bluuebunny/crossref_metadata_2025 huggingface dataset:
Variation in P‐content in aquatic plant tissues offers an efficient tool for determining plant growth strategies along a resource gradient
Authors: Garbey C. , Murphy K.J. , Thiébaut G. , Muller S.
Published: February 2004

1. The application of functional ecology models to aquatic plants often relies on morphological and life‐history traits which may reflect, in part, the phenotypic plasticity displayed by aquatic plants. The present study was designed to evaluate the use of physiological traits, such as nutrition patterns, to describe aquatic plant strategies along a gradient of increasing resource availablity.

2. Taking phosphorus (P) as an example, nutrition‐use efficiencies were evaluated in five species, through the P‐content in plant tissues, the variations in P‐content according to nutrient availability and the perenniality of P‐storage. Plasticity in P‐storage was also investigated in Ranunculus peltatus, a morphologically highly plastic species.

3. In 2001, P‐content was analysed in Callitriche hamulata, C. obtusangula, C. platycarpa, Elodea nuttallii and R. peltatus tissue samples. These five species were sampled at nine different sites in streams along an increasing resource gradient in the Northern Vosges Biosphere Reserve (NE France). Variations of P‐content in the roots, stems and dissected and floating leaves of R. peltatus were also studied.

4. Only C. platycarpa and R. peltatus were found to occur in low nutrient availability conditions. Callitriche hamulata, C. obtusangula and E. nuttallii were restricted to mesotrophic and eutrophic sites. The highest nutrient‐use efficiency was found for E. nuttallii which was able to adapt its P‐storage to varying resource availabilities. Ranunculus peltatus was able to store high concentrations of P, but its P‐integration within the vegetative structure was less efficient under eutrophic conditions. Callitriche spp. appeared to have relatively low nutrient‐use efficiencies, although C. obtusangula displayed a high P‐content. While P was stored preferentially in roots in R. peltatus populations occurring in nutrient‐rich sites, there was no particular P‐storage organ for populations from nutrient‐poor sites.

5. On the basis of P‐usage, R. peltatus and E. nuttallii presented competitor traits, C. hamulata and C. platycarpa displayed stress‐tolerant nutrient signatures and for C. obtusangula, ruderal or competitor characteristics dominated. The use of physiological traits, such as nutrition patterns, may provide valuable, complementary information about aquatic plant strategies, independent from the influence of morphological trait plasticity often displayed by these plants.
Physico‐chemical properties of flour and starch from jackfruit seeds (Artocarpus heterophyllus Lam.) compared with modified starches
Authors: Mukprasirt Amornrat , Sajjaanantakul Kamontip
Published: February 2004

Jackfruit (Artocarpus heterophyllus Lam.) is one of the most popular tropical fruits grown in Asia. The objective of this study was to compare physico‐chemical properties of native flour and starch from jackfruit seeds (A. heterophyllus) to commercially modified starches (Novation 2300 and Purity 4). The colour of jackfruit seed starch was lighter than the Novation 2300 starch but darker than the Purity 4 starch. The jackfruit seed starch had a narrower gelatinization temperature range than Purity 4 and required less gelatinization energy compared with modified starches. The peak viscosity of jackfruit seed starch was lower than commercially modified starches. Likewise, setback viscosity, swelling power and solubility of jackfruit seed starch showed similar trends. Results from this study suggest that native starch from jackfruit seed could be used as an alternative for modified starches in a system needing starch with a high thermal and/or mechanical shear stability.
Pulsed‐vacuum immersion of chicken meat and skin in acid solutions. Effects on mass transfers, colour and microbial quality
Authors: Deumier François
Published: February 2004

Decontaminating chicken skin by immersion in an organic acid solution at low temperature (7 °C) led to 3.7, 2.5, 2.4 and 2.0 decimal reductions in total viable counts, Enterobacteriaceae, total coliforms and Escherichia coli, respectively. Lactic acid was a more effective decontaminating agent than citric acid. This decontaminating effect was very closely correlated with solution pH and chicken‐skin pH variations. The use of pulsed vacuum immersion (PVI) increased mass transfers (water gain and acidification) and the decontamination effect, especially with respect to total viable counts. In PVI conditions, the skin‐solution contact area was increased as a result of solution infiltration into microfolds and feather follicles, but the skin colour was modified (yellowing and greening) as the intensity of the acid treatment increased. In contrast, the results of a triangular test revealed that chicken sausages produced with acid‐treated skins did not differ from those made with untreated skins.
Characteristics of edible films made from dairy proteins and zein hydrolysate cross‐linked with transglutaminase
Authors: Oh Jun‐Hyun , Wang Baowu , Field Perris D. , Aglan Heshmat A.
Published: February 2004

The main objectives of this research were to develop whey protein or casein films incorporating zein hydrolysate and also cross‐linked by transglutaminase as to well as characterize the physical and mechanical properties of the film. Zein hydrolysate decreased the solubility of the whey protein film (P < 0.05), while treatment with transglutaminase did not change the solubility of the film significantly. Electrophoresis patterns demonstrated that casein molecules were cross‐linked by transglutaminase and the extent of this cross‐linkage was further increased when zein hydrolysate was added. In addition, the use of zein hydrolysate decreased the tensile strength of the whey protein film by 35–45%. The elongation of the casein film was increased by 41% because of the action of transglutaminase and zein hydrolysate (P < 0.05). The water vapour permeability of the films was not significantly different. As the addition of zein hydrolysate and treatment with transglutaminase improved the flexibility of the films, the level of plasticizer required to maintain film flexibility could be reduced without sacrificing their water vapour permeability.
Colour of post‐harvest ripened and vine ripened tomatoes (Lycopersicon esculentum Mill.) as related to total antioxidant capacity and chemical composition
Authors: Wold Anne‐Berit , Rosenfeld Hans J. , Holte Kari , Baugerød Halvard , Blomhoff Rune , Haffner Karin
Published: February 2004

Greenhouse grown tomatoes (cvs ‘Durinta’, ‘Favorita’ and ‘Liberto’) were harvested green and vine ripe. Colour measurements and chemical analyses were performed on green, post‐harvest red and vine‐ripe fruits. Partial least square (PLS) regression was applied to predict the ferric reducing ability of plasma (FRAP) by means of colour measurements (L*, a* and b*). The PLS showed that 78% of the variation in colour were able to predict 74% of the variation in FRAP value. Chemical variables and colour variables together were able to predict 88% of the variation in FRAP by means of PLS regression. Linear regression showed a high negative correlation between high values of L*, b* and FRAP, and a high positive correlation between titratable acidity, a*, hue, a*/b*, soluble solids, vitamin C, dry matter and FRAP values. Thus, colour measurements of intact tomatoes can be used as a non‐destructive method to assess total antioxidant capacity of tomatoes.
Reduction of chlorine concentration and microbial load during washing‐disinfection of shredded lettuce
Authors: Pirovani María , Piagentini Andrea , Güemes Daniel , Arkwright Silvana
Published: February 2004

Disinfection of shredded lettuce by washing is an essential operation to eliminate foreign matter and cellular fluids produced by cutting, and to reduce the initial microbial contamination. The response surface methodology was used to evaluate the effect of initial chlorine concentration (50–150 ppm), water‐to‐produce ratio (20–50 L kg−1) and time (2–8 min) on the depletion of total and available chlorine and also the microbial count reduction on shredded lettuce. The predictive models obtained, validated through additional confirmatory experiments, were shown to be adequate and could be used as a way of improving the operation.
Effects of short‐term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase
Authors: Pasqualini S. , Batini P. , Ederli L. , Porceddu A. , Piccioni C. , De Marchis F. , Antonielli M.
Published: February 2004

The role that the constituents of the ascorbate–glutathione cycle play in the mechanism of contrasting ozone sensitivities was examined in mature and old tobacco leaves after acute ozone‐fumigation (150 p.p.b., 5 h). Levels of the enzyme activities associated with the detoxifying system were lower in ozone‐sensitive Bel W3 control plants than in unfumigated ozone‐tolerant Bel B plants. In particular, the endogenous activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the metabolites ascorbic acid (AA) and reduced glutathione (GSH) were more abundant in Bel B than Bel W3 control plants. These results suggest that the higher tolerance of Bel B to O3 is associated with a greater initial content of the antioxidant enzymes or metabolites. Only in the mature leaves of the ozone‐tolerant Bel B cv. did fumigation trigger activation of APX and, weakly, of dehydroascorbate reductase (DHAR). The activity of these enzymes was significantly lower after ozone treatment in both mature and old leaves of Bel W3 than in control plants. Fumigation had little effect on the ascorbate content. Its main effects on the glutathione pool were that it boosted the oxidized form and lowered the reduced form, particularly in mature Bel W3 leaves. Extractable GR activity remained unchanged in both Bel B and Bel W3 immediately after fumigation, but increased slightly 24 h later, particularly in mature leaves of Bel W3. Exposure to O3 caused a sharp decline in chloroplastic GR mRNA levels in both cultivars. However, as Western blot analysis failed to detect any major changes in GR protein content at this time, the protein must be highly stable. There is therefore a good correlation between tolerance to O3 and high endogenous levels of antioxidant metabolites such as AA and GSH in tobacco. In addition, the degree of inducibility of the system discriminates the two cultivars investigated.
Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide
Authors: Monzani Enrico , Roncone Raffaella , Galliano Monica , Koppenol Willem H. , Casella Luigi
Published: February 2004

Peroxidases perform the nitration of tyrosine and tyrosyl residues in proteins, in the presence of nitrite and hydrogen peroxide. The nitrating species is still unknown but it is usually assumed to be nitrogen dioxide. In the present investigation, the nitration of phenolic compounds derived from tyrosine by lactoperoxidase and horseradish peroxidase was studied, with the aim of elucidating the mechanism of the reaction. The results indicate that nitrogen dioxide cannot be the only nitrating species and suggest the presence of two simultaneously operative pathways, one proceeding through enzyme‐generated nitrogen dioxide and another through a more reactive species, assumed to be complexed peroxynitrite, which is generated by reaction of hydrogen peroxide with the enzyme–nitrite complex. The importance of the two pathways depends on peroxide and nitrite concentrations. With lactoperoxidase, nitration through the highly reactive intermediate is preferred except at very low nitrite concentration, while with horseradish peroxidase, the nitrogen dioxide driven mechanism is preferred except at very high nitrite concentration. The preferred mechanism for the two enzymes is that operative in the physiological nitrite concentration range.
N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster ovary cell line
Authors: Forno Guillermina , Bollati Fogolin Mariela , Oggero Marcos , Kratje Ricardo , Etcheverrigaray Marina , Conradt Harald S. , Nimtz Manfred
Published: February 2004

GM‐CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM‐CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N‐glycosylation sites occupied. Minor low molecular mass forms completely lacked N‐linked oligosaccharides but contained 1–3 O‐linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex‐HexNAc units (Δm 365 Da), were detected in MALDI‐TOF MS spectra of the desialylated GM‐CSFs. MALDI‐TOF MS and HPAEC‐PAD analysis indicated the presence of predominantly tri‐ and tetraantennary N‐linked oligosaccharide chains with and without N‐acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal α1–6‐linked fucose. The oligosaccharide patterns of both GM‐CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N‐glycans were found α2–3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM‐CSF form more than 90% of the Asn37 were occupied by N‐glycans. O‐glycosylation at the N‐terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O‐glycosylated glycoprotein form. In the triply modified GM‐CSF molecules, Ser5 was additionally O‐glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N‐glycosylation site occupied in the preparation derived from suspension culture. ESI‐MS and MALDI‐MS analysis of endoproteolytically cleaved peptides as well as MALDI‐TOF MS of the intact glycoprotein demonstrated the N‐ and C‐termini integrity of the GM‐CSF preparations.
Characterization of novel structural features in the lipopolysaccharide of nondisease associated nontypeable Haemophilus influenzae
Authors: Landerholm Malin K. , Li Jianjun , Richards James C. , Hood Derek W. , Moxon E. Richard , Schweda Elke K. H.
Published: February 2004

Nontypeable Haemophilus influenzae (NTHi) is a common commensal of the human upper respiratory tract and is associated with otitis media in children. The structures of the oligosaccharide portions of NTHi lipopolysaccharide (LPS) from several otitis media isolates are now well characterized but it is not known whether there are structural differences in LPS from colonizing, nondisease associated strains. Structural analysis of LPS from nondisease associated NTHi strains 11 and 16 has been achieved by the application of high‐field NMR techniques, ESI‐MS, ESI‐MSn, capillary electrophoresis coupled to ESI‐MS, composition and linkage analyses on O‐deacylated LPS and core oligosaccharide material. This is the first study to report structural details on LPS from strains taken from the nasopharynx from healthy individuals. Both strains express identical structures and contain the common element of H. influenzae LPS, l‐α‐d‐Hepp‐(1→2)‐[PEtn→6]‐l‐α‐d‐Hepp‐(1→3)‐[β‐d‐Glcp‐(1→4)]‐l‐α‐d‐Hepp‐(1→5)‐[PPEtn→4]‐α‐Kdop‐(2→6)‐lipid A, in which each heptose is elongated by a single hexose residue with no further oligosaccharide extensions. In the major Hex3 glycoform, the terminal Hepp residue (HepIII) is substituted at the O‐2 position by a β‐d‐Galp residue and the central Hepp residue (HepII) is substituted at O‐3 by a α‐d‐Glcp residue. Notably, the strains express two phosphocholine (PCho) substituents, one at the O‐6 position of α‐d‐Glcp and the other at the O‐6 position of β‐d‐Galp. Major acetylation sites were identified at O‐4 of Gal and O‐3 of HepIII. Additionally, both strains express glycine, and strain 11 also expresses detectable amounts of N‐acetylneuraminic acid.
Membrane transport of fatty acylcarnitine and free l‐carnitine by rat liver microsomes
Authors: Gooding Jason M. , Shayeghi Majid , Saggerson E. David
Published: February 2004

Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long‐chain fatty acyl‐CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl‐CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free l‐carnitine – processes that are necessary for an indirect route of provision of ER luminal fatty acyl‐CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [14C]palmitoylcarnitine into microsomes through the use of the luminal CAT and acyl‐CoA:ethanol acyltransferase as a reporter system to detect formation of luminal [14C]palmitoyl‐CoA. Rapid, unidirectional transport of free L‐[3H]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N‐ethylmaleimide or sulfobetaine – properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/re‐esterification cycle.
Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II
Authors: Tohri Akihiko , Dohmae Naoshi , Suzuki Takehiro , Ohta Hisataka , Inoue Yasunori , Enami Isao
Published: February 2004

To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N‐succinimidyl propionate or glycine methyl ester in the presence of a water‐soluble carbodiimide, respectively. The N‐succinimidyl propionate‐modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester‐modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N‐succinimidyl propionate‐modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI‐TOF MS. The results showed that six domains (Lys11–Lys14, Lys27–Lys38, Lys40, Lys90–Lys96, Lys143–Lys152, Lys166–Lys174) were modified with N‐succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056–2061].
NUB1‐mediated targeting of the ubiquitin precursor UbC1 for its C‐terminal hydrolysis
Authors: Tanaka Tomoaki , Yeh Edward T. H. , Kamitani Tetsu
Published: February 2004

NEDD8 is a ubiquitin‐like protein that controls vital biological events through its conjugation to target proteins. Previously, we identified a negative regulator of the NEDD8 conjugation system, NEDD8 ultimate buster‐1 (NUB1), that recruits NEDD8 and its conjugates to the proteasome for degradation. Recently, we performed yeast two‐hybrid screening with NUB1 as bait and isolated a ubiquitin precursor UbC1 that is composed of nine tandem repeats of a ubiquitin unit through α‐peptide bonds. Interestingly, NUB1 interacted with UbC1 through its UBA domain. Further study revealed that the UBA domain interacted with α‐peptide bond‐linked polyubiquitin, but not with isopeptide bond‐linked polyubiquitin, indicating that the UBA domain of NUB1 is a specific acceptor for the linear ubiquitin precursor. A functional study revealed that an unidentified protein that was immunoprecipitated with NUB1 served as a ubiquitin C‐terminal hydrolase for UbC1. Thus, NUB1 seems to form a protein complex with the unidentified ubiquitin C‐terminal hydrolase and recruit UbC1 to this complex. This might allow the ubiquitin C‐terminal hydrolase to hydrolyze UbC1, in order to generate ubiquitin monomers. Northern blot analysis showed that the mRNAs of both NUB1 and UbC1 were enriched in the testis. Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules of the testis. These results may imply that the UbC1 hydrolysis mediated by NUB1 is involved in cellular functions in the seminiferous tubules such as spermatogenesis.
The structure–function relationship in the clostripain family of peptidases
Authors: Labrou Nikolaos E. , Rigden Daniel J.
Published: February 2004

In this study we investigate the active‐site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site‐directed mutagenesis and molecular modelling. A benzamidinyl‐diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site‐directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time‐dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme–BDD complex is KD = 74.6 ± 2.1 µm and the maximal rate constant of inactivation is k3 = 0.21·min−1. Effective protection against inactivation by BDD is provided by the substrate N‐benzoyl‐l‐arginine ethyl ester (BAEE). Cleavage of BDD‐modified enzyme with trypsin and subsequent separation of peptides by reverse‐phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site‐directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition‐derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto‐activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.
Metal‐binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae
Authors: Urvoas Agathe , Moutiez Mireille , Estienne Clément , Couprie Joël , Mintz Elisabeth , Le Clainche Loïc
Published: February 2004

We studied the interaction of several metal ions with the copper chaperone from Enterococcus hirae (EhCopZ). We show that the stoichiometry of the protein–metal complex varies with the experimental conditions used. At high concentration of the protein in a noncoordinating buffer, a dimer, (EhCopZ)2–metal, was formed. The presence of a potentially coordinating molecule L in the solution leads to the formation of a monomeric ternary complex, EhCopZ–Cu–L, where L can be a buffer or a coordinating molecule (glutathione, tris(2‐carboxyethyl)phosphine). This was demonstrated in the presence of glutathione by electrospray ionization MS. The presence of a tyrosine close to the metal‐binding site allowed us to follow the binding of cadmium to EhCopZ by fluorescence spectroscopy and to determine the corresponding dissociation constant (Kd = 30 nm). Competition experiments were performed with mercury, copper and cobalt, and the corresponding dissociation constants were calculated. A high preference for copper was found, with an upper limit for the dissociation constant of 10−12 m. These results confirm the capacity of EhCopZ to bind copper at very low concentrations in living cells and may provide new clues in the determination of the mechanism of the uptake and transport of copper by the chaperone EhCopZ.
Three cyclin‐dependent kinases preferentially phosphorylate different parts of the C‐terminal domain of the large subunit of RNA polymerase II
Authors: Pinhero Reena , Liaw Peter , Bertens Kimberly , Yankulov Krassimir
Published: February 2004

The C‐terminal domain (CTD) of the largest subunit of RNA polymerase II plays critical roles in the initiation, elongation and processing of primary transcripts. These activities are at least partially regulated by the phosphorylation of the CTD by three cyclin‐dependent protein kinases (CDKs), namely CDK7, CDK8 and CDK9. In this study, we systematically compared the phosphorylation of different recombinant CTD substrates by recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 kinases. We showed that CDK7, CDK8 and CDK9 produce different patterns of phosphorylation of the CTD. CDK7/CycH/MAT1 generates mostly hyperphosphorylated full‐length and truncated CTD peptides, while CDK8/CycC and CDK9/CycT1 generate predominantly hypophosphorylated peptides. Total activity towards different parts of the CTD also differs between the three kinases; however, these differences did not correlate with their ability to hyperphosphorylate the substrates. The last 10 repeats of the CTD can act as a suppressor of the activity of the kinases in the context of longer peptides. Our results indicate that the three kinases possess different biochemical properties that could reflect their actions in vivo.
CD38 is expressed as noncovalently associated homodimers on the surface of murine B lymphocytes
Authors: Moreno‐García Miguel E. , Partida‐Sánchez Santiago , Primack Julie , Sumoza‐Toledo Adriana , Muller‐Steffner Hélène , Schuber Francis , Oppenheimer Norman , Lund Frances E. , Santos‐Argumedo Leopoldo
Published: February 2004

CD38 is a transmembrane glycoprotein that functions as an ectoenzyme and as a receptor. Based on the structural similarity between CD38 and ADP‐ribosyl cyclase from Aplysia californica, it was hypothesized that CD38 is expressed as a homodimer on the surface of cells. Indeed, CD38 dimers have been reported, however, the structural requirements for their stabilization on the plasma membrane are unknown. We demonstrate that the majority of CD38 is assembled as noncovalently associated homodimers on the surface of B cells. Analysis of CD38 mutants, expressed in Ba/F3 cells, revealed that truncation of the cytoplasmic region or mutation of a single amino acid within the α1‐helix of CD38 decreased the stability of the CD38 homodimers when solubilized in detergent. Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half‐lives of these CD38 mutant proteins on the plasma membrane were significantly reduced. Together, these results show that CD38 is expressed as noncovalently associated homodimers on the surface of murine B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells.
Carboxydipeptidase activities of recombinant cysteine peptidases
Authors: Judice Wagner A. S. , Puzer Luciano , Cotrin Simone S. , Carmona Adriana K. , Coombs Graham H. , Juliano Luiz , Juliano Maria A.
Published: February 2004

The recombinant cysteine peptidases, cruzain from Trypanosoma cruzi and CPB2.8ΔCTE from Leishmania mexicana, are cathepsin L‐like and characteristically endopeptidases. In this study, we characterized the carboxydipeptidase activities of these enzymes and compared them with those of human recombinant cathepsin B and cathepsin L. The analysis used the internally quenched fluorescent peptide Abz‐FRFK*‐OH and some of its analogues, where Abz is ortho‐aminobenzoic acid and K* is (2,4‐dinitrophenyl)‐ε‐NH2‐lysine. These peptides were demonstrated to be very sensitive substrates, due to the strong quenching effect of K* on the fluorescence of the Abz group. The carboxydipeptidase activity of cruzain was shown to be very similar to that of cathepsin B, while that of CPB2.8ΔCTE is closer to the carboxydipeptidase activity of cathepsin L. The S2 subsite architecture of cruzain and the nature of the amino acid at the P2 position of the substrates determine its carboxydipeptidase activity and gives further and direct support to the notion that the carboxydipeptidase activity of the papain family cysteine peptidases rely on the S2–P2 interaction [Nägler D. K., Tam, W., Storer, A.C., Krupa, J.C., Mort, J.S. & Menard, R. (1999) Biochemistry38, 4868–4874]. Cruzain and CPB2.8ΔCTE presented a broad pH‐range for both the endo‐ and exo‐peptidase activities, although the later is approximately one order of magnitude lower. This feature, that is not common in related mammalian cysteine peptidases, is consistent with the enzymes being exposed to different environmental conditions and having different locations during parasite development.
Ouabain stimulates endothelin release and expression in human endothelial cells without inhibiting the sodium pump
Authors: Saunders Robert , Scheiner‐Bobis Georgios
Published: February 2004

Ouabain, a sodium pump (Na+/ K+‐ATPase) inhibitor, has been shown to act as a hormone and is possibly involved in the pathogenesis of hypertension. The mechanism by which ouabain may act was investigated using primary cultures of human umbilical artery endothelial cells (HUAECs), which are known to express and release the vasoconstrictive hormone endothelin (ET‐1). Five minutes after application, low concentrations of ouabain induced Ca2+ oscillations and stimulated ET‐1 release from endothelial cells into the medium. To investigate whether the observed effects were due to inhibition of the sodium pump, the effects of ouabain on the uptake of 86Rb+ by HUAECs were examined. Unexpectedly, ouabain concentrations below 10 nm stimulated 86Rb+ uptake by 15–20%, and in some experiments by 50%, results that are consistent with a stimulation of the pump. Within the concentration range 1–10 nm, ouabain induced a 2.5‐fold stimulation (phosphorylation) of mitogen‐activated protein kinase (MAP kinase). After incubation of HUAECs with ouabain for 12 h, the glycoside stimulated cell growth by 49 ± 4%, as measured by cell number, with a maximum response at 5 nm. At similar concentrations, ouabain also increased ET‐1 mRNA abundance by 19.5 ± 3.1%. The results indicate that, by influencing ET‐1 expression and release, ouabain may contribute to the regulation of vascular tone. The data also confirm that it is not a global inhibition of the sodium pump that is involved in the mechanism of action of this cardiac glycoside.
Unique cell membrane expression of topoisomerase‐II alpha as a useful diagnostic marker of liposarcoma
Authors: Endo Hideko , Hirokawa Mitsuyoshi , Ishimaru Naozumi , Tanaka Yasushi , Yamashita Michiko , Sakaki Mika , Hayashi Yoshio , Sano Toshiaki
Published: February 2004

Topoisomerase‐II alpha (Topo‐II alpha) is known as a cell cycle‐related intranuclear marker. To the best of our knowledge, the expression of Topo‐II alpha on extranuclear sites has not been reported. The aim of the present study was to determine the usefulness of Topo‐II alpha immunostaining for detecting the lipoblasts that are essential to diagnosing liposarcoma. Surgical specimens, including benign lipomatous tumors (four cases), well‐differentiated liposarcomas (three cases), myxoid liposarcomas (six cases), pleomorphic liposarcomas (two cases), dedifferentiated liposarcomas (two cases), myxoid malignant fibrous histiocytomas (six cases), and one case of mesenteric panniculitis, were studied. Samples were immunostained using antibodies for Topo‐II alpha, S‐100 protein and Ki‐67. In addition, we used the western blot method to investigate immunohistochemical‐affinity in adipocytes. Mature adipocytes and lipoblasts in all of the benign and malignant lipomatous tumors intensively expressed cell contours positivity for Topo‐II alpha. Cytoplasm of the lipoblasts occasionally reacted to the antibody and highlighted intracytoplasmic small unilocular, multivacuolated, or bubble‐like patterns. Western blot analysis confirmed a 70 kDa product reactive to Topo‐II alpha in the cell membrane fragment of mature adipocytes. S‐100 protein expressed adipocytes and lipoblasts, but the detection of lipoblasts was not as easy as in Topo‐II alpha immunostaining. Immunoreactivity of Ki‐67 was limited to the nuclei, and the nuclear labeling index of Ki‐67 correlated with that of Topo‐II alpha. The immunoreactivity of Topo‐II alpha for lipoblasts was more sensitive and obvious than those of S‐100 protein. Immunostaining using the antibody for Topo‐II alpha seems to be useful in recognizing lipoblasts that have been overlooked in hematoxylin–eosin‐stained preparations, and is a useful marker for diagnosing liposarcoma.
Pattern of Epstein–Barr virus association in childhood non‐Hodgkin's lymphoma: Experience of University of Malaya Medical Center
Authors: Peh Suat‐Cheng , Nadarajah Veera S. , Tai Yan‐Chin , Kim Lian‐Hua , Abdullah Wan Ariffin B.
Published: February 2004

The pattern of childhood non‐Hodgkin's lymphoma (NHL) usually differs in adults. The most common subtypes are lymphoblastic, Burkitt's and anaplastic large cell lymphoma. Recent data indicate that a higher risk of developing lymphoma is associated in children of certain ethnic origins. The difference is probably related to the underlying etiological factors of these diseases, and Epstein–Barr virus (EBV) is a strong candidate. The present study aims to determine the disease pattern of childhood lymphomas in the University Hospital Kuala Lumpur, for a direct comparison to the reported data of adults from the same medical center. A total of 69 and 34 childhood NHL and Hodgkin's lymphomas, respectively, were retrieved. The most common subtypes were lymphoblastic (23 cases), Burkitt's (25 cases) and anaplastic large cell lymphomas (9 cases). Epstein–Barr virus association was more prevalent in B‐cell (23%) than T‐cell (12%) lymphomas. The most common EBV‐associated tumor was Burkitt's lymphoma, and there was an increased risk of EBV association for Burkitt's lymphoma in Chinese patients. In conclusion, the pattern of childhood lymphoma in Malaysia is relatively similar to children elsewhere in the world. The EBV association of B‐ and T‐NHL differs between children and adults from the same medical center because of differences in the subtype composition in these two age groups.
High frequency of EBV association and 30‐bp deletion in the LMP‐1 gene in CD56+ lymphomas of the upper aerodigestive tract
Authors: Tai Yan‐Chin , Kim Lian‐Hua , Peh Suat‐Cheng
Published: February 2004

Natural killer (NK)/T‐cell lymphomas are frequently associated with Epstein–Barr virus (EBV), and usually lack TCR gene rearrangement. Studies from Asia have reported frequent deletion in the LMP‐1 gene in EBV‐associated nasopharyngeal carcinoma (NPC). The present study aims to investigate LMP‐1 and TCRγ gene status in upper aerodigestive tract lymphomas. A total of 43 cases were classified into T‐, B‐, and NK/T‐cell tumors based on the phenotype expressions of CD3+/CD20–/CD56–, CD3–/CD20+/CD56–, and CD3+/CD20–/CD56+, respectively. The presence of EBV in the tumor was confirmed by EBV early RNA‐in situ hybridization. LMP‐1 gene deletion and TCR γ gene rearrangement were analyzed by polymerase chain reaciton on paraffin‐embedded tissues. There were 20 NK/T‐, eight T‐, and 15 B‐cell phenotype lymphomas in the present series, and EBV was detected in 19 (95%), two (25%), and three (20%) cases in the respective groups. All EBV+ cases carried 30‐bp deletion in the LMP‐1 gene, and two of the NK/T‐cell cases were infected by both the wild type and deleted strains. Five (25%) of the NK/T‐cell phenotype lymphomas showed rearranged TCR γ gene. The present study revealed a high frequency of EBV association, and a high frequency of 30‐bp deletion in the LMP‐1 gene in the virus in the present series of lymphoma. The NK/T‐phenotype lymphomas are comprised of both NK‐cell and cytotoxic T‐lymphocyte‐derived tumors.
Senescence marker protein‐30 knockout mouse as a novel murine model of senile lung
Authors: Mori Takanori , Ishigami Akihito , Seyama Kuniaki , Onai Reiko , Kubo Sachiho , Shimizu Kazue , Maruyama Naoki , Fukuchi Yoshinosuke
Published: February 2004

Senescence marker protein‐30 (SMP30) was originally identified as a novel protein of which expression decreases in an androgen‐independent manner with aging in the rat liver and functions to protect cells from apoptosis. By reverse transcription–polymerase chain reaction analysis, SMP30 mRNA transcripts were found in the mouse lung, liver, kidney, testis and cerebrum. We examined SMP30 expression in the mouse liver, kidney and lung during aging and a distinct temporal profile of SMP30 expression was found in each tissue; the SMP30 mRNA level peaked at 1–3 months of age and decreased thereafter in the liver (the highest at 1 month of age followed by a rapid decline and consistently low thereafter in the kidney), and peaked at 12 months of age in the lung. To investigate the physiological role of SMP30 in the lung, immunohistochemical studies of wild‐type (SMP30Y/+) mice and histopathological examinations of SMP30 knockout (SMP30Y/–) mice were performed. Immunoreactivity against anti‐SMP30 antibody was mainly detected in bronchial epithelial cells and strongly detected at 6–12 months of age. Morphometric analysis was performed to measure the mean linear intercept and destructive index, and found peripheral airspace enlargement without alveolar destruction in SMP30Y/– mice at 1, 3 and 6 months of age compared with the SMP30Y/+ mice. Our results strongly suggest that SMP30Y/– mice could be a novel model for a senile lung and further examinations of SMP30Y/– mice may offer clues to elucidate the mechanisms of the development of pulmonary diseases in the elderly.
Clinicopathological correlation of childhood IgA glomerulonephritis presenting diffuse endocapillary proliferation
Authors: Hisano Satoshi , Kiyoshi Yasuhiro , Tanaka Ikuko , Tokieda Keiko , Niimi Kazuhiko , Tsuru Noboru , Takebayashii Shigeo , Iwasaki Hiroshi
Published: February 2004

The aim of the present study is to clarify the clinicopathological correlation of childhood IgA glomerulonephritis (GN) presenting diffuse endocapillary proliferation. Twenty‐seven patients were used in the present study. The 27 patients were divided into three groups (mild, moderate and severe) according to the percentage of glomeruli displaying global endocapillary proliferation per total glomeruli at the first biopsy. The degree of both cellular crescent and lysis of the glomerular basement membrane (GBM) at the first biopsy was semiquantitatively evaluated. The degree of cellular crescent and lysis in the GBM was greater in the severe group than in the mild group. The degree of lysis of the GBM positively correlated with the degree of distribution of endocapillary proliferation. The degree of glomerular sclerosis at the second biopsy was greater in the severe group compared with the other two groups. The severity of cellular crescent at the first biopsy positively correlated with that of glomerular sclerosis at the second biopsy. Multiple logistic regression analysis indicated that the risk of glomerular sclerosis at the second biopsy was 19‐fold higher in the odds ratio in the severe group compared with the mild group. In conclusion, the progression of glomerular sclerosis in serial biopsy is dependent on the degree of distribution of endocapillary proliferation and the severity of cellular crescents at the first biopsy in childhood IgA GN presenting diffuse endocapillary proliferation.
Expression of MAGE‐A3 in intrahepatic cholangiocarcinoma and its precursor lesions
Authors: Tsuneyama Koichi , Sasaki Motoko , Shimonishi Tomonori , Nakanuma Yasuni
Published: February 2004

MAGE‐A3 antigen is known to be neo‐expressed in a large proportion of tumors but not detectable in normal tissues, and could be a target antigen recognized by autologous cytotoxic T lymphocytes. In the present study, the expression of MAGE‐A3 at protein and mRNA levels was examined in intrahepatic cholangiocarcinoma (ICC) and its precursor lesions. Carcinomatous and dysplastic biliary cells expressed MAGE‐A3 in their cytoplasm diffusely, although there was no MAGE‐A3 expression in normal and hyperplastic biliary cells. MAGE‐A3 was expressed in one of 10 cases (10%) of low‐grade dysplasia, four of 13 (31%) cases of high‐grade dysplasia/in situ carcinoma, and 32 of 68 invasive ICC cases (47%), respectively. The MAGE‐A3 mRNA expression pattern was similar to that of MAGE‐A3 protein. The incidence and intensity of MAGE‐A3 expression increased along the progression of biliary neoplasia (P < 0.05). There was no correlation between MAGE‐3 expression and histological differentiation or anatomical locations of invasive ICC. MAGE‐A3 is a promising target molecule for the specific immunotherapy of ICC.
Immunohistopathological and molecular genetic features of a case in which gastrointestinal stromal tumor recurred five times
Authors: Inoue Tsukasa , Suzuki Takashi , Nakagawa Kunitoshi , Kurokawa Yoshimochi , Satomi Susumu , Moriya Takuya , Sasano Nobuaki , Sasano Hironobu
Published: February 2004

Gastrointestinal stromal tumor (GIST) is a distinct group of mesenchymal neoplasms recently shown to exhibit differentiation toward interstitial cells of Cajal. Although previous studies have shown that the clinical outcome of patients with GIST is associated with mitotic activity, the proliferation index determined by the Ki‐67 labeling index, immunophenotype (CD34 and/or p53) and mutation in exon 11 of the c‐kit, a definitive discrimination between benign and malignant GIST has not yet been established. We report a patient in whom malignant GIST in the abdomen recurred five times. In this case, the primary GIST and the five recurrent GIST were associated with c‐kit immunoreactivity, but the mitotic index of the GIST tended to be increasingly higher with subsequent recurrences. Mutational analysis of the c‐kit revealed that the primary and recurrent GIST were mutant‐negative. These data indicated that ‘morphologically appearing benign’ tumors with lower proliferative parameters may also have the capacity of metastasis and recurrence.
Low‐grade tubular‐mucinous renal neoplasm with neuroendocrine differentiation: A histological, immunohistochemical and ultrastructural study
Authors: Kuroda Naoto , Nakamura Shoichiro , Miyazaki Eriko , Hayashi Yoshihiro , Taguchi Takahiro , Hiroi Makoto , Yamasaki Yoshikazu , Shuin Taro , Enzan Hideaki
Published: February 2004

Low‐grade tubular‐mucinous renal neoplasm (LGTMRN) was recently established as a distinct carcinoma classification. A 70‐year‐old, female traffic accident victim underwent a detailed examination that disclosed a huge mass in the lower pole of the left kidney. The patient underwent a nephrectomy based on a diagnosis of renal tumor. Macroscopically, the tumor was well demarcated and a whitish color with focal hemorrhage. Histological examination showed that tumor cells proliferated through tubular, trabecular, and solid growth patterns in the mucinous background. Focally, foci of clear cells or the proliferation of spindle cells was also observed. Nuclei were generally round and uniform in size. No abnormal mitotic figures were identified. Immunohistochemically, tumor cells were diffusely positive for AE1/AE3, vimentin and chromogranin A, and focally positive for cytokeratin (CK) 18, CK19, Ulex europaeus agglutinin‐1, epithelial membrane antigen, neuron‐specific enolase (NSE), CD9 and CD57. Ultrastructurally, tumor cells contained a moderate number of mitochondria, rough endoplasmic reticulum and dense‐core granules. No renin granules or glycogen were observed. Microvilli were focally seen. Our results render further evidence that LGTMRN is a distinct entity from the hitherto established renal neoplasms. Foci of clear cells and neuroendocrine differentiation should be added to the histological spectrum of LGTMRN.
Urinary tract and genito‐urinary suppurative infections due to anaerobic bacteria
Authors: BROOK ITZHAK
Published: February 2004

Abstract Anaerobes have been involved in many different types of urinary tract infection. This review describes the microbiology, diagnosis and management of urinary tract and genito‐urinary suppurative infections caused by anaerobic bacteria. The types of infections of the urinary tract in which anaerobes have been involved include para‐ or periurethral cellulitis or abscess, acute and chronic urethritis, cystitis, acute and chronic prostatitis, prostatic and scrotal abscesses, periprostatic phlegmon, ureteritis, periureteritis, pyelitis, pyelonephritis, renal abscess, scrotal gangrene, metastatic renal infection pyonephrosis, perinephric abscess, retroperitoneal abscess and other infections. The anaerobes recovered in these studies were Gram‐negative bacilli (including Bacteroides fragilis and pigmented Prevotella and Porphyromonas sp.), Clostridium sp., anaerobic Gram‐positive cocci and Actinomyces sp. In many cases, they were recovered mixed with coliforms or streptococci. The recovery of anaerobes requires the administration of antimicrobial therapy that is effective against these organisms. These antimicrobials include metronidazole, chloramphenicol, clindamycin, a carbapenem, cefoxitin and the combination of a penicillin and a beta‐lactamase inhibitor. Percutaneous drainage, open surgical drainage or nephectomy might be indicated for abscesses.
Fibroepithelial polyp originating from the glans penis in a child
Authors: YILDIRIM IBRAHIM , IRKILATA CEM , SÜMER FAHRI , AYDUR EMIN , ÖZCAN AYHAN , DAYANÇ MURAT
Published: February 2004

Abstract A fibroepithelial polyp is a benign neoplasm that occurs throughout the urinary tract. In this article, we report a pediatric case of a fibroepithelial polyp originating from the glans penis. To our knowledge, this is a previously unreported lesion arising from the glans penis.