bluuebunny/crossref_metadata_2025 huggingface dataset:
A global calculation of the δ13C of soil respired carbon: Implications for the biospheric uptake of anthropogenic CO2
Authors: Ciais P. , Friedlingstein P. , Schimel D. S. , Tans P. P.
Published: December 2003

The continuing emissions of fossil CO2 depleted in 13C have been causing a gradual decrease in atmospheric δ13C by roughly 1.4‰ since preindustrial times. The progressive penetration of this perturbation into the land biota causes the soil organic matter to be enriched in 13C with respect to recently formed plant material. This effect which we call the “biotic isotope disequilibrium” is important when it comes to deducing the terrestrial carbon fluxes by using δ13C in atmospheric CO2. We have estimated the geographical distribution of the isotopic disequilibrium, which is primarily influenced by the turnover of carbon in the various ecosystems, from the output of two biospheric models, (SLAVE and CENTURY). The disequilibrium is estimated to shift up the δ13C of atmospheric CO2 by the same amount as a net sink of 0.6 Gt C yr−1 in the land biota. This “fake” terrestrial sink due to the isotopic disequilibrium is distributed mainly in northern midlatitudes (0.2 Gt C yr−1) and tropical forests (0.3 Gt C yr−1).
Ocean primary production derived from satellite data: An evaluation with atmospheric oxygen measurements
Authors: Balkanski Yves , Monfray Patrick , Battle Mark , Heimann Martin
Published: December 2003

Recently, very precise measurements have detected the seasonal variability in the atmospheric O2/N2 ratio at several sites in the northern and southern hemispheres. In this paper, we derive marine primary productivity (PP) from satellite ocean color data. To infer air‐sea oxygen fluxes, a simple one‐dimensional diagnostic model of ocean biology has been developed that depends on only two parameters: a time delay between organic production and oxidation (set to 2 weeks) and an export scale length (50 m). This model gives a global net community production of 4.3 mol C m−2 yr−1 in the euphotic zone and 3.2 mol C m−2 yr−1 in the mixed layer. This last value corresponds to a global f ratio (net community production (NCP)/PP) at the base of the mixed layer of 0.37. The air‐sea fluxes derived from this model are then used at the base of a three‐dimensional atmospheric model to compare the atmospheric seasonal cycle of O2/N2 at five sites: Cape Grim (40.6°S, 144.6E), Baring Head (41.3°S, 174.8°E), Mauna Loa (19.5°N,154.8°W), La Jolla (32.9°N, 117.3°W), and Barrow (71.3°N, 156.6°W). The agreement between model and observations is very encouraging. We infer from the agreement that the seasonal variations in O2/N2 are largely controlled by the photosynthesis rate but also by the remineralization linked to the deepening and shoaling of the mixed layer. Lateral ventilation to high latitudes may also be an important factor controlling the amplitude of the seasonal cycle.
A model function for the ocean‐normalized radar cross section at 14 GHz derived from NSCAT observations
Authors: Wentz Frank J. , Smith Deborah K.
Published: December 2003

A model for the ocean surface normalized radar cross section σo is derived from 3 months of NASA scatterometer (NSCAT) observations (September 15 to December 18, 1996). The model expresses σo as a function of wind speed, relative wind direction, incidence angle, and polarization. The dependence of σo on wind speed is based on collocated special sensor microwave/imager (SSM/I) satellite wind retrievals and European Centre for Medium‐Range Weather Forecasts (ECMWF) model winds. We find that at low winds (<5 ms−1), the SSM/I winds are more reliable than ECWMF, probably owing to small location errors in the ECMWF wind features. The primary wind direction dependence of σo (i.e., the second harmonic) is found from histograms of the σo difference between the forward and aft antennas. The σo versus wind speed relationship is adjusted for cross‐swath incidence angle differences and is then incorporated into the NSCAT 1 model used to process the 10‐month (September 15, 1996, to June 29, 1997) NSCAT data set. The resulting NSCAT 1 wind vectors are compared to ECMWF wind fields and buoys. The mean and standard deviation of the NSCAT minus ECMWF (buoy) wind speed difference are 0.05 and 1.78 ms−1 (−0.29 and 1.26 ms−1), respectively. The wind direction mean and standard deviation differences are 0.8° and 18.5° (7.9° and 15.7°), respectively. The difference between the NSCAT and the ECMWF (buoy) direction exceeds 90° only 1.1% (1.2%) of the time. We have no explanation for why the buoy wind directions are biased 8° relative to both NSCAT and ECMWF.
Fitting a regional ocean model with adjustable open boundaries to TOPEX/POSEIDON data
Authors: Vogeler Armin , Schröter Jens
Published: December 2003

TOPEX/POSEIDON altimeter data referenced to geoid JGM2 are assimilated into an eddy‐resolving quasi‐geostrophic ocean model. The location of the model domain is the African sector of the Antarctic Circumpolar Current. All lateral boundaries of the model domain are open. The model has three layers, a spatial resolution of approximately 20 km and realistic bottom topography. The adjoint method (4DVAR) is used to optimize control variables which are the initial state of the stream function and the time dependent boundary values of the stream function and the relative vorticity. Both the numerical implementation of the model and the assimilation method are formulated such that the model can be placed in any ocean location without difficulties. A two‐step procedure is applied where first the initial values are optimized. In the second step the time dependent boundary values are adjusted for assimilation periods which are several times longer than the decorrelation times of the initial field. A number of experiments are reported with assimilation periods of different length up to 60 days. For short assimilation periods the error can be reduced from a first guess error of 34 cm root mean square to 5 cm. However, our results indicate that in a long time limit the lowest achievable error lies between 10 and 12 cm. This seems to be due to the model's inability to represent the dynamics of the data sufficiently well.
Distribution of oxygen isotopes in the water masses of Drake Passage and the South Atlantic
Authors: Meredith Michael P. , Grose Katie E. , McDonagh Elaine L. , Heywood Karen J. , Frew Russell D. , Dennis Paul F.
Published: December 2003

Measurements of the ratio of stable isotopes of oxygen (18O and 16O) from samples collected on World Ocean Circulation Experiment sections SR1b (eastern Drake Passage) and A11 (Punta Arenas to Cape Town) are used, together with hydrographic data, to deduce information about the formation and variability of South Atlantic and Southern Ocean water masses. The Drake Passage surface waters south of the Polar Front (PF) are isotopically light (δ18O around −0.4‰) owing to the influence of meteoric waters. The salinity and δ18O of the A11 surface waters yield an apparent freshwater end‐member which is much isotopically lighter than the local precipitation, thus advection of these waters from farther south dominates over local effects in determining the surface water properties. The Drake Passage section shows unusual proximity of the two main fronts of the Antarctic Circumpolar Current (the PF and Subantarctic Front (SAF)), and we observe cold, fresh, and isotopically light water derived from the temperature‐minimum Winter Water at the SAF. This water is of the correct density to freshen the intermediate water north of the SAF and thus play a role in the formation of the comparatively fresh Antarctic Intermediate Water (AAIW) of the South Atlantic. This confirms the role of Antarctic water in forming the South Atlantic variety of AAIW. Across the A11 section the oxygen isotope and salinity data at the AAIW core show very similar traces, with waters in the Malvinas Current loop showing lowest values of both. At the eastern boundary of the South Atlantic, the input of Red Sea Water from east of South Africa is observed via the presence of anomalously isotopically heavy AAIW. We deduce potentially significant temporal variability in the isotopic composition of Weddell Sea Deep Water (WSDW) by comparing the Drake Passage data to earlier data covering the outflow of the Weddell Sea. The A11 data show WSDW consistent with such variability, indicating that its effects could persist in the waters as they flow north into the western South Atlantic. We speculate that such variability could be due to small changes in the amount of glacial ice melt in WSDW.
Surface water characteristics in NCEP global spectral model and reanalysis
Authors: Roads J. O. , Chen S.‐C. , Kanamitsu M. , Juang H.
Published: December 2003

Characteristics of the National Centers for Environmental Prediction and National Center for Atmospheric Research (NCEP/NCAR) reanalysis surface water budget are compared to the surface water budget characteristics in a long‐term simulation with the NCEP global spectral model (GSM) used in the reanalysis. There are many geographic similarities. There are a few differences though, mainly because the reanalysis has a source (artificial) of water from a seasonally varying damping (forcing) term. Because the GSM does not have this (forcing) term, the GSM surface becomes dryer and ultimately has decreased land evaporation and precipitation. Since evaporation variations are strongly related to surface air and skin temperature variations, this decreased GSM surface water is also associated with increased land temperature. However, the GSM surface water variability is greater because the reanalysis time constant (60 days) used to damp (force) the surface water back to the assumed climatology is shorter than characteristic land surface parameterization hydrologic times, which are of the order of 6 months. In agreement with the decreased surface water variability, the reanalysis evaporation variability is also less than the evaporation variability in the GSM. This artificial time constraint may be a reason that the reanalysis temperature predictability by surface water is less than the demonstrated GSM predictability.
Optimal design of aquifer cleanup systems under uncertainty using a neural network and a genetic algorithm
Authors: Aly Alaa H. , Peralta Richard C.
Published: December 2003

We present a methodology to account for the stochastic nature of hydraulic conductivity during the design of pump‐and‐treat systems for aquifer cleanup. The methodology (1) uses a genetic algorithm to find the global optimal solution and (2) incorporates a neural network to model the response surface within the genetic algorithm. We apply the methodology for a real example and different optimization scenarios. The employed optimization formulation requires few hydraulic conductivity realizations. The presented approach produces a trade‐off curve between reliability and treatment facility size.
Thiol compounds from a free‐living pathogenic opportunistic amoeba, Acanthamoeba polyphaga
Authors: Ondarza Raúl N. , Iturbe Angélica , Hernández Eva , Hurtado Gerardo
Published: December 2003

New bimane‐reacting compounds from perchloric acid extracts have been detected by HPLC from Acanthamoeba polyphaga. The main compounds detected are cysteine, glutathione and other novel thiol compounds. All of these compounds must be thiols, since they disappear or decrease substantially when treated by N‐ethylmaleimide prior to acetonitrile/bimane derivatization. Cysteine and glutathione increase in quantity when dithiothreitol reduction is applied to the fresh extract. This means that they are likely to be present in their oxidized and reduced form and indicates the possible presence of a corresponding thiol/disulphide enzymic system. There are other compounds that have a different behaviour, since although they can react with bimane, they do not disappear if treated previously by N‐ethylmaleimide. This shows that they are not thiols but can react with bimane. The main thiol compounds found to be present, in both the parasite and the host lymphocyte cells, were cysteine and glutathione. We were unable to detect ovothiol A in Acanthamoeba but instead we found another thiol compound that could be structurally related to trypanothione. The new thiol compounds unique to this parasite and not present in lymphocytes will permit the study of disulphide‐reducing enzymes as potential drug targets.
Kinetic study of the appearance of an anti‐bacterial peptide in the course of bovine haemoglobin peptic hydrolysis
Authors: Choisnard Luc , Froidevaux Renato , Nedjar‐Arroume Naïma , Lignot Brigitte , Vercaigne‐Marko Dominique , Krier François , Dhulster Pascal , Guillochon Didier
Published: December 2003

The kinetics of the α(1–23) peptide, which is the first anti‐bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 °C in an homogenous‐phase system. A one‐step reversed‐phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti‐bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin‐denaturing agent. Two different mechanisms, ‘one‐by‐one’ for native haemoglobin hydrolysis and ‘zipper’ for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one‐by‐one or zipper, the anti‐bacterial α(1–23) peptide is a transient peptide. To prepare the α(1–23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DHc) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of α(1–23) peptide with respect to haemoglobin at the optimal DHc values were 55 and 25% respectively.
Immobilized lipase‐catalysed production of alkyl esters of restaurant grease as biodiesel
Authors: Hsu An‐Fei , Jones Kerby , Foglia Thomas A. , Marmer William N.
Published: December 2003

Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran‐T.l. and gran‐C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol‐gel matrix (IM PS‐30). All alcoholysis reactions were carried out in solvent‐free media employing a one‐step addition of the alcohol to the reaction system. Of the lipases studied, IM PS‐30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran‐T.l. and gran‐C.a. lipases gave poor conversions to esters, and the SP435‐catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (aw) was an important factor in the methanolysis reactions; reaction media with aw<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS‐30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched‐chain alcohols.
Development of an operational synaptobrevin‐based fluorescent substrate for tetanus neurotoxin quantification
Authors: Perpetuo Elen A. , Juliano Luis , Prado Sally M. , Fratelli Fernando , Fernandes Irene , Lebrun Ivo
Published: December 2003

Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two‐chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low‐molecular‐mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73–82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti‐recombinant fragment C antibody, and the cleavage was in a single bond (Gln‐Phe) with purified and crude TTx. Besides, ELISA applied to the anti‐TTx serum produced at our Institute showed cross‐reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. The results obtained suggest that the use of the new fluorescent substrate, Abz‐synaptobrevin73–82‐EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).
The calcium‐binding protein of Entamoeba histolytica as a fusion partner for expression of peptides in Escherichia coli
Authors: Reddi Honey , Bhattacharya Alok , Kumar Vijay
Published: December 2003

We describe the construction of an Escherichia coli expression vector, CBP that allows the C‐terminal fusion of heterologous proteins to the calcium‐binding protein (CaBP) of the parasitic protozoan Entamoeba histolytica. The intrinsic nature of this protein to remain soluble on heat treatment has been exploited in its use as a novel fusion partner. The presence of a histidine tag and an enterokinase recognition site, aid in the affinity purification and proteolytic cleavage of the fusion protein. The efficacy of the vector was tested using the preS1 region of the envelope protein of the hepatitis B virus. The CaBP‐preS1 fusion protein partitioned in the soluble fraction on heat treatment and this facilitated its rapid purification.
Effect of promoters on cellular immune response induced by recombinant fowlpox virus expressing multi‐epitope polypeptides from HIV‐1
Authors: Vázquez‐Blomquist Dania , González Syduet , Duarte Carlos A.
Published: December 2003

HIV stimulates strong immune CD8+ cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi‐epitope polypeptide (MEP) from HIV‐1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9‐fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon‐γ‐secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.
Modified high amylose starch for immobilization of uricase for therapeutic application
Authors: Mulhbacher Jérome , McGeeney Kevin , Ispas‐Szabo Pompilia , Lenaerts Vincent , Mateescu Mircea‐Alexandru
Published: December 2003

Urate oxidase (uricase) was immobilized on carboxymethyl high amylose starch cross‐linked 35 (CM‐HASCL‐35), on aminoethyl high amylose starch cross‐linked 35, as well as on commercial supports, CNBr‐activated Sepharose and diaminodipropylamine agarose. The N‐ethyl‐5‐phenylisoxazolium‐3′‐sulphonate (Woodward reagent K) gave a high binding but totally inhibited the enzyme activity. Best results were obtained with CM‐HASCL‐35 using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodi‐imide as a coupling agent. The immobilized enzyme retained 88% of its initial limit rate [Vmax(app)=16 EU/mg for immobilized uricase versus Vmax=18 EU/mg for the free enzyme], with an apparent decrease of affinity for urate substrate [Km(app)=0.17 mM versus Km=0.03 mM for the free enzyme]. The coupling yield was 60% and the modified uricase was found more resistant to proteolysis than the free enzyme. The immobilized uricase retained 25% of its initial activity after 60 min in pancreatic proteolysis medium (pancreatin), whereas the free enzyme retained only 5% of its initial activity. The best immobilization yield was obtained with the polymeric support based on CM‐HASCL‐35 (53%), which gave better results than commercial supports based on agarose.
The effect of chitin and chitosan on fibroblast‐populated collagen lattice contraction
Authors: Howling Graeme I. , Dettmar Peter W. , Goddard Paul A. , Hampson Frank C. , Dornish Michael , Wood Edward J.
Published: December 2003

The effects of chitin [(1→4)‐2‐acetamido‐2‐deoxy‐β‐d‐glucan] and its partially deacetylated derivatives, chitosans, on the human dermal fibroblast‐mediated contraction of collagen lattices were examined in vitro as a model for the contraction of cutaneous wounds in vivo. Chitosan CL313A, a short‐chain‐length 89% deacetylated chitosan chloride, inhibited fibroblast‐populated collagen lattice (FPCL) contraction at higher initial concentrations (500 and 1000 μg/ml) in FPCLs fabricated with responsive dermal fibroblasts, while in FPCLs containing non‐responsive fibroblasts inhibition of contraction was reduced. The responsive and non‐responsive phenotype of human dermal fibroblasts to treatment with chitosan CL313A has been reported previously by us. The inhibition of fibroblast‐mediated collagen lattice contraction by chitosan appeared to be strongly correlated with whether the cells were responsive or non‐responsive. The effect of chitin‐50A on fibroblast‐mediated collagen lattice contraction was also examined to investigate whether the level of deacetylation was important for its inhibitory effect on contraction. However, this had no effect on contraction at the concentrations tested, supporting previous work that only chitosan samples with higher levels of deacetylation showed any biological activity. This work indicates that highly deacetylated chitosan inhibits fibroblast‐mediated contraction of collagen lattices and may therefore be useful as a therapeutic agent to reduce contraction and therefore scarring in wound healing in vivo.
Purification and characterization of a novel enantioselective hydrolase from Bacillussubtilis
Authors: Maqbool Qurrat A. , Johri Sarojini , Verma Lata , Riyaz‐ul‐Hassan S. , Verma Vijeshwar , Koul Surrinder , Taneja Subhash C. , Parshad Rajinder , Qazi Ghulam N.
Published: December 2003

Screening of the micro‐organisms from an in‐house microbial culture repository, identified a bacterial strain bearing membrane‐bound, inducible ester hydrolase activity. The strain designated as RRL‐BB1 has been identified as Bacillus subtilis by 16 S rRNA typing. Its application in the kinetic resolution of several racemates, including drug intermediates, showed moderate to high enantioselectivity. The enzyme, designated as BBL, exhibited high enantioselectivity (ee ≈99%) with acyl derivatives of unsubstituted and substituted 1‐(phenyl)ethanols and 1‐(6‐methoxy‐2‐naphthyl)ethanols. With acyl derivatives of 2‐(6‐methoxy‐2‐naphthyl)propan‐1‐ol, moderate enantioselectivity was observed. The enzyme also showed moderate enantioselectivity with alkyl esters of carboxylic acids i.e. 2‐bromopropanoic acid and 2‐hydroxy‐4‐phenylbutanoic acid. The enzyme was purified to >90% purity from cell‐free extract of RRL‐BB1 with 26% overall yield. The purified enzyme exhibited hydrolase activity without any noticeable decrease in the rate of hydrolysis or the enantioselectivity profile. A specific activity of 450 units/mg protein resulted after at least a 200‐fold purification of the crude cell‐free extract. The key purification step was the irreversible adsorption of the salt‐precipitated crude enzyme on hydrophobic resin, in the presence of a low salt concentration, and desorption of the enzyme with a linear gradient of 1% sodium cholate. The purified enzyme was a 45 kD monomer as shown by SDS/PAGE. The N‐terminal amino acid sequence of the purified enzyme was determined as Thr‐Lys‐Leu‐Thr‐Val‐Gln‐Thr‐Arg‐Asp‐Gly‐Ala‐Leu‐Arg‐Gly‐Thr. The N‐terminal sequence did not bear any homology with other known bacterial lipases. BBL is maximally active at 37 °C, pH 8.0 and fairly stable up to 40 °C, pH 6–10. The enzyme is insensitive to EDTA but inhibited by serine protease inhibitor PMSF. Its activity (72%) was retained in the presence of the anionic detergent SDS at a concentration of 0.2% (w/v).
Stabilization of α‐chymotrypsin by modification with β‐cyclodextrin derivatives
Authors: Fernández Michael , Villalonga María de Lourdes , Fragoso Alex , Cao Roberto , Villalonga Reynaldo
Published: December 2003

Bovine pancreatic α‐chymotrypsin was chemically modified with two different β‐cyclodextrin derivatives, named mono‐6‐formyl‐β‐cyclodextrin and mono‐6‐succinyl‐6‐deoxy‐β‐cyclodextrin. The modified enzymes contained approx. 3–5 mol of oligosaccharide/mol of protein, and retained full proteolytic and esterolytic activity. The optimum temperature for α‐chymotrypsin was increased by 8 °C and its thermostability was enhanced by about 4–6 °C after modification. The conjugated enzymes were also more resistant to thermal inactivation at temperatures ranging from 45 to 55 °C. Additionally, the modified enzymes were 7‐fold more stable against incubation at pH 9.0. The possible influence of supramolecular interactions on the thermal stabilization of modified α‐chymotrypsins was also studied.
Effects of bovine colostral ultrafiltrates on growth and differentiation of 3T3‐L1 preadipocytes
Authors: Lee Seong‐Ho , Hossner Kim L.
Published: December 2003

This study was designed to compare the effects of whole and size‐fractionated bovine colostrum with bovine calf serum (BCS) on the growth and differentiation of 3T3‐L1 fibroblasts. High (HMW) and low (LMW)‐molecular‐mass ultrafiltrate fractions of colostrum were prepared from defatted colostrum (COL) by diafiltration through membranes with a molecular‐mass cut‐off of 30 kDa. Incorporation of [3H]thymidine into the cells was used as a reflection of DNA synthesis/cell proliferation. The growth‐promoting activity of LMW was 2.3‐ and 2.5‐fold higher than COL and HMW, respectively (P<0.05), and 185 μg/ml LMW stimulated cell proliferation equivalent to 10% BCS. Although insulin‐like growth factor (IGF)‐I, IGF‐II and platelet‐derived growth factor AB stimulated 3T3‐L1 cells, antibodies to these factors did not inhibit the LMW effects. The LMW fraction was about twice as effective as COL and HMW in stimulating differentiation of the cells into adipocytes, but maximal differentiation was only 60% of that seen with 10% fetal bovine serum (FBS). Treatment with COL, HMW, IGF‐I and insulin induced peroxisome‐proliferator‐activated receptor γ RNA, but levels were about half of that with 10% FBS treatment and LMW induction was 80% of FBS. Low amounts of leptin mRNA were detected in adipocytes and abundance did not differ between treatments with BCS, hormones or COL fractions. This study showed that bovine colostral LMW stimulated the growth and differentiation of 3T3‐L1 preadipocytes and may be a useful serum substitute to support the growth of these cells.
Evaluation of meningococcal C oligosaccharide conjugate vaccines by size‐exclusion chromatography/multi‐angle laser light scattering
Authors: Jumel Kornelia , Ho Mei M. , Bolgiano Barbara
Published: December 2003

The mean molecular masses of three different meningococcal C saccharide (MenC)–protein conjugate vaccines and their constituent proteins were estimated using HPLC size‐exclusion chromatography (SEC) with multi‐angle laser light scattering (MALLS) and refractive‐index (RI) detection (SEC/MALLS). Chromatography of two CRM197 conjugates (MenC–CRM197‐A and MenC–CRM197‐B) and one tetanus toxoid (TT) conjugate (MenC–TT) was performed in PBS, pH 7.4, on TSK‐Gel® (TosoHaas) analytical columns [CRM197 is a non‐catalytic cross‐reacting mutant (CRM) of diphtheria toxin]. Analysis of the light‐scattering signal measured at 18 angles simultaneously, using the RI signal as a measure of concentration, gave absolute weight‐average‐molecular‐mass (M̄w) values for the CRM197 conjugates as follows: MenC‐CRM197‐A, ≈75000 g·mol−1 and MenC–CRM197‐B, ≈350000 g·mol−1, suggesting that MenC–CRM197‐A is a monomer (one carrier protein per conjugate molecule), while MenC‐CRM197‐B is largely composed of conjugates containing three or four CRM197 molecules. The MenC–TT conjugate eluted as a two‐component system with M̄w of 1.63×106 and 395000 g· mol−1, suggesting that some cross‐linked complexes contain up to six TT molecules. Comparison of results from MALLS/RI with those obtained using UV detection highlights the differences in size and relative composition of the various subpopulations of the MenC conjugates that can be obtained using different detection systems.
Protein phosphatase 1 binds to phospho-Ser-1394 of the macrophage-stimulating protein receptor
Authors: SANTORO Massimo M. , GAUDINO Giovanni , VILLA-MORUZZI Emma
Published: December 2003

The tyrosine kinase Ron, receptor for MSP (macrophage-stimulating protein), displays several serine residues of unknown functions. Using [32P]H3PO4 metabolic labelling, we found that Ron is serine-phosphorylated and dephosphorylated in vitro by PP1 (protein phosphatase 1). PP1 associates with Ron obtained from cells of different origins. The association is stimulated by MSP or serum and is prevented by wortmannin, an inhibitor of the Akt/PKB (protein serine/threonine kinase B) pathway. Akt/PKB phosphorylates Ron Ser-1394, thus providing a docking site for 14-3-3 (scaffold proteins binding to phosphoserine/phosphothreonine-containing sequences). In living cells, PP1 binds to the Ron mutant S1394A, but the association is no longer regulated by serum, MSP or wortmannin. The role of PP1 association with Ron is highlighted by (1) Ser-1394 dephosphorylation by PP1 in vitro and in living cells, (2) loss of 14-3-3 association with Ron after Ser-1394 dephosphorylation by PP1 in vitro and (3) an increase in 14-3-3 association after PP1 inactivation in living cells. These results suggest that PP1 can modulate the downstream Ron signalling generated by MSP via Akt/PKB and 14-3-3 binding. This is the first report on ligand-regulated association of PP1 with a growth factor receptor.
Peroxisome-proliferator-activated receptor γ suppresses Wnt/β-catenin signalling during adipogenesis
Authors: MOLDES Marthe , ZUO Ying , MORRISON Ron F. , SILVA David , PARK Bae-Hang , LIU Jiajian , FARMER Stephen R.
Published: December 2003

The Wnt/β-catenin signalling pathway appears to operate to maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. To define the mechanisms regulating suppression of Wnt/β-catenin signalling, we analysed the β-catenin expression in response to activation of transcription factors that regulate adipogenesis. The results show an extensive down-regulation of nuclear β-catenin that occurs during the first few days of differentiation of 3T3-L1 preadipocytes and coincides with the induction of the adipogenic transcription factors, C/EBPβ (CCAAT-enhancer-binding protein) and PPARγ (peroxisome-proliferator-activated receptor). To assess the role of each of these factors in this process, we conditionally overexpressed C/EBPβ in Swiss mouse fibroblasts using the TET-off system. Abundant expression of C/EBPβ alone had minimal effect on β-catenin expression, whereas expression of C/EBPβ, in the presence of dexamethasone, induced PPARγ expression and caused a measurable decrease in β-catenin. In addition, exposure of cells expressing both C/EBPβ and PPARγ to a potent PPARγ ligand resulted in an even greater decrease in β-catenin by mechanisms that involve the proteasome. Our studies also suggest a reciprocal relationship between PPARγ activity and β-catenin expression, since ectopic production of Wnt-1 in preadipocytes blocked the induction of PPARγ gene expression. Moreover, by suppressing β-catenin expression, ectopic expression of PPARγ in Wnt-1-expressing preadipocytes rescued the block in adipogenesis after their exposure to the PPARγ ligand, troglitazone.
Use of the transport specificity ratio and cysteine-scanning mutagenesis to detect multiple substrate specificity determinants in the consensus amphipathic region of the Escherichia coli GABA (γ-aminobutyric acid) transporter encoded by gabP
Authors: KING Steven C. , BROWN-ISTVAN Lisa
Published: December 2003

The Escherichia coli GABA (γ-aminobutyric acid) permease, GabP, and other members of the APC (amine/polyamine/choline) transporter superfamily share a CAR (consensus amphipathic region) that probably contributes to solute translocation. If true, then the CAR should contain structural features that act as determinants of substrate specificity (kcat/Km). In order to address this question, we have developed a novel, expression-independent TSR (transport specificity ratio) analysis, and applied it to a series of 69 cysteine-scanning (single-cysteine) variants. The results indicate that GabP has multiple specificity determinants (i.e. residues at which an amino acid substitution substantially perturbs the TSR). Specificity determinants were found: (i) on a hydrophobic surface of the CAR (from Leu-267 to Ala-285), (ii) on a hydrophilic surface of the CAR (from Ser-299 to Arg-318), and (iii) in a cytoplasmic loop (His-233) between transmembrane segments 6 and 7. Overall, these observations show that (i) structural features within the CAR have a role in substrate discrimination (as might be anticipated for a transport conduit) and, interestingly, (ii) the substrate discrimination task is shared among specificity determinants that appear too widely dispersed across the GabP molecule to be in simultaneous contact with the substrates. We conclude that GabP exhibits behaviour consistent with a broadly applicable specificity delocalization principle, which is demonstrated to follow naturally from the classical notion that translocation occurs synchronously with conformational transitions that change the chemical potential of the bound ligand [Tanford (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2882–2884].
Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (γ-aminobutyric acid) transporter encoded by gabP
Authors: KING Steven C. , HU Liaoyuan A. , PUGH Amy
Published: December 2003

The Escherichia coli GABA (γ-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [3H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [3H]nipecotic acid and [14C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in kcat/Km). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [3H]nipecotic acid relative to [14C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity (kcat/Km) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842–5847] that have a functionally significant CAR-like domain.
Oligomerization of the cardiac ryanodine receptor C-terminal tail
Authors: STEWART Richard , ZISSIMOPOULOS Spyros , LAI F. Anthony
Published: December 2003

The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function. Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex [Gao, Tripathy, Lu and Meissner (1997) FEBS Lett. 412, 223–226]. To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization. Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure. We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking. The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization. Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate. These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel.
Calpain facilitates GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes
Authors: PAUL David S. , HARMON Anne W. , WINSTON Courtney P. , PATEL Yashomati M.
Published: December 2003

Calpains are a family of non-lysosomal cysteine proteases. Recent studies have identified a member of the calpain family of proteases, calpain 10, as a putative diabetes-susceptibility gene that may be involved in the development of type 2 diabetes. Inhibition of calpain activity has been shown to reduce insulinstimulated glucose uptake in isolated rat-muscle strips and adipocytes. In this report, we examine the mechanism by which calpain affects insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Inhibition of calpain activity resulted in approx. a 60% decrease in insulin-stimulated glucose uptake. Furthermore, inhibition of calpain activity prevented the translocation of insulin-responsive glucose transporter 4 (GLUT4) vesicles to the plasma membrane, as demonstrated by fluorescent microscopy of whole cells and isolated plasma membranes; it did not, however, alter the total GLUT4 protein content. While inhibition of calpain did not affect the insulin-mediated proximal steps of the phosphoinositide 3-kinase pathway, it did prevent the insulin-stimulated cortical actin reorganization required for GLUT4 translocation. Specific inhibition of calpain 10 by antisense expression reduced insulin-stimulated GLUT4 translocation and actin reorganization. Based on these findings, we propose a role for calpain in the actin reorganization required for insulin-stimulated GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. These studies identify calpain as a novel factor involved in GLUT4 vesicle trafficking and suggest a link between calpain activity and the development of type 2 diabetes.
Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis
Authors: GUL Sheraz , SONKARIA Sanjiv , PINITGLANG Surapong , FLOREZ-ALVAREZ José , HUSSAIN Syeed , THOMAS Emrys W. , OSTLER Elizabeth L. , GALLACHER Gerard , RESMINI Marina , BROCKLEHURST Keith
Published: December 2003

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in kcat/knon-cat and a 100-fold advantage in the proficiency constant, kcat/knon-cat·Km. Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.
Human aldehyde dehydrogenase 3A1 (ALDH3A1): biochemical characterization and immunohistochemical localization in the cornea
Authors: PAPPA Aglaia , ESTEY Tia , MANZER Rizwan , BROWN Donald , VASILIOU Vasilis
Published: December 2003

ALDH3A1 (aldehyde dehydrogenase 3A1) is expressed at high concentrations in the mammalian cornea and it is believed that it protects this vital tissue and the rest of the eye against UV-light-induced damage. The precise biological function(s) and cellular distribution of ALDH3A1 in the corneal tissue remain to be elucidated. Among the hypotheses proposed for ALDH3A1 function in cornea is detoxification of aldehydes formed during UV-induced lipid peroxidation. To investigate in detail the biochemical properties and distribution of this protein in the human cornea, we expressed human ALDH3A1 in Sf9 insect cells using a baculovirus vector and raised monoclonal antibodies against ALDH3A1. Recombinant ALDH3A1 protein was purified to homogeneity with a single-step affinity chromatography method using 5´-AMP–Sepharose 4B. Human ALDH3A1 demonstrated high substrate specificity for medium-chain (6 carbons and more) saturated and unsaturated aldehydes, including 4-hydroxy-2-nonenal, which are generated by the peroxidation of cellular lipids. Short-chain aliphatic aldehydes, such as acetaldehyde, propionaldehyde and malondialdehyde, were found to be very poor substrates for human ALDH3A1. In addition, ALDH3A1 metabolized glyceraldehyde poorly and did not metabolize glucose 6-phosphate, 6-phosphoglucono-δ-lactone and 6-phosphogluconate at all, suggesting that this enzyme is not involved in either glycolysis or the pentose phosphate pathway. Immunohistochemistry in human corneas, using the monoclonal antibodies described herein, revealed ALDH3A1 expression in epithelial cells and stromal keratocytes, but not in endothelial cells. Overall, these cumulative findings support the metabolic function of ALDH3A1 as a part of a corneal cellular defence mechanism against oxidative damage caused by aldehydic products of lipid peroxidation. Both recombinant human ALDH3A1 and the highly specific monoclonal antibodies described in the present paper may prove to be useful in probing biological functions of this protein in ocular tissue.
Class III phosphoinositide 3-kinase–Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes
Authors: TASSA Amina , ROUX Marie Paule , ATTAIX Didier , BECHET Daniel M.
Published: December 2003

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C2C12 myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K–Beclin1 autophagic complexes.
Secretory phospholipases A2 induce neurite outgrowth in PC12 cells
Authors: NAKASHIMA Satoru , IKENO Yutaka , YOKOYAMA Tatsuya , KUWANA Masakazu , BOLCHI Angelo , OTTONELLO Simone , KITAMOTO Katsuhiko , ARIOKA Manabu
Published: December 2003

sPLA2s (secretory phospholipases A2) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn-2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA2s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA2 activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA2 family, bee venom sPLA2, also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA2s, but not by group IB and IIA sPLA2s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca2+ channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca2+- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA2s in the nervous system.