bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of vitronectin expression in human HepG2 hepatoma cells by transforming growth factor‐β1
Authors: KOLI Katri , LOHI Jouko , HAUTANEN Aarno , KESKI‐OJA Jorma
Published: March 2005

Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the regulation of vitronectin by transforming growth factor‐β1 (TGFβ1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGFβ‐regulated gene, plasminogen activator inhibitor (PAI‐1). Rabbit antibodies raised against human plasma‐derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGFβ1‐induced production and extracellular deposition of vitronectin. Accordingly, TGFβ1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2–20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGFβ induction profiles of vitronectin and PAI‐1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet‐derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGFβ1 enhanced the expression of PAI‐1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI‐1 in these cells. The results indicate that TGFβ1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI‐1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the α subunit of the receptor in response to TGFβ1. These effects of TGFβ are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair.
Ca2+‐induced reversible translocation of phospholipase A2 between the cytosol and the membrane fraction of rat liver macrophages
Authors: KRAUSE Hellmut , DIETER Peter , SCHULZE‐SPECKING Agnes , BALLHORN Annette , DECKER Karl
Published: March 2005

In cell‐free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be rapidly associated with the particulate fraction in a Ca2+‐dependent manner at Ca2+ concentrations of 0.1–1.0 μM. This is also the range of the levels of intracellular Ca2+ reported for basal and various stimulated conditions. After translocation, phospholipase A2 could be released from the membranes in the presence of Ca2+ chelators, increasing the specific activity of phospholipase A2 in the supernatant fraction. These findings support the view that translocation is a regulatory mechanism of phospholipase A2 by bringing the enzyme to its substrate. Unlike the situation with protein kinase C, Mg2+ exerted little effect on phospholipase A2 translocation, indicating that this process is regulated in vivo mainly by fluctuations of the intracellular Ca2+ content.
Molecular analysis of a human interferon‐inducible gene family
Authors: LEWIN Andrew R. , REID Laurence E. , McMAHON Martin , STARK George R. , KERR Ian M.
Published: March 2005

Three functional members of the 1–8 gene family have been isolated on a single human genomic DNA fragment of less than 18 kb. The 1–8U and 1–8D genes are extremely similar: each is contained within a <2‐kb fragment, has in its 5′flanking region two adjacent 14‐base‐pair sequences showing high similarity to interferon‐stimulable response elements (ISREs) and has two highly related exons. The third gene (9–27) has a similar overall structure, shows substantial similarity to the 1–8s but has only one ISRE which is 3′ of two CCAAT boxes not present in the 1–8U and D genes. The cDNAs corresponding to the three genes share 120 nucleotides of identical sequence and show > 90% identity over 70% of the coding sequence. For the 1–8U and D genes the high similarity extends into the 5′ non‐coding and flanking regions. The open reading frames encode polypeptides that are likely to be of very similar structure. Antiserum to a conserved peptide detects a polypeptide(s) of about 14 kDa on PAGE which separates into three components on isoelectric focussing. The 9–27 and 1–8U genes are highly interferon‐inducible, the 1–8D gene is much less so. These differences are mimicked by the activities of the corresponding ISREs placed 5′ of a marker gene in expression constructs. They presumably reflect differences in the interaction of the ISREs with the various interferon‐inducible and constitutive factors that govern the interferon response.
Inhibition of hepatic proteolysis by insulin
Authors: HALLBRUCKER Christian , vom DAHL Stephan , LANG Florian , GEROK Wolfgang , HÄUSSINGER Dieter
Published: March 2005

Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 ± 1.3% (n= 15) and led to an amiloride‐sensitive, bumetanide‐sensitive and furosemide‐sensitive net K+ uptake of 5.53 ± 0.31 μmol · g−1 (n= 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 μM), respectively. The insulin‐induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM).

In perfused livers from 24‐h‐starved rats, both the insulin‐stimulated net K+ uptake and the insulin‐induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 ± 0.3% (n= 4) and 13.8 ± 1.2% (n= 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol · l−1), both the insulin‐induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50–60%.

During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 ± 0.24 μmol · g−1), which was accompanied by stimulation of [3H]leucine release by 16.4 ± 4.6% (n= 4).

Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 ± 0.2 μmol · g−1 (n= 4) and simultaneously inhibited [3H]leucine release by 12.4 ± 1.7% (n= 4).

There was a close relationship between the Ba2+ or insulin‐induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine‐induced cell swelling or the nutritional state.

The data suggest that the insulin‐induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange. The findings further suggest that modification of insulin‐induced intracellular net K+ accumulation parallels the antiproteolytic potency of the hormone, consistent with a role of hormone‐induced ion movements across the plasma membrane in mediating the antiproteolytic effect of insulin in liver.
Kluyveromyces lactis toxin has an essential chitinase activity
Authors: BUTLER Andrew R. , O'DONNELL Ray W. , MARTIN Vera J. , GOODAY Graham W. , STARK Michael J. R.
Published: March 2005

The Kluyveromyces lactis toxin is a protein containing three subunits (α, β and γ) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle. Despite the toxin's complex structure, the γ subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the γ polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin. The toxin α subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase. The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin. However, since the α subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.
Correlations between the conformations elucidated by CD spectroscopy and the antigenic properties of four peptides of the foot‐and‐mouth disease virus
Authors: SILIGARDI Giuliano , DRAKE Alex F. , MASCAGNI Paolo , ROWLANDS David , BROWN Fred , GIBBONS William A.
Published: March 2005

The conformational features of four related antigenic peptides (A, B, C and USA) from the foot‐and‐mouth disease virus (FMDV) (VP1; 141–160 of serotype A, subtype 12), assessed by CD, were found to correlate with the serological properties of these peptides. The CD spectra of the four peptides, obtained under cryogenic and solvent titration conditions, were consistent with three conformational components (a left‐handed extended helix, an α‐helix and a 310 helix) for peptides A and C and four components (a β‐turn of type II, an α‐helix, a γ‐turn and a 310 helix) for peptides B and USA. The amino acid substitutions at positions 148 and 153, which distinguish the peptides, are therefore responsible for both their conformational and antigenic differences.
Assignment of the five disulfide bridges in an α‐amylase inhibitor from wheat kernel by fast‐atom‐bombardment mass spectrometry and Edman degradation
Authors: POERIO Elia , CAPORALE Carlo , CARRANO Lucia , PUCCI Piero , BUONOCORE Vincenzo
Published: March 2005

The assignment of the five disulfide bridges in an α‐amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast‐atom‐bombardment mass spectrometry (FAB‐MS) and automatic sequencing based on Edman degradation. Direct FAB‐MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.
Influence of ganglioside GM3 and its breakdown products on lymphoblastic transformation and T‐suppressor activity
Authors: DYATLOVITSKAYA Ella V. , KOROLEVA Alla B. , SUSKOVA Victoriya S. , ROZYNOV Boris V. , BERGELSON Lev D.
Published: March 2005

The influence of ganglioside GM3 and some of its breakdown products on phytohemagglutinin‐induced blast transformation of human lymphocytes and concanavalin‐A‐induced T‐suppressor activity was studied. The structures of two major hydrolysis products of GM3 were established by negative‐ion fast‐atom‐bombardment mass spectrometry as neuraminyllactosylsphingosine (NeuLacSph) and neuraminyllactosylceramide (NeuLacCer). Both substances were shown to be potent inhibitors of mitogen‐induced lymphoblastic transformation whereas their acetylation products NeuAcLacSphAc and GM3 did not affect the proliferative response of lymphocytes to phytohemagglutinin. On the other hand, only GM3 and NeuLacSph were able to enhance concanavalin‐A‐induced T‐suppressor activity. On the basis of these data, it is suggested that the effects of GM3 and its breakdown products on lymphoblastic transformation and T‐suppressor activity must rest on different mechanisms and that N‐deacylation of GM3 appears to be an essential step in conversion of the ganglioside into an inhibitor of lymphocyte blast transformation.
Localization on the mitochondrial F1 ATPase α subunit of an epitope masked in the membrane‐bound enzyme using a monoclonal antibody and synthetic peptides
Authors: MORADI‐AMÉLI Mahnaz , CLERC François Frédéric , CIEUR Franck , SEIBERRAS Ghislaine , GODINOT Catherine
Published: March 2005

The epitope of the monoclonal antibody 20D6 was localized by N‐terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1α subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven‐amino‐acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the α subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti‐F1 antisera showed that this region is highly immunodominant.

In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the α subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane.
On the role of polysialoglycosphingolipids as tetanus toxin receptors
Authors: SCHIAVO Giampietro , DEMEL Rudy , MONTECUCCO Cesare
Published: March 2005

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors.

At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect.

The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin (light chain plus the 50‐kDa N‐terminal part of the heavy chain) is involved in the low‐pH‐driven lipid interaction of the toxin.

On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.
The molecular mechanism by which adrenalin inhibits glycogen synthesis
Authors: NAKIELNY Sara , CAMPBELL David G. , COHEN Philip
Published: March 2005

Improved methodology was used to establish that the phosphorylation of a serine located 10 residues from the N‐terminus of glycogen synthase (N10) increases from 0.12 mol · mol−1 to 0.54 mol · mol−1in vivo in response to adrenalin. The only ‘N10 kinase’ detected in muscle extracts was casein kinase‐1 (CK1), although its activity was unaffected by injection of adrenalin in vivo or by incubation with cyclic‐AMP‐dependent protein kinase and MgATP in vitro. Prior phosphorylation of the serine residue N7 by phosphorylase kinase increased sixfold the rate of phosphorylation of glycogen synthase by CK1, and altered the specificity of CK1 so that it phosphorylated the serine residue N10 specifically. Stoichiometric phosphorylation of N7 decreased the activity ratio (∓ glucose 6‐phosphate) of glycogen synthase from 0.80 to 0.45, and subsequent phosphorylation of N10 to 0.8 mol · mol−1 produced a further decrease to 0.17, demonstrating that N10 phosphorylation inhibits glycogen synthase. The major ‘N10 phosphatase’ in skeletal muscle extracts was identified as the glycogen‐associated form of protein phosphatase‐1 (PP1G), accounting for approximately 75% of the N10 phosphatase activity in the extracts and about 90% of the activity in isolated glycogen particles. Phosphorylation of N10, after prior phosphorylation of N7, decreased the rate of dephosphorylation of N7. These results, in conjunction with previous findings, establish that adrenalin inhibits glycogen synthase by increasing the phosphorylation of N7, N10 and three further serines located 30, 34 and 38 residues from the start of the C‐terminal CNBr peptide (termed the region C30–C38). They also indicate that increased phosphorylation of N10, the region C30–C38, and perhaps N7, is initiated through the inhibition of PP1G by adrenalin, which results from phosphorylation of its glycogen‐targetting subunit by cyclic‐AMP‐dependent protein kinase [Hubbard, M. J. & Cohen, P. (1989) Eur. J. Biochem. 186, 711–716]. The conclusion that direct phosphorylation of glycogen synthase by cyclic‐AMP‐dependent protein kinase makes little contribution to inhibition by adrenalin, is at variance with the teachings of the major textbooks of biochemistry.
Purification and characterisation of the insulin‐stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II
Authors: LAVOINNE Alain , ERIKSON Eleanor , MALLER James L. , PRICE Daniel J. , AVRUCH Joseph , COHEN Philip
Published: March 2005

The insulin‐stimulated protein kinase (ISPK) was purified over 50000‐fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE‐cellulose, phenyl‐Superose, Mono S and Mono Q. About 10 μg enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein‐staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 kinase II. The substrate specificities of ISPK and S6 kinase II were also very similar and like S6 kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen‐activated protein kinase and MgATP. ISPK was distinct from an insulin‐stimulated 70‐kDa S6 kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.
Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization
Authors: YOSHIDA Tadashi , ISHIKAWA Kazunobu , SATO Michihiko
Published: March 2005

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE.

The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous‐CO and ferrous‐O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other.

An NADPH‐cytochrome‐P‐450 reductase system composed of NADPH and detergent‐solubilized NADPH‐cytochrome‐P‐450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.
Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptophan‐tryptophan quinone
Authors: DUINE Johannis A.
Published: March 2005

The presently best known and largest group of quinoproteins consists of enzymes using the cofactor 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐f]quinoline‐4,5‐dione (PQQ), a compound having a pyrrole ring fused to a quinoline ring with an o‐quinone group in it. Representatives of this group are found among the bacterial, NAD(P)‐independent, periplasmic dehydrogenases. Despite their high midpoint redox potential, the overall behaviour of quinoprotein dehydrogenases is similar to that of their counterparts, those using a flavin cofactor or a nicotinamide coenzyme. Apart from an exceptional Gram‐positive one, the sole organisms where the presence of PQQ has really been established are Gram‐negative bacteria. Evidence for the occurrence of covalently bound PQQ is lacking since it has now been shown that several enzymes previously considered to contain this prosthetic group do not in fact do so.

Another group of quinoproteins, consisting of amine oxidoreductases, has a protein chain containing one of the following quinonoid aromatic amino acids: 6‐hydroxy‐phenylalanine‐3,4‐dione (TPQ) or 4‐(2′‐tryptophyl)‐tryptophan‐6,7‐dione (TTQ). There is no doubt that these o‐quinones play a role as cofactor, in the case of TPQ in prokaryotic as well as eukaryotic amine oxidases. It appears, therefore, that a novel calass of amino‐acid‐derived cofactors is emerging, ranging from the free radical form of tyrosine and tryptophan to those containing a dicarbonyl group (like the already known pyryvoyl group and the o‐quinones here described).
The ATP requirement for initiation of eukaryotic translation varies according to the mRNA species
Authors: JACKSON Richard J.
Published: March 2005

The requirement for ATP for initiation of eukaryotic mRNA translation was tested using gel‐filtered rabbit reticulocyte lysates incubated with labelled Met‐tRNAMetf and exogenous RNA templates, and assaying the formation of labelled 80S initiation complexes in the presence of GTP, or labelled 40S initiation complexes in the presence of a non‐hydrolysable analogue of GTP. Initiation complex formation on globin mRNA, or on capped viral RNAs such as papaya mosaic virus RNA and tobacco mosaic virus RNA, was strongly stimulated by ATP. In contrast, initiation complex formation on (uncapped) encephalomyocarditis virus RNA was uninfluenced by the presence or absence of ATP, which may be correlated with the recent evidence for scanning‐independent internal initiation on this viral RNA. In addition, initiation complex formation on uncapped cowpea mosaic virus RNA and on poly(A,U,G) was only slightly stimulated by ATP, much less than in the case of the capped RNAs. These results suggest that most of the ATP hydrolysed during translation initiation is consumed in cap‐dependent processes, probably in unwinding the mRNA, and relatively little in the actual migration or scanning of 40S subunits along the mRNA.
κ‐Casein micelles: structure, interaction and gelling studied by small‐angle neutron scattering
Authors: de KRUIF Cees G. , MAY Roland P.
Published: March 2005

Small‐angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of κ‐casein micelles in a buffer at pH = 6.7 were made using the D11 diffractometer in Grenoble. Results indicate that the micelles have a dense core with a fluffy outer layer. This outer layer. This outer layer appears to give rise to a steeply repulsive interaction on contact. In fact, the hard‐sphere model best fits the measured scattering intensities. Adding chymosin to the dispersion initiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles. This unexpected second process resembled that of spinodal demixing. The dispersion phase thus separates into a water and a protein phase on a time scale of hours. The observed phenomona contribute to the understanding of the cheese‐making process.
Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin
Authors: PIDARD Dominique , FRELINGER Andrew L. , BOUILLOT Colette , NURDEN Alan T.
Published: March 2005

The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane‐bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb‐IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb‐IIIa and fibrinogen‐binding‐site expression has yet been established. We have now evaluated the proteolysis of GPIIb‐IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I‐labelled purified fibrinogen and to the capacity of CT‐treated platelets to aggregate. Treatment of platelets with CT at 22°C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr∼ 90000) into a previously described, major membrane‐bound fragment with Mr 60000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr∼ 120000), which was converted into a faster migrating species with Mr∼ 115000. This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37°C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy‐terminal‐cleaved form. However, incubation at 37°C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60‐kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60‐kDa fragment results in loss of receptor activity.
The acyl‐carrier protein in Neurospora crassa mitochondria is a subunit of NADH: ubiquinone reductase (complex I)
Authors: SACKMANN Uwe , ZENSEN Ralf , RÖHLEN Dirk , JAHNKE Ulrike , WEISS Hanns
Published: March 2005

We determined the primary structure of a 9.6‐kDa subunit of the respiratory chain NADH:ubiquinone reductase (complex I) fromNeurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl‐carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N‐terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan‐2 mutant of N. crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6‐kDa subunit of complex I. We cultivated N. crassa in the presence of chloramphenicol to accumulate the nuclear‐encoded peripheral arm of complex I. This pre‐assembled arm also contains the 9.6‐kDa subunit. These results demonstrate that an acyl‐carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N. crassa.
Kinetic studies of the variations of cytoplasmic pH, nucleotide triphosphates (31P‐NMR) and lactate during normoxic and anoxic transitions in maize root tips
Authors: SAINT‐GES Véronique , ROBY Claude , BLIGNY Richard , PRADET Alain , DOUCE Roland
Published: March 2005

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P‐NMR chemical shift of the cytoplasmic Pi peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady‐state value of 7.1 ‐ 7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cytoplasmic Pi peak drifted gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady‐state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis.
Induction, regulation and messenger half‐life of cytochromes P450 IA1, IA2 and IIIA6 in primary cultures of rabbit hepatocytes
Authors: DAUJAT Martine , CLAIR Philippe , ASTIER Catherine , FABRE Isabelle , PINEAU Thierry , YERLE Martine , GELLIN Joel , MAUREL Patrick
Published: March 2005

A study on the regulation and induction of expression of cytochromes P450‐IA1, IA2 and IIIA6 genes has been undertaken using primary cultures of adult rabbit hepatocytes grown in a serum‐free chemically and hormonally defined medium. In 72‐h‐old cultures, 50 μM β‐naphthoflavone induced both IA1 and IA2 mRNA, the maximum level being reached after 4 h and 12 h, respectively. This was shown to result from an increase in the rate of transcription of gene IA1. In contrast, gene IA2 was constitutively transcribed in untreated cells, but mRNA only accumulated in the presence of β‐naphthoflavone which, however, did not affect the rate of transcription. Actinomycin D fully blocked induction of both IA1 and IA2 mRNA in response to their inducer. In untreated cells the presence of cycloheximide allowed a ‘constitutive’ expression of gene IA1, while in β‐naphthoflavone‐treated cells, it produced a super‐induction of IA1 but no modification of IA2 gene expression. Rifampicin (50 μM) strongly increased the IA1 mRNA level and rate of transcription only in cycloheximide‐treated cells. Rifampicin and dexamethasone, two prototypical inducers of P450‐IIIAs, induced both large and small IIIA6 mRNAs in a time‐dependent fashion, the maximum level being reached after 24 h. This was related to a large increase in the rate of transcription of the gene. Cycloheximide significantly decreased the accumulation of both IIIA6 mRNAs in response to rifampicin, while actinomycin D fully blocked induction. The half‐lives of IA1, IA2 and IIIA6 mRNAs were determined by two different methods, namely actinomycin D and [3H]uridine‐chase experiments. In untreated cells, the half‐lives for IA1, IA2 and IIIA6 mRNAs were 14 h, 16 h and 19 h, respectively when determined by the uridine chase and 18 h, 25 h and 22 h when determined by the actinomycin‐D chase. These values were not modified significantly in cells treated with β‐naphthoflavone or rifampicin, indicating that neither of these inducers affected the stability of IA1 and IA2 or IIIA6 messages, respectively. We conclude that: (a) gene IA1 is primarily regulated at the transcriptional level in response to β‐naphthoflavone, derepression of this gene being the most critical step controlling its expression; (b) gene IA2 expression does not require protein synthesis and appears to be placed under the control of both transcriptional and post‐transcriptional effects (stabilization of cytoplasmic IA2 mRNA by the inducer, suspected by several authors to account for message accumulation, was not detected in this work); (c) gene IIIA6 expression is primarily regulated at the transcriptional level in response to rifampicin and dexamethasone and requires protein synthesis. Finally, CYP 1A1 and 1A2 were located on rabbit chromosome 17 p1.2 and CYP 3A6 on 6 q‐ter.
Recombinant expression and properties of the human calcium‐binding extracellular matrix protein BM‐40
Authors: NISCHT Roswitha , POTTGIESSER Jutta , KRIEG Thomas , MAYER Ulrike , AUMAILLEY Monique , TIMPL Rupert
Published: March 2005

A cDNA construct (∼1 kb) of human BM‐40 in a plasmid with the cytomegalovirus promoter and enhancer was used to produce several stable clones by transfecting two human cell lines (293, HT 1080). These clones showed a high expression of exogenous 1‐kb BM‐40 mRNA and no or only little endogenous 2.2‐kb mRNA. These clones also secreted BM‐40 at high rates (5–50 μg ml‐1 day1) into serum‐free culture medium as shown by electrophoresis, radioimmunoassay and metabolic labelling. Transfection with the plasmid and overexpression of BM‐40 had no effect on cell spreading, proliferation rate and adhesion patterns to extracellular matrix substrates. Recombinant human BM‐40 was purified by anion‐exchange chromatography and showed the expected N‐terminal sequence and amino acid composition. The protein was also identical or similar to authentic BM‐40 purified from the mouse Engelbreth‐Holm‐Swarm tumor in hexosamine content, electrophoretic mobility, circular dichroism and binding activity for calcium and collagen IV. Reduction of both authentic and recombinant BM‐40 decreased binding activity which indicates correct formation of disulfide bonds in the recombinant protein. A specific and sensitive radioimmunoassay for human BM‐40 was shown to be useful for detecting small quantities of the protein in human cell culture medium and blood. No significant cross‐reaction was, however, detected between human and mouse BM‐40.
Cell‐cycle‐dependent, differential prenylation of proteins
Authors: SEPP‐LORENZINO Laura , RAO Srinivasa , COLEMAN Peter S.
Published: March 2005

Isoprenylated proteins related to cell growth have been detected during proliferation. Since cholesterogenesis (isoprenoid synthesis) is mandatory for cell proliferation, the observation of a temporally coordinated protein prenylation during the cell division cycle might constitute obligatory processes in the signalling pathway for initiating DNA replication and/or in maintaining the growing state. We have found such a definitive cell‐cycle‐phase‐dependent pattern of prenylation for various classes of cytosolic and nuclear matrix proteins in synchronized HepG2 cells. Characteristic [3H]mevalonate incorporation began to increase during mid‐to‐late G1, just after cholesterol synthesis reached its apex, and peaked just prior to or coincident with mid S. Incorporation then declined subsequent to S (during G2) as cells approached mitosis. Prior to the rise in mevalonate incorporation into proteins, during early‐to‐mid G1, steady‐state [14C]acetate incorporation into chromatographically resolved cholesterogenic lipid intermediates displayed a maximum only into cholesterol. However, during the late‐G1/S interval, a singular peak of 14C incorporation was found for the farnesyl moiety (farnesol/nerolidol plus farnesyl diphosphate). Except for the farnesyl moiety, none of the other polyisoprenoids detected by our procedures showed any fluctuation in 14C incorporation subsequent to mid G1. These results support the proposal that subsequent to peak cholesterol synthesis in early‐to‐mid G1, the generation of a cholesterol‐pathway‐dependent set of post‐translationally modified, polyisoprenylated proteins could constitute an obligatory step leading to the duplication of the cellular genome, thereby impelling transit through the cell cycle. The well known high flux through cholesterogenesis in tumors, which manifests an intrinsic lack of sensitivity to feedback inhibition and operates continuously, is consonant with this proposal.
Glycosphingolipid specifity of the human sulfatide activator protein
Authors: VOGEL A. , SCHWARZMANN G. , SANDHOFF K.
Published: March 2005

The interaction of the sulfatide activator protein with different glycosphingolipids has been studied in detail. The following findings were made.

1. The sulfatide activator protein forms water‐soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe‐Seyler's Z. Physiol. Chem. 356, 6588–6591] and various other glycosphingolipids.

2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules.

3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein.

4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by β‐galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long‐chain fatty acids in their hydrophobic ceramide anchor.
Nuclear skeleton, DNA domains and control of replication and transcription
Authors: GEORGIEV Georgii P. , VASSETZKY Yegor S. , LUCHNIK Andrey N. , CHERNOKHVOSTOV Victor V. , RAZIN Sergey V.
Published: March 2005

Chromosomal DNA is organized in loops or domains of about 100 kb. Their ends seem to be attached to special protein skeletal structures. The DNA‐attachment sites can be subdivided into permanent and transient types. The permanent or constitutive attachment sites, which are retained in all types of cells (including those inactive in replication and transcription), either coincide with or are located close to replication origins. This observation provides a simple way for isolation of DNA fragments containing replication origins. Such fragments from the chicken α‐globin gene domain and other regions of the chicken genome contain DNA sequences which interact with nuclear proteins present in dividing cells, but absent from non‐dividing cells. Several new consensus sequences interacting with nuclear proteins were detected. The 5′ end region of the α‐globin gene domain containing a replication origin was found to possess enhancer activity lacking tissue specificity. Hence, the domain organization of DNA is related to the organization of replication process. Other sets of data indicate that the integrity of DNA domains is important for maintaining transcription within the domain. According to these data, even a single nick at an distance of about 100 kbp seems to be sufficient for blocking transcription within the whole domain at the stage of RNA elongation. Thus, topological integrity of DNA may be an important factor involved in formation of active chromatin.
σ receptors
Authors: SU Tsung‐Ping
Published: March 2005

The σ receptor is a neuronal substrate that binds several psychoactive compounds. These include cocaine, some steroids, dextromethorphan, phencyclidine (PCP), and benzomorphans such as pentazocine and N‐allyl‐normatezocine (SKF‐10047). Many newer atypical antipsychotic drugs also bind to the σ receptor. The σ receptor, however, is not the PCP receptor. The σ receptor exists in the central nervous system, endocrine, immune and certain peripheral tissues. Progesterone and certain steroids have been shown to represent endogenous ligands for the σ receptor. The σ receptor resides likely in the nonsynaptic region of the plasma membrane. The σ receptor exists in two forms: high‐affinity and low‐affinity. The solubilized σ receptor retains all of the pharmacological characteristics of a membrane‐bound receptor. A major physiological role of the σ receptor may involve the modulation of a tonic potassium channel.
Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain
Authors: CONEJERO‐LARA Francisco , SÁNCHEZ‐RUIZ José M. , MATEO Pedro L. , BURGOS Francisco J. , VENDRELL Josep , AVILÉS Francesc X.
Published: March 2005

High‐sensitivity differential scanning calorimetry has been applied to the study of porcine pancreatic carboxypeptidase B, the proenzyme and its 81‐residue activation domain. The thermal study has been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has been found to be reversible and corresponds to that of a typical compact globular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversible, highly scan‐rate‐dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two‐state irreversible model [Sánchez‐Ruiz, J. M., López‐Lacomba, J. L., Cortijo, M. & Mateo, P. L. (1988) Biochemistry 27, 1648–1652]. Thus the kinetic constants and activation parameters of the denaturation process could be obtained and compared to those for other proteins, particularly those of the closely related carboxypeptidase A system.
Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin
Authors: DUAN Wei , ACHEN Marc G. , RICHARDSON Samantha J. , LAWRENCE Michael C. , WETTENHALL Richard E. H. , JAWOROWSKI Anthony , SCHREIBER Gerhard
Published: March 2005

A chicken liver cDNA library was constructed in bacteriophage λgt10. A full‐length transthyretin cDNA clone was identified by screening with rat transthyretin cDNA and was sequenced. A three‐dimensional model of chicken transthyretin was obtained by computer‐graphics‐based prediction from the derived amino acid sequence for chicken transthyretin and from the structure of human transthyretin determined by X‐ray diffraction analysis [Blake, C. C. F., Geisow, M. J., Oatley, S. J., Rérat, B. & Rérat, C. (1978) J. Mol. Biol. 121, 339–356]. The similarity of the amino acid sequences of chicken and human transthyretins was 75% overall and 100% for the central channel containing the thyroxine‐binding site. Also, the organization of the transthyretin gene into exons and introns and the tissue specificity of expression of the transthyretin gene were similar in chicken and mammals, despite an evolutionary distance of about 3 × 108 years from their common ancestor, the Cotylosaurus. By far the highest levels of transthyretin mRNA were found in choroid plexus. The data suggest a fundamental role for the cerebral expression of transthyretin in all vertebrates. It has been proposed that this role is the transport of thyroxine from the bloodstream to the brain [Schreiber, G., Aldred, A. R., Jaworowski, A., Nilsson, C., Achen, M. G. & Segal, M. B. (1990) Am. J. Physiol. 258, R338–R345].
Protein‐kinase‐C‐independent activation of arachidonate release and prostaglandin E2 formation in macrophages interacting with certain bacteria
Authors: SVENSSON Ulf , HOLST Elisabet , SUNDLER Roger
Published: March 2005

Certain bacterial species, of which we selected Fusobacterium nucleatum, Gardnerella vaginalis, Peptostreptococcus anaerobius and Propionibacterium acnes, were found to induce release of arachidonic acid in a dose‐ and time‐dependent manner in mouse macrophages. The release of arachidonic acid showed a characteristic lag period of approximately 10 min and was accompanied by selective transformation into prostaglandin E2. Bacteria killed by various methods caused a similar response, indicating that bacteiral surface structures rather than secreted products were involved. Down‐regulation of protein kinase C by treatment of macrophages with 4β‐phorbol 12‐myristate 13‐acetate hardly affected the response to bacteria at all, except for a partial inhibition in the case of P. acnes. Furthermore, the generation of prostaglandin E2 was synergistically enhanced when macrophages were exposed to both bacteria and phorbol ester. It is also unlikely that bacterial activation was mediated exclusively via a rise in cytosolic [Ca2+], since bacteria stimulated the release of arachidonic acid also when [Ca2+] was clamped at various levels and since the response to bacteria was enhanced in an additive to synergistic manner when combined with calcium ionophore.

Changes in protein phosphorylation in macrophages exposed to F. nucleatum (Gram‐negative) were virtually identical to those seen with bacterial lipopolysaccharide, while P. anaerobius (Gram‐positive) induced enhanced labeling of a single detectable phosphoprotein. In both cases, the changes in protein phosphorylation showed a time lag of 4–8 min and occurred independently of protein kinase C, consistent with a possible role in signal transduction. These results demonstrate that certain bacteria cause activation of arachidonic acid release and prostaglandin E2 formation in mouse macrophages; that the response is independent of protein kinase C and that it is not wholly mediated via a rise in cytosolic [Ca2+].
Heat‐shock response of rat hepatoma variant cells
Authors: BARANYI Maria , HEVÉR‐SZABÓ Anna , VENETIANER Anikó
Published: March 2005

The effect of mild heat shock on protein synthesis was examined in differentiated and dedifferentiated, glucocorticoid‐sensitive and resistant clones of H4IIEC3 rat hepatoma cells by one‐ and two‐dimensional gel electrophoresis of [35S]methionine‐labeled proteins. Among the major heat‐shock proteins, five were induced in all hepatoma clones. Certain members of the HSP70 family and the corresponding mRNAs were only slightly inducible in the glucocorticoid‐resistant variants, but were strongly inducible in the sensitive ones. Three other proteins lacked heat inducibility in the dedifferentiated clones. The constitutive level of one major heat‐shock protein was elevated in all dedifferentiated variants. These results show that the stage of differentiation influences the expression of heat‐shock genes of hepatoma cells. We found no correlation between the elevated constitutive or induced level of heat‐shock proteins and heat resistance.