bluuebunny/crossref_metadata_2025 huggingface dataset:
Rho GTPases regulate rhabdom morphology in octopus photoreceptors
Authors: MILLER ARIA M. , RAMIREZ TERESA , ZUNIGA FREDDI I. , OCHOA GINA H. , GRAY SHAUNTE , KELLY SHANNON D. , MATSUMOTO BRIAN , ROBLES LAURA J.
Published: August 2005

In the cephalopod retina, light/dark adaptation is accompanied by a decrease/increase in rhabdom size and redistribution of rhodopsin and retinochrome. Rearrangements in the actin cytoskeleton probably govern changes in rhabdom size by regulating the degradation/formation of rhabdomere microvilli. Photopigment movements may be directed by microtubules present in the outer segment core cytoplasm. We believe that rhodopsin activation by light stimulates Rho and Rac signaling pathways, affecting these cytoskeletal systems and their possible functions in controlling rhabdom morphology and protein movements. In this study, we localized cytoskeletal and signaling proteins in octopus photoreceptors to determine their concurrence between the lighting conditions. We used toxin B fromClostridium difficileto inhibit the activity of Rho/Rac and observed its effect on the location of signaling proteins and actin and tubulin. In both lighting conditions, we found Rho in specific sets of juxtaposed rhabdomeres in embryonic and adult retinas. In the light, Rho and actin were localized along the length of the rhabdomere, but, in the dark, both proteins were absent from a space beneath the inner limiting membrane. Rac colocalized with tubulin in the outer segment core cytoplasm and, like Rho, the two proteins were also absent beneath the inner limiting membrane in the dark. The distribution of actin and Rho was affected by toxin B and, in dark-adapted retinas, actin and Rho distribution was similar to that observed in the light. Our results suggest that the Rho/Rac GTPases are candidates for the regulation of rhabdomere size and protein movements in light-dark-adapted octopus photoreceptors.
GABA-mediated component in the feedback response of turtle retinal
cones
Authors: TATSUKAWA T. , HIRASAWA H. , KANEKO A. , KANEDA M.
Published: August 2005

The negative feedback from horizontal cells to cone photoreceptors
contributes to the formation of the receptive-field surround in cone
photoreceptors. Recently, studies on the modulation of voltage-gated
Ca2+ currents in cone photoreceptors have led to great progress
in our understanding of the mechanism of horizontal-cone feedback. Another
highly probable hypothesis is that GABA mediates this feedback. This
hypothesis is supported by the facts that cone photoreceptors respond to
GABA and that horizontal cells release GABA. However, GABA-mediated
synaptic inputs from horizontal cells to cone photoreceptors have not been
demonstrated. In the present study, we examined whether cone
photoreceptors receive GABAergic inputs from horizontal cells using a
slice patch technique in the turtle retina. When 1 mM of GABA was applied
to the cone photoreceptors, GABA-induced currents were activated.
GABA-induced currents reversed their polarity at the equilibrium potential
of Cl−. The application of 30 μM of SR95531, an
antagonist of GABAA receptors, alone did not produce any change
in the holding currents. When 200 μM of pentobarbital was introduced
to potentiate the GABAergic inputs to the cone photoreceptors, however,
the inhibitory action of SR95531 on GABAergic inputs became detectable.
The amplitude of the GABAergic inputs, potentiated by pentobarbital,
increased when the horizontal cells were depolarized by the application of
20 μM of kainate, while the amplitude decreased when the horizontal
cells were hyperpolarized by the application of 10 μM of CNQX. When
the cone photoreceptors were voltage clamped at a potential at which the
voltage-gated Ca2+ current was inactive, horizontal-cone
feedback was not observed. However, the horizontal-cone feedback became
detectable when the GABAergic inputs to the cone photoreceptors were
potentiated by pentobarbital. We concluded that the contribution of
GABAergic inputs from horizontal cells to cone pedicles in the formation
of the receptive-field surround in cone photoreceptors is very limited but
that the modulation of voltage-gated Ca2+ currents in cone
photoreceptors is a physiologically relevant mechanism for horizontal-cone
feedback.
Sign-conserving amacrine neurons in the fly's external plexiform layer
Authors: DOUGLASS JOHN K. , STRAUSFELD NICHOLAS J.
Published: August 2005

Amacrine cells in the external plexiform layer of the fly's lamina have been intracellulary recorded and dye-filled for the first time. The recordings demonstrate that like the lamina's short photoreceptors R1–R6, type 1 lamina amacrine neurons exhibit nonspiking, “sign-conserving” sustained depolarizations in response to illumination. This contrasts with the sign-inverting responses that typify first-order retinotopic relay neurons: monopolar cells L1–L5 and the T1 efferent neuron. The contrast frequency tuning of amacrine neurons is similar to that of photoreceptors and large lamina monopolar cells. Initial observations indicate that lamina amacrine receptive fields are also photoreceptor-like, suggesting either that their inputs originate from a small number of neighboring visual sampling units (VSUs), or that locally generated potentials decay rapidly with displacement. Lamina amacrines also respond to motion, and in one recording these responses were selective for the orientation of moving edges. This functional organization corresponds to the anatomy of amacrine cells, in which postsynaptic inputs from several neighboring photoreceptor endings are linked by a network of very thin distal processes. In this way, each VSU can receive convergent inputs from a surround of amacrine processes. This arrangement is well suited for relaying responses to local intensity fluctuations from neighboring VSUs to a central VSU where amacrines are known to be presynaptic to the dendrites of the T1 efferent. The T1 terminal converges at a deeper level with that of the L2 monopolar cell relaying from the same optic cartridge. Thus, the localized spatial responses and receptor-like temporal response properties of amacrines are consistent with possible roles in lateral inhibition, motion processing, or orientation processing.
Whole-cell recording of light-evoked photoreceptor responses in a slice preparation of the cuttlefish retina
Authors: CHRACHRI ABDESSLAM , NELSON LISA , WILLIAMSON RODDY
Published: August 2005

A new tissue slice preparation of the cuttlefish eye is described that permits patch-clamp recordings to be acquired from intact photoreceptors during stimulation of the retina with controlled light flashes. Whole-cell recordings using this preparation, from the retinas of very youngSepia officinalisdemonstrated that the magnitude, latency, and kinetics of the flash-induced photocurrent are closely dependent on the magnitude of the flash intensity. Depolarizing steps to voltages more positive than −40 mV, from a membrane holding potential of −60 mV, induced a transient inward current followed by a larger, more sustained outward current in these early-stage photoreceptors. The latter current resembled the delayed rectifier (IK) already identified in many other nerve cells, including photoreceptors. This current was activated at −30 mV from a holding potential of −60 mV, had a sustained time course, and was blocked in a dose-dependent manner by tetraethylammonium chloride (TEA). The smaller, transient, inward current appeared at potentials more positive than −50 mV, reached peak amplitude at −30 mV and decreased with further depolarization. This current was characterized as the sodium current (INa) on the basis that it was inactivated at holding potentials above −40 mV, was blocked by tetrodotoxin (TTX) and was insensitive to cobalt.

Intracellular perfusion of the photoreceptors,viathe patch pipette, demonstrated that U-73122 and heparin blocked the evoked photocurrent in a dose-dependent manner, suggesting the involvement of the phospholipase C (PLC) and inositol 1,4,5-triphosphate (InsP3), respectively, in the phototransduction cascade. Perfusion with cyclic GMP increased significantly the evoked photocurrent, while the inclusion of phorbol-12,13-dibutyrate reduced significantly the evoked photocurrent, supporting the involvement of cGMP and the diacylglycerol (DAG) pathways, respectively, in the cuttlefish transduction process.
Inducible nitric oxide synthase subserves cholinergic vasodilation in retina
Authors: BERRA ALEJANDRO , GANZINELLI SABRINA , SARAVIA MARIO , BORDA ENRI , STERIN-BORDA LEONOR
Published: August 2005

In this paper, we investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of inducible (i) nitric oxide synthase (iNOS) expression and activity. The signaling pathway involved is also examined. These experiments also provide a link between mAChR activation and the nitric oxide (NO)-dependent regulation of retinal vascular diameter. The diameter of the retinal vessels at a distance of 1 disc diameter from the center of the optic disc was measured in rats using digital retinal photography, and both iNOS-mRNA gene expression and NOS were specifically measured using RT-PCR and [U-14C] citrulline assays, respectively. Stimulation of M1and M3mAChR with carbachol caused an increase in vessel diameter, in iNOS-mRNA levels and in NOS activity in the retina. Aminoguanidine, an inhibitor of iNOS, attenuated all these effects. Inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not calcium/calmodulin (CaM) prevented the muscarinic-dependent increase in iNOS-mRNA levels. The results obtained suggest that the activation of mAChR increases retinal vessel diameters by increasing the production of nitric oxide (NO) through iNOS activation and iNOS-mRNA gene expression. The mechanism appears to occur secondarily to stimulation of PLC and PKC enzymatic activity.
Characteristics of hornbill-dispersed fruits in a tropical seasonal forest in Thailand
Authors: KITAMURA SHUMPEI , YUMOTO TAKAKAZU , POONSWAD PILAI , CHUAILUA PHITAYA , PLONGMAI KAMOL
Published: August 2005

Asian hornbills are primarily frugivorous. We studied the characteristics of fruits consumed by four sympatric hornbill species in Thailand: Great Hornbill (Buceros bicornis), Wreathed Hornbill (Aceros undulatus), Austin's Brown Hornbill (Anorrhinus austeni) and Oriental Pied Hornbill (Anthracoceros albirostris). We compared the frequency of distribution of 11 variables for all fruit species collected in the study area (n = 259) and fruit species consumed by hornbills (n = 73). Our analysis revealed that fruits consumed by hornbills are: (1) large, (2) easily accessible within the canopy, (3) red, purple or black and (4) dehiscent or indehiscent with a thin husk. The range of fruit sizes eaten by hornbills in our study is comparable to that reported from other sites in Southeast Asia and Africa. The large gape width of hornbills enables them to consume large fruits that small frugivores would find difficult to consume.
Checks, Balances, and Thresholds: State Regulatory Re-enforcement and Federal Preemption
Authors: Teske Paul
Published: August 2005

In the last two decades, many cases of actual or attempted federal preemption of state regulation have followed an intriguing new interactive pattern. While this pattern is not comprehensive enough to define a new theory of federalism (see Zimmerman 2004), it now occurs regularly enough to merit close attention and an attempted understanding of its dynamics (see Walters 2004). It is also an example of the kind of complicated federalism, with vertical checks, balances and thresholds, that has emerged in a 21stcentury America closely divided upon whether and how to use government policy to address various social and economic problems.
The Politics of Preemption: Prospects for the States
Authors: Posner Paul L.
Published: August 2005

Woodrow Wilson told us that the federal-state relationship is the cardinal question of our system that is destined to be reformulated for each generation. His prophetic words aptly characterize the challenges federal and state governments face in satisfying both the requisites of an increasingly global economy and nationalized political culture while at the same time reflecting the differential values and interests of states. States need to retain their diversity and capacity to innovate and respond to different values and interests that may not get a hearing in Washington; conversely, Washington needs to be alert and reactive when states prove unresponsive to key values and interests or collectively incapable of solving national economic or social problems.
Shell of the Great Scallop Pecten maximus as a high‐frequency archive of paleoenvironmental changes
Authors: Chauvaud Laurent , Lorrain Anne , Dunbar Robert B. , Paulet Yves‐Marie , Thouzeau Gérard , Jean Frédéric , Guarini Jean‐Marc , Mucciarone David
Published: August 2005

We investigate the environmental and biological controls on oxygen isotope ratios in the shells of modern Pecten maximus bivalves collected alive in the Bay of Brest (France). Seasonal δ18O profiles, obtained by drilling the daily calcite ridges deposited at the surface of the left valve, were compared with in situ measurements of temperature and salinity. We show that P. maximus δ18O values accurately track seasonal variations in bottom water temperature. Shell growth rate has no significant effect on shell δ18O values. Our study demonstrates that daily variations in water temperature can be reconstructed within about 0.5°C. Temperatures estimated with the paleotemperature equation established in this study were compared with temperatures derived from previously published equations. The comparison indicates that the most commonly used paleotemperature equation for biogenic calcite (Epstein et al., 1953) provides inaccurate temperature estimates, but the Kim and O'Neil (1997) equation, established from abiogenic calcite precipitation, provides results very similar to ours and should therefore be used for scallop individuals coming from populations where proper empirical calibration cannot be done. Pecten maximus bivalves precipitate calcite in isotopic equilibrium with seawater, produce large daily growth striae, are stenohaline, and are well preserved in archeological and geological deposits, making them an excellent high‐frequency archive of paleoenvironmental change.
Neotectonic architecture of Taiwan and its implications for future large earthquakes
Authors: Shyu J. Bruce H. , Sieh Kerry , Chen Yue‐Gau , Liu Char‐Shine
Published: August 2005

The disastrous effects of the 1999 Chi‐Chi earthquake in Taiwan demonstrated an urgent need for better knowledge of the island's potential earthquake sources. Toward this end, we have prepared a neotectonic map of Taiwan. The map and related cross sections are based upon structural and geomorphic expression of active faults and folds both in the field and on shaded relief maps prepared from a 40‐m resolution digital elevation model, augmented by geodetic and seismologic data. The active tandem suturing and tandem disengagement of a volcanic arc and a continental sliver to and from the Eurasian continental margin have created two neotectonic belts in Taiwan. In the southern part of the orogen both belts are in the final stage of consuming oceanic crust. Collision and suturing occur in the middle part of both belts, and postcollisional collapse and extension dominate the island's northern and northeastern flanks. Both belts consist of several distinct neotectonic domains. Seven domains (Kaoping, Chiayi, Taichung, Miaoli, Hsinchu, Ilan, and Taipei) constitute the western belt, and four domains (Lutao‐Lanyu, Taitung, Hualien, and Ryukyu) make up the eastern belt. Each domain is defined by a distinct suite of active structures. For example, the Chelungpu fault (source of the 1999 earthquake) and its western neighbor, the Changhua fault, are the principal components of the Taichung Domain, whereas both its neighboring domains, the Chiayi and Miaoli Domains, are dominated by major blind faults. In most of the domains the size of the principal active fault is large enough to produce future earthquakes with magnitudes in the mid‐7 values.
An overview of the impulsive geomagnetic field disturbances and power grid impacts associated with the violent Sun‐Earth connection events of 29–31 October 2003 and a comparative evaluation with other contemporary storms
Authors: Kappenman John G.
Published: August 2005

An overview is provided of the geomagnetic storms and impacts on electric power grids associated with the violent Sun‐Earth events of October 2003. During the period from 29 to 31 October 2003, two large geomagnetic storms were observed, as measured by periods of high Kp, Ap, and Dst indices. In fact, these storms had Ap rankings of 6th and 16th all time. This ranking would suggest that the October 2003 storms would be significant with regard to geomagnetically induced currents (GIC) in power grids. However, the resulting geomagnetic storms were much lower in delta B and dB/dt intensity than other historically large geomagnetic storms. A variety of geomagnetic storm processes drove observed GIC. For example, ground observations indicated the presence of large dB/dt pulsations and GIC at North American midlatitude locations on 29 October 2003 that may be due to unusually intense Kelvin‐Helmholtz shearing. Sustained disturbance conditions at low‐latitude and equatorial latitude locations that are likely linked to ring current intensifications may be the source of sustained GIC at these locations and the cause of large power transformer failures. Comparative evaluations will be provided for the 29–31 October 2003 storms and other important and contemporary storms, such as those observed on 13–14 March 1989, 13–14 July 1982, and 15–16 July 2001. Rather than an index‐based evaluation method, the comparative evaluations presented in this paper are based on comparisons of storm morphology. This approach provides a more meaningful comparison of geomagnetic field disturbance dynamics that are important to characterize large GIC threats to power grid infrastructures.
Modeling data relationships with a local variance reducing technique: Applications in hydrology
Authors: Bárdossy András , Pegram Geoffrey G. S. , Samaniego Luis
Published: August 2005

The assessment of an appropriate function describing the relationship between hydrological variables is a frequent problem. The usual way of estimating an overall function is a difficult task if the relationship between the variables is highly nonlinear. Nearest neighbor methods provide an alternative. In this paper different generalizations of the nearest neighbor method are suggested. Besides using different estimators the problem of finding an appropriate distance measure is discussed. Linear embedding of the observation space into an appropriate Euclidean space can improve the distance‐based methods substantially. The suggested transformations and the corresponding local linear estimators are applied to two different examples: the prediction of local discharge characteristics from catchment properties and a simple 1 day flood forecasting problem.
Reevaluation of global emissions from rice paddies of methyl iodide and other species
Authors: Lee‐Taylor J. , Redeker K. R.
Published: August 2005

We present new modeled estimates of global methyl halide emissions from rice cultivation. We extrapolate existing data on rice behavior, including temperature, seasonality and soil moisture effects, to the global scale and on a monthly basis. Our best estimates are 2.4 to 4.9 Gg yr−1 for methyl chloride, 0.5 to 0.9 Gg yr−1 for methyl bromide, and 16 to 29 Gg yr−1 for methyl iodide, with additional uncertainty ranges of ±73%, ±87% and ±75%, respectively. Our central range of estimates reflects the greatest remaining model uncertainty, emission response to soil halide concentrations. We find that peak emissions of methyl halides, especially methyl iodide, are sharply defined in regional and temporal extent. Ambient temperature strongly influences rice emissions of methyl bromide and methyl iodide, which rise by ∼10% for an increase of 1°C.
Deformation of interplanetary shock fronts in the magnetosheath
Authors: Koval A. , Šafránková J. , Němeček Z. , Přech L. , Samsonov A. A. , Richardson J. D.
Published: August 2005

We present in detail the propagation of an IP shock observed by multiple spacecraft in the solar wind and by INTERBALL (IB) in the magnetosheath (hereafter MSH). Our investigation brings an evidence that the observed IP shock slightly decelerates in the MSH contrary to gasdynamic prediction. This result motivates a statistical study of a small number of similar shocks observed in the solar wind and in the MSH that confirmed the case study result. Moreover, we model the interaction of IP shocks with the magnetosphere using an MHD model and show that the observation of shock deceleration agrees with the model prediction. The MHD model suggests that the speed of the shock front propagation in the MSH decreases from the bow shock toward the magnetopause and the front of the IP shock deforms. However, the experimental data are not sufficient to undoubtedly confirm this trend.
Decadal changes in ocean properties revealed by ARGO floats
Authors: King Brian A. , McDonagh Elaine L.
Published: August 2005

This paper demonstrates that CTD data from autonomous profiling floats can be used to detect basin‐scale changes of water properties on decadal, and potentially shorter, timescales. In the western Indian Ocean at 32°S, the properties of Subantarctic Mode Water have changed such that between 1987 and 2002, salinity has increased by 0.04 at certain temperature levels. Although in this case detailed and high‐accuracy data are available from a research cruise, we demonstrate that floats have the following value: real‐time float data are of sufficient accuracy to reveal changes in ocean properties on timescales of a decade or less; floats achieve spatial and temporal coverage not possible with research ships, providing vital supporting evidence for the interpretation of cruise CTD data. Floats therefore provide a mechanism for monitoring changes during the interval between dedicated research cruises and for identifying areas in which further cruises are likely to be fruitful.
The production of hydrogen peroxide during early-stage protein aggregation: a common pathological mechanism in different neurodegenerative diseases?
Authors: Tabner B.J. , Turnbull S. , Fullwood N.J. , German M. , Allsop D.
Published: August 2005

By means of an ESR spin-trapping method, we have shown that Aβ (amyloid β), α-synuclein and various toxic forms of the prion protein all appear to generate H2O2in vitro. A fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of H2O2 during the early stages of protein aggregation.
Expression of normal sequence pathogenic proteins for neurodegenerative disease contributes to disease risk: ‘permissive templating’ as a general mechanism underlying neurodegeneration
Authors: Hardy J.
Published: August 2005

Loci underlying autosomal dominant forms of most neurodegenerative disease have been identified: prion mutations cause Gerstmann Straussler syndrome and hereditary Creutzfeldt–Jakob disease, tau mutations cause autosomal dominant frontal temporal dementia and α-synuclein mutations cause autosomal dominant Parkinson's disease. In these cases, the pathogenic mutation is in the protein that is deposited in the diseased tissue and the whole protein is deposited. In Alzheimer's disease, mutations in amyloid precursor protein or in the presenilins cause autosomal dominant disease. These are the substrate and proteases responsible for the production of the deposited peptide Aβ. Thus, in all the cases, the mutations lead to the disease by a mechanism that involves the deposition process. Furthermore, sporadic forms of all these diseases are predisposed by genetic variability at the same loci, implying that the quantity of the normal protein influences the risk of this form of disease. These results show that the amount of pathogenic protein expression is a key factor in determining disease initiation. Recent work on transgenic models of these diseases is consistent with the view that there are two stages of pathogenesis: a concentration-dependent formation of a pathogenic protein oligomer followed by aggregation on to this oligomeric template by a process that is less dependent on the concentration of the protein.
Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation
Authors: Antonny B. , Bigay J. , Casella J.-F. , Drin G. , Mesmin B. , Gounon P.
Published: August 2005

The GTP switch of the small G-protein Arf1 (ADP-ribosylation factor 1) on lipid membranes promotes the polymerization of the COPI (coat protein complex I) coat, which acts as a membrane deforming shell to form transport vesicles. Real-time measurements for coat assembly on liposomes gives insights into how the GTPase cycle of Arf1 is coupled in time with the polymerization of the COPI coat and the resulting membrane deformation. One key parameter seems to be the membrane curvature. Arf-GAP1 (where GAP stands for GTPase-activating protein), which promotes GTP hydrolysis in the Arf1–COPI complex is highly sensitive to lipid packing. Its activity on Arf1-GTP increases by two orders of magnitude as the diameter of the liposomes approaches that of authentic transport vesicles (60 nm). This suggests that during membrane budding, Arf1-GTP molecules are progressively eliminated from the coated area where the membrane curvature is positive, but are protected from Arf-GAP1 at the bud neck due to the negative curvature of this region. As a result, the coat should be stable as long as the bud remains attached and should disassemble as soon as membrane fission occurs.
On the action of Brefeldin A on Sec7-stimulated membrane-recruitment and GDP/GTP exchange of Arf proteins
Authors: Cherfils J. , Melançon P.
Published: August 2005

Arf (ADP-ribosylation factor) proteins form a special class of small GTP-binding proteins in that their activation by GDP/GTP exchange is coupled to their recruitment to membranes using a built-in structural mechanism. These coupled processes are stimulated by GEFs (guanine nucleotide-exchange factors) that carry a catalytic Sec7 domain, whose basic mechanism has been uncovered by biochemical and structural studies. Crystal structures of intermediates of the GDP/GTP exchange reaction, from which GDP has not dissociated, notably allowed a movie of the exchange reaction to be reconstituted. They showed that Sec7 domains secure Arf-GDP to membranes before they proceed to nucleotide dissociation, and thus are active participants to the coupling of membrane-recruitment to nucleotide exchange. The drug BFA (Brefeldin A) was used to trap the complex that initiates the exchange reaction, providing a structural basis for its inhibition of Arf and its action on the membrane-recruitment of isolated Sec7 domains. Based on the dissection of this basic mechanism, the survey of reported BFA effects in cells on large multidomain ArfGEFs of the BIG1/2 and GBF1 families shows that the levels and compartmental distribution of BFA-induced recruitment of ArfGEFs to membranes cannot be explained from isolated Sec7 domains acting as independent domains. This leads to the hypothesis that Sec7 activity is inhibited in these ArfGEFs by an intramolecular interaction, which would be released by interaction with a compartment-specific receptor.
Localization and function of Arf family GTPases
Authors: Donaldson J.G. , Honda A.
Published: August 2005

Arfs are a family of Ras-related GTP-binding proteins that function in the regulation of membrane trafficking and structure. The six mammalian Arf proteins are expressed ubiquitously and so it is anticipated that each will have a distinct localization and function within the cell. It has been assumed that much of this specificity will be defined by determining which regulators of Arfs, the GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins) function with which Arf proteins. Although in vitro assays may indicate Arf preferences for the numerous Arf GEFs and GAPs that have been identified, in the cell the different Arfs, GEFs and GAPs are targeted to specific compartments where they carry out their functions. We have embarked on studies to define regions of the Arf1 and Arf6 proteins that determine their sites of action and specific activities at the Golgi and plasma membrane respectively. Chimaeras were made between Arf1 and Arf6 in order to identify regions of the protein that contributed to targeting and function. Whereas Arf6 is targeted to the plasma membrane through multiple regions along the protein, we have found a Golgi-targeting region in Arf1 that is sufficient to target Arf6 to the Golgi complex.
Spatiotemporal regulation of the Pak1 kinase
Authors: Parrini M.C. , Matsuda M. , de Gunzburg J.
Published: August 2005

Pak1 (p21-activated kinase 1) is a key regulator of the actin cytoskeleton, adhesion and cell motility. Such biological roles require a tight spatial and kinetic control of its localization and activity. We summarize here the current knowledge on Pak1 dynamics in vivo. Inactive dimeric Pak1 is mainly cytosolic. Localized interaction with the activators Cdc42-GTP and Rac1-GTP stimulates the kinase at the sites of cellular protrusions. Moreover, Pak1 is dynamically engaged into multiprotein complexes forming adhesions to the extracellular matrix. Cutting edge microscopy technologies on living cells are finally shedding light on the intricate spatiotemporal mechanisms regulating Pak1.
Targeting of Rab GTPases to cellular membranes
Authors: Ali B.R. , Seabra M.C.
Published: August 2005

Rab proteins are members of the superfamily of Ras-like small GTPases and are involved in several cellular processes relating to membrane trafficking and organelle mobility throughout the cell. Like other small GTPases, Rab proteins are initially synthesized as soluble proteins and for membrane attachment they require the addition of lipid moiety(ies) to specific residues of their polypeptide chain. Despite their well-documented roles in regulating cellular trafficking, Rab proteins own trafficking is still poorly understood. We still need to elucidate the molecular mechanisms of their recruitment to cellular membranes and the structural determinants for their specific cellular localization. Recent results indicate that Rab cellular targeting might be Rab-dependent, and this paper briefly reviews our current knowledge of this process.
Functional consequences of DNA mismatch repair missense mutations in murine models and their impact on cancer predisposition
Authors: Scherer S.J. , Avdievich E. , Edelmann W.
Published: August 2005

Mutations in MMR (DNA mismatch repair) genes underlie HNPCC (hereditary non-polyposis colon cancer) and also a significant proportion of sporadic colorectal cancers. MMR maintains genome stability and suppresses tumour formation by correcting DNA replication errors and by mediating an apoptotic response to DNA damage. Analysis of mouse lines with MMR missense mutations demonstrates that these MMR functions can be separated and allows the assessment of their individual roles in tumour suppression. These studies in mice indicate that, although the increased mutation rates caused by MMR defects are sufficient to drive tumorigenesis, both functions co-operate in tumour suppression.
Molecular targets of nitric-oxide-donating aspirin in cancer
Authors: Kashfi K. , Rigas B.
Published: August 2005

Nitric-oxide-donating aspirin (NO-ASA), consisting of ASA (aspirin) plus an -ONO2 moiety linked to it via a molecular spacer, is a new drug for cancer prevention. NO-ASA seems to overcome the low potency and toxicity of traditional ASA. The -ONO2 moiety is responsible for releasing NO, and it appears to be required for biological activity. In studies in vitro, NO-ASA inhibits the growth of colon, pancreatic, prostate, lung, skin, leukaemia and breast cancer cells, and is up to 6000-fold more potent than traditional ASA. This effect is owing to cell kinetics [inhibition of proliferation, induction of apoptosis (multiple criteria) and blocking the G1 to S cell-cycle transition] and cell signalling [inhibition of Wnt signalling (IC50=0.2 μM), inhibition of NF-κB (nuclear factor κB) activation (IC50=7.5 μM), inhibition of nitric oxide synthase-2 expression (IC50=48 μM), inhibition of MAPK (mitogen-activated protein kinase) signalling (IC50=10 μM) and induction of cyclo-oxygenase-2 at approx. 10 μM]. In studies in vivo, NO-ASA inhibits intestinal carcinogenesis in Min mice (tumour multiplicity was reduced by 59% after 3 weeks, with no effect in control animals and no side effects) and in the N-nitrosobis(2-oxopropyl)amine model of pancreatic cancer, where there was an 89% reduction in NO-ASA (3000 p.p.m. in the diet)-treated animals (P<0.001). There was no statistically significant effect by traditional ASA at equimolar doses. Our data indicate that NO-ASA is a highly promising agent for the prevention and/or treatment of cancer.
Methylation of theESR1CpG island in the colorectal mucosa is an ‘all or nothing’ process in healthy human colon, and is accelerated by dietary folate supplementation in the mouse
Authors: Belshaw N.J. , Elliott G.O. , Williams E.A. , Mathers J.C. , Buckley L. , Bahari B. , Johnson I.T.
Published: August 2005

ESR1 is frequently silenced by CGI (CpG island) methylation, both in human colorectal tumours and, in an age-dependent manner, in healthy mucosa. It is not clear, however, whether methylation of individual cytosines occurs randomly within the epithelial genome, or preferentially within individual cells as an ‘all-or-nothing’ phenomenon. CGI methylation can be quantified in human DNA residues recovered from faecal samples. We used bisulphite genomic sequencing of human DNA from this source and from a colorectal cancer cell line (SW48) to show that the ESR1 CGI is methylated in an allele-specific manner. This provides support for the ‘all or none’ mechanism for methylation of this gene, and shows how age-dependent methylation of the ESR1 CGI leads rapidly to silencing of the gene within the cells, and hence the colonic crypt within which it occurs. Preliminary studies with a rodent model suggest the rate of age-dependent methylation of ESR1 is modifiable by dietary folate.
Is COX-2 a ‘collateral’ target in cancer prevention?
Authors: Kashfi K. , Rigas B.
Published: August 2005

NSAIDs (non-steroidal anti-inflammatory drugs) prevent colon and other cancers. The fact that NSAIDs inhibit the eicosanoid pathway prompted mechanistic drug-developmental work focusing on COX (cyclo-oxygenase) and its products. The increased prostaglandin E2 levels and the overexpression of COX-2 in colon and many other cancers provided the rationale for clinical trials with COX-2 inhibitors for cancer prevention or treatment. However, one COX-2 inhibitor has been withdrawn from the market because of cardiovascular side effects, and there are concerns about a class effect. Evidence suggests that COX-2 may not be the only, or the ideal, target for cancer prevention; for example, COX-2 is not expressed in human aberrant crypt foci, the earliest recognizable pre-malignant lesion in the colon; COX-2 is expressed in less than half of the adenomas; in vitro data show that NSAIDs do not require the presence of COX-2 to prevent cancer; in familial adenomatous polyposis, the COX-2 inhibitor, celecoxib, had a modest effect, which was weaker than that of a traditional NSAID; and COX-2-specific inhibitors have several COX-2-independent activities, which may account for part of their cancer-preventive properties. The multiple COX-2-indpendent targets, and the limitations of COX-2 inhibitors, suggest the need to explore targets other than COX-2.
DMBT1 expression and glycosylation during the adenoma–carcinoma sequence in colorectal cancer
Authors: Robbe C. , Paraskeva C. , Mollenhauer J. , Michalski J.C. , Sergi C. , Corfield A.
Published: August 2005

The gene DMBT1 (deleted in malignant brain tumour-1) has been proposed to play a role in brain and epithelial cancer, but shows unusual features for a classical tumour-suppressor gene. On the one hand, DMBT1 has been linked to mucosal protection, whereas, on the other, it potentially plays a role in epithelial differentiation. Thus its function in a particular tissue is of mechanistic importance for its role in cancer. Because the former function requires secretion to the lumen and the latter function may depend on its presence in the extracellular matrix, we decided to investigate DMBT1 expression, location and its mode of secretion during malignant transformation in colorectal cancer. Using human colorectal PC/AA cell lines and tissue sections from individual patients, we have examined the expression of DMBT1 and its glycosylation in the adenoma–carcinoma sequence leading to the adenocarcinoma phenotype.
The importance of colonic butyrate transport to the regulation of genes associated with colonic tissue homoeostasis
Authors: Daly K. , Cuff M.A. , Fung F. , Shirazi-Beechey S.P.
Published: August 2005

The transition from normality to malignancy in colorectal cancer is characterized by alterations in the expression of genes associated with the maintenance of tissue homoeostasis. Butyrate, a product of microbial fermentation of dietary fibre in the colon, is known to regulate a number of genes associated with the processes of proliferation, differentiation and apoptosis of colonic epithelial cells, and, hence, homoeostasis of colonic tissue. We have shown previously that the transport of butyrate into colonocytes is of fundamental importance to butyrate's regulatory ability, and therefore sought to assess the expression profile of butyrate-responsive genes in colon cancer tissue, where the expression of the colonic luminal-membrane butyrate transporter, MCT1 (monocarboxylate transporter 1), is significantly down-regulated. In the present paper, we first employed microarray analysis to assess global changes in butyrate-responsive genes using HT29 human colon carcinoma cells treated with butyrate. There was consistency in the butyrate response of selected genes in two other human colonic cell lines (HCT116 and AA/C1) using quantitative real-time PCR. Furthermore, we report that expression levels of selected butyrate-responsive genes involved in the processes of proliferation, differentiation and apoptosis, are deregulated in colon cancer tissue, correlating with decreased expression of MCT1. These findings support our hypothesis that a reduction in MCT1 expression, and hence butyrate transport, can lead to a reduction in the intracellular butyrate levels required to regulate gene expression. Collectively, our results highlight the important contribution of butyrate transport to the maintenance of tissue homoeostasis and disease prevention.
Pantothenate biosynthesis in higher plants
Authors: Coxon K.M. , Chakauya E. , Ottenhof H.H. , Whitney H.M. , Blundell T.L. , Abell C. , Smith A.G.
Published: August 2005

Pantothenate (vitamin B5) is a water-soluble vitamin essential for the synthesis of CoA and ACP (acyl-carrier protein, cofactors in energy yielding reactions including carbohydrate metabolism and fatty acid synthesis. Pantothenate is synthesized de novo by plants and micro-organisms; however, animals obtain the vitamin through their diet. Utilizing our knowledge of the pathway in Escherichia coli, we have discovered and cloned genes encoding the first and last enzymes of the pathway from Arabidopsis, panB1, panB2 and panC. It is unlikely that there is a homologue of the E. coli panD gene, therefore plants must make β-alanine by an alternative route. Possible candidates for the remaining gene, panE, are being investigated. GFP (green fluorescent protein) fusions of the three identified plant enzymes have been generated and the subcellular localization of the enzymes studied. Work is now being performed to elucidate expression patterns of the transcripts and characterize the proteins encoded by these genes.
Mechanistic and inhibition studies of chorismate-utilizing enzymes
Authors: Kerbarh O. , Bulloch E.M.M. , Payne R.J. , Sahr T. , Rébeillé F. , Abell C.
Published: August 2005

The shikimate biosynthetic pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites; it involves seven enzymatic steps in which phosphoenolpyruvate and erythrose 4-phosphate are converted into chorismate. In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin. As mammals do not possess such a biosynthetic system, the enzymes involved in the pathway have aroused considerable interest as potential targets for the development of antimicrobial drugs and herbicides. As an initiative to investigate the mechanism of some of these enzymes, we showed that the antimicrobial effect of (6S)-6-fluoroshikimate is the result of irreversible inhibition of 4-amino-4-deoxychorismate synthase by 2-fluorochorismate. Based on this study, a catalytic mechanism for this enzyme was proposed, in which the residue Lys-274 is involved in the formation of a covalent intermediate. In another study, Yersinia enterocolitica Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin, was for the first time biochemically characterized and shown to catalyse the formation of salicylate from chorismate via isochorismate as a reaction intermediate. A three-dimensional model for this enzyme was constructed that will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake.
Structures and reaction mechanisms of riboflavin synthases of eubacterial and archaeal origin
Authors: Fischer M. , Römisch W. , Illarionov B. , Eisenreich W. , Bacher A.
Published: August 2005

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The dismutation of 6,7-dimethyl-8-ribityllumazine catalysed by riboflavin synthase produces riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, devoid of similarity to riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the CD spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers. Whereas the riboflavin synthase of M. jannaschii is devoid of similarity with those of eubacteria and eukaryotes, it has significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalysing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor.
Role of electron-transfer quenching of chlorophyll fluorescence by carotenoids in non-photochemical quenching of green plants
Authors: Dreuw A. , Fleming G.R. , Head-Gordon M.
Published: August 2005

NPQ (non-photochemical quenching) is a fundamental photosynthetic mechanism by which plants protect themselves against excess excitation energy and the resulting photodamage. A discussed molecular mechanism of the so-called feedback de-excitation component (qE) of NPQ involves the formation of a quenching complex. Recently, we have studied the influence of formation of a zeaxanthin–chlorophyll complex on the excited states of the pigments using high-level quantum chemical methodology. In the case of complex formation, electron-transfer quenching of chlorophyll-excited states by carotenoids is a relevant quenching mechanism. Furthermore, additionally occurring charge-transfer excited states can be exploited experimentally to prove the existence of the quenching complex during NPQ.
Active membrane transport and receptor proteins from bacteria
Authors: Saidijam M. , Bettaney K.E. , Szakonyi G. , Psakis G. , Shibayama K. , Suzuki S. , Clough J.L. , Blessie V. , Abu-bakr A. , Baumberg S. , Meuller J. , Hoyle C.K. , Palmer S.L. , Butaye P. , Walravens K. , Patching S.G. , O'Reilly J. , Rutherford N.G. , Bill R.M. , Roper D.I. , Phillips-Jones M.K. , Henderson P.J.F.
Published: August 2005

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5–35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides.
Subunit movement in individual H+-ATP synthases during ATP synthesis and hydrolysis revealed by fluorescence resonance energy transfer
Authors: Börsch M. , Gräber P.
Published: August 2005

F-type H+-ATP synthases synthesize ATP from ADP and phosphate using the energy supplied by a transmembrane electrochemical potential difference of protons. Rotary subunit movements within the enzyme drive catalysis in either an ATP hydrolysis or an ATP synthesis direction respectively. To monitor these subunit movements and associated conformational changes in real time and with subnanometre resolution, a single-molecule FRET (fluorescence resonance energy transfer) approach has been developed using the double-labelled H+-ATP synthase from Escherichia coli. After reconstitution into a liposome, this enzyme was able to catalyse ATP synthesis when the membrane was energized.
Spectroscopic and theoretical characterization of the AΔ2-XΠ2 transition of CH–Ne
Authors: Kerenskaya Galina , Schnupf Udo , Basinger William H. , Heaven Michael C.
Published: August 2005

The AΔ2-XΠ2 transition of CH–Ne was examined using laser-induced fluorescence and fluorescence depletion techniques. The spectrum was found to be particularly congested due to the large number of bound states derived from the CH(A,n=2)+Ne interaction, and the small energy spacings between these states resulting from the relatively weak anisotropy of the van der Waals bond. High-level ab initio calculations were used to generate two-dimensional potential energy surfaces for CH(X)−Ne and CH(A)−Ne. The equilibrium structures from these surfaces were bent and linear for the X and A states, respectively. Variational calculations were used to predict the bound states supported by the ab initio surfaces. Empirical modification of the potential energy surfaces for the A state was used to obtain energy-level predictions that were in good agreement with the experimental results. Transitions to all of the optically accessible internal rotor states of CH(A,n=2)−Ne were identified, indicating that CH performs hindered internal rotations in the lowest-energy levels of the A and X states. The characteristics of the potential energy surfaces for CH–Ne in the X,A,B, and C states suggest that dispersion and exchange repulsion forces dominate the van der Waals interaction.