bluuebunny/crossref_metadata_2025 huggingface dataset:
SENSATIONS, SWATCHES, AND SPECKLED HENS
Authors: Fantl Jeremy , Howell Robert
Published: November 2003

Abstract:  We argue that there is a interesting connection between the old problem of the Speckled Hen and an argument that can be traced from Russell to Armstrong to Putnam that we call the “gradation argument.” Both arguments have been used to show that there is no “Highest Common Factor” between appearances we judge the same – no such thing as “real” sensations. But, we argue, both only impugn the assumption of epistemic certainty regarding introspective reports.
VAGUE SIMPLES
Authors: McKinnon Neil
Published: November 2003

Abstract:  Gareth Evans has argued influentially against vague identities. David Lewis and Theodore Sider have argued against vague parthood. Much of the distaste among philosophers for metaphysical vagueness is sourced in these arguments. I argue that even if the considerations adduced by Evans, Lewis and Sider are conclusive, metaphysical boundary vagueness remains possible.
AGAINST LEWIS's NEW THEORY OF CAUSATION: A STORY WITH THREE MORALS
Authors: Strevens Michael
Published: November 2003

Abstract:   A recent paper by David Lewis, “Causation as Influence”, provides a new theory of causation. This paper presents an argument against the theory, using a series of counterexamples that are, I think, of independent interest to philosophers of causation. I argue that (a) the relation asserted by a claim of the form “C was a cause of E” is distinct from the relation of causal influence, (b) the former relation depends very much, contra Lewis, on the individuation conditions for the event E, and (c) Lewis's account is unsatisfactory as an analysis of either kind of relation. The counterexamples presented in this paper provide, I suggest, some insight into the reasons for the failure of counterfactual accounts of causal relations.
Spontaneous ‘baroreflex sequences’ occur as deterministic functions of breathing phase
Authors: Rothlisberger Brian W. , Badra Leslie J. , Hoag Jeffrey B. , Cooke William H. , Kuusela Tom A. , Tahvanainen Kari U.O. , Eckberg Dwain L.
Published: November 2003

Parallel increases or decreases of systolic pressures and R–R intervals occur spontaneously in healthy resting humans, and are thought to be expressions of vagal baroreflex physiology. We studied ten healthy supine young adults, and tested the null hypothesis that spontaneous baroreflex sequences are distributed uniformly throughout the breathing cycle. We recorded the electrocardiogram, photoplethysmographic arterial pressure, respiration (pneumobelt), and peroneal nerve muscle sympathetic activity in supine subjects who breathed spontaneously, or held their breaths in inspiration after 2 min of hyperventilation with 100% oxygen. We analysed pairs of three or more increasing or decreasing systolic pressures and R–R intervals with linear regression, and related the gain and timing of the onset of such sequences to the phase of respiration, and to preceding muscle sympathetic nerve activity. We found that baroreflex sequences occur erratically, at a frequency about one‐third that of breathing. However, when baroreflex sequences do occur, the timing of their onset is dictated by the phase of respiration. Parallel increases of systolic pressures and R–R intervals (‘up’ sequences) begin just before and after the beginning of expiration, and parallel decreases of systolic pressures and R–R intervals (‘down’ sequences) begin during late expiration and inspiration. Average gains of up and down baroreflex sequences triggered by muscle sympathetic bursts are comparable during breathing and apnoea. However, the latencies between sympathetic bursts and baroreflex sequences are less during breathing than during apnoea. We propose that parallel systolic pressure – R–R interval sequences are expressions of arterial baroreflex physiology, and that the nearly fixed timing of such sequences within breaths reflects simply respiratory gating of muscle sympathetic bursts.
Circulating norepinephrine and cerebrovascular control in conscious humans
Authors: Kimmerly D. S. , Tutungi E. , Wilson T. D. , Serrador J. M. , Gelb A. W. , Hughson R. L. , Shoemaker J. K.
Published: November 2003

Background:  Cerebral vasoconstriction without concurrent changes in systemic arterial blood pressure has been observed in both normal individuals and those with idiopathic orthostatic intolerance following several minutes of postural stress when circulating catecholamines are elevated. Therefore, we tested the hypothesis that α‐adrenergic activation with and without elevated circulating norepinephrine (NE) directly affects cerebrovascular tone in healthy individuals.

Methods:  Mean arterial pressure (MAP; tonometry) and cerebral blood flow velocity (MFV) in the middle cerebral artery (transcranial Doppler) were measured in seven healthy individuals during 15 min periods of saline and of 50 (low NE) and 100 (high NE) ng kg−1 min−1 infusions of NE. Following this, phentolamine (PHO) was administered to return MAP back to baseline while high NE infusion continued (high NE + PHO). Finally, NE infusion was stopped allowing the persistent effects of PHO to dominate.

Results:  Circulating NE caused a dose‐dependent increase in MAP (P<0·05). During combined high NE + PHO, blood pressure was initially reduced to baseline levels but then increased a second time (P<0·05) during the final ∼5 min of this phase. MFV remained constant during both low NE and high NE. In contrast, the secondary increase in BP during the late high NE + PHO phase was associated with elevated MFV. Cerebral vascular resistance (CVR) increased during high NE but was reduced to baseline during both early and late portions of the combined high NE + PHO phase (i.e. despite the late‐phase increase in blood pressure).

Conclusions:  The increase in CVR during NE infusion was explained by an autoregulatory response to the increased blood pressure and not an α‐mediated constriction. However, PHO appeared to interfere with the normal autoregulatory response to increasing blood pressure.
Prognostic predictors for survival in patients with COPD using cardiopulmonary exercise testing
Authors: Hiraga Toru , Maekura Ryoji , Okuda Yoshinari , Okamoto Takashi , Hirotani Atsushi , Kitada Seigo , Yoshimura Kenji , Yokota Soichiro , Ito Masami , Ogura Takeshi
Published: November 2003

We studied the relationship between physiologic parameters in cardiopulmonary exercise testing (CPET) and prognosis in terms of survival time in patients with chronic obstructive pulmonary disease (COPD) in order to accurately assess the severity of the disease. From a group of 195 patients with COPD who had consecutively undergone CPET between July 1989 and October 1997, we enrolled 120 subjects (mean age 67·6 years, 104 males) with exertional dyspnoea into a cohort protocol. Of these subjects, 34 (28·3%) died during the 3–5‐year follow‐up period after CPET. By univariate analysis, the following factors were significantly associated with survival time: age, body mass index, %FVC, %FEV1, FEV1%, Paco2 at rest, severity of exercise‐induced hypoxemia evaluated by ΔPao2/ΔV̇o2 (Pao2‐slope), oxygen uptake, ventilation, tidal volume, Paco2 and oxygen pulse at maximum exercise, as well as prescribing long‐term oxygen therapy. By multivariate analysis, age and the Pao2‐slope showed significance as independent prognostic factors, and the Pao2‐slope was most closely associated with the survival time. These results reveal that CPET is a useful technique to accurately assess the relationship between the functional impairments and the prognosis of patients with COPD.
Influence of oxygen administration on pulmonary haemodynamics and tissue oxygenation during exercise in COPD patients with different ACE genotypes
Authors: Kanazawa Hiroshi , Hirata Kazuto , Yoshikawa Junichi
Published: November 2003

We previously found that the angiotensin‐converting enzyme (ACE) DD genotype is associated with exaggerated pulmonary hypertension and disturbance of tissue oxygenation during exercise in chronic obstructive pulmonary disease (COPD) patients. This study was designed to compare the effect of oxygen administration on pulmonary haemodynamics and tissue oxygenation during exercise in COPD patients with different ACE genotypes. Forty‐three COPD patients (II = 16, ID = 12, DD = 15) underwent right heart catheterization, and then performed an exercise test with room air or oxygen. We measured pulmonary haemodynamic variables and indices of tissue oxygenation such as mixed venous oxygen tension (Pvo2) and arterial lactate concentration, both at rest and after exercise. The magnitude of difference in mean pulmonary arterial pressure and pulmonary vascular resistance after exercise between breathing of room air and breathing of oxygen did not significantly differ among the three groups. Pvo2 after exercise with room air or oxygen was significantly higher in patients with the II genotype than in those with the ID or DD genotype. In contrast, lactate concentration after exercise with room air or oxygen was significantly lower in patients with the II genotype than in those with the ID or DD genotype. Moreover, the magnitude of difference in Pvo2 and lactate concentration after exercise between breathing of room air and breathing of oxygen was the II > ID > DD genotype. These findings suggest that the ability of oxygen administration to improve tissue oxygenation during exercise is associated with the ACE genotypes in COPD patients.
Quantification of inspiratory‐induced vasoconstrictive episodes: a comparison of laser Doppler fluxmetry and photoplethysmography
Authors: Rauh Robert , Posfay Alexander , Mück‐Weymann Michael
Published: November 2003

Laser Doppler fluxmetry (LDF) or photoplethysmography (PPG) are frequently used as non‐invasive tools to assess skin blood flow. They are both thought to be suitable and interchangeable methods for the detection of sympathetically activated vasoconstrictive episodes that can be provoked by a voluntary deep inspiration (DI), the so‐called inspiratory gasp response (IGR). However, a rigorous comparison of the two methods has not been reported in the literature. The aim of this study was to compare the detection of IGR with LDF and PPG. We investigated 14 healthy volunteers (five female, nine male, 25·4 ± 2·7 years). A PPG probe was applied to the tip of the thumb of the dominant hand, a LDF sensor to the tip of the adjacent index finger. After adaptation and baseline measurements the subjects were asked to perform a DI with time intervals of 90, 60, 30 and 15 s. We found that both methods are useful to detect individual IGR. Overall correlation of IGR amplitude detected with LDF and PPG was significant (r = 0·433). Surprisingly, there was a continuous increase of the correlation coefficient from the first (r = 0·105) or second (r = −0·184) IGR to the fifth (r = 0·727) IGR. This implies that experimental data obtained with PPG and LDF are not equivalent and that one has to be cautious regarding the comparison and interpretation of results obtained with these two different methods.
Maximum rate of oxygen consumption related to succinate dehydrogenase activity in skeletal muscle fibres of chronic heart failure patients and controls
Authors: Bekedam Martijn A. , Van Beek‐Harmsen Brechje J. , Boonstra Anco , Van Mechelen Willem , Visser Frans C. , Van Der Laarse Willem J.
Published: November 2003

Previous studies indicate that the low maximum rate of oxygen consumption (VO2max) of chronic heart failure (CHF) patients is not because of impaired pump function of the heart. We hypothesize that VO2 during maximum exercise is determined by the total oxidative capacity of skeletal muscle. VO2max of six controls and 14 CHF patients, New York Heart Association class I–III, was determined using an incremental bicycle ergometer test. Cryostat sections of a biopsy from the quadriceps femoris muscle were incubated for succinate dehydrogenase (SDH) using quantitative histochemistry. VO2max (range: 29 ml O2 kg min−1 in a class III patient to 118 ml O2 kg min−1 in a control subject) correlates with the mean SDH activity of skeletal muscle fibres (r = 0·79 or r = 0·81, including or excluding oxygen uptake at rest, respectively; P<0·001). The relationship between VO2max and SDH activity is similar to that determined previously using isolated single muscle fibres and myocardial trabeculae under hyperoxic conditions. From the product of SDH activity and the cross‐sectional area of the fibre (i.e. spatially integrated SDH activity), it is possible to calculate the maximum oxygen uptake rate per unit muscle fibre length. This uptake rate is linearly related to the number of capillaries per fibre (r = 0·76, P<0·001) in all subjects, suggesting that oxidative capacity of skeletal muscle fibres in CHF patients decreases in proportion to the oxygen supply capacity of the microcirculation.
Injurious ventilation strategies cause systemic release of IL‐6 and MIP‐2 in rats in vivo*
Authors: Haitsma Jack J. , Uhlig Stefan , Verbrugge Serge J. , Göggel Rolf , Poelma Davey L. H. , Lachmann Burkhard
Published: November 2003

In vivo experiments showed no increased production of tumour necrosis factor (TNF) in response to injurious ventilation strategies in otherwise untreated animals. Because interleukin‐6 (IL‐6) and macrophage inflammatory protein‐2 (MIP‐2) are more sensitive markers of ventilation‐induced cytokine release, serum and bronchoalveolar lavage (BAL) samples were examined for these mediators. Eighty‐five adult rats were randomized to three different ventilation strategies. Rats were ventilated with low pressures and low tidal volumes [13/3; peak inspiratory pressure (PIP)/positive end‐expiratory pressure (PEEP) in cmH2O], the second group of rats was ventilated with high pressures and low PEEP resulting in high tidal volumes (32/6), and the third group was ventilated with the same high pressures but without PEEP (32/0). Animals were ventilated either for 90 or 240 min, subsequently serum and BAL were collected for analyses on IL‐6 and MIP‐2 content. Non‐ventilated animals served as healthy controls. Ventilation with 32/0 for 90 or 240 min, led to increased serum IL‐6 levels. Serum MIP‐2 levels were increased by ventilation with 32/6 (90 min) and 32/0 (240 min). Ventilation under any condition, even at 13/3, resulted in elevated MIP‐2 levels in the BAL fluid. Even at normal pressures pulmonary MIP‐2 levels were increased, suggesting that ventilation may promote pro‐inflammatory responses in healthy subjects.
Heel skin hyperaemia: direct compression versus vascular occlusion
Authors: Mayrovitz Harvey N. , Sims Nancy , Dribin Lori
Published: November 2003

Vulnerability of the heel to ulceration in bed‐bound persons is related to direct pressure‐induced blood flow decreases. Periodic pressure reduction is a clinical strategy to help prevent ulcers by allowing flow‐repayment hyperaemia that has a magnitude and duration thought to be related to the duration of the prior interval of ischaemia. However, there are reasons to question whether effects of flow stoppages caused by direct tissue loading are similar to those because of ischaemia without superimposed direct pressure. This question was investigated by comparing posterior heel skin blood flow responses via laser‐Doppler perfusion monitoring of 27 supine‐lying subjects in whom blood flow was reduced by 5‐min of direct heel loading on a support surface and by 5‐min of ankle‐cuff compression. Results showed that blood flow reductions were the same for both methods but the hyperaemia was significantly greater when flow reduction was produced by direct heel loading. This was true for ratio of peak hyperaemic flow to baseline (8·20 ± 1·32 s versus 4·68 ± 0·80 s, P≤0·001), hyperaemic to baseline 3‐min flow‐time area ratios (4·70 ± 0·65 s versus 1·95 ± 0·29 s, P≤0·001) and for total hyperaemia durations (352 ± 39 s versus 181 ± 14 s, P<0·001). These findings raise new questions regarding the precise physiological effects of heel and tissue loading in general, the factors that contribute to the hyperaemic response and their clinical impact and interpretation. Possible sources of the observed greater post‐loading hyperaemia responses are discussed.
Is the International Convergence of Capital Adequacy Regulation Desirable?
Authors: Acharya Viral V.
Published: November 2003

The merit of international convergence of bank capital requirements in the presence of divergent closure policies of different central banks is examined. The lack of a complementary variation between minimum bank capital requirements and regulatory forbearance leads to a spillover from more forbearing to less forbearing economies and reduces the competitive advantage of banks in less forbearing economies. Linking the central bank's forbearance to its alignment with domestic bank owners, it is shown that in equilibrium, a regression toward the worst closure policy may result: The central banks of initially less forbearing economies also adopt greater forbearance.
Operational stability of electrophosphorescent devices containing p and n doped transport layers
Authors: D’Andrade Brian W. , Forrest Stephen R. , Chwang Anna B.
Published: November 2003

The operational stability of low-operating voltage p-i-n electrophosphorescent devices containing fac-tris(2-phenylpyridine) iridium as the emissive dopant is investigated. In these devices, Li-doped 4,7-diphenyl-1,10-phenanthroline (BPhen) served as an n-type electron transport layer, or as an undoped hole blocking layer (HBL), and 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane doped 4,4′,4″-tris(3-methylphenylphenylamino) triphenylamine served as a p-type hole transport layer. The glass transition temperature of BPhen can be increased by the addition of aluminum(III)bis(2-methyl-8-quinolinato)4-phenylphenolate (BAlq), resulting in improved morphological stability, thereby reducing device degradation. When thermally stable BAlq was used as a HBL in both p-i-n and undoped devices, the extrapolated operational lifetime (normalized to an initial luminance of 100 cd/m2) of the p-i-n and undoped devices are 18 000 and 60 000 h, respectively, indicating that the presence of p and n dopants can accelerate device degradation.
Duration of employment is not a predictor of disability of cleaners: a longitudinal study
Authors: Gamperiene Migle , Nygård Jan F. , Brage Sören , Bjerkedal Tor , Bruusgaard Dag
Published: November 2003

Aims: Cleaning is a high-risk occupation for developing musculoskeletal disorders. Sickness absence is twice as high as in other occupations. Disability pensions for musculoskeletal disorders are twice as high in cleaners as in other employed women. However, a result from Norwegian and Danish studies shows that female cleaners do not report higher morbidity of musculoskeletal disorders than other women. The objective was to analyse whether female cleaners have a higher risk of obtaining a disability pension than women in other unskilled occupations and whether the length of employment influences the risk. Methods: The material is from the National Census in 1980 and 1990 and supplemented with disability pensioning data from the National Insurance Administration and the Population registry. Women aged 20—49, working as cleaners, seamstresses, nursing, kitchen, or shop assistants in 1980 were followed until 1990 or until receiving disability pension. Female cleaners aged 30—59 years in 1990 were categorized into two cohorts by occupation in 1980. They were followed from 1991 to 1994, to the date they died, or received disability pension. Incidence rates and incidence rate ratio for disability pension and mortality was calculated by Poisson regression. Cox regression calculated the relative risk of obtaining disability pension. Results: Disability pension rates were higher among cleaners than among other women in unskilled occupations (1.4 per 1,000 person years (CI 95% 1.35—1.46)), but the risk of obtaining disability pension did not increase with increasing exposure to cleaning (HR 0.8 (CI 95% 0.6—1.2)). Conclusions: The cleaning occupation has high disability rates compared with other unskilled occupations. A contribution factor to these high rates is a selection of women with poor health into the occupation.
Proteolytic release of the carboxy-terminal fragment of proHB-EGF causes nuclear export of PLZF
Authors: Nanba Daisuke , Mammoto Akiko , Hashimoto Koji , Higashiyama Shigeki
Published: November 2003

Cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) via metalloprotease activation yields amino- and carboxy-terminal regions (HB-EGF and HB-EGF-C, respectively), with HB-EGF widely recognized as a key element of epidermal growth factor receptor transactivation in G protein–coupled receptor signaling. Here, we show a biological role of HB-EGF-C in cells. Subsequent to proteolytic cleavage of proHB-EGF, HB-EGF-C translocated from the plasma membrane into the nucleus. This translocation triggered nuclear export of the transcriptional repressor, promyelocytic leukemia zinc finger (PLZF), which we identify as an HB-EGF-C binding protein. Suppression of cyclin A and delayed entry of S-phase in cells expressing PLZF were reversed by the production of HB-EGF-C. These results indicate that released HB-EGF-C functions as an intracellular signal and coordinates cell cycle progression with HB-EGF.
Evidence for a two membrane–spanning autonomous mitochondrial DNA replisome
Authors: Meeusen Shelly , Nunnari Jodi
Published: November 2003

The unit of inheritance for mitochondrial DNA (mtDNA) is a complex nucleoprotein structure termed the nucleoid. The organization of the nucleoid as well as its role in mtDNA replication remain largely unknown. Here, we show in Saccharomyces cerevisiae that at least two populations of nucleoids exist within the same mitochondrion and can be distinguished by their association with a discrete proteinaceous structure that spans the outer and inner mitochondrial membranes. Surprisingly, this two membrane–spanning structure (TMS) persists and self-replicates in the absence of mtDNA. We tested whether TMS functions to direct the replication of mtDNA. By monitoring BrdU incorporation, we observed that actively replicating nucleoids are associated exclusively with TMS. Consistent with TMS's role in mtDNA replication, we found that Mip1, the mtDNA polymerase, is also a stable component of TMS. Taken together, our observations reveal the existence of an autonomous two membrane–spanning mitochondrial replisome as well as provide a mechanism for how mtDNA replication and inheritance may be physically linked.
SUMO-2/3 regulates topoisomerase II in mitosis
Authors: Azuma Yoshiaki , Arnaoutov Alexei , Dasso Mary
Published: November 2003

We have analyzed the abundance of SUMO-conjugated species during the cell cycle in Xenopus egg extracts. The predominant SUMO conjugation products associated with mitotic chromosomes arose from SUMO conjugation of topoisomerase II. Topoisomerase II was modified exclusively by SUMO-2/3 during mitosis under normal circumstances, although we observed conjugation of topoisomerase II to SUMO-1 in extracts with exogenous SUMO-1 protein. Inhibition of SUMO modification by a dominant-negative mutant of the SUMO-conjugating enzyme Ubc9 (dnUbc9) did not detectably alter topoisomerase II activity, but it did increase the amount of unmodified topoisomerase II retained on mitotic chromosomes after high salt washing. dnUbc9 did not disrupt the assembly of condensed mitotic chromosomes or block progression of extracts through mitosis, but it did block the dissociation of sister chromatids at the metaphase–anaphase transition. Together, our results suggest that SUMO conjugation is important for chromosome segregation in metazoan systems, and that mobilization of topoisomerase II from mitotic chromatin may be a key target of this modification.
A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae
Authors: Kaufman Brett A. , Kolesar Jill E. , Perlman Philip S. , Butow Ronald A.
Published: November 2003

The yeast mitochondrial chaperonin Hsp60 has previously been implicated in mitochondrial DNA (mtDNA) transactions: it is found in mtDNA nucleoids associated with single-stranded DNA; it binds preferentially to the template strand of active mtDNA ori sequences in vitro; and wild-type (ρ+) mtDNA is unstable in hsp60 temperature-sensitive (ts) mutants grown at the permissive temperature. Here we show that the mtDNA instability is caused by a defect in mtDNA transmission to daughter cells. Using high resolution, fluorescence deconvolution microscopy, we observe a striking alteration in the morphology of mtDNA nucleoids in ρ+ cells of an hsp60-ts mutant that suggests a defect in nucleoid division. We show that ρ− petite mtDNA consisting of active ori repeats is uniquely unstable in the hsp60-ts mutant. This instability of ori ρ− mtDNA requires transcription from the canonical promoter within the ori element. Our data suggest that the nucleoid dynamics underlying mtDNA transmission are regulated by the interaction between Hsp60 and mtDNA ori sequences.
Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities
Authors: Yamazaki Satoru , Iwamoto Ryo , Saeki Kazuko , Asakura Masanori , Takashima Seiji , Yamazaki Ayano , Kimura Rina , Mizushima Hiroto , Moribe Hiroki , Higashiyama Shigeki , Endoh Masayuki , Kaneda Yasufumi , Takagi Satoshi , Itami Satoshi , Takeda Naoki , Yamada Gen , Mekada Eisuke
Published: November 2003

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain–truncated form (HBΔtm) of the molecule. HBuc/uc mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBΔtm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.
The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling inDictyostelium
Authors: Noegel Angelika A. , Blau-Wasser Rosemarie , Sultana Hameeda , Müller Rolf , Israel Lars , Schleicher Michael , Patel Hitesh , Weijer Cornelis J.
Published: November 2003

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.
Two Phases of Actin Polymerization Display Different Dependencies on PI(3,4,5)P3Accumulation and Have Unique Roles during Chemotaxis
Authors: Chen Lingfeng , Janetopoulos Chris , Huang Yi Elaine , Iijima Miho , Borleis Jane , Devreotes Peter N.
Published: November 2003

The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of Dictyostelium discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P3/PI(3,4)P2reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild-type cells but partially restore polarity and chemotactic response in pten-cells. Surprisingly, the fast phase of PI(3,4,5)P3accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis.
Permissive Roles of Phosphatidyl Inositol 3-Kinase and Akt in Skeletal Myocyte Maturation
Authors: Wilson Elizabeth M. , Tureckova Jolana , Rotwein Peter
Published: November 2003

Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between signaling pathways activated by hormones and growth factors, and intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and myocyte enhancer factor 2 (MEF2) families. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo, and in the maintenance and hypertrophy of mature muscle in the adult, but the precise signaling pathways responsible for these effects remain incompletely defined. To study mechanisms of IGF action in muscle, we have developed a mouse myoblast cell line termed C2BP5 that is dependent on activation of the IGF-I receptor and the phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathway for initiation of differentiation. Here, we show that differentiation of C2BP5 myoblasts could be induced in the absence of IGF action by recombinant adenoviruses expressing MyoD or myogenin, but it was reversibly impaired by the PI3-kinase inhibitor LY294002. Similar results were observed using a dominant-negative version of Akt, a key downstream component of PI3-kinase signaling, and also were seen in C3H 10T1/2 fibroblasts. Inhibition of PI3-kinase did not prevent accumulation of muscle differentiation-specific proteins (myogenin, troponin T, or myosin heavy chain), did not block transcriptional activation of E-box containing muscle reporter genes by MyoD or myogenin, and did not inhibit the expression or function of endogenous MEF2C or MEF2D. An adenovirus encoding active Akt could partially restore terminal differentiation of MyoD-expressing and LY294002-treated myoblasts, but the resultant myofibers contained fewer nuclei and were smaller and thinner than normal, indicating that another PI3-kinase-stimulated pathway in addition to Akt is required for full myocyte maturation. Our results support the idea that an IGF-regulated PI3-kinase pathway functions downstream of or in parallel with MyoD, myogenin, and MEF2 in muscle development to govern the late steps of differentiation that lead to multinucleated myotubes.
Mechanism of Human Dermal Fibroblast Migration Driven by Type I Collagen and Platelet-derived Growth Factor-BB
Authors: Li Wei , Fan Jianhua , Chen Mei , Guan Shengxi , Sawcer David , Bokoch Gary M. , Woodley David T.
Published: November 2003

Migration of human dermal fibroblasts (HDFs) is critical for skin wound healing. The mechanism remains unclear. We report here that platelet-derived growth factor-BB (PDGF-BB) is the major promotility factor in human serum for HDF motility on type I collagen. PDGF-BB recapitulates the full promotility activity of human serum and anti-PDGF neutralizing antibodies completely block it. Although collagen matrix initiates HDF migration without growth factors, PDGF-BB–stimulated migration depends upon attachment of the cells to a collagen matrix. The PDGF-BB's role is to provide directionality and further enhancement for the collagen-initiated HDF motility. To study the collagen and PDGF-BB “dual signaling” in primary HDF, we establish “gene cassettes” plus lentiviral gene delivery approach, in which groups of genes are studied individually or in combination for their roles in HDF migration. Focal adhesion kinase, p21Rac,CDC42-activated kinase and Akt are grouped into an upstream kinase gene cassette, and the four major mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 5) are grouped into a downstream kinase gene cassette. The experiments demonstrate 1) the genes' individual roles and specificities, 2) their combined effects and sufficiency, and 3) the mechanisms of their intermolecular connections in HDF migration driven by collagen and PDGF-BB.
Sites of Interaction between Aldolase and Thrombospondin-related Anonymous Protein inPlasmodium
Authors: Buscaglia Carlos A. , Coppens Isabelle , Hol Wim G. J. , Nussenzweig Victor
Published: November 2003

Gliding motility and host cell invasion by apicomplexan parasites are empowered by an acto-myosin motor located underneath the parasite plasma membrane. The motor is connected to host cell receptors through trans-membrane invasins belonging to the thrombospondin-related anonymous protein (TRAP) family. A recent study indicates that aldolase bridges the cytoplasmic tail of MIC2, the homologous TRAP protein in Toxoplasma, and actin. Here, we confirm these unexpected findings in Plasmodium sporozoites and identify conserved features of the TRAP family cytoplasmic tail required to bind aldolase: a subterminal tryptophan residue and two noncontiguous stretches of negatively charged amino acids. The aldolase substrate and other compounds that bind to the active site inhibit its interaction with TRAP and with F-actin, suggesting that the function of the motor is metabolically regulated. Ultrastructural studies in salivary gland sporozoites localize aldolase to the periphery of the secretory micronemes containing TRAP. Thus, the interaction between aldolase and the TRAP tail takes place during or preceding the biogenesis of the micronemes. The release of their contents in the anterior pole of the parasite upon contact with the target cells should bring simultaneously aldolase, TRAP and perhaps F-actin to the proper subcellular location where the motor is engaged.
A Role for Intermediate Filaments in Determining and Maintaining the Shape of Nerve Cells
Authors: Helfand Brian T. , Mendez Melissa G. , Pugh Jason , Delsert Claude , Goldman Robert D.
Published: November 2003

To date, the functions of most neural intermediate filament (IF) proteins have remained elusive. Peripherin is a type III intermediate filament (IF) protein that is expressed in developing and in differentiated neurons of the peripheral and enteric nervous systems. It is also the major IF protein expressed in PC12 cells, a widely used model for studies of peripheral neurons. Dramatic increases in peripherin expression have been shown to coincide with the initiation and outgrowth of axons during development and regeneration, suggesting that peripherin plays an important role in axon formation. Recently, small interfering RNAs (siRNA) have provided efficient ways to deplete specific proteins within mammalian cells. In this study, it has been found that peripherin-siRNA depletes peripherin and inhibits the initiation, extension, and maintenance of neurites in PC12 cells. Furthermore, the results of these experiments demonstrate that peripherin IF are critical determinants of the overall shape and architecture of neurons.
ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression
Authors: Okiyoneda Tsukasa , Harada Kazutsune , Takeya Motohiro , Yamahira Kaori , Wada Ikuo , Shuto Tsuyoshi , Suico Mary Ann , Hashimoto Yasuaki , Kai Hirofumi
Published: November 2003

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.
Endocytosed Cation-Independent Mannose 6-Phosphate Receptor Traffics via the Endocytic Recycling Compartment en Route to thetrans-Golgi Network and a Subpopulation of Late Endosomes
Authors: Lin Sharron X. , Mallet William G. , Huang Amy Y. , Maxfield Frederick R.
Published: November 2003

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.
Mek1 Kinase Activity Functions Downstream ofRED1in the Regulation of Meiotic Double Strand Break Repair in Budding Yeast
Authors: Wan Lihong , de los Santos Teresa , Zhang Chao , Shokat Kevan , Hollingsworth Nancy M.
Published: November 2003

Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint. Mek1 is a protein kinase. Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors. Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways. Contrary to previous reports, Red1 is not a substrate for Mek1. Instead, RED1 is required for wild-type levels of Mek1 kinase activity. In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain. The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mek1 kinase. We propose that Red1 is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair.
GLUT4 Is Retained by an Intracellular Cycle of Vesicle Formation and Fusion with Endosomes
Authors: Karylowski Ola , Zeigerer Anja , Cohen Alona , McGraw Timothy E.
Published: November 2003

The intracellularly stored GLUT4 glucose transporter is rapidly translocated to the cell surface upon insulin stimulation. Regulation of GLUT4 distribution is key for the maintenance of whole body glucose homeostasis. We find that GLUT4 is excluded from the plasma membrane of adipocytes by a dynamic retention/retrieval mechanism. Our kinetic studies indicate that GLUT4-containing vesicles continually bud and fuse with endosomes in the absence of insulin and that these GLUT4 vesicles are 5 times as likely to fuse with an endosome as with the plasma membrane. We hypothesize that this intracellular cycle of vesicle budding and fusion is an element of the active mechanism by which GLUT4 is retained. The GLUT4 trafficking pathway does not extensively overlap with that of furin, indicating that the trans-Golgi network, a compartment in which furin accumulates, is not a significant storage reservoir of GLUT4. An intact microtubule cytoskeleton is required for insulin-stimulated recruitment to the cell surface, although it is not required for the basal budding/fusion cycle. Nocodazole disruption of the microtubule cytoskeleton reduces the insulin-stimulated exocytosis of GLUT4, accounting for the reduced insulin-stimulated translocation of GLUT4 to the cell surface.
Epithelial Cell Polarity Alters Rho-GTPase Responses toPseudomonas aeruginosa
Authors: Kazmierczak Barbara I. , Mostov Keith , Engel Joanne N.
Published: November 2003

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.
Tor Pathway Regulates Rrn3p-dependent Recruitment of Yeast RNA Polymerase I to the Promoter but Does Not Participate in Alteration of the Number of Active Genes
Authors: Claypool Jonathan A. , French Sarah L. , Johzuka Katsuki , Eliason Kristilyn , Vu Loan , Dodd Jonathan A. , Beyer Ann L. , Nomura Masayasu
Published: November 2003

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.
Fast Evaluation of Fluctuations in Biochemical Networks With the Linear Noise Approximation
Authors: Elf Johan , Ehrenberg Måns
Published: November 2003

Biochemical networks in single cells can display large fluctuations in molecule numbers, making mesoscopic approaches necessary for correct system descriptions. We present a general method that allows rapid characterization of the stochastic properties of intracellular networks. The starting point is a macroscopic description that identifies the system's elementary reactions in terms of rate laws and stoichiometries. From this formulation follows directly the stationary solution of the linear noise approximation (LNA) of the Master equation for all the components in the network. The method complements bifurcation studies of the system's parameter dependence by providing estimates of sizes, correlations, and time scales of stochastic fluctuations. We describe how the LNA can give precise system descriptions also near macroscopic instabilities by suitable variable changes and elimination of fast variables.
Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks
Authors: Shannon Paul , Markiel Andrew , Ozier Owen , Baliga Nitin S. , Wang Jonathan T. , Ramage Daniel , Amin Nada , Schwikowski Benno , Ideker Trey
Published: November 2003

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network forHalobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.