bluuebunny/crossref_metadata_2025 huggingface dataset:
Some reconciliation of glacial‐interglacial calcite flux reconstructions for the eastern equatorial Pacific
Authors: Loubere Paul , Richaud Mathieu
Published: March 2007

Carbonate production in the open ocean plays a significant role in fixing upper water column pCO2, and carbonate flux from the surface transfers alkalinity to the deep sea. A key region for this is in the eastern equatorial Pacific (EEP). Reconstructions of calcite accumulation rates with traditional methods (mass accumulation rate (MAR)) have been used to infer a large Last Glacial Maximum (LGM) increase in fluxes for the EEP. The opposite result has been obtained using thorium normalization (Th‐norm) of accumulation rates. New 14C dating, combined with detailed correlation among EEP cores based on planktonic and benthic stable isotope records, emphasizes the sensitivity of the MAR approach to the age model and brings some reconciliation to the methodologies. The new results lead to LGM MAR based accumulation rates that are less than previously estimated, and to lower LGM paleofluxes. Both the MAR and thorium normalized methods show a flux maximum during the deglacial with the new age model. Although the signals show some convergence, the magnitudes of MAR accumulation rates appear to be between about 2 and 4 times those from Th normalization for the LGM at the sites examined. Thus sediment focusing inferred from Th normalization remains significant for the EEP.
Segmentation and along‐strike asymmetry of the passive margin in Socotra, eastern Gulf of Aden: Are they controlled by detachment faults?
Authors: Fournier Marc , Huchon Philippe , Khanbari Khaled , Leroy Sylvie
Published: March 2007

On the island of Socotra, the southern passive margin of the Gulf of Aden displays along its strike two different types of asymmetric structures. Western Socotra is made up of a series of southward tilted blocks bounded by consistently northward dipping normal faults. Eastern Socotra consists of a broad asymmetric anticline with a steep northern limb and a gently dipping southern limb. A zone of NE–SW striking strike‐slip and normal faults separates the two areas. The overall structure is interpreted as representing two rift segments separated by a transfer zone. The along‐strike juxtaposition of crustal‐scale asymmetric structures on the southern margin of the Gulf of Aden is complemented by the asymmetry of the conjugate margins on either side of the gulf. Whereas the western Socotra margin is narrow and characterized by oceanward dipping normal faults, the conjugate Oman margin is broader and dominated by horsts and graben. Considering that asymmetric structures in the upper crust are often associated with synthetic shear zones at deeper ductile levels, we propose that the western and eastern Socotra margin segments were controlled at depth by two detachment faults with opposite dips and senses of shear. The normal faults of western Socotra would sole out into a top‐to‐the‐north ductile shear zone, whereas the asymmetric anticline of eastern Socotra would be associated with a top‐to‐the‐south detachment fault.
Shear at the critical diurnal latitude
Authors: van Haren Hans
Published: March 2007

Around latitudes ∣ϕ∣ ≈ 30° where diurnal D1 equals the local inertial frequency f = 2Ωsinϕ, Ω denoting the Earth's rotational vector, several mechanisms can enhance shear at f due to a reduction in vertical scales. This would imply locally enhanced deep‐ocean mixing. Here, recent 1.5 years of acoustic Doppler current profiler (ADCP) observations from the Canary Basin demonstrate largest kinetic energy at semidiurnal tides (D2), but a complete absence of D2‐shear. Instead, shear is peaking at subinertial 0.97 ± 0.01f and terdiurnal 3f(≈D2 + f ≈ D3 here), and vertical scales Δz(f) < 0.1Δz(D2). However, the f‐band is broader than deterministic tidal frequencies and the smallest vertical scales, organizing shear in thin layers, are found at the lower inertio‐gravity wave limit, which equals 0.97f for the weakest stratification observed (N = 6f, using Δz = 10 m). Hence, besides possibly subharmonic resonance, other mechanisms must be involved in enhancing f‐shear, including non‐linear harmonic interactions and wave trapping at the critical latitude's poleward shift.
Interplanetary origin of intense geomagnetic storms (Dst < −100 nT) during solar cycle 23
Authors: Gonzalez W. D. , Echer E. , Clua‐Gonzalez A. L. , Tsurutani B. T.
Published: March 2007

We study the interplanetary causes of intense geomagnetic storms (Dst < −100 nT) that occurred during solar cycle 23 (1997–2005). It was found that the most common interplanetary structures leading to the development of an intense storm were: magnetic clouds, sheath fields, sheath fields followed by a magnetic cloud and corotating interaction regions leading high speed streams. However, the relative importance of each of those driving structures was found to vary with the solar cycle phase. We divide the cycle in three phases (rising, maximum and declining) and explain the differences. We also discuss about the geoeffectiveness of each of the four main interplanetary driving structures.
Winter accumulation in the percolation zone of Greenland measured by airborne radar altimeter
Authors: Helm V. , Rack W. , Cullen R. , Nienow P. , Mair D. , Parry V. , Wingham D. J.
Published: March 2007

We here determine the surface elevation and the winter snow accumulation rate along a profile in the percolation zone of the Greenland Ice Sheet from data collected with ESA's Airborne SAR/Interferometric Radar Altimeter System (ASIRAS) in spring 2004. The altimeter data show that in addition to a backscatter peak at the air‐snow interface a dominant second peak occurs. This second peak appears due to the strong scattering properties of the last summer surface layer. A robust re‐tracking algorithm was developed that enables the tracking of both interfaces. Utilizing this algorithm, the winter snow thickness is estimated to 1.50 ± 0.13 m. This compares favorably with field measurements (1.43 ± 0.04 m). The snow depth estimates in combination with snow‐density measurements of 420 kg m−3 give a mean winter mass accumulation rate of 63 cm water equivalent (w.e.) and a spatial variation of ±6 cm w.e. Furthermore a strong correlation is found between surface gradient and accumulation rate, with higher accumulation rate in flatter areas. The approach adopted here has significant potential for remote measurements of winter snow accumulation rate across ice sheets at larger spatial scales.
Three‐layer view of transports and hydraulics in the Strait of Gibraltar: A three‐dimensional model study
Authors: Sannino G. , Carillo A. , Artale V.
Published: March 2007

The ability of a three‐dimensional sigma coordinate free surface model to reproduce the interfacial layer between the Atlantic and Mediterranean waters in the Strait of Gibraltar is verified. The model uses a coastal‐following curvilinear orthogonal grid that includes the Gulf of Cadiz and the Alboran Sea, with very high resolution in the strait (less than 500 m). It is forced by imposing the two major semidiurnal tidal components (M2 and S2) along the Atlantic and the Mediterranean open boundaries. The main experiment covers an entire spring‐neap cycle (fortnight period). Results show that the model is able to reproduce a thick interfacial layer that carries a substantial fraction of the total transport. The interfacial thickness is strongly modulated by tidal forcing that enhances the vertical exchanges between layers. In order to take into account the thick interfacial layer, a three‐layer approximation is used to study the hydraulics. Results show that the exchange regime is intermittently maximal due to cyclically controlled regions over Camarinal Sill and within Tarifa Narrows. The analysis has been repeated in a two‐layer framework to evaluate the impact of the introduction of an interfacial layer on the calculation of hydraulics. In the two‐layer approximation, there is not any controlled region extending on the whole cross section, so the exchange is always submaximal. Concluding, the two‐layer approximation underestimates hydraulic controls in the strait respect to the three‐layer approach.
An investigation into axisymmetrization of a vortex embedded in horizontal shearing currents
Authors: Luo Zhexian , Liu Chongjian
Published: March 2007

The problem of tropical cyclogenesis in a triple‐scale coexisting system which includes a subtropical high ridge, a preexisting cyclonic circulation with the initial maximum tangential wind Vbmax of 5 m s−1, and single‐, double‐, or four‐cluster anomalies is examined in this paper, using a shallow water primitive equation (SWPE) model. This work validates the recent results concerning the upscale cascade mechanism suggested for tropical cyclogenesis in the double‐scale coexisting system containing the basic state vortex and anomalies, and it is shown that, for the double‐cluster anomaly, the mean azimuthal maximum tangential wind of the final vortex is increased and the radius of the maximum tangential wind (RMW) is contracted in the case of 1.388 ≥ Kut ≥ 0.0 in which Kut = ∣usmax∣/Vbmax with usmax being the maximum of the easterlies south of the initial subtropical ridge, which demonstrates an amplification of the vortex within this range of parameter. For the single‐cluster anomaly of large amplitude the anomaly will become a master vortex, and for the four‐cluster one the spin‐up of the initial basic state vortex will be decreased. Compared with the case of still environmental currents, introducing the subtropical ridge will have the following impact on axisymmetrization in horizontal shearing currents: (1) The interaction of a single‐cluster anomaly with the basic state vortex will occur in the environmental currents with cyclonic vorticity when the anomaly is located initially in the SW/SE quadrant of the basic state vortex; the interaction between the anomaly and the basic state vortex makes the spin‐up double that in the case of still environmental currents. (2) Although axisymmetrization of vortex configuration may occur, it is mainly limited to the inner region of the vortex with a distinct new nonaxisymmetry in its outer region, and there exist a series of cells of smaller scales in the northeastern quadrant of the outer region. (3) The total kinetic energy for the inner region of the vortex in the mean increases but fluctuates with time, unlike the still environmental currents case in which it increases monotonically.
Aerosol indirect effects as a function of cloud top pressure
Authors: Massie Steven T. , Heymsfield Andrew , Schmitt Carl , Müller Detlef , Seifert Patric
Published: March 2007

Aerosol indirect effects are quantified as a function of cloud top pressure over India and the Indian Ocean in February and March 2003–2005 from analyses of moderate resolution imaging spectroradiometer (MODIS) radiances, reflectance, and aerosol optical depth data. We test the hypothesis that aerosol indirect effects are present throughout the troposphere. Theoretical cloud reflectance values are calculated by application of the discrete‐ordinate DISORT multiple scattering program, as a function of cloud top pressure, aerosol optical depth, and cloud phase (i.e., liquid droplets and cloud ice), and are compared to MODIS cloud reflectance data at 1.38 and 3.75 μm. Aerosol indirect effects are apparent for pressure levels associated with liquid droplets; that is, derivatives of cloud reflectance with respect to aerosol optical depth at 3.75 μm for liquid droplets over land and ocean are larger than corresponding derivatives associated with theoretical aerosol direct effects. Aerosol indirect effects are not apparent for pressure levels associated with tropospheric ice crystals.
Tropical western Pacific warm pool and maritime continent precipitation rates and their contrasting relationships with the Walker Circulation
Authors: Dayem Katherine E. , Noone David C. , Molnar Peter
Published: March 2007

The Cenozoic reorganization of islands in the Maritime Continent region of the tropical western Pacific may have increased the amount of land surface area, changed the distribution of sea surface temperature (SST), and consequently provided a necessary condition for the onset of the Walker Circulation. Precipitation rates over the Maritime Continent, measured with the Tropical Rainfall Measurement Mission (TRMM) satellite and generated from the Climate Prediction Center Merged Analysis of Precipitation (CMAP), correlate positively with a variety of measures of the Walker Circulation's strength (surface easterlies and high‐level westerlies over the western Pacific) and hence with La Niña conditions. Similarly measured precipitation rates over the western Pacific warm pool correlate positively with the strength of tropical zonal circulation shifted to the east, and thus a weakened Walker Circulation and stronger El Niño‐like conditions. Areas of high precipitation rates correlate with areas of ascent in the atmosphere and with high SST. Condensation associated with precipitation over the warm pool provides about twice the amount of latent heating as that over the Maritime Continent largely because the surface area of the warm pool is about 1.9 times larger than that of the Maritime Continent. These results support the conjecture that tectonic expansion and reorganization of the Maritime Continent may have led to a strengthening of the Walker Circulation, and may be related to a change in tropical Pacific climate from a permanent and zonally uniform (El Niño‐like) state in Pre‐Ice Age time to the present day El Niño‐Southern Oscillation (ENSO) state.
Power series solution for falling head ponded infiltration with evaporation
Authors: Mollerup M. , Hansen S.
Published: March 2007

Assumptions for many of the derived analytical infiltration models are either a constant infiltration rate or a constant head at the soil surface. For many realistic infiltration situations a falling head boundary condition is more appropriate. Philip showed that both the wetting profiles and the cumulated infiltration, in one‐dimensional infiltration into homogenous soil with constant head at the upper boundary, can be written as a power series in t. Presently, it is shown that a power series solution can be applied for falling head infiltration with evaporation. When comparing with the constant head solution, the falling head solution differs only from the second term, i.e., the sorptivity remains unchanged, a result which can be applied to falling head infiltrometer technique. The effect of a constant evaporation rate in the falling head scenario appears from the third term. In the presented numerical examples the retention model by van Genuchten and the hydraulic conductivity model by Mualem are applied. By comparing the wetting profiles obtained with a FEM solution it is shown that the discrepancies between the models are very small. Also, the estimation of the time at which the pond is emptied is concordant. The infiltration model is compared with a model by Parlange and colleagues, and a simple infiltrometer method for determining the saturated hydraulic conductivity is proposed.
Hydrologic and atmospheric controls on initiation of convective precipitation events
Authors: Juang Jehn‐Yih , Porporato Amilcare , Stoy Paul C. , Siqueira Mario S. , Oishi A. Christopher , Detto Matteo , Kim Hyun‐Seok , Katul Gabriel G.
Published: March 2007

The pathway to summertime convective precipitation remains a vexing research problem because of the nonlinear feedback between soil moisture content and the atmosphere. Understanding this feedback is important to the southeastern U. S. region, given the high productivity of the timberland area and the role of summertime convective precipitation in maintaining this productivity. Here we explore triggers of convective precipitation for a wide range of soil moisture states and air relative humidity in a mosaic landscape primarily dominated by hardwood forests, pine plantations, and abandoned old field grassland. Using half‐hourly sensible heat flux, micrometeorological, hydrological time series measurements collected at adjacent HW, PP, and OF ecosystems, and a simplified mixed layer slab model, we developed a conditional sampling scheme to separate convective from nonconvective precipitation events in the observed precipitation time series. The series analyzed (2001–2004) includes some of the wettest and driest periods within the past 57 years. We found that convective precipitation events have significantly larger intensities (mean of 2.1 mm per 30 min) when compared to their nonconvective counterparts (mean of 1.1 mm per 30 min). Interestingly, the statistics of convective precipitation events, including total precipitation, mean intensity, and maximum intensity, are statistically different when convective precipitation is triggered by moist and dry soil conditions but are robust in duration. Using the data, we also showed that a “boundary line” emerges such that for a given soil moisture state, air relative humidity must exceed a defined minimum threshold before convective precipitation is realized.
Stretch‐activated calcium channels relay fast calcium waves propagated by calcium‐induced calcium influx
Authors: Jaffe Lionel F.
Published: March 2007

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium‐induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium‐induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 μm/s (at 20°C), rather than the order of 3 to 30 μm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch‐activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non‐flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.
Lipid rafts in health and disease
Authors: Michel Vera , Bakovic Marica
Published: March 2007

Lipid rafts are sphingolipid‐ and cholesterol‐rich domains of the plasma membrane which contain a variety of signalling and transport proteins. Different subtypes of lipid rafts can be distinguished according to their protein and lipid composition. Caveolae are types of rafts that are rich in proteins of the caveolin family (caveolin‐1, −2 and −3) which present a distinct signalling platform. The importance of lipid raft signalling in the pathogenesis of a variety of conditions, such as Alzheimer's, Parkinson's, cardiovascular and prion diseases, systemic lupus erythematosus and HIV, has been elucidated over recent years and makes these specific membrane domains an interesting target for pharmacological approaches in the cure and prevention of these diseases. This Review analyses the importance of lipid raft proteins and lipids in health and disease, with a focus on the current state of knowledge.
FGF4 regulates blood and muscle specification in Xenopus laevis
Authors: Isaacs Harry V. , Deconinck Anne E. , Pownall Mary E.
Published: March 2007

Background information. FGF (fibroblast growth factor) signalling is known to be required for many aspects of mesoderm formation and patterning during Xenopus development and has been implicated in regulating genes required for the specification of both blood and skeletal muscle lineages.

Results. In the present study, we have specifically knocked down the expression of FGF4 using AMO (antisense morpholino oligonucleotide)‐mediated inhibition and demonstrate that FGF4 acts in the dorsal marginal zone to restrict blood development and promote the development of skeletal muscle. In addition, we used a drug inhibitor of FGF signalling and an inducible form of FGFR1 (FGF receptor 1) to identify a period of competence during late blastula and gastrula stages when FGF signalling acts to regulate blood versus muscle specification. Notably, we found that it is the dorsal activity of FGF that is required to restrict the expression of SCL (stem cell leukaemia) to the ventral blood island.

Conclusions. Our data indicate that FGF4 is a key organizer‐derived signal involved in the process of dorsoventral patterning of the mesoderm.
Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids
Authors: Gallego Oriol , Belyaeva Olga V. , Porté Sergio , Ruiz F. Xavier , Stetsenko Anton V. , Shabrova Elena V. , Kostereva Natalia V. , Farrés Jaume , Parés Xavier , Kedishvili Natalia Y.
Published: March 2007

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low Km values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low Km values for retinoids (0.12–1.1 μM), whilst they strongly differ in their kcat values, which range from 0.35 min−1 for AKR1B1 to 302 min−1 for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.
Hydrolysis of (1,4)-β-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-β-D-mannan endohydrolase and β-D-mannosidase
Authors: Hrmova Maria , Burton Rachel A. , Biely Peter , Lahnstein Jelle , Fincher Geoffrey B.
Published: March 2007

A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2–6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has Km constants of 0.16 mg·ml−1 and 5.3 mM and kcat constants of 12.9 and 3.9 s−1 respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-β-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-β-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-β-D-manno-oligosaccharides to Man is a family GH1 β-D-mannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134–11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase.
Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation
Authors: Masson Kristina , Heiss Elke , Band Hamid , Rönnstrand Lars
Published: March 2007

The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr568 and Tyr936 in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.
Age-related accumulation of pentosidine in aggrecan and collagen from normal and degenerate human intervertebral discs
Authors: Sivan Sarit Sara , Tsitron Eve , Wachtel Ellen , Roughley Peter , Sakkee Nico , van der Ham Frits , Degroot Jeroen , Maroudas Alice
Published: March 2007

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1–A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol·(g of protein)−1·year−1. Using previously reported protein turnover rate constants (kT) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (kF) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009–13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81±0.25 compared with 3.71±0.26 μmol of pentosidine·(mol of lysine)−1·year−1 respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year−1 compared with 0.134 year−1 for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective kF values of 1.81±0.25 and 3.18±0.37 μmol of pentosidine·(mol of lysine)−1·year−1. We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.
Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions
Authors: Piromjitpong Juthamart , Wongsantichon Jantana , Ketterman Albert J.
Published: March 2007

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.
Protein phosphatase 1 regulates the stability of the circadian protein PER2
Authors: Gallego Monica , Kang Heeseog , Virshup David M.
Published: March 2007

The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKIϵ (casein kinase I ϵ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKIϵ-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.
Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin
Authors: Shi Zhen , Ferreira Gloria C.
Published: March 2007

Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248–257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (Kiapp values of 1 μM and 2.3 μM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/Km, protoporhyrin IX) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.
Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3
Authors: Nakano Yoko , Banfi Botond , Jesaitis Algirdas J. , Dinauer Mary C. , Allen Lee-Ann H. , Nauseef William M.
Published: March 2007

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.
Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
Authors: Dardente Hugues , Fortier Erin E. , Martineau Vincent , Cermakian Nicolas
Published: March 2007

CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix–loop–helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK–BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1–2 (cryptochromes 1–2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)–BMAL1(BMAL2) circadian transcriptional activation by CRY1–2.
Clustering of monosialyl-Gb5 initiates downstream signalling events leading to invasion of MCF-7 breast cancer cells
Authors: van Slambrouck Severine , Steelant Wim F. A.
Published: March 2007

Invasion is a complex process controlled by secretion and activation of proteases, alteration of integrin levels and GSL (glycosphingolipid) patterns. Differential organization of GSLs with specific membrane proteins and signal transducers in GEMs (GSL-enriched microdomains), initiates signalling events to modify cellular phenotype. Although the GSL monosialyl-Gb5 has been linked with invasion, its functional role in invasion is poorly described and understood. To investigate this problem, we induced the invasion of human breast cancer cells and subsequently explored the underlying mechanism. In the present study, the invasion of human MCF-7 breast cancer cells is highly dependent on clustering of monosialyl-Gb5, and the subsequent activation of monosialyl-Gb5-associated focal adhesion kinase and cSrc in GEM leading to the downstream activation of extracellular-signal-regulated kinase (ERK). As a result, we observed increased expression levels and activity of matrix metalloproteinases-2 and -9, which correlated with decreased expression of integrins α1 and β1. Together these results suggest that the organization of crucial molecules in GEMs of MCF-7 cells is critical for their invasive properties.
Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC–MS/MS method
Authors: Harwood D. Tim , Kettle Anthony J. , Winterbourn Christine C.
Published: March 2007

GSH is rapidly oxidized by HOCl (hypochlorous acid), which is produced physiologically by the neutrophil enzyme myeloperoxidase. It is converted into, mainly, oxidized glutathione. Glutathione sulfonamide is an additional product that is proposed to be covalently bonded between the cysteinyl thiol and amino group of the γ-glutamyl residue of GSH. We have developed a sensitive liquid chromatography–tandem MS assay for the detection and quantification of glutathione sulfonamide as well as GSH and GSSG. The assay was used to determine whether glutathione sulfonamide is a major product of the reaction between GSH and HOCl, and whether it is formed by other two-electron oxidants. At sub-stoichiometric ratios of HOCl to GSH, glutathione sulfonamide accounted for up to 32% of the GSH that was oxidized. It was also formed when HOCl was generated by myeloperoxidase and its yield increased with the flux of oxidant. Of the other oxidants tested, only hypobromous acid and peroxynitrite produced substantial amounts of glutathione sulfonamide, but much less than with HOCl. Chloramines were able to generate detectable levels only when at a stoichiometric excess over GSH. We conclude that glutathione sulfonamide is sufficiently selective for HOCl to be useful as a biomarker for myeloperoxidase activity in biological systems. We have also identified a novel oxidation product of GSH with a molecular weight two mass units less than GSH, which we have consequently named dehydroglutathione. Dehydroglutathione represented a few percent of the total products and was formed with all of the oxidants except H2O2.
p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4–p56lck association-dependent manner
Authors: Barbat Christiane , Trucy Maylis , Sorice Maurizio , Garofalo Tina , Manganelli Valeria , Fischer Alain , Mazerolles Fabienne
Published: March 2007

We previously showed that the association of CD4 and GM3 ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the GM1 ganglioside that partially co-localized with GM3 in these domains. In the present study, we show that CD4–p56lck association in CD4 signalling is required for the redistribution of p56lck, PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56lck. In addition, we show that although these proteins associated in different ways with GM1 and GM3, all of the associations were dependent on CD4–p56lck association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56lck in detergent-insoluble membranes without association with GM1 or GM3. We propose that CD4 ligation and binding with p56lck and their interaction with GM3 and/or GM1 gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.
Structural and functional analysis of the Na+/H+ exchanger
Authors: Slepkov Emily R. , Rainey Jan K. , Sykes Brian D. , Fliegel Larry
Published: March 2007

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.
Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen
Authors: Monaco Susanna , Gioia Magda , Rodriguez Janet , Fasciglione Giovanni Francesco , Di Pierro Donato , Lupidi Giulio , Krippahl Ludwig , Marini Stefano , Coletta Massimo
Published: March 2007

The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37 °C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the β-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation intermediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour, which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism.