BBA - General Subjects (v.1526, #1)

Occurrence of ovalbumin in ovarian yolk of chicken during oogenesis by Yasushi Sugimoto; Shinya Sanuki; Hisham R. Ibrahim; Takayoshi Aoki; Takahiro Kusakabe; Katsumi Koga (1-4).
Yolk specimens from chicken ovaries during oogenesis gave a positive signal for ovalbumin as analyzed by Western blotting, indicating that the ovarian yolk contains ovalbumin. Northern blotting and reverse transcriptase-polymerase chain reaction gave a negative signal for ovalbumin mRNA in the liver and other organs except oviduct, whereas the laying hen serum was found to indicate immunologically the presence of ovalbumin. It was therefore assumed that ovalbumin synthesized in the oviduct might partly be secreted into the blood circular system, from which it is taken up into the oocyte.
Keywords: Ovalbumin; Ovarian yolk; Oviduct; Gallus gallus;

A rapid and sensitive method for the analysis of cyanophycin by Nora A. Erickson; Nancy H. Kolodny; Mary M. Allen (5-9).
A method has been devised for the quantitative analysis of cyanophycin, based on 1H nuclear magnetic resonance (NMR) spectroscopy, allowing determination of the nitrogen status of cyanobacteria. Cyanophycin is extracted with minimal washing from small volumes of cells and quantified by integration of the NMR peak attributed to the protons attached to the δ-carbon of arginine. Linear relationships were found between the amount of cyanophycin determined by this method and both known concentrations of cyanophycin solutions and the amount of cyanophycin determined using the standard chemical arginine assay.
Keywords: Cyanobacterium; Cyanophycin; Nitrogen reserve; 1H nuclear magnetic resonance spectroscopy; Arginine analysis;

Oxidative stress increases MICA and MICB gene expression in the human colon carcinoma cell line (CaCo-2) by Kazuo Yamamoto; Yoshihide Fujiyama; Akira Andoh; Tadao Bamba; Hidetoshi Okabe (10-12).
The human major histocompatibility complex class I chain-related A gene (MICA) and the MICB gene are newly identified members of the major histocompatibility complex class I chain-related gene family. We demonstrate here that oxidative stress, induced by H2O2, promoted MICA (2.2-fold) and MICB (3.8-fold) gene expression using the human colon carcinoma cell line (CaCo-2) and semi-quantitative RT-PCR.
Keywords: Oxidative stress; H2O2; MICA; MICB;

Unstimulated RAW 264.7 macrophages express negligible heme oxygenase-1 (HO-1) protein but incubation with the nitric oxide (NO) donor spermine nonoate (SPNO) induced HO-1 and weakly cyclo-oxygenase-2 (COX-2) protein. This effect was potentiated by coincubation with the COX-2 selective inhibitor, SC58125. Cells incubated with SPNO showed a strong increase in HO-1 mRNA levels after 4 h with a significant potentiation in the presence of SC58125, which did not modify HO-1 mRNA stability. The induction of HO-1 by NO and its potentiation by anti-inflammatory agents may play a role in inflammatory and immune responses.
Keywords: Heme oxygenase; Nitric oxide; Macrophage; Non-steroidal anti-inflammatory drug;

Interleukin-15 mediates reciprocal regulation of adipose and muscle mass: a potential role in body weight control by Neus Carbó; Joaquı́n López-Soriano; Paola Costelli; Belén Alvarez; Sı́lvia Busquets; Francesco M Baccino; LeBris S Quinn; Francisco J López-Soriano; Josep M Argilés (17-24).
Interleukin (IL)-15 is a cytokine which is highly expressed in skeletal muscle. Cell culture studies have indicated that IL-15 may have an important role in muscle fiber growth and anabolism. However, data concerning the metabolic effects of this cytokine in vivo are lacking. In the present study, IL-15 was administered to adult rats for 7 days. While IL-15 did not cause changes in either muscle mass or muscle protein content, it induced significant changes in the fractional rates of both muscle protein synthesis and degradation, with no net changes in protein accumulation. Additionally, IL-15 administration resulted in a 33% decrease in white adipose tissue mass and a 20% decrease in circulating triacylglycerols; this was associated with a 47% lower hepatic lipogenic rate and a 36% lower plasma VLDL triacylglycerol content. The decrease in white fat induced by IL-15 was in adipose tissue. No changes were observed in the rate of lipolysis as a result of cytokine administration. These findings indicate that IL-15 has significant effects on both protein and lipid metabolism, and suggest that this cytokine may participate in reciprocal regulation of muscle and adipose tissue mass.
Keywords: Interleukin-15; Protein turnover; Lipoprotein lipase; Lipogenesis; Cytokine;

Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3 by Margaret Barkett; Julia E Dooher; Lori Lemonnier; LaVone Simmons; Jennifer N Scarpati; Yuan Wang; Thomas D Gilmore (25-36).
The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-κB family. v-Rel causes rapidly fatal lymphomas in young chickens, and transforms and immortalizes chicken lymphoid cells in vitro. Several mutations that have enhanced the oncogenicity of v-Rel have been selected during in vitro and in vivo passage of v-Rel-containing retroviruses. In this report, we show that the C-terminal deletion and two point mutations (Asp→Gly at residue 91 and Asp→Asn at residue 437) in v-Rel make it resistant to cleavage by the cell-death protease caspase-3. In contrast, c-Rel, which has Asp residues at these sites, can be cleaved by caspase-3 in vitro as well as in vivo in cells induced to undergo apoptosis. We have characterized activities of v-Rel mutants with recreated single caspase-3 cleavage sites, two cleavage sites, or an introduced artificial cleavage site. All of these mutant v-Rel proteins are sensitive to caspase-3 cleavage in vitro, and show wild-type activity in terms of nuclear localization in chicken fibroblasts and DNA binding in vitro. Moreover, all caspase-3-sensitive v-Rel mutants transform chicken spleen cells in vitro and induce fatal lymphoid tumors in vivo to approximately the same extent as wild-type v-Rel. As with v-Rel mutants, caspase-3-resistant c-Rel mutants behave similarly to caspase-3-sensitive wild-type c-Rel in terms of DNA binding, transcriptional activation, in vitro transformation, and tumorigenicity. Mammalian c-Rel proteins can also be cleaved by caspase-3 in vitro, and a c-Rel mutant from a human pre-T lymphoma cell line is less sensitive than wild-type human c-Rel to cleavage by caspase-3. Taken together, these results demonstrate that specific mutations render oncogenic forms of Rel proteins resistant to cleavage by a cell-death caspase; however, the biological relevance of this resistance remains unclear. Nevertheless, to our knowledge, this is the first demonstration of mutations in caspase-3 recognition sites occurring during the evolution of an oncogenic protein.
Keywords: Rel; Nuclear factor κB; Caspase; Apoptosis; Retroviral oncogene; Transcription factor;

Hybrid glycoprotein and neoproteoglycan probes were prepared by coupling various glycoproteins or polysaccharides to peroxidase or biotinyl bovine serum albumin, respectively. Lectins recognizable by the neoglycoconjugate probes were extracted from 16 cultivable mushrooms. Dot-blot assay revealed five extracts to be reactive with only hybrid glycoprotein probes, but others also reacted with neoproteoglycan probes. According to the reactivity pattern with probe screening, the one lectin from Oudemansiella platyphylla extract (OPL) bound best with asialotransferrin– and asialoagalactotransferrin–peroxidase probes and was isolated using an asialotransferrin column, but it did not bind with other hybrid glycoprotein or neoproteoglycan probes. OPL, consisting of two polypeptides with high homology in the N-terminal amino acid sequences, exhibited weak hemagglutinating activity. Purified OPL specifically bound the β-GlcNAc probe among various biotinylated polymeric sugar probes, while it exhibited essentially the same binding specificity toward neoglycoconjugate probes as that of the crude extract, showing a preference for the asialobiantennary complex type of N-linked glycans. These results indicate that the neoglycoconjugate probes are valuable in lectin screening.
Keywords: Lectin screening; Neoproteoglycan; Hybrid glycoprotein; Biotin–glycoconjugate; Mushroom lectin; Peroxidase–glycoprotein;

Metabolism and effects on cholestasis of isoursodeoxycholic and ursodeoxycholic acids in bile duct ligated rats by Edmund Purucker; Hanns-Ulrich Marschall; Ron Winograd; Siegfried Matern (44-52).
Isoursodeoxycholic acid (isoUDCA), the 3β-epimer of ursodeoxycholic acid (UDCA), may have pharmaceutical potential because of its similar hydrophilicity and in vitro cytoprotection as compared with UDCA. We compared metabolism and effects on cholestasis of UDCA and isoUDCA in experimental cholestasis in rats. Cholestasis was induced by bile duct ligation. For bile flow and biliary bile acid analysis, UDCA or isoUDCA were infused intraduodenally. For the study of chronic effects, chow was supplemented with 2.5 g/kg UDCA or isoUDCA for 3 weeks. Sham-operated animals served as controls. IsoUDCA became completely converted to UDCA in the liver. Choleresis and biliary bile acids were the same after the intraduodenal administration of either compound. Oral administration of UDCA or isoUDCA significantly improved liver biochemistry but not clinical and histological parameters in chronic cholestasis. The decrease of serum cholic acid in control animals was more pronounced after isoUDCA (−93%) than after UDCA (−76%). Only after UDCA, this decrease was compensated by increases of UDCA, β-muricholic acid (MCA), and Δ22-β-MCA. Our results show that isoUDCA has the same effect on choleresis and liver biochemistry as UDCA. IsoUDCA features pro-drug characteristics of UDCA and causes compared to the latter lower serum bile acid concentrations in non-cholestatic animals.
Keywords: Bile acid metabolism; Bile duct ligation; Cholestasis; Isoursodeoxycholic acid; Ursodeoxycholic acid;

Effect of albumin on the kinetics of ascorbate oxidation by Evgenia Lozinsky; Artem Novoselsky; Alexander I. Shames; Oshra Saphier; Gertz I. Likhtenshtein; Dan Meyerstein (53-60).
The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steady-state fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule.
Keywords: Fluorophore-nitroxide; Ascorbate oxidation; Bovine serum albumin;

Adsorption of IgG onto hydrophobic teflon. Differences between the Fab and Fc domains by Arnoldus W.P Vermeer; Carla E Giacomelli; Willem Norde (61-69).
The effect of differences in the degree of hydrophobicity of protein patches/fragments on the adsorption behaviour of the protein is investigated. The adsorption isotherm of a monoclonal mouse anti-human immunoglobulin G (isotype 2b) onto hydrophobic Teflon particles is measured using a depletion method. The adsorption-induced denaturation of the immunoglobulin as a function of the adsorbed amount is studied by differential scanning calorimetry, and the corresponding rearrangements in the secondary structure of the whole IgG molecule and its Fab and Fc fragments are determined by circular dichroism spectroscopy. The effects of adsorption on the Fab and Fc fragments in the intact IgG molecule occur independently. Adsorption of the whole IgG molecule leads to denaturation of the Fab fragments, whereas the Fc fragment remains unperturbed; adsorption of the isolated fragments results in structural changes in both Fab and Fc. The surface hydrophobicity of the isolated fragments was studied by HPLC. These experiments support the hypothesis that differences in the degree of denaturation between Fab and Fc are due to the higher degree of hydrophobicity of the Fab fragment. The adsorption-induced changes in the secondary structure are more prominent for the isolated fragments as compared to intact IgG. This is ascribed to the higher flexibility of the isolated fragment, as compared to the fragment in the whole molecule.
Keywords: Immunoglobulin; Fab; Fc fragment; Protein hydrophobicity; Hydrophobic surface; Preferential adsorption; Differential scanning calorimetry; Circular dichroism;

UDP hydrolase activity associated with the porcine liver annexin fraction by Malgorzata Danieluk; Marcin Golczak; Slawomir Pikula; Joanna Bandorowicz-Pikula (70-76).
In the crude fraction of porcine liver annexins, we identified annexin IV (AnxIV), AnxII and AnxVI of MW (molecular weight) of 32, 36 and 68 kDa, respectively, an albumin of MW of 61.5 kDa and an UDP hydrolase (UDPase) of MW of 62 kDa, related to the human UDPase from Golgi membranes. The latter enzyme exhibits its highest specificity towards UDP and GDP but not ADP and CDP, and it is stimulated by Mg2+ and Ca2+. AnxVI itself, although it binds purine nucleotides, does not exhibit hydrolytic activity towards nucleotides. Taken together, these results suggest that AnxVI may interact in vivo with a nucleotide-utilizing enzyme, UDPase. This is in line with observations made by other investigators that various annexins are able to interact with nucleotide-utilizing proteins, such as protein kinases, GTPases, cytoskeletal proteins and p120GAP. Such interactions could be of particular importance in modulating the biological activities of these proteins in vivo.
Keywords: Annexin; Ecto-ATPase; Uridine diphosphohydrolase; UDP; GDP; Calcium;

Developmental expression and distribution of amphibian glutathione transferases by Fernanda Amicarelli; Anna Maria Ragnelli; Pierpaolo Aimola; Franca Cattani; Antonella Bonfigli; Osvaldo Zarivi; Michele Miranda; Carmine Di Ilio (77-85).
This work is aimed at detecting the expression and location of embryonic Bufo bufo GST (bbGSTP1-1) and adult B. bufo GST (bbGSTP2-2) during toad development, in order to assign a putative role to these enzymes also on the basis of their compartmentalization and to verify whether during the premetamorphic liver ontogeny the bbGSTP2-2 form appears. This study was also performed in the adult liver (the primary site of Pi class GST expression) and in the mature ovary, to discern if the embryonic form derives from maternal form. The results show that the embryos and the ovary express only bbGSTP1-1. Moreover, bbGSTP1-1 distribution is the same both in the early embryos and in the ovary: this strongly suggests that bbGSTP1-1 is of maternal origin. As development goes on, a wide distribution of bbGSTP1-1 all over the differentiating organs is observed. The embryonic liver expresses exclusively the bbGSTP1-1 form, while the adult liver is highly positive only towards the bbGSTP2-2 form. This implies that the switch towards the adult bbGSTP2-2 form occurs in metamorphic or postmetamorphic phases and that the detoxication metabolic requirements of the embryo may be completely fulfilled by the bbGSTP1-1 isoenzyme.
Keywords: Amphibia; Development; Glutathione transferase; Liver; Immunolocalization;

Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase by João Meireles Ribeiro; António Carloto; Marı́a Jesús Costas; José Carlos Cameselle (86-94).
Free ADP-ribose has a reducing ribose moiety and it is hazardous due to its nonenzymic reactivity toward protein side chains. ADP-ribose hydrolases are putative protective agents to avoid the intracellular accumulation of ADP-ribose. In mammalian sources, two types of enzymes with ADP-ribose hydrolase activity are known: (i) highly specific ADP-ribose pyrophosphatases, which in a Mg2+-dependent fashion hydrolyse only ADP-ribose and the nonphysiological analogue IDP-ribose, and (ii) less specific nucleoside diphosphosugar or diphosphoalcohol (NDP-X) pyrophosphatases, which besides A(I)DP-ribose hydrolyse also some nonreducing NDP-X substrates. So far, of these two enzyme types only the less specific one has been reported in human sources: an ADP-sugar pyrophosphatase purified from erythrocytes or expressed from cDNA clones. Here we report that human placenta extracts contain two ADP-ribose hydrolases, which were characterised after a near 1000-fold purification. One is an ADP-sugar pyrophosphatase: it hydrolysed ADP-ribose, ADP-glucose and ADP-mannose, but not e.g. UDP-glucose, at similar rates. It resembles the erythrocyte and recombinant enzyme(s), but showed a 5–20-fold lower K m for ADP-ribose (7 μM). The other enzyme is a highly specific ADP-ribose pyrophosphatase (the first of this kind to be reported in humans): it hydrolysed only ADP-ribose and IDP-ribose at similar rates, with a very low, 0.4 μM K m for the former. This is a major candidate to control the accumulation of free ADP-ribose in humans. It remains to be seen whether it belongs to the ‘nudix’ protein family, which includes several ADP-ribose hydrolases and other ‘housecleaning’ enzymes (M.J. Bessman, D.N. Frick, S.F. O’Handley, J. Biol. Chem. 271 (1996) 25059-25062).
Keywords: Adenosine diphosphate ribose; Adenosine diphosphate ribose pyrophosphatase; Adenosine diphosphate sugar pyrophosphatase; Sugar nucleotide; Nudix hydrolase; Protein glycation;

Involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation by Supatra Porasuphatana; Pei Tsai; Sovitj Pou; Gerald M. Rosen (95-104).
Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO) and superoxide (O⋅− 2) during enzymatic cycling, and the ratio of each free radical is dependent upon the concentration of l-arginine. Using spin trapping and electron paramagnetic resonance spectroscopy, we detected α-hydroxyethyl radical (CH3 CHOH), produced during the NOS I metabolism of ethanol (EtOH). The generation of CH3 CHOH by NOS I was found to be Ca2+/calmodulin dependent. Superoxide dismutase prevented CH3 CHOH formation in the absence of l-arginine. However, in the presence of l-arginine, the production of CH3 CHOH was independent of O⋅− 2 but dependent upon the concentration of l-arginine. Formation of CH3 CHOH was inhibited by substituting d-arginine for l-arginine, or inclusion of the NOS inhibitors N G-nitro-l-arginine methyl ester, N G-monomethyl-l-arginine and the heme blocker, sodium cyanide. The addition of potassium hydrogen persulfate to NOS I, generating the perferryl complex (NOS-[Fe5+=O]3+) in the absence of oxygen and Ca2+/calmodulin, and EtOH resulted in the formation of CH3 CHOH. NOS I was found to produce the corresponding α-hydroxyalkyl radical from 1-propanol and 2-propanol, but not from 2-methyl-2-propanol. Data demonstrated that the perferryl complex of NOS I in the presence of l-arginine was responsible for catalyses of these secondary reactions.
Keywords: Nitric oxide synthase; Nitric oxide; Superoxide; Perferryl complex;

Fluorescence and stopped flow methods were used to compare clinically used heparins with regard to their ability to bind to antithrombin and to accelerate the inactivation of factor Xa. Titration of antithrombin with both low molecular weight heparin (LMWH) (enoxaparin, fragmin and ardeparin) and unfractionated heparin (UFH) produced an equivalent fluorescence increase and indicates similar affinity of all heparin preparations to antithrombin. However, relative to UFH enoxaparin, the LMWH with the smallest average molecular mass, contained only 12% material with high affinity for antithrombin. The rate of factor Xa inhibition by antithrombin increased with the concentration of the examined heparins to the same limiting value, but the concentration required for maximal acceleration depended on the preparation. According to these data the high affinity fraction of the heparin preparations increased the intrinsic fluorescence and inhibitory activity equally without additional effects by variations in chain length and chemical composition. In contrast, in the presence of Ca UFH accelerated the inhibition of factor Xa by antithrombin 10-fold more efficiently than comparable concentrations of the high affinity fractions of enoxaparin and fragmin. The bell-shaped dependence of this accelerating effect suggests simultaneous binding of both proteins to heparin. In conclusion, under physiologic conditions the anti-factor Xa activity of heparin results from a composite effect of chain length and the content of material with high affinity to antithrombin. Thus, the reduced antithrombotic activity of LMWH relative to UFH results from a smaller content of high affinity material and the absence of a stimulating effect of calcium.
Keywords: Antithrombin; Factor Xa; Low molecular weight heparin;