bluuebunny/crossref_metadata_2025 huggingface dataset:
The evaluation of noise- and threshold-induced bias in the integration of single-fish echoes
Authors: Bethke Eckhard
Published: April 2004

The echo integration of single-fish echoes shows that characteristically the received-signal energy of single targets is small compared with the echo energy of schooled fish. The measuring error is minimized by the application of an integration threshold. The echo energy, however, is often only slightly larger than the noise and reverberation level making the determination of the optimal integration threshold difficult. During the evaluation of data from the echo-integration surveys on redfish in the Irminger Sea it was observed that the integration value of single fish increased steadily with decreasing integration threshold. There is no way to determine the integration threshold by eye as for schooled fish.

The approach taken in all past publications for the estimation of the influence of the integration threshold on the integration result has been based on the computation of an equivalent beam angle. The influence of “environment noise” was not considered. Here a model is presented, which considers both influences on the integration result during the integration of single-fish echoes. It is assumed that the targets are distributed evenly in the observed volume and that in each pulse volume of the beam only one target is present. The starting point of the computations is the equation for signal processing implemented in the EK500. The echo signal power received is converted pixel-by-pixel into the appropriate volume-backscattering coefficients, sv and stored as echograms. These echograms are available for post-processing in the Bergen Integrator BI500. To exclude noise and reverberation from the subsequent processing a threshold was introduced.

We assume that the fish echoes are always larger than the noise and reverberation. This is a very common situation and the largest part of the energy of noise and reverberation can be eliminated in this way. When a fish echo is received outside the centre of the beam it is attenuated by the beam pattern of the transducer and may therefore lie below the threshold and could be cut off. This leads to the reduction of the measured values and a measuring error. An opposite error arises when the signal crosses the threshold. In practice the signal received always consists of noise and echo signals and has more power than it should have. The measured value is larger and this is another error of the measurement process per se.

The object of this paper is to calculate the influence of the threshold level on the result of measurement and to derive a practicable rule for the determination of the threshold level. The noise is modelled as a constant input power. The computations are carried out with model functions of the transducer beam and the received-echo pulse. General statements can be met by defining a signal-to-noise ratio and a signal-to-threshold ratio. The results of the theoretical investigations are applied on acoustic data obtained during redfish survey WH229 in the Irminger Sea and adjacent waters.
Dynamics of fisheries, and the flexibility of vessel activity in Denmark between 1989 and 2001
Authors: Ulrich Clara , Andersen Bo Sølgaard
Published: April 2004

Danish fishing vessels can be characterized by their diversity of fishing practice in terms of fishing gear and target species, and by their operational flexibility in respect of these fishing practices throughout the year. We describe the temporal fluctuations in this flexibility by following the activity of individual fishing vessels between 1989 and 2001. Initially, a typology of fisheries (classification of fishing trips) and vessel groups (classification of fishing vessels) was established through multivariate analyses of catch and effort data for 1999. In all, 54 fisheries and 25 vessel groups were identified. These typologies were then applied to all data for the whole time period, and the dynamics of fisheries and vessel groups investigated. The dynamics of vessels groups are studied both within groups (main and secondary fisheries, changes in activity patterns) and between groups (tracking of vessels shifting between groups). Results show average stability of vessel activity in terms of the main fishery, along with a great diversity of secondary fisheries and some possibilities for shifting between gears and areas. We conclude that the level of technical interactions is high, and that separation into distinct management units is difficult.
Detecting drizzle in marine warm clouds using combined visible, infrared, and microwave satellite data
Authors: Shao Hongfei , Liu Guosheng
Published: April 2004

We present a method to detect drizzle in marine warm clouds by combining visible, near‐infrared, and microwave measurements from the Tropical Rainfall Measuring Mission (TRMM) satellite. A visible/near‐infrared algorithm is used to simultaneously retrieve cloud optical depth (τ) and effective radius (re). A new microwave algorithm is developed to retrieve liquid water path (LWPm). First, the relationship among LWPm, re and τ are investigated by using radiative transfer model simulations and by analyzing satellite observations. Results from both the modeling and the analysis indicate that LWPm, re and τ satisfy a relation in the form of LWPm = αreβτ(3β−1)/2, and that α and β stay relatively constant within a satellite scene for nondrizzle clouds, while they are different from those for drizzle clouds. On the basis of this result, a drizzle detection index (DI) is defined by the ratio of LWPm to the liquid water path calculated from τ and re using a relation derived for nondrizzle clouds. Model simulation results show that for nondrizzle clouds DI is close to 1, and for drizzle/rain clouds DI is greater than 1. Case studies using satellite data indicate that values of DI are greater in the regions with large re but relatively small τ, and/or near the center of convective cells. The relation among LWPm, re and τ derived from satellite retrievals is consistent with that of radiative transfer simulation results. Finally, coincident airborne cloud radar measurements were used to verify the capability of DI for drizzle detection. The result indicates that DI is useful in differentiating between drizzle and nondrizzle clouds, and suggesting that DI of ∼1.1 is the drizzle threshold, which is also consistent with our radiative transfer model simulations.
Geochemical precursors of the activity of an open‐conduit volcano: The Stromboli 2002–2003 eruptive events
Authors: Carapezza Maria Luisa , Inguaggiato Salvatore , Brusca Lorenzo , Longo Manfredi
Published: April 2004

Marked increases of CO2, H2 and He dissolved in thermal waters and changes in the dissolved carbon isotopic composition, were observed at Stromboli before the 28 December 2002 eruption and before a violent explosive paroxysm occurred on 5 April 2003. High anomalous CO2 flux values were recorded at the crater rim since a week before the eruption onset. The first anomalies in the thermal waters (dissolved CO2 amount) appeared some months before the eruption, when magma column rose at a very high level in the conduit. High peaks of dissolved H2 and He were recorded a few days before the paroxysm. Carbon isotopic composition indicates a magmatic origin of the dissolved CO2 whose increase, together with those of H2 and He, is attributed to an increasing output of deep gases likely produced by depressurization of a rising batch of a deep gas‐rich magma, whose fragments have been emitted during the explosion.
Modified alginate matrices for the immobilization of bioactive agents
Authors: Le‐Tien Canh , Millette Mathieu , Lacroix Monique , Mateescu Mircea‐Alexandru
Published: April 2004

Bioactive agents (catalase – an enzyme, and nisin – a bacteriocin) were covalently immobilized on alginate activated with sodium periodate (oxidatively converting 2,3‐dihydroxy groups into dialdehyde residues), followed or preceded by ionotropic gelation. For the same protein coupling yield, the retained enzyme activity of the immobilized enzyme (ImE) can be markedly increased by diminishing the bead diameter, a phenomenon that illustrates the role of substrate/product diffusion through the bead gel layer. When the amount of enzyme introduced for coupling was about 15 mg/100 mg of support and the bead diameter was about 100 μm, a high retained specific activity (95–98%) was obtained. Diffusion phenomena can be markedly decreased by enzyme immobilization on the surface of microbeads (obtained by gelation of activated alginate prior to immobilization). In this case, the retained activity was approx. 75% of that of the free enzyme. A slightly higher Km value of ImE suggested that the enzyme–substrate affinity was almost maintained. The profiles of ImE activities at various pH values, at various temperatures and when undergoing proteolysis showed a overall higher stability for the immbolized than that for the free enzyme. Nisin immobilized on the microbead surface, when submitted to proteolysis, conserved its bacteriocin activity, strongly inhibiting the growth of Lactobacillus sake when subjected to an agar spot test, whereas free nisin totally lost its activity.
Immunization with a DNA vaccine encoding the hepatitis‐C‐virus structural antigens elicits a specific immune response against the capsid and envelope proteins in rabbits andMacaca irus(crab‐eating macaque monkeys)
Authors: Dueñas‐Carrera Santiago , Viña Ariel , Martínez Rafael , Alvarez‐Lajonchere Liz , Alvarez‐Obregón Julio César , Marante Jeny , Pérez Anna , Mosqueda Omar , Martínez Gillian , Morales Juan
Published: April 2004

In the present study, we evaluated the capability of the plasmid pIDKE2, encoding the HCV (hepatitis C virus) structural proteins Core, E1 and E2, to induce immune response against HCV antigens after injection into rabbits andMacaca irus(crab‐eating macaque). Animals were immunized intramuscularly with different amounts of plasmid on weeks 0, 3 and 8. Monkeys received a booster dose on week 46. All rabbits immunized with pIDKE2 generated a positive antibody response and, particularly in rabbits immunized with 2 mg, antibody titres reached values above 1:1500 and 1:400 against the core and the envelope proteins, respectively, 28 weeks after primary immunization. The antibody response in monkeys developed slowly, but antibody titres greater than 1:3500 against HCV structural antigens were detected at week 52. Moreover, anti‐E2 antibodies recognized synthetic peptides covering the HVR‐1 (hypervariable region‐1) from different isolates corresponding to different genotypes. Additionally, a specific lymphoproliferative response against Core and E2 was detected in two out of three monkeys immunized with pIDKE2. The other monkey had a specific proliferative response to E1. Taking all these data together, immunization with pIDKE2 is able to elicit both humoral and cellular immunity against HCV structural antigens in animal models other than mice.
Separation of immunoglobulin G precipitate from contaminating proteins using microfiltration
Authors: Neal Greg , Francis Richard , Shamlou Parviz Ayazi , Keshavarz‐Moore Eli
Published: April 2004

A small‐scale stirred‐cell device was used to separate an antivenom antibody precipitate from a suspension containing contaminating soluble proteins. The device has a total volume of 200 ml and is equipped with a filter membrane with a cut‐off size of 3 μm. About 90% of the antibody‐precipitate particles in the feed were 29 μm or smaller, and the concentration of solids was 12% (w/w). The microfiltration cell was operated in a constant‐volume (continuous/diafiltration) mode, and its performance was compared with an industrial disc‐stack centrifuge currently used in the manufacture of antibody precipitate. In terms of product purity, the separation performance of the microfiltration operation was found to be comparable with the disc‐stack centrifuge, whereas the overall yield was 10% better than that obtained from the centrifuge. This was attributed to the ability of microfiltration to reduce material losses by integrating a number of operations in a single piece of equipment. These included separation, concentration and buffer exchange, as well as dissolution and final recovery of the antibody in an appropriate buffer. The results obtained from the stirred cell are potentially scaleable, and dynamic microfiltration is shown to be an attractive process option.
Freeze‐drying of proteins: some emerging concerns
Authors: Roy Ipsita , Gupta Munishwar Nath
Published: April 2004

Freeze‐drying (lyophilization) removes water from a frozen sample by sublimation and desorption. It can be viewed as a three‐step process consisting of freezing, primary drying and secondary drying. While cryoprotectants can protect the protein from denaturation during early stages, lyoprotectants are needed to prevent protein inactivation during drying. The structural changes as a result of freeze‐drying have been investigated, especially by FTIR (Fourier‐transform IR) spectroscopy. In general, drying results in a decrease of α‐helix and random structure and an increase in β‐sheet structure. In the case of basic fibroblast growth factor and γ‐interferon, enhanced FTIR showed large conformational changes and aggregation during freeze‐drying, which could be prevented by using sucrose as a lyoprotectant. It is now well established that structural changes during freeze‐drying are responsible for low activity of freeze‐dried powders in nearly anhydrous media. Strategies such as salt activation can give ‘activated’ enzyme powders, e.g. salt‐activated thermolysin‐catalysed regioselective acylation of taxol to give a more soluble derivative for therapeutic use. In the presence of moisture, freeze‐dried proteins can undergo disulphide interchange and other reactions which lead to inactivation. Such molecular changes during storage have been described for human insulin, tetanus toxoid and interleukin‐2. Some successful preventive strategies in these cases have also been mentioned as illustrations. Finally, it is emphasized that freeze‐drying is not an innocuous process and needs to be understood and used carefully.
Changes in diffusion through the brain extracellular space
Authors: Mota Manuel , Teixeira José A. , Keating José B. , Yelshin Alexander
Published: April 2004

ECS (extracellular space) works as the microenvironment of brain cells. Diffusion through ECS may be described through an effective diffusion coefficient, De, which in turn depends on ECS porosity, ɛ, and tortuosity, T. In the present research, diffusion data together with ɛ and T were collected from the specialized literature and analysed to seek a correlation of T versus ɛ. On the basis of De data, upper and lower T boundaries were defined and related to topologically ‘dense’ and ‘loose’ cell arrangement. A possible range for T variation was obtained for ECS, with ɛ ranging from 0.05 to 0.6. A tortuosity index (n) in the form of T and ɛ logarithmic ratio was introduced. This index may be adopted for recalculation of T or ɛ if only one of these parameters is known. As a result of data analysis and modelling, it was concluded that, upon different external conditions, for instance oxygen depletion, the ECS porosity decreases and cells (presumably through membrane rearrangements) adjust the void space to keep the diffusion within a defined range, which gives the living tissue the ability to maintain the diffusion level up to two or more times higher than in conventional granular bed packing. Thus, even with a dramatic ECS decrease, the cellular system is still able to support a given diffusion by decreasing the value of T. The obtained results clearly show the existence of three data clusters: a region of normal brain functioning, both for young and adult brains, for values of ɛ comprised between 0.15 and 0.30, and two regions of abnormal brain behaviour to the left and to the right of the normal region, corresponding to different states (aging, tumours, anoxia, brain death, etc.). The present approach allows defining the optimal range of ɛ and T to assure the best ECS diffusion efficiency for a specified macromolecule. This might be important in brain clinical treatment.
Activation of the DNA-binding ability of latent p53 protein by protein kinase C is abolished by protein kinase CK2
Authors: POSPÍŠILOVÁ Šárka , BRÁZDA Václav , KUCHAŘÍKOVÁ Kateřina , LUCIANI M. Gloria , HUPP Ted R. , SKLÁDAL Petr , PALEČEK Emil , VOJTĚŠEK Bořivoj
Published: April 2004

p53 is one of the most important regulators of cell proliferation and differentiation and of programmed cell death, triggering growth arrest and/or apoptosis in response to different cellular stress signals. The sequence-specific DNA-binding function of p53 protein can be activated by several different stimuli that modulate the C-terminal domain of this protein. The predominant mechanism of activation of p53 sequence-specific DNA binding is phosphorylation at specific sites. For example, phosphorylation of p53 by PKC (protein kinase C) occurs in undamaged cells, resulting in masking of the epitope recognized by monoclonal antibody PAb421, and presumably promotes steady-state levels of p53 activity in cycling cells. In contrast, phosphorylation by cdk2 (cyclin-dependent kinase 2)/cyclin A and by the protein kinase CK2 are both enhanced in DNA-damaged cells. We determined whether one mechanism to account for this mutually exclusive phosphorylation may be that each phosphorylation event prevents modification by the other kinase. We used non-radioactive electrophoretic mobility shift assays to show that C-terminal phosphorylation of p53 protein by cdk2/cyclin A on Ser315 or by PKC on Ser378 can efficiently stimulate p53 binding to DNA in vitro, as well as binding of the monoclonal antibody Bp53-10, which recognizes residues 371–380 in the C-terminus of p53. Phosphorylation of p53 by CK2 on Ser392 induces its DNA-binding activity to a much lower extent than phosphorylation by cdk2/cyclin A or PKC. In addition, phosphorylation by CK2 strongly inhibits PKC-induced activation of p53 DNA binding, while the activation of p53 by cdk2/cyclin A is not affected by CK2. The presence of CK2-mediated phosphorylation promotes PKC binding to its docking site within the p53 oligomerization domain, but decreases phosphorylation by PKC, suggesting that competition between CK2 and PKC does not rely on the inhibition of PKC–p53 complex formation. These results indicate the crucial role of p53 C-terminal phosphorylation in the regulation of its DNA-binding activity, but also suggest that antagonistic relationships exist between different stress signalling pathways.
Regulation of glycogen metabolism in cultured human muscles by the glycogen phosphorylase inhibitor CP-91149
Authors: LERÍN Carlos , MONTELL Eulàlia , NOLASCO Teresa , GARCÍA-ROCHA Mar , GUINOVART Joan J. , GÓMEZ-FOIX Anna M.
Published: April 2004

Pharmacological inhibition of liver GP (glycogen phosphorylase), which is currently being studied as a treatment for Type II (non-insulin-dependent) diabetes, may affect muscle glycogen metabolism. In the present study, we analysed the effects of the GP inhibitor CP-91149 on non-engineered or GP-overexpressing cultured human muscle cells. We found that CP-91149 treatment decreased muscle GP activity by (1) converting the phosphorylated AMP-independent a form into the dephosphorylated AMP-dependent b form and (2) inhibiting GP a activity and AMP-mediated GP b activation. Dephosphorylation of GP was exerted, irrespective of incubation of the cells with glucose, whereas inhibition of its activity was synergic with glucose. As expected, CP-91149 impaired the glycogenolysis induced by glucose deprivation. CP-91149 also promoted the dephosphorylation and activation of GS (glycogen synthase) in non-engineered or GP-overexpressing cultured human muscle cells, but exclusively in glucose-deprived cells. However, this inhibitor did not activate GS in glucose-deprived but glycogen-replete cells overexpressing PTG (protein targeting to glycogen), thus suggesting that glycogen inhibits the CP-91149-mediated activation of GS. Consistently, CP-91149 promoted glycogen resynthesis, but not its overaccumulation. Hence, treatment with CP-91149 impairs muscle glycogen breakdown, but enhances its recovery, which may be useful for the treatment of Type II (insulin-dependent) diabetes.
Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels
Authors: RAINBOW Richard D. , JAMES Marian , HUDMAN Diane , JOHI Mohammed AL , SINGH Harprit , WATSON Peter J. , ASHMOLE Ian , DAVIES Noel W. , LODWICK David , NORMAN Robert I.
Published: April 2004

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein–SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254–1545), rSUR2A-CTB (residues 1254–1403) and rSUR2A-CTC (residues 1294–1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358–1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294–1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294–1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.
Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release
Authors: DULHUNTY Angela F. , CURTIS Suzanne M. , CENGIA Louise , SAKOWSKA Magdalena , CASAROTTO Marco G.
Published: April 2004

We show that peptide fragments of the dihydropyridine receptor II–III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793–Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671–Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at ≥10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 µM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 µM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II–III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers.
Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4
Authors: BOULANGER Marie-Chloé , MIRANDA Tina Branscombe , CLARKE Steven , di FRUSCIO Marco , SUTER Beat , LASKO Paul , RICHARD Stéphane
Published: April 2004

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 (Drosophilaarginine methyltransferases 1–9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.
Functional refolding of the Campylobacter jejuni MOMP (major outer membrane protein) porin by GroEL from the same species
Authors: GOULHEN Florence , DÉ Emmanuelle , PAGÈS Jean-Marie , BOLLA Jean-Michel
Published: April 2004

Functional and structural studies of outer membrane proteins from Gram-negative bacteria are frequently carried out using refolded proteins. Recombinant proteins are produced in Escherichia coli as inclusion bodies and then tediously refolded by dilution in buffered detergent solutions. In the present work, we obtained the refolding of MOMP (major outer membrane protein) from Campylobacter assisted by the molecular chaperone GroEL. Refolded MOMP recovered its native pore-forming activity when reconstituted in planar lipid bilayers. Both proteins were purified from the Campylobacter jejuni strain 85H. The purity of GroEL was assessed by silver staining and MS. Its native ultrastructure was observed by the use of transmission electron microscopy. Denaturation of MOMP was performed in urea at 65 °C followed by dialysis against 100 mM acetic acid, and was assessed by CD analysis. MOMP refolding reached a maximum efficiency in the presence of GroEL (at a MOMP/GroEL molar ratio of 9:1) and ATP. Under these conditions, 95% of denatured MOMP was refolded after a 15 min incubation. This approach represents an alternative method in studies of membrane protein refolding.
Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates
Authors: INOUE Katsuhisa , FEI You-Jun , ZHUANG Lina , GOPAL Elangovan , MIYAUCHI Seiji , GANAPATHY Vadivel
Published: April 2004

In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis–Menten constant for the transport process is 38±5 µM for citrate and 37±6 µM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis–Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.
Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy
Authors: DOWLING Paul , DORAN Philip , OHLENDIECK Kay
Published: April 2004

Although the primary abnormality in dystrophin is the underlying cause for mdx (X-chromosome-linked muscular dystrophy), abnormal Ca2+ handling after sarcolemmal microrupturing appears to be the pathophysiological mechanism leading to muscle weakness. To develop novel pharmacological strategies for eliminating Ca2+-dependent proteolysis, it is crucial to determine the fate of Ca2+-handling proteins in dystrophin-deficient fibres. In the present study, we show that a key luminal Ca2+-binding protein SAR (sarcalumenin) is affected in mdx skeletal-muscle fibres. One- and two-dimensional immunoblot analyses revealed the relative expression of the 160 kDa SR (sarcoplasmic reticulum) protein to be approx. 70% lower in mdx fibres when compared with normal skeletal muscles. This drastic reduction in SAR was confirmed by immunofluorescence microscopy. Patchy internal labelling of SAR in dystrophic fibres suggests an abnormal formation of SAR domains. Differential co-immunoprecipitation experiments and chemical cross-linking demonstrated a tight linkage between SAR and the SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1) isoform of the SR Ca2+-ATPase. However, the relative expression of the fast Ca2+ pump was not decreased in dystrophic membrane preparations. This implies that the reduction in SAR and calsequestrin-like proteins plays a central role in the previously reported impairment of Ca2+ buffering in the dystrophic SR [Culligan, Banville, Dowling and Ohlendieck (2002) J. Appl. Physiol. 92, 435–445]. Impaired Ca2+ shuttling between the Ca2+-uptake SERCA units and calsequestrin clusters via SAR, as well as an overall decreased luminal ion-binding capacity, might indirectly amplify the Ca2+-leak-channel-induced increase in cytosolic Ca2+ levels. This confirms the idea that abnormal Ca2+ cycling is involved in Ca2+-induced myonecrosis. Hence, manipulating disturbed Ca2+ handling might represent new modes of abolishing proteolytic degradation in muscular dystrophy.
A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma
Authors: GIUDICI Maria-Luisa , EMSON Piers C. , IRVINE Robin F.
Published: April 2004

Type I PIPkins (phosphatidylinositol 4-phosphate 5-kinases) are the enzymes that catalyse the major cellular route of synthesis of PtdIns(4,5)P2, and three isoforms (α, β and γ) with several splice variants have been found to date. In the present paper, we describe the discovery of a novel splice variant of the γ isoform, which we call PIPkin Iγc, and which is characterized by the inclusion of a 26-amino-acid insert near the C-terminus. Its transcript appears to be selectively expressed in brain, where it locates in the neurons of restricted regions, such as cerebellum, hippocampus, cortex and olfactory bulb, as indicated by in situ hybridization studies. Overexpression of two different catalytically inactive constructs of PIPkin Iγc in rat cerebellar granule cells causes a progressive loss of their neuronal processes, whereas equivalent kinase-dead versions of PIPkin Iγa did not induce any such effect, suggesting the possible existence of a specific PtdIns(4,5)P2 pool synthesized by PIPkin Iγc, which is involved in the maintenance of some neuronal cellular processes.
Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms
Authors: ZHAO Xiangshan , GAN Lixia , PAN Haiyun , KAN Donghui , MAJESKI Michael , ADAM Stephen A. , UNTERMAN Terry G.
Published: April 2004

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1–149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1–50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.
Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles
Authors: CELEJ M. Soledad , D'ANDREA Mariana G. , CAMPANA Patricia T. , FIDELIO Gerardo D. , BIANCONI M. Lucia
Published: April 2004

The catalytic behaviour of α-CT (α-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme–micelle interaction leads to an increase in both the Vmax and the affinity for the substrate p-nitrophenyl acetate, indicating higher catalytic efficiency for bound α-CT. The bell-shaped profile of α-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in α-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the α-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in α-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of α-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased α-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm−1, corresponding to the α-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the β-sheet content in micelle-bound α-CT. Our data suggest that the higher catalytic efficiency of micelle-bound α-CT results from significant conformational changes.
Identification of glycogen synthase as a new substrate for stress-activated protein kinase 2b/p38beta
Authors: KUMA Yvonne , CAMPBELL David G. , CUENDA Ana
Published: April 2004

The endogenous glycogen synthase in extracts from mouse skeletal muscle, liver and brain bound specifically to SAPK2b (stress-activated protein kinase 2b)/p38β, but not to other members of the group of SAPK/p38 kinases. Glycogen synthase was phosphorylated in vitro more efficiently by SAPK2b/p38β than by SAPK2a/p38α, SAPK3/p38γ or SAPK4/p38δ. SAPK2b/p38β phosphorylated glycogen synthase in vitro at residues Ser644, Ser652, Thr718 and Ser724, two of which (Ser644 and Ser652) are also phosphorylated by glycogen synthase kinase 3. Thr718 and Ser724 are novel sites not known to be phosphorylated by other protein kinases. Glycogen synthase becomes phosphorylated at Ser644 in response to osmotic shock; this phosphorylation is prevented by pretreatment of the cells with SB 203580, which inhibits SAPK2a/p38α and SAPK2b/p38β activity. In vitro, phosphorylation of glycogen synthase by SAPK2b/p38β alone had no significant effect on its activity, indicating that phosphorylation at residue Ser644 itself is insufficient to decrease glycogen synthase activity. However, after phosphorylation by SAPK2b/p38β, subsequent phosphorylation at Ser640 by glycogen synthase kinase 3 decreased the activity of glycogen synthase. This decrease was not observed when SAPK2b/p38β activity was blocked with SB 203580. These results suggest that SAPK2b/p38β may be a priming kinase that allows glycogen synthase kinase 3 to phosphorylate Ser640 and thereby inhibit glycogen synthase activity.
Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila
Authors: CHOI Tae-Yeong , PARK S. Young , KANG Ho-Sung , CHEONG Jae-Hun , KIM Han-Do , LEE Bok Luel , HIROSE Fumiko , YAMAGUCHI Masamitsu , YOO Mi-Ae
Published: April 2004

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16–115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.
A new ephrin-A1 isoform (ephrin-A1b) with altered receptor binding properties abrogates the cleavage of ephrin-A1a
Authors: FINNE Eivind F. , MUNTHE Else , AASHEIM Hans-Christian
Published: April 2004

Ephrins are ligands for the Eph receptor tyrosine kinases, which play important roles in patterning nervous and vascular systems. Ephrin-A1 is a glycosylphosphatidylinositol-anchored ligand that binds to the EphA receptor tyrosine kinases. In the present study, we have identified a new ephrin-A1 isoform, denoted ephrin-A1b (ephrin-A1 isoform b). Compared with the originally described ephrin-A1 sequence, ephrin-A1a [Holzman, Marks and Dixit (1990) Mol. Cell. Biol. 10, 5830–5838], ephrin-A1b lacks a segment of 22 amino acids (residues 131–152). At the transcript level, exon 3 is spliced out in the transcript encoding ephrin-A1b. Transfection of HEK-293T cells (human embryonic kidney 293 cells) with an ephrin-A1b-expressing plasmid resulted in a significant expression of the protein on the cell surface. However, soluble EphA2 receptor (EphA2-Fc) bound weakly to ephrin-A1b-expressing transfectants, but bound strongly to ephrin-A1a-expressing transfectants. Ephrins have been shown to undergo regulated cleavage after interaction with their receptors. This process is inhibited by co-expression of ephrin-A1a and ephrin-A1b, indicating that ephrin-A1b influences the cleavage process. Taken together, these findings indicate that this newly described isoform may regulate the function of its ephrin-A1a counterpart.
The porcine taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21) gene: evolution by gene duplication and gene conversion
Authors: LUNDELL Kerstin
Published: April 2004

Porcine taurochenodeoxycholic acid 6α-hydroxylase, cytochrome P450 4A21 (CYP4A21), differs from other members of the CYP4A subfamily in terms of structural features and catalytic activity. CYP4A21 participates in the formation of hyocholic acid, a species-specific primary bile acid in the pig. The CYP4A21 gene was investigated and found to be approx. 13 kb in size and split into 12 exons. The intron–exon organization of the CYP4A21 gene corresponds to that of CYP4A fatty acid hydroxylase genes in other species. Comparison with a genomic segment of a pig CYP4A fatty acid hydroxylase gene (CYP4A24) revealed a sequence identity with CYP4A21 that extends beyond the exons, indicating a common origin by gene duplication. A pronounced sequence identity was found also within the proximal 5´-flanking regions, whereas the patterns of mRNA expression of CYP4A21 and CYP4A fatty acid hydroxylases in pig liver differ. Sequence comparison aiming to elucidate the origin of the unique features of CYP4A21 revealed a region of decreased sequence identity from exon 6 to exon 8, strongly suggesting that gene conversion could have contributed to the evolution of CYP4A21.
Critical cysteine residues for regulation of integrin alphaIIbbeta3 are clustered in the epidermal growth factor domains of the beta3 subunit
Authors: KAMATA Tetsuji , AMBO Hironobu , PUZON-McLAUGHLIN Wilma , TIEU Kenneth Khiem , HANDA Makoto , IKEDA Yasuo , TAKADA Yoshikazu
Published: April 2004

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin β3 subunit are involved in the regulation of αIIbβ3. In the present study, we studied systematically the role of disulphide bonds in β3 on the ligand-binding function of αIIbβ3 by mutating individual cysteine residues of β3 to serine. We found that the disulphide bonds that are critical for αIIbβ3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate αIIbβ3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated αIIbβ3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep αIIbβ3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.
Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail
Authors: YAMANOUCHI Jun , HATO Takaaki , TAMURA Tatsushiro , FUJITA Shigeru
Published: April 2004

Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the α and β tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the α tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membranedistal region of the αIIb tail. Alanine substitution of the RPP residues in the αIIb tail rendered αIIbβ3 constitutively active in a metabolic energy-dependent manner. Although an αIIb/α6Aβ3 chimaeric integrin, in which the αIIb tail was replaced by the α6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the α6A tail inhibited binding of an activation-dependent antibody PAC1. In αIIb/α6Aβ3, deleting the TSDA sequence from the α6A tail or single amino acid substitutions of the TSDA residues inhibited αIIb/α6Aβ3 activation and replacing the membrane-distal region of the αIIb tail with TSDA rendered αIIbβ3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the αIIb tail containing the RPP sequence of the membrane-distal region failed to activate αIIbβ3. These results suggest that the RPP sequence after the GFFKR motif of the αIIb tail suppresses energy-dependent αIIbβ3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by α subunit tails.
pH-dependent autocleavage of lambda repressor occurs in the operator-bound form: characterization of lambda repressor autocleavage
Authors: GHOSH Kaushik , PAL Atasi , CHATTOPADHYAYA Rajagopal
Published: April 2004

The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the λ cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 °C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the λ repressor. The difficulty in obtaining this covalent modification is rationalized using our recent λ repressor models. Bimolecular type II trans-cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165–1173], but it could still be cleaved by an 85–202 ‘enzyme’ fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans-cleavage was not observed with the covalently modified intact λ repressor used as substrate and the purified wild-type λ repressor 112–236 fragment used as the ‘enzyme’. All these results show that for the wild-type λ repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.