evguenia S. , Barr Rebekah , Pathak Vinay K.
Published: July 2002

Using
in vivo
fidelity assays in which bacterial β-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate ≈5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine–thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template–primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adenine–thymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template–primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template–primer conformation at the polymerase active site and that the MLV Y586 residue and template–primer conformation are important determinants of RT fidelity.
Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes
Authors: Paquin Joanne , Danalache Bogdan A. , Jankowski Marek , McCann Samuel M. , Gutkowska Jolanta
Published: July 2002

We recently discovered the existence of the oxytocin/oxytocin receptor (OT/OTR) system in the heart. Activation of cardiac OTR stimulates the release of atrial natriuretic peptide (ANP), which is involved in regulation of blood pressure and cell growth. Having observed elevated OT levels in the fetal and newborn heart at a stage of intense cardiomyocyte hyperplasia, we hypothesized a role for OT in cardiomyocyte differentiation. We used mouse P19 embryonic stem cells to substantiate this potential role. P19 cells give rise to the formation of cell derivatives of all germ layers. Treatment of P19 cell aggregates with dimethyl sulfoxide (DMSO) induces differentiation to cardiomyocytes. In this work, P19 cells were allowed to aggregate from day 0 to day 4 in the presence of 0.5% DMSO, 10
−7
M OT and/or 10
−7
M OT antagonist (OTA), and then cultured in the absence of these factors until day 14. OT alone stimulated the production of beating cell colonies in all 24 independently growing cultures by day 8 of the differentiation protocol, whereas the same result was obtained in cells induced by DMSO only after 12 days. Cells induced with OT exhibited increased ANP mRNA, had abundant mitochondria (i.e., they strongly absorbed rhodamine 123), and expressed sarcomeric myosin heavy chain and dihydropyridine receptor-α1, confirming a cardiomyocyte phenotype. In addition, OT as well as DMSO increased OTR protein and OTR mRNA, and OTA completely inhibited the formation of cardiomyocytes in OT- and DMSO-supplemented cultures. These results suggest that the OT/OTR system plays an important role in cardiogenesis by promoting cardiomyocyte differentiation.
Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array
Authors: McKendry Rachel , Zhang Jiayun , Arntz Youri , Strunz Torsten , Hegner Martin , Lang Hans Peter , Baller Marko K. , Certa Ulrich , Meyer Ernst , Güntherodt Hans-Joachim , Gerber Christoph
Published: July 2002

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically
in situ
. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3′ and 5′ overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.
Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death
Authors: Kim Min-Jung , Jo Dong-Gyu , Hong Gil-Sun , Kim Byung Ju , Lai Michael , Cho Dong-Hyung , Kim Ki-Woo , Bandyopadhyay Arun , Hong Yeon-Mi , Kim Do Han , Cho Chunghee , Liu Jun O. , Snyder Solomon H. , Jung Yong-Keun
Published: July 2002

Cain/cabin1 is an endogenous inhibitor of calcineurin (Cn), a calcium-dependent serine/threonine phosphatase involved in various cellular functions including apoptosis. We show here that during apoptosis cain/cabin1 is cleaved by calpain at the carboxyl terminus to generate a cleavage product with a molecular mass of 32 kDa as a necessary step leading to Cn-mediated cell death. Mouse cain/cabin1 was identified from a thymus cDNA library by anin vitrosubstrate-screening assay with calpain. Exposure of Jurkat cells to the calcium ionophore,A23187, induced cain/cabin1 cleavage and cell death, accompanied by activation of calpain and Cn. The calpain inhibitors, calpeptin and zLLY, suppressed bothA23187-induced cain/cabin1 cleavage and Cn activation, indicating that Cn activation and cain/cabin1 cleavage are calpain-dependent. Expression of cain/cabin1 or a catalytically inactive Cn mutant [CnAβ2(1–401/H160N)] and treatment with FK506 reducedA23187-induced cell death.In vitrocalpain cleavage and immunoprecipitation assays with deletion mutants of cain/cabin1 showed that cleavage occurred in the Cn-binding domain of cain/cabin1, indicating that the cleavage at its C terminus by calpain prevented cain/cabin1 from binding to Cn. In addition,in vitrobinding assays showed that cain/cabin1 bound to the Cn B-binding domain of Cn A. Taken together, these results indicate that calpain cleaves the calcineurin-binding domain of cain/cabin1 to activate Cn and elicit calcium-triggered cell death.
Crystal structure of monomeric human β-2-microglobulin reveals clues to its amyloidogenic properties
Authors: Trinh Chi H. , Smith David P. , Kalverda Arnout P. , Phillips Simon E. V. , Radford Sheena E.
Published: July 2002

Dissociation of human β-2-microglobulin (β
2
m) from the heavy chain of the class I HLA complex is a critical first step in the formation of amyloid fibrils from this protein. As a consequence of renal failure, the concentration of circulating monomeric β
2
m increases, ultimately leading to deposition of the protein into amyloid fibrils and development of the disorder, dialysis-related amyloidosis. Here we present the crystal structure of a monomeric form of human β
2
m determined at 1.8-Å resolution that reveals remarkable structural changes relative to the HLA-bound protein. These involve the restructuring of a β bulge that separates two short β strands to form a new six-residue β strand at one edge of this β sandwich protein. These structural changes remove key features proposed to have evolved to protect β sheet proteins from aggregation [Richardson, J. & Richardson, D. (2002)
Proc. Natl. Acad. Sci. USA
99, 2754–2759] and replaces them with an aggregation-competent surface. In combination with solution studies using
1
H NMR, we show that the crystal structure presented here represents a rare species in solution that could provide important clues about the mechanism of amyloid formation from the normally highly soluble native protein.
A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity
Authors: Kao Richard Y. T. , Jenkins Jeremy L. , Olson Karen A. , Key Marc E. , Fett James W. , Shapiro Robert
Published: July 2002

The results of previous preclinical and clinical studies have identified angiogenin (ANG) as a potentially important target for anticancer therapy. Here we report the design and implementation of a high-throughput screening assay to identify small molecules that bind to the ribonucleolytic active site of ANG, which is critically involved in the induction of angiogenesis by this protein. Screening of 18,310 compounds from the National Cancer Institute (NCI) Diversity Set and ChemBridge DIVERSet yielded 15 hits that inhibit the enzymatic activity of ANG with
K
i
values <100 μM. One of these, NCI compound 65828 [8-amino-5-(4′-hydroxybiphenyl-4ylazo)naphthalene-2-sulfonate;
K
i
= 81 μM], was selected for more detailed studies. Minor changes in ANG or ligand structure markedly reduced potency, demonstrating that inhibition reflects active-site rather than nonspecific binding; these observations are consistent with a computationally generated model of the ANG⋅65828 complex. Local treatment with modest doses of 65828 significantly delayed the formation of s.c. tumors from two distinct human cancer cell types in athymic mice. ANG is the likely target involved because (
i
) a 65828 analogue with much lower potency against the enzymatic activity of ANG failed to exert any antitumor effect, (
ii
) tumors from 65828-treated mice had fewer interior blood vessels than those from control mice, and (
iii
) 65828 appears to have no direct effect on the tumor cells. Our findings provide considerable support for the targeting of the enzymatic active site of ANG as a strategy for developing new anticancer drugs.
Characterization of three alternatively spliced isoforms of the Rel/NF-κB transcription factor Relish from the mosquito
Aedes
aegypti
Authors: Shin Sang Woon , Kokoza Vladimir , Ahmed Abduelaziz , Raikhel Alexander S.
Published: July 2002

The Rel/NF-κB transcription factor Relish performs a central role in the acute-phase response to microbial challenge by activating immune antibacterial peptides. We cloned and molecularly characterized the gene homologous to
Drosophila
Relish from the mosquito
Aedes aegypti
. Unlike
Drosophila
Relish,
Aedes
Relish has three alternatively spliced transcripts encoding different proteins. First, the predominant
Aedes
Relish transcript of 3.9 kb contains both the Rel-homology domains and the inhibitor κB (IκB)-like domain, which is similar to
Drosophila
Relish and to the mammalian p105 and p100 Rel/NF-κB transcription factors. Second,
Aedes
Relish transcript contains Rel-homology domains identical to those of the major transcript but it completely lacks the IκB-like domain-coding region, which has been replaced by a unique 3′-untranslated region sequence. In the third transcript, a deletion replaces most of the N-terminal sequence and Rel-homology domains; however, the IκB-like domain is intact. All three
Aedes
Relish transcripts were induced by bacterial injection but not by blood feeding.
In vitro
-translated protein from the Rel-only construct specifically binds to the κB motif from
Drosophila
cecropin A1 and
Aedes
defensin genes. PCR and Southern blot hybridization analyses show that these three transcripts originated from the same large inducible mRNA encoded by a single Relish gene.
Atm
heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts
Authors: Worgul Basil V. , Smilenov Lubomir , Brenner David J. , Junk Anna , Zhou Wei , Hall Eric J.
Published: July 2002

It is important to know whether the human population includes genetically predisposed radiosensitive subsets.
In vitro
studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the
ATM
gene (
ATM
+/−
) may be slightly more radiosensitive
in vitro
, it remained to be determined whether the greater susceptibility of
ATM
+/−
cells translates into an increased sensitivity for late effects
in vivo
, though there is a suggestion that radiotherapy patients that are heterozygous for the
ATM
gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type,
Atm
heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that
ATM
heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are
ATM
heterozygotes could be predisposed to increased late normal tissue damage.
Insecticide use and organophosphate resistance in the coffee leaf miner Leucoptera coffeella (Lepidoptera: Lyonetiidae)
Authors: Fragoso D.B. , Guedes R.N.C. , Picanço M.C. , Zambolim L.
Published: July 2002

Increasing rates of insecticide use against the coffee leaf miner Leucoptera coffeella (Guérin-Méneville) and field reports on insecticide resistance led to an investigation of the possible occurrence of resistance of this species to some of the oldest insecticides used against it in Brazil: chlorpyrifos, disulfoton, ethion and methyl parathion. Insect populations were collected from ten sites in the state of Minas Gerais, Brazil and these populations were subjected to discriminating concentrations established from insecticide LC99s estimated for a susceptible standard population. Eight of the field-collected populations showed resistance to disulfoton, five showed resistance to ethion, four showed resistance to methyl parathion, and one showed resistance to chlorpyrifos. The frequency of resistant individuals in each population ranged from 10 to 93% for disulfoton, 53 to 75% for ethion, 23 to 76% for methyl parathion, and the frequency of resistant individuals in the chlorpyrifos resistant population was 35%. A higher frequency of individuals resistant to chlorpyrifos, disulfoton and ethion was associated with greater use of insecticides, especially other organophosphates. This finding suggests that cross-selection, mainly between organophosphates, played a major role in the evolution of insecticide resistance in Brazilian populations of L. coffeella. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide and triphenyl phosphate) suggested that cytochrome P450-dependent monooxygenases may play a major role in resistance with minor involvement of esterases and glutathione S-transferases.
Tunnel specificity and forager movement in subterranean termites (Isoptera: Rhinotermitidae and Termitidae)
Authors: Evans T.A.
Published: July 2002

The movement of foragers of two species of Australian, subterranean, mound-building termites, Coptotermes lacteus (Froggatt) (Rhinotermitidae) and Nasutitermes exitiosus (Hill) (Termitidae), was investigated in their natural habitat using artificial feeding sites along trenches dug to mimic natural forager tunnels that radiate out from the central mound-nests. Termites were dyed by self-feeding on cardboard soaked with histological fat-stains on one or two trenches and then termites were collected from other feeding sites at two and four weeks after the fat-stains were placed. At two and four weeks after marking commenced, 60–75% of marked termites were found in trenches containing the marked paper, and 2–16% were found in trenches on the opposite side of the nest. The proportion of marked termites in a sample was three to eight times greater in the trenches containing the marked paper relative to other trenches. Although difficulties with fat-stains used as markers might explain some of the observed patterns, it is evident that C. lacteus and N. exitiosus foragers do not move randomly between feeding sites in their natural habitat.
Alternative antecedents, probabilities, and the suppression of fallacies in Wason's selection task
Authors: Handley Simon J. , Feeney Aidan , Harper Catherine
Published: July 2002

Three experiments examined the influence of a second rule on the pattern of card selections on Wason's selection task. In Experiment 1 participants received a version of the task with a single test rule or one of two versions of the task with the same original test rule together with a second rule. The probability of q was manipulated in the two-rules conditions by varying the size of the antecedent set in the second rule. The results showed a significant suppression of q card and not-p card selections in the alternative-rule conditions, but no difference as a function of antecedent set size. In Experiment 2 the size of the antecendent set in the two-rules conditions was manipulated using the context of a computer printing double-sided cards. The results showed a significant reduction of q card selections in the two-rules conditions, but no effect of p set size. In Experiment 3 the scenario accompanying the rule was manipulated, and it specified a single alternative antecedent or a number of alternative antecedents. The q card selection rates were not affected by the scenario manipulation but again were suppressed by the presence of a second rule. Our results suggest that people make inferences about the unseen side of the cards when engaging with the task and that these inferences are systematically influenced by the presence of a second rule, but are not influenced by the probabilistic characteristics of this rule. These findings are discussed in the context of decision theoretic views of selection task performance (Oaksford & Chater, 1994).
Insensitivity of visual short-term memory to irrelevant visual information
Authors: Andrade Jackie , Kemps Eva , Werniers Yves , May Jon , Szmalec Arnaud
Published: July 2002

Several authors have hypothesized that visuo-spatial working memory is functionally analogous to verbal working memory. Irrelevant background speech impairs verbal short-term memory. We investigated whether irrelevant visual information has an analogous effect on visual short-term memory, using a dynamic visual noise (DVN) technique known to disrupt visual imagery (Quinn & McConnell, 1996b). Experiment 1 replicated the effect of DVN on pegword imagery. Experiments 2 and 3 showed no effect of DVN on recall of static matrix patterns, despite a significant effect of a concurrent spatial tapping task. Experiment 4 showed no effect of DVN on encoding or maintenance of arrays of matrix patterns, despite testing memory by a recognition procedure to encourage visual rather than spatial processing. Serial position curves showed a one-item recency effect typical of visual short-term memory. Experiment 5 showed no effect of DVN on short-term recognition of Chinese characters, despite effects of visual similarity and a concurrent colour memory task that confirmed visual processing of the characters. We conclude that irrelevant visual noise does not impair visual short-term memory. Visual working memory may not be functionally analogous to verbal working memory, and different cognitive processes may underlie visual short-term memory and visual imagery.
Representation and retrieval of arithmetical facts: Developmental difficulties
Authors: Temple Christine M. , Sherwood Susan
Published: July 2002

One form of developmental difficulty with arithmetic affects the storage or retrieval of arithmetical facts, such as tables, which are required to implement arithmetical computations (Temple, 1991, 1994). Such difficulties may arise because of impairment in a specialized system for the storage of arithmetical facts or as a result of causally linked impairment in another cognitive domain. This study explored issues concerning the representation and retrieval of arithmetical facts in children with number fact disorders (NF) and in normal children, in particular the status of hypothesized linked impairments: short-term memory (STM) spans, counting skills, speed of speech, and speed of number fact and lexical retrieval. There was no evidence that NF children had weak STM spans on any span measure or that STM spans related to arithmetical fact skills. There was also no evidence that NF children had weak counting abilities or free counting speeds. The NF children were slower in speeded counting, which also correlated with number fact skill. The significance or not of this is discussed. The NF children were also slower than controls in speed of speech and on some measures of speed of access. However, the absence of correlation with number fact skill, the absence of generality across tasks, and the possibility that delayed speeds in fact retrieval reflect the use of alternative strategies, together suggest that the increased speeds are not causally linked to number fact skill. The results are consistent with modular accounts, in which there is a specialized system for the storage and retrieval of arithmetical facts.
Outcome and Cue Properties Modulate Blocking
Authors: De Houwer Jan , Beckers Tom , Glautier Steven
Published: July 2002

Participants saw a series of situations in which a cue (a light appearing at a certain position) could be followed by an outcome (a drawing of a tank that exploded) and were afterwards asked to rate the likelihood of the outcome in the presence of the cue. In Experiments 1 and 2, the compound cues AT and KL were always followed by the outcome (AT+, KL+). During an elemental phase that either preceded or followed the compound phase, Cue A was also paired with the outcome (A+). Cue T elicited a lower rating than Cues K and L when cues were described as being weapons but not when the cues were said to be indicators. The magnitude of this blocking effect was also influenced by whether the outcome occurred to a maximal or submaximal extent. Experiment 3 replicated the effect of cue instructions on blocking (A+, AT+) but showed that cue instructions had no impact on reduced overshadowing (B-, BT+). The results shed new light on previous findings and support probabilistic contrast models of human contingency judgements.
Effect of mental imagery on realizing affordances
Authors: Boschker Marc S. J. , Bakker Frank C. , Michaels Claire F.
Published: July 2002

Using a reaction time experiment, we examined whether imagining a response would lead to an increase in the frequency of its execution. During a pre-test and a post-test, participants had to respond as quickly as possible with either their left or their right hand, as they preferred, to the illumination of one of 17 target positions arrayed in front of them in a semicircle. Between these two phases, participants performed a practice condition. Each of 40 right-handed participants was assigned to one of four groups that differed in their practice condition: One group made only dominant-hand responses to all target locations, two imagery groups imagined dominant hand responses to all target locations, and the last group received a no-practice, control task. One imagery group received instructions emphasizing that imagery has a strong effect; the second group received instructions suggesting that imagery was not effective. The results showed an increased incidence of the practised response for both imagery groups during the post-test. No effect was found for the physical performance group and the control group. The change in performance for the imagery groups was not accompanied by a change in reaction time. The results are discussed in terms of imagining the realization of action possibilities and from a neuropsychological point of view.
Authorship effects in the prediction of handwriting strokes: Evidence for action simulation during action perception
Authors: Knoblich Günther , Seigerschmidt Eva , Flach Rüdiger , Prinz Wolfgang
Published: July 2002

Does the action system contribute to action perception? Recent evidence suggests that actions are simulated while being observed. Given that the planning and simulating system are the same only when one observes one's own actions, it might be easier to predict the future outcomes of actions when one has carried them out oneself earlier on. In order to test this hypothesis, three experiments were conducted in which participants observed parts of earlier self- and other-produced trajectories and judged whether another stroke would follow or not. When the trajectories were produced without constraints, participants accomplished this task only for self-produced trajectories. When the trajectories were produced under narrow constraints, the predictions were equally accurate for self- and for other-generated trajectories. These results support the action simulation assumption. The more the actions that one observes resemble the way one would carry them out oneself, the more accurate the simulation.
Human agency and associative learning: Pavlovian principles govern social process in causal relationship detection
Authors: Cramer Robert Ervin , Weiss Robert Frank , William Robin , Reid Suzanne , Nieri Lia , Manning-Ryan Barbara
Published: July 2002

Estimates of a worker's causal relationship (CR) to production obeyed associative principles, despite the participants’ a priori beliefs that workers are responsible or “at cause” for production. In three experiments, social analogues of conditioned stimuli (workers) and unconditioned stimuli (company production information) were manipulated in familiar Pavlovian paradigms. The findings included (1) CR acquisition, (2) unconditioned stimulus-intensity effects, and (3) CR blocking. The research plan employed an approach that Neal Miller (1959) termed “extension of liberalized S-R theory” and drew on the Rescorla-Wagner model to integrate the experimental results, to illuminate the empirical data of social attribution research, and to guide the study of causal relationship detection using social stimuli.
The Prospects of Heterodox Economics: a Comment
Authors: Rutherford Malcolm
Published: July 2002

My comments on the papers given by Bob Coats, Roger Backhouse, and Sheila Dow will focus on the history and prospects of institutional economics, as that is the part of the heterodox world I know most about.
A Rejoinder to Abraham Hirsch
Authors: Hollander Samuel , Peart Sandra
Published: July 2002

The dispute between Hollander and Peart, and Hirsch, turns on the nature and role of verification in Mill's perception of the appropriate method for Political Economy. Professor Hirsch maintains against us that, for Mill, the models constructed by political economists are insulated from verification. His case is based on two counterclaims. First, that when Mill writes of “verification” in Book III of the Logic, he has in mind a procedure differing from that appropriate for Political Economy, which allows only “indirect verification” (outlined in Book VI). Hirsch finds that Hollander and Peart confuse the two. Secondly, since the contexts of our case studies often relate to policy formulation, Hirsch finds our elucidations of an appeal to experience of a more basic order to be unconvincing.
A novel mechanism for the regulation of amyloid precursor protein metabolism
Authors: Chen Qi , Kimura Hideo , Schubert David
Published: July 2002

Modifier of cell adhesion protein (MOCA; previously called presenilin [PS] binding protein) is a DOCK180-related molecule, which interacts with PS1 and PS2, is localized to brain areas involved in Alzheimer's disease (AD) pathology, and is lost from the soluble fraction of sporadic Alzheimer's disease (AD) brains. Because PS1 has been associated with γ-secretase activity, MOCA may be involved in the regulation of β-amyloid precursor protein (APP) processing. Here we show that the expression of MOCA decreases both APP and amyloid β-peptide secretion and lowers the rate of cell-substratum adhesion. In contrast, MOCA does not lower the secretion of amyloid precursor-like protein (APLP) or several additional type 1 membrane proteins. The phenotypic changes caused by MOCA are due to an acceleration in the rate of intracellular APP degradation. The effect of MOCA expression on the secretion of APP and cellular adhesion is reversed by proteasome inhibitors, suggesting that MOCA directs nascent APP to proteasomes for destruction. It is concluded that MOCA plays a major role in APP metabolism and that the effect of MOCA on APP secretion and cell adhesion is a downstream consequence of MOCA-directed APP catabolism. This is a new mechanism by which the expression of APP is regulated.
The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export
Authors: Prudovsky Igor , Bagala Cinzia , Tarantini Francesca , Mandinova Anna , Soldi Raffaella , Bellum Stephen , Maciag Thomas
Published: July 2002

The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy to study the spatio-temporal aspects of this nonclassical export pathway and demonstrate that heat shock stimulates the redistribution of FGF1 from a diffuse cytosolic pattern to a locale near the inner surface of the plasma membrane where it colocalized with S100A13 and Syt1. In addition, coexpression of dominant-negative mutant forms of S100A13 and Syt1, which both repress the release of FGF1, failed to inhibit the stress-induced peripheral redistribution of intracellular FGF1. However, amlexanox, a compound that is known to attenuate actin stress fiber formation and FGF1 release, was able to repress this process. These data suggest that the assembly of the intracellular complex involved in the release of FGF1 occurs near the inner surface of the plasma membrane and is dependent on the F-actin cytoskeleton.
Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER
Authors: Molinari Maurizio , Galli Carmela , Piccaluga Verena , Pieren Michel , Paganetti Paolo
Published: July 2002

BACE457 is a recently identified pancreatic isoform of human β-secretase. We report that this membrane glycoprotein and its soluble variant are characterized by inefficient folding in the ER, leading to proteasome-mediated ER-associated degradation (ERAD). Dissection of the degradation process revealed that upon release from calnexin, extensively oxidized BACE457 transiently entered in disulfide-bonded complexes associated with the lumenal chaperones BiP and protein disulfide isomerase (PDI) before unfolding and dislocation into the cytosol for degradation. BACE457 and its lumenal variant accumulated in disulfide-bonded complexes, in the ER lumen, also when protein degradation was inhibited. The complexes were disassembled and the misfolded polypeptides were cleared from the ER upon reactivation of the degradation machinery. Our data offer new insights into the mechanism of ERAD by showing a sequential involvement of the calnexin and BiP/PDI chaperone systems. We report the unexpected transient formation of covalent complexes in the ER lumen during the ERAD process, and we show that PDI participates as an oxidoreductase and a redox-driven chaperone in the preparation of proteins for degradation from the mammalian ER.
Transepithelial Migration ofToxoplasma gondiiIs Linked to Parasite Motility and Virulence
Authors: Barragan Antonio , Sibley L. David
Published: July 2002

After oral ingestion, Toxoplasma gondii crosses the intestinal epithelium, disseminates into the deep tissues, and traverses biological barriers such as the placenta and the blood-brain barrier to reach sites where it causes severe pathology. To examine the cellular basis of these processes, migration of T. gondii was studied in vitro using polarized host cell monolayers and extracellular matrix. Transmigration required active parasite motility and the highly virulent type I strains consistently exhibited a superior migratory capacity than the nonvirulent type II and type III strains. Type I strain parasites also demonstrated a greater capacity for transmigration across mouse intestine ex vivo, and directly penetrated into the lamina propria and vascular endothelium. A subpopulation of virulent type I parasites exhibited a long distance migration (LDM) phenotype in vitro, that was not expressed by nonvirulent type II and type III strains. Cloning of parasites expressing the LDM phenotype resulted in substantial increase of migratory capacity in vitro and in vivo. The potential to up-regulate migratory capacity in T. gondii likely plays an important role in establishing new infections and in dissemination upon reactivation of chronic infections.
Molecular Adaptation of Borrelia burgdorferi in the Murine Host
Authors: Liang Fang Ting , Nelson F. Kenneth , Fikrig Erol
Published: July 2002

An analysis of expression of 137 lipoprotein genes on the course of murine infection revealed a two-step molecular adaptation by Borrelia burgdorferi, the Lyme disease spirochete. For the first step, regardless whether the initial inocula of B. burgdorferi expressed either all (cultured spirochetes) or less than 40 (host-adapted spirochetes) of the 137 lipoprotein genes, the spirochetes were modulated to transcribe 116 of the genes within 10 d after being introduced to the murine host. This step of adaptation was induced by the microenvironment of the host tissue. During the second step, which was forced by host immune selection pressure and occurred between 17 and 30 d after infection, B. burgdorferi down-regulated most of the lipoprotein genes and expressed less than 40 of the 137 genes. This novel adaptation mechanism could be a critical step for B. burgdorferi to proceed to chronic infection, as the pathogen would be cleared at the early stage of infection if the spirochetes failed to undergo this process.
cAMP Increases Density of ENaC Subunits in the Apical Membrane of MDCK Cells in Direct Proportion to Amiloride-sensitive Na+ Transport
Authors: Morris Ryan G. , Schafer James A.
Published: July 2002

Antidiuretic hormone and/or cAMP increase Na+ transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na+ transport could be explained quantitatively by an increased density of ENaC Na+ channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat α-, β-, and γENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. Proc. Natl. Acad. Sci. USA. 93:15370–15375). The density of ENaC subunits was quantified by specific binding of 125I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4–8 nM. Transepithelial Na+ transport was measured as the amiloride-sensitive short-circuit current (AS-Isc) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-Isc measured in the same experiments. Stimulation with cAMP (20 μM 8-p-chlorothio-cAMP plus 200 μM IBMX) significantly increased AS-Isc from 11.2 ± 1.3 to 18.1 ± 1.3 μA/cm2. M2 binding (at 1.7 nM M2) increased in direct proportion to AS-Isc from 0.62 ± 0.13 to 1.16 ± 0.18 fmol/cm2. Based on the concentration dependence of M2 binding, the quantity of Na+ channels per unit of AS-Isc was calculated to be the same in the presence and absence of cAMP, 0.23 ± 0.04 and 0.21 ±0.05 fmol/μA, respectively. These values would be consistent with a single channel conductance of ∼5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-Isc to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the Isc increase produced by cAMP.
Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling
Authors: Yu Xiaochun , Sharma Kailash D. , Takahashi Tsuyoshi , Iwamoto Ryo , Mekada Eisuke
Published: July 2002

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between835Ala and918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.
Expression of Caveolin-1 Induces Premature Cellular Senescence in Primary Cultures of Murine Fibroblasts
Authors: Volonte Daniela , Zhang Kun , Lisanti Michael P. , Galbiati Ferruccio
Published: July 2002

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a “transformation suppressor” protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G0/G1phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in β-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.
Role for YakA, cAMP, and Protein Kinase A in Regulation of Stress Responses ofDictyostelium discoideumCells
Authors: Taminato Alexandre , Bagattini Raquel , Gorjão Renata , Chen Guokai , Kuspa Adam , Souza Glaucia Mendes
Published: July 2002

The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyosteliumcells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found thatyakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation inpkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress.
Molecular Dissection of Cytokinesis by RNA Interference inDrosophilaCultured Cells
Authors: Somma Maria Patrizia , Fasulo Barbara , Cenci Giovanni , Cundari Enrico , Gatti Maurizio
Published: July 2002

We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP,pavarotti, rho1, pebble,spaghetti squash, syntaxin1A, andtwinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved inDrosophila mitotic cytokinesis.
DictyosteliumEB1 Is a Genuine Centrosomal Component Required for Proper Spindle Formation
Authors: Rehberg Markus , Gräf Ralph
Published: July 2002

EB1 proteins are ubiquitous microtubule-associated proteins involved in microtubule search and capture, regulation of microtubule dynamics, cell polarity, and chromosome stability. We have cloned a complete cDNA of Dictyostelium EB1 (DdEB1), the largest known EB1 homolog (57 kDa). Immunofluorescence analysis and expression of a green fluorescent protein-DdEB1 fusion protein revealed that DdEB1 localizes along microtubules, at microtubule tips, centrosomes, and protruding pseudopods. During mitosis, it was found at the spindle, spindle poles, and kinetochores. DdEB1 is the first EB1-homolog that is also a genuine centrosomal component, because it was localized at isolated centrosomes that are free of microtubules. Furthermore, centrosomal DdEB1 distribution was unaffected by nocodazole treatment. DdEB1 colocalized with DdCP224, the XMAP215 homolog, at microtubule tips, the centrosome, and kinetochores. Furthermore, both proteins were part of the same cytosolic protein complex, suggesting that they may act together in their functions. DdEB1 deletion mutants expressed as green fluorescent protein or maltose-binding fusion proteins indicated that microtubule binding requires homo-oligomerization, which is mediated by a coiled-coil domain. A DdEB1 null mutant was viable but retarded in prometaphase progression due to a defect in spindle formation. Because spindle elongation was normal, DdEB1 seems to be required for the initiation of the outgrowth of spindle microtubules.