bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of interleukin 1β on the biosynthesis of cholesterol, phosphatidylcholine, and sphingomyelin in fibroblasts, and on their efflux from cells to lipid‐free apolipoprotein A‐I
Authors: Kronqvist Robert , Leppimäki Petra , Mehto Pia , Slotte J. Peter
Published: March 2003

In this study we have investigated the effect of interleukin 1β (IL‐1β) on the metabolism of cholesterol and choline‐phospholipids in cultured fibroblasts, and also measured efflux of these lipids to lipid‐free apo A‐I as a function of IL‐1β treatment. Long‐term exposure (up to 48 h) of cells to IL‐1β (1 ng·mL−1) markedly increased the rate of cholesterol esterification, as determined by the incorporation of [3H]oleic acid into cholesteryl esters. This treatment also led to a substantially increased mass of cholesteryl esters in the cells. The accumulation of cholesteryl esters in IL‐1β‐treated cells could be blocked using compound 58‐035 to inhibit the activity of acyl‐CoA cholesterol acyl transferase. The activation of cholesterol esterification by IL‐1β was evident within a few hours after initiation of the IL‐1β treatment. Cholesterol biosynthesis was inhibited by 25% by IL‐1β (after 48 h exposure), and this eventually led to a 20% decrease in cell cholesterol mass. Treatment of cells with IL‐1β for 48 h also reduced the synthesis of sphingomyelin and caused a 30% decrease in cell sphingomyelin mass (after 48 h at 1 ng·mL−1 of IL‐1β). IL‐1β did not stimulate an acute (within a few minutes up to an hour) degradation of cell [3H]sphingomyelin. This suggests that IL‐1β did not activate an endogenous sphingomyelinase in these cells, but only affected rates of synthesis. The rate of phosphatidylcholine synthesis was barely affected, but mass was moderately reduced by a 48‐h treatment of cells with IL‐1β. Finally, the efflux of cell [3H]cholesterol, [3H]sphingomyelin, and [3H]phosphatidylcholine to lipid‐free apolipoprotein A‐I was markedly increased from cells treated with IL‐1β for 24 and 48 h. We conclude that long‐term exposure of cells to IL‐1β had marked effects on the cellular homeostasis of cholesterol and choline‐containing phospholipids.
Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+channels containing Kv1.1 and 1.2 α subunits
Authors: Wang Fan C. , Bell Natalie , Reid Paul , Smith Leonard A. , McIntosh Paul , Robertson Brian , Dolly J. Oliver
Published: March 2003

Dendrotoxin (DTX) homologues are powerful blockers of K+channels that contain certain subfamily Kv1 (1.1–1.6) α‐ and β‐subunits, in (α)4(β)4stoichiometry. DTXkinhibits potently Kv1.1‐containing channels only, whereas αDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTXkwith native K+channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site‐directed mutants in which amino acids were altered in the 310‐helix, β‐turn, α‐helix and random‐coil regions. The mutants’ antagonism of high‐affinity [125I]DTXkbinding to Kv1.1‐possessing channels in rat brain membranes and blockade of the Kv1.1 current expressed in oocytes were quantified. Also, the levels of inhibition of [125I]αDTX binding to brain membranes by the DTXkmutants were used to measure their high‐ and low‐affinity interactions, respectively, with neuronal Kv1.2‐containing channels that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random‐coil region, or R52 or R53 in the α‐helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled αDTX or DTXkwas dramatically reduced by alanine substitution of K26 or W25 in the β‐turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo‐oligomer. The 310‐helical N‐terminal region of DTXkwas found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to ≈1246‐fold reduction in the inhibitory potency for [125I]DTXkbinding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTXkfor Kv1.2‐possessing oligomers was much less dramatic (≈16‐fold).
Post‐translational modifications of the insect sulfakinins
Authors: Predel Reinhard , Brandt Wolf , Kellner Roland , Rapus Jürgen , Nachman Ronald J. , Gäde Gerd
Published: March 2003

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea‐SK [EQFDDY(SO3H)GHMRFamide] and Lem‐SK‐2 [pQSDDY(SO3H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post‐translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N‐terminally blocked Pea‐SK and the nonblocked Lem‐SK‐2 occur naturally in neurohaemal release sites. Thirdly, modified Pea‐SK with O‐methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of ≈ 5 × 10−10 m (heart) and 2 × 10−9 m (hindgut).
PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis
Authors: Besson Arnaud , Robbins Stephen M. , Wee Yong V.
Published: March 2003

The level of phosphorylation within cells is tightly regulated by the concerted action of protein kinases and protein phosphatases [Hunter, T. (1995) Cell80, 225–236]. Disregulation in the activity of either of these players can lead to cellular transformation. Many protein tyrosine kinases are proto‐oncogenes and it has been postulated that some protein phosphatases may act as tumor suppressors. Herein we will review the recent findings addressing the roles the candidate tumor suppressor PTEN/MMAC1/TEP1 (PTEN, phosphatase and tensin homologue deleted from chromosome 10; MMAC 1, mutated in multiple advanced cancers 1; TEP1, TGF β regulated and epithelial cell enriched phosphatase 1) plays in signal transduction and tumorigenesis. PTEN is a dual specificity protein phosphatase (towards phospho‐Ser/Thr and phospho‐Tyr) and, unexpectedly, also has a phosphoinositide 3‐phosphatase activity. PTEN plays an important role in the modulation of the 1‐phosphatidylinositol 3‐kinase (PtdIns 3‐kinase) pathway, by catalyzing the degradation of the PtdIns(3,4,5)P3 generated by PtdIns 3‐kinase; this inhibits the downstream functions mediated by the PtdIns 3‐kinase pathway, such as activation of protein kinase B (PKB, also known as Akt), cell survival and cell proliferation. Furthermore, PTEN modulates cell migration and invasion by negatively regulating the signals generated at the focal adhesions, through the direct dephosphorylation and inhibition of focal adhesion kinase (FAK). Growth factor receptor signaling is also negatively regulated by PTEN, through the inhibition of the adaptor protein Shc. While some of the functions of PTEN have been elucidated, it is clear that there is much more to discover about the roles of this unique protein.
Human chemokine receptors CCR5, CCR3 and CCR2B share common polarity motif in the first extracellular loop with other human G‐protein coupled receptors
Authors: Efremov Roman , Truong Marie‐José , Darcissac Edith Christine A. , Zeng Jin , Grau Odile , Vergoten Gérard , Debard Christine , Capron André , Bahr George M.
Published: March 2003

Chemokine receptors (CRs) are 7‐helix membrane proteins from the family of G‐protein coupled receptors (GPCRs). A few human CRs act as cofactors for macrophage–tropic (M‐tropic) human immunodeficiency virus type‐1 (HIV‐1) entry into cells, while others do not. In this study, we describe an application of molecular modeling techniques to delineate common molecular determinants that might be related to coreceptor activity, and the use of the data to identify other GPCRs as putative cofactors for M‐tropic HIV‐1 entry. Subsequently, the results were confirmed by an experimental approach. The sequences of extracellular domains (ECDs) of CRs were employed in a compatibility search against a database of environmental profiles derived for proteins with known spatial structure. The best‐scoring sequence–profile alignments obtained for each ECD were compared in pairs to check for common patterns in residue environments, and consensus sequence–profile fits for ECDs were also derived. Similar hydrophobicity motifs were found in the first extracellular loops of the CRs CCR5, CCR3, and CCR2B, and are all used by M‐tropic HIV‐1 for cell entry. In contrast, other CRs did not reveal common motifs. However, the same environmental pattern was also delineated in the first extracellular loop of some human GPCRs showing either high (group 1) or low (group 2) degree of similarity of their polarity patterns with those in HIV‐1 coreceptors. To address the question of whether the delineated molecular determinant plays a critical role in the receptor–virus binding, three of the identified GPCRs, bradykinin receptor (BRB2) and G‐protein receptor (GPR)‐CY6 from group 1, and GPR8 from group 2, were cloned and transfected into HeLa‐CD4 cells, which are nonpermissive to M‐tropic HIV‐1 infection. We demonstrate that, similar to CCR5, the two selected GPCRs from group 1 were capable of mediating M‐tropic HIV‐1 entry, whereas GPR8 from group 2 did not serve as HIV‐1 coreceptor. The potential biological significance of the identified structural motif shared by the human CCR5, CCR3, CCR2B and other GPCRs is discussed.
p55‐hGRF, a short natural form of the Ras–GDP exchange factor
Authors: Meyer Philippe , Janin Joël , Baudet‐Nessler Sylvie
Published: March 2003

p55‐hGRF, a natural short form of the guanine‐nucleotide‐releasing factor for p21‐Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39‐hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H‐Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55‐hGRF and p39‐hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1 : 1 and an equilibrium between dimeric and monomeric forms of the complex.
Apolipoprotein A‐IMilano unfolds via an intermediate state as studied by differential scanning calorimetry and circular dichroism
Authors: Suurkuusk Malin , Hallén Dan
Published: March 2003

In this study the thermal and denaturant induced denaturation behaviors of apolipoprotein A‐IMilano (apo A‐IM) have been studied by differential scanning calorimetry and circular dichroism spectroscopy, as well as solution properties by analytical ultracentrifugation. Thermal denaturation is dependent on pH, sodium phosphate concentration and NaCl concentration. The protein is highly self‐associated at the protein concentrations used in this study. Denaturation of apo A‐IM at pH 7.4 and 8.0 occurs in two steps. The midpoint between the transition is at 37 °C. The first step at 31 °C involves melting of tertiary structure and rearrangement of protein association complexes, i.e. a transition into an intermediate molten globular‐like state. Subsequent melting of this intermediate state into an unfolded state occurs at 52 °C. At pH 2.8 the protein lacks all tertiary structure and denaturation occurs over a large temperature interval, indicating the induction of a molten globular‐like state at low pH.
Mimicking lipid‐binding‐induced conformational changes in the human apolipoprotein E N‐terminal receptor binding domain
Authors: Clément‐Collin Vanessa , Leroy Arnaud , Monteilhet Claude , Aggerbeck Lawrence P.
Published: March 2003

We studied the effects of n‐propanol and pH on the structure of the apolipoprotein E3 N‐terminal receptor binding domain, apo E3(1–191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid‐induced conformational changes in apo E3. The addition of 30% n‐propanol, at pH 7, induces a conformational change in apo E3(1–191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross‐linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu‐C, as is the case for apo E3(1–191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n‐propanol increases the amount of calculated α‐helical structure (42–65%) and alters the tertiary structure of the protein although not as much as when apo E3(1–191) is incorporated into lipid bicelles. In the absence of n‐propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n‐propanol at pH 4.5 increases the Stokes radius of apo E3(1–191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0–4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu‐C. As assessed by far and near UV circular dichroism, the addition of 30% n‐propanol at pH 4.5, in contrast to pH 7, markedly increases the α‐helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1–191) into lipid bicelles. The results suggest that a combination of n‐propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.
Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius
Authors: Durbecq Virginie , Thia‐Toong Thia‐Lin , Charlier Daniel , Villeret Vincent , Roovers Martine , Wattiez Ruddy , Legrain Christianne , Glansdorff Nicolas
Published: March 2003

The genes coding for aspartate carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial‐like pyrBI operon encoding a catalytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI genes showed 27.6–50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of l‐aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase. Only monomers could be found in extracts of recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.
Structure–function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes
Authors: Solovicová Adriana , Christensen Trine , Hostinová Eva , Gašperík Juraj , Ševčík Jozef , Svensson Birte
Published: March 2003

The mutation Gly467→Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild‐type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild‐type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka ≅ 1013·m−1) than the Glu enzyme (Ka ≅ 1012 ·m−1). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1‐deoxynojirimycin, was about 8–9 orders of magnitude weaker (Ka ≅ 104·m−1) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.
Cloning of the mistletoe lectin gene and characterization of the recombinant A‐chain
Authors: Eck Jürgen , Langer Martin , Möckel Babette , Baur Axel , Rothe Markus , Zinke Holger , Lentzen Hans
Published: March 2003

Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66‐kDa heterodimeric disulphide‐linked glycoprotein is classified as type II ribosome‐inactivating protein (RIP) due to the rRNA‐cleaving enzyme activity of the A‐subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two‐chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post‐translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full‐length 1923 nucleotide DNA sequence coding for the prepro‐protein was obtained showing the existence of a single intron‐free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A‐subunit was expressed in a heterologous system. Expression of the A‐chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20–30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome‐inactivating activity of the unglycosylated recombinant A‐chain (IC50 20.5 pm) protein was in the same range as that of the glycosylated plant‐derived ML A‐chain (IC50 3.7 pm), which was very similar to that of ricin A‐chain (IC50 4.9 pm). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A‐chains.
High‐resolution solution structure of gurmarin, a sweet‐taste‐suppressing plant polypeptide
Authors: Fletcher Jamie I. , Dingley Andrew J. , Smith Ross , Connor Mark , Christie MacDonald J. , King Glenn F.
Published: March 2003

Gurmarin is a 35‐residue polypeptide from the Asclepiad vine Gymnema sylvestre. It has been utilised as a pharmacological tool in the study of sweet‐taste transduction because of its ability to selectively inhibit the neural response to sweet tastants in rats. We have chemically synthesised and folded gurmarin and determined its three‐dimensional solution structure to high resolution using two‐dimensional NMR spectroscopy. Structure calculations utilised 612 interproton‐distance, 19 dihedral‐angle, and 18 hydrogen‐bond restraints. The structure is well defined for residues 3–34, with backbone and heavy atom rms differences of 0.27 ± 0.09 Å and 0.73 ± 0.09 Å, respectively. Gurmarin adopts a compact structure containing an antiparallel β‐hairpin (residues 22–34), several well‐defined β‐turns, and a cystine‐knot motif commonly observed in toxic and inhibitory polypeptides. Despite striking structural homology with δ‐atracotoxin, a spider neurotoxin known to slow the inactivation of voltage‐gated Na+ channels, we show that gurmarin has no effect on a variety of voltage‐sensitive channels.
Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp.
Authors: Corsaro M. Michela , Grant William D. , Grant Susan , Marciano Carmela E. , Parrilli Michelangelo
Published: March 2003

An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp. was found to contain d‐galactopyranuronic acid (GalpA), 2,4‐diacetamido‐2,4,6‐trideoxy‐d‐glucopyranose (QuipNAc4NAc), 2‐acetamido‐2‐deoxy d‐mannopyranuronic acid (ManpNAcA) and one uncommon unit of d‐galactopyranuronic acid with the carboxyl group amide‐linked to glycine [GalpA(Gly)]. The polysaccharide was studied by one‐dimensional and two‐dimensional 1H‐NMR and 13C‐NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis. The following linear structure of the repeating unit was established:

→3)‐α‐d‐GalpA(Gly)‐(1→4)‐β‐d‐ManpNAcA‐(1→4)‐α‐d‐GalpA‐(1→3)‐α‐d‐QuipNAc4NAc‐(1→.

A preliminary phylogenetic assignment for the bacterium is also reported.
Active site determination of yeast geranylgeranyl protein transferase type I expressed inEscherichia coli
Authors: Kim Hyunkyung , Yang Chul‐Hak
Published: March 2003

Theram2andcal1genes encode the α and β subunits of yeast geranylgeranyl protein transferase type I (GGPT‐I), respectively. Arginine 166 of the β subunit was changed to isoleucine (βR166I), histidine 216 to aspartic acid (βH216D), and asparagine 282 to alanine (βN282A) by sequential PCR using mutagenic primers. The mutants were expressed under the same conditions as the wild‐type and were assayed for GGPT‐I activity. Wild‐type yeast GGPT‐I, αH145D, αD140N, βR166I, βH216D and βN282A mutant GGPT‐Is were partially purified by ammonium sulfate fractionation followed by a Q‐Sepharose column. Characterization studies were performed using the active fraction of the Q‐Sepharose column. In the chemical modification reactions, the catalytic activity of purified enzyme decreased in proportion to the concentration of modifying reagents, such as phenylglyoxal and diethyl pyrocarbonate (DEPC). Geranylgeranyl pyrophosphate (GGPP) protected the enzyme activity from the modification with phenylglyoxal. The measurement of GGPP binding to wild‐type and five mutant GGPT‐Is was performed by a gel‐filtration assay. The binding of GGPP to the βR166I mutant was low and theKmvalue for GGPP in the βR166I mutant increased about 29‐fold. Therefore, the results suggest a role for this arginine residue that directly influences the GGPP binding. The activity of the DEPC‐modified GGPT‐I was inhibited by 80% at 5 mmDEPC. The differential absorption at 242 nm may suggest that at this concentration the modified histidine residues were 1.5 mol per GGPT‐I. The protein substrate, glutathione S‐transferase fused undecapeptide (GST‐CAIL) protected the enzyme from inactivation by DEPC, and theKmvalue for GST‐CAIL in the βH216D mutant increased about 12‐fold. The trypsin digestion of [14C]DEPC‐modified enzyme yielded a single radioactive peptide. As a result of the sequence of this radioactive peptide, the histidine 216 residue was assumed to be an essential part of binding of peptide substrate.
Cyclosporin A inhibits Ca2+‐mediated upregulation of the DNA repair enzyme DNA polymerase β in human peripheral blood mononuclear cells
Authors: Ahlers Christoph , Kreideweiß Stefan , Nordheim Alfred , Rühlmann Andreas
Published: March 2003

Alterations in gene expression may represent an underlying cause of undesired side‐effects mediated by the immunosuppressant cyclosporin A (CsA). We employed the method of differential display PCR to identify new genes whose expression is modulated by CsA. Human peripheral blood mononuclear cells (PBMCs), or subpopulations thereof, were simultaneously stimulated with the phorbol ester 4β‐phorbol 12‐myristate 13‐acetate (PMA) and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. We identify the gene encoding the DNA repair enzyme DNA polymerase β (Pol β) as a novel CsA‐sensitive transcription unit. Our data show that transcription of pol β mRNA is induced by Ca2+ and that CsA significantly inhibits PMA/ionomycin‐ and ionomycin‐mediated upregulation of both pol β mRNA and Pol β protein. The CsA‐mediated inhibition of pol β upregulation is maintained for at least 21 h after gene activation and is exerted via the phosphatase calcineurin. FK506, another immunosuppressant that targets calcineurin, also inhibits pol β upregulation, while rapamycin competes with FK506 action. This work identifies Ca2+ as an inducer of pol β gene activity in primary blood cells. The demonstrated CsA sensitivity of this process suggests a novel molecular mechanism that may contribute to the increased tumor incidence in patients receiving CsA treatment.
Mechanisms and kinetics of procathepsin D activation
Authors: Wittlin Sergio , Rösel Johannes , Hofmann Francesco , Stover David R.
Published: March 2003

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the ‘autoproteolysis‐site’ (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild‐type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two‐phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.
Human TRH‐degrading ectoenzyme
Authors: Schomburg Lutz , Turwitt Stefan , Prescher Gabriele , Lohmann Dietmar , Horsthemke Bernhard , Bauer Karl
Published: March 2003

Thyrotropin‐Releasing Hormone (TRH) is an important extracellular signal substance that acts as a stimulator of hormone secretion from adenohypophyseal target cells and fulfills many criteria for the function of a neuromodulator/neurotransmitter within the central and peripheral nervous systems. The inactivation of TRH‐signals is catalysed by a highly specific ectoenzyme. Here, we characterize the human TRH‐degrading ectoenzyme (TRH‐DE) by primary sequence, functional expression, genomic structure and chromosomal assignment. By screening a cDNA‐library constructed from human lung, 5.7 kb of cDNA were identified. The longest open reading frame predicts a type II integral membrane protein of 117 kDa. The extracellular domain contains the HEXXH + E motif that is characteristic of a certain family of Zn‐dependent aminopeptidases. Within this family, the sequences of human and rat TRH‐DE reveal an unusual high degree of conservation (96% identical residues). Specific enzymatic activity was observed after transfecting COS‐7 cells with human TRH‐DE cDNA yielding a Km for TRH hydrolysis of 29.7 µm. Northern blot analysis demonstrated a restricted tissue distribution with highest transcript levels in the brain. Using fluorescent in situ hybridization with the cDNA and a genomic λ clone, respectively, we localized the TRH‐DE gene to the long arm of human chromosome 12. Five independent P1 artificial chromosome clones were required to span the complete cDNA sequence and revealed that it is distributed on 19 exons. Interspecies Southern analysis suggests that the gene is present as a single copy in human, monkey, rat, mouse, dog, bovine, rabbit and chicken DNA. All of these data further the notion that the TRH‐DE is not an ordinary enzyme but a specific neuropeptidase that has been highly conserved among species.
Differential in vitro activation and deactivation of cysteine proteinases isolated during spore germination and vegetative growth of Dictyostelium discoideum
Authors: Cavallo Dora , Cervi David , Sands Todd W. , Cotter David A.
Published: March 2003

Acid‐activatable cysteine proteinases of Dictyostelium discoideum were first identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto‐induced and heat‐induced spore germination of D. discoideum strain SG2 and SG1. The major acid‐activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases which generally require the cleavage of a pro‐peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4‐[(2S,3S)‐3‐carboxyoxiran‐2‐ylcarbonyl‐l‐leucylamido]butylguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+‐ATPases and ammonia.
Structural elucidation of the O‐antigenic polysaccharides from Escherichia coli O21 and the enteroaggregative Escherichia coli strain 105
Authors: Staaf Mikael , Urbina Felipe , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigen polysaccharide of the lipopolysaccharide from an enteroaggregative Escherichia coli (strain 105) has been elucidated, using primarily one‐dimensional and two‐dimensional NMR experiments. The sequence of residues was deduced with heteronuclear multiple‐bond correlation and NOESY experiments. The structure of the repeating unit of the polysaccharide from the enteroaggregative E. coli is as follows:

The structure of the O‐antigen from enteroaggregative E. coli strain 105 was shown to be identical with that of E. coli O21 by sugar and methylation analyses as well as by 1H‐NMR and 13C‐NMR spectroscopy.
Biochemical properties of a minimal functional domain with ATP‐binding activity of the NTPase/helicase of hepatitis C virus
Authors: Borowski Peter , Kuehl Reinhard , Mueller Oliver , Hwang Lih‐Hwa , Schulze zur Wiesch Julian , Schmitz Herbert
Published: March 2003

The RNA‐stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP‐binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N‐ and C‐terminal sequencing and by its capability to bind 5′‐fluorosulfonylbenzoyladenosine (FSBA).

The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 µm. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP‐binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides.

Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 µm), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 µm). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP‐binding domain.
RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes
Authors: Drew Horace R. , Lewy Denise , Conaty Jason , Rand Keith N. , Hendry Philip , Lockett Trevor
Published: March 2003

A general study has been carried out to determine how well hammerhead ribozymes might reduce levels of specific protein synthesis in living cells, compared with RNA hairpin loops as stable but noncleaving controls. Four different experiments are described. First, a wide variety of hammerhead ribozymes, as well as hairpin loops, was cloned into a gene‐expression cassette for β‐galactosidase, upstream of the coding sequences for that reporter gene, and expressed from plasmids in several strains of Escherichia coli. The results show that ribozymes, when acting intramolecularly in E. coli, do not significantly reduce the amount of protein synthesized from any construct. As a control, long RNA hairpin loops do greatly reduce the amount of protein made. Secondly, we studied the transcription–translation of these same plasmids in a cell extract from E. coli. Once again, hammerhead ribozymes show no effect on levels of β‐galactosidase, whereas long RNA hairpin loops produce a strong reduction, by apparent attentuation at the level of translation. Thirdly, we added an SV40 promoter to each plasmid, in order to study the effects of these gene‐regulators on protein synthesis in Chinese hamster ovary cells. Here active intramolecular ribozymes produce a slight reduction in β‐galactosidase, whereas long RNA hairpin loops produce an even stronger reduction than before. Those hairpin loops apparently induce degradation of their own mRNA in Chinese hamster ovary cells, by a mechanism not seen in E. coli. Finally, analyses of total RNA by S1‐trimming show that hammerhead ribozymes will self‐cleave a mRNA by a total of no more than 45–50% in E. coli, compared with 70–80% in vitro. Other analyses using Northern blotting were unable to detect any ribozyme cleavage in E. coli or Chinese hamster ovary cells. In summary, the ability of hammerhead ribozymes to reduce protein synthesis appears weak or nonexistent in all the cellular systems tested. By comparison, long RNA hairpin loops reduce protein synthesis strongly: by an apparent attentuation mechanism in E. coli or by a novel degradation of their own mRNA in Chinese hamster ovary cells.
DNA interactions of cisplatin tethered to the DNA minor groove binder distamycin
Authors: Loskotová Hana , Brabec Viktor
Published: March 2003

Modifications of natural DNA in a cell‐free medium using cisplatin tethered to the AT‐specific, minor groove binder distamycin, were studied using various methods of biochemical analysis or molecular biophysics. These methods include: binding studies using differential pulse polarography and flameless atomic absorption spectrophotometry, mapping DNA adducts using a transcription assay, use of ethidium bromide as a fluorescent probe for DNA adducts of platinum, measurement of DNA unwinding by gel electrophoresis, measurement of CD spectra, an interstrand cross‐linking assay using gel electrophoresis under denaturing conditions, measurement of melting curves with the aid of absorption spectrophotometry and the use of terbium ions as a fluorescent probe for distorted base pairs in DNA. The results indicate that attachment of distamycin to cisplatin changes several features of the DNA‐binding mode of the parent platinum drug. Major differences comprise different conformational alterations in DNA and a considerably higher efficiency of the conjugated drug to form in DNA interstrand cross‐links. Cisplatin tethered to distamycin, however, coordinates to DNA with similar base sequence preferences as the untargeted platinum drug. The results point to a unique profile of DNA binding for cisplatin–distamycin conjugates, suggesting that tethering cisplatin to minor groove oligopeptide binders may also lead to an altered biological activity profile.
Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases
Authors: Shibuya Masaaki , Zhang Hong , Endo Aki , Shishikura Kaori , Kushiro Tetsuo , Ebizuka Yutaka
Published: March 2003

Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (β‐amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75–77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (≈ 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.
Azide binding to the trinuclear copper center in laccase and ascorbate oxidase
Authors: Gromov I. , Marchesini A. , Farver O. , Pecht I. , Goldfarb D.
Published: March 2003

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron‐nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1 : 1 to 1 : 7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g ≈ 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g ≈ 1.97 signal, with similar saturation characteristics was found in the AO : azide (1 : 7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti‐ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti‐ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1 : 7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g∥ region that are attributed to a perturbed T2 in protein molecules where the anti‐ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6–90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.
Inhibition of HIV‐1 enhancer‐controlled transcription by artificial enhancer‐binding peptides derived from bacteriophage 434 repressor
Authors: Caderas Georg , Klauser Stephan , Liu Niankun , Bienz Alexander , Gutte Bernd
Published: March 2003

An artificial HIV‐1 enhancer‐binding 42‐residue peptide (R42) that had been derived from bacteriophage 434 repressor inhibited the cell‐free in vitro transcription of HIV‐1 enhancer‐containing plasmids [Hehlgans, T., Stolz, M., Klauser, S., Cui, T., Salgam, P., Brenz Verca, S., Widmann, M., Leiser, A., Städler, K. & Gutte, B. (1993) FEBS Lett.315, 51–55; Caderas, G. (1997) PhD Thesis, University of Zürich]. Here we show that, after N‐terminal extension of R42 with a viral nuclear localization signal, the resulting nucR42 peptide was active in intact cells. NucR42 could be detected immunologically in nuclear extracts and produced a 60–70% reduction of the rate of transcription of an HIV‐1 enhancer‐carrying plasmid in COS‐1 cells that had been cotransfected with the HIV enhancer plasmid, an expression plasmid for nucR42, and a control. NucR42 was also synthesized chemically and the synthetic product characterized by HPLC, mass spectrometry, and quantitative amino acid analysis. Band shift, footprint, and in vitro transcription assays in the presence of exogenous NF‐κBp50 indicated that the binding sites of nucR42 and NF‐κB on the HIV enhancers overlapped and that a relatively small excess of nucR42 sufficed to displace NF‐κBp50. Band shift and in vitro transcription experiments showed also that exchange of the 434 repressor‐derived nine‐residue recognition helix of nucR42 for four glycines abolished the HIV enhancer binding specificity whereas leucine zipper‐ or retro‐leucine zipper‐mediated dimerization of R42 analogues increased it suggesting the potential application of such dimeric HIV enhancer‐binding peptides as intracellular inhibitors of HIV replication.
Functional evaluation of the genes involved in malonate decarboxylation by Acinetobacter calcoaceticus
Authors: Koo Jae Hyung , Kim Yu Sam
Published: March 2003

The genomic locus containing the potential repressor gene mdcY (inactivated by a putative IS3 element) and the mdcLMACDEGBH genes from Acinetobacter calcoaceticus was cloned and sequenced. In order to evaluate the biochemical function of the protein components, the genes were expressed independently and their activities predicted by database analysis. The mdcA gene product, the α subunit, was found to be malonate/acetyl‐CoA transferase and the mdcD gene product, the β subunit, was found to be malonyl‐CoA decarboxylase. The mdcE gene product, the γ subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the δ subunit, was an acyl‐carrier protein, which has a unique CoA‐like prosthetic group. Various combinations of malonate decarboxylase subunits allowed us to estimate their contribution to malonyl‐CoA decarboxylase activity. The prosthetic group was identified as carboxymethylated 2′‐(5″‐phosphoribosyl)‐3′‐dephospho‐CoA by mass spectrometry. The mdcH gene product was determined to have malonyl‐CoA/dephospho‐CoA acyltransferase activity. Using database analysis mdcLM, mdcG, mdcB and mdcI were estimated to be the genes for a malonate transporter, a holo‐acyl carrier synthase, protein for the formation of precursor of the prosthetic group and a regulatory protein, respectively. From the data shown above we propose a metabolic pathway for malonate in A. calcoaceticus.