bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunobiological properties of chemically defined lipid A from lipopolysaccharide of Porphyromonas (Bacteroides) gingivalis
Authors: OGAWA Tomohiko
Published: March 2005

Porphyromonas gingivalis 381 lipid A, characterized by β‐(1–6)‐linked glucosamine disaccharide 1‐phosphate, with one hydroxyacyl group and one acyloxyacyl group, i.e., 3‐hydroxy‐15‐methyl‐hexadecanoyl and 3‐hexadecanoyloxy‐15‐methylhexadecanoyl groups at the 2 and 2′ positions, respectively, was less endotoxically active than the synthetic Escherichia‐coli‐type lipid A (compound 506), which possesses β‐(1–6)‐linked glucosamine disaccharide 1,4′‐bisphosphate, with two acyloxyacyl groups at the 2′ and 3′ positions and two 3‐hydroxytetradecanoyl group at the 2 and 3 positions and the synthetic Salmonella‐type lipid A (compound 516), which has three acyloxyacyl groups at the 2, 2′ and 3′ positions and one hydroxyacyl group at the 3 position. P. gingivalis lipid A exhibited no or very low endotoxic activities, i.e., lethal toxicity in galactosamine‐loaded mice, preparative ability for local Shwartzman reaction, pyrogenicity and Limulus test as compared with compounds 506 and 516. However, polyclonal B‐cell activation of BALB/c mouse splenocytes was as strong as that of compound 506. Furthermore, P. gingivalis lipid A had stronger immunoadjuvant and hemagglutinating activities than compound 506. The absence of ester‐linked phosphate at the 4′ position and ester‐linked fatty acids, and the presence and positions of fatty acids possessing considerable lengths of acyl chains are unique features of P. gingivalis lipid A, and they differentiate this lipid A from enterobacterial and other lipids A. The good balance between endotoxic properties and beneficial immunobiological activities of P. gingivalis lipid A may be attributable to these features.
Biochemical characterization of two forms of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase kinase from cauliflower (Brassica oleracia)
Authors: BALL Kathryn L. , DALE Susan , WEEKES John , HARDIE D. Grahame
Published: March 2005

We recently reported the existence of a protein kinase cascade in higher plants, of which the central component is a 3‐hydroxy‐3‐methylglutaryl(HMG‐)‐CoA reductase kinase functionally related to mammalian AMP‐activated protein kinase [MacKintosh, R. W., Davies, S. P., Clarke P. R., Weekes, J., Gillespie, S. G., Gibb, B. J. & Hardie, D. G. (1992) Eur. J. Biochem. 209, 923–931]. We have now purified this protein kinase 9000‐fold from cauliflower inflorescences. During the course of this work we noticed a second minor form (form B) which separated from the major form (A) on ion exchange and gel filtration. Both forms phosphorylate the catalytic fragment of mammalian HMG‐CoA reductase. Both forms are markedly inactivated by incubation with the reactive ATP analogue p‐fluorosulphonylbenzoyl adenosine (FSO2PhCOAdo), and also by mammalian protein phosphatase 2C, indicating that form B, like form A, is activated by phosphorylation. Form A has an apparent native molecular mass of 200 kDa by gel filtration and, after labelling with [14C]FSO2PhCOAdo, of 150 kDa by electrophoresis in non‐denaturing gels. The catalytic subunit was identified as a polypeptide of 58 kDa after labelling with [14C]FSO2PhCOAdo. Form B has an apparent native molecular mass of 45 kDa by gel filtration, and was identified as a polypeptide of 45 kDa after labelling with [14C]FSO2PhCOAdo and [γ‐32P]ATP. Using a series of variants of the synthetic peptide substrate, the substrate specificities of the two forms are similar but not identical. Form B does not appear to be a proteolytic fragment of form A, and we therefore propose that it represents a closely related member of the same protein kinase sub‐family.
Activation of rat liver AMP‐activated protein kinase by kinase kinase in a purified, reconstituted system
Authors: WEEKES John , HAWLEY Simon A. , CORTON Julia , SHUGAR David , HARDIE D. Grahame
Published: March 2005

AMP‐activated protein kinase, purified from rat liver as far as the diethylaminoethyl–Sepharose step, is inactivated by treatment with protein phosphatase 2C, and reactivated by an endogenous ‘kinase kinase’. Further purification of AMP‐activated protein kinase on Blue Sepharose removes the kinase kinase, but the system can be reconstituted by adding back the flow‐through from the Blue‐Sepharose column. The kinase kinase can be further purified by subjecting the flow‐through from the Blue‐Sepharose column to chromatography on a Mono‐Q column. A single peak of kinase kinase activity is obtained. Using this fraction, and the most highly purified preparation of AMP‐activated protein kinase, phosphorylation of the 63‐kDa polypeptide, previously identified as the catalytic subunit of AMP‐activated protein kinase, can be demonstrated. As previously shown in the partially purified system, phosphorylation of the 63‐kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concentration. The structurally related AMP analogue, 8‐aza‐9‐deazaadenosine‐5′‐monophosphate, mimics the effect of AMP on both allosteric activation and phosphorylation of the kinase, while adenosine (5′)tetraphospho(5′)adenosine antagonizes both effects. These results suggest that both the allosteric effect of AMP, and the promotion of phosphorylation and activation by the kinase kinase, are due to binding of AMP to a single site on the kinase.
The 30‐kDa rat liver transcription factor nuclear factor 1 binds the rat growth‐hormone proximal silencer
Authors: ROY René J. , GUÉRIN Sylvain L.
Published: March 2005

Transcription of the gene encoding rat growth hormone is under the influence of cis‐acting negative regulatory elements termed silencers. We showed previously that one such element, designated the rat growth hormone proximal silencer‐1 site, binds a nuclear protein, the nuclear‐factor‐1‐like protein that is probably a member of the CAAT transcription factor/nuclear‐factor‐1 (CTF/NF‐I) family of transcription factors. This nuclear protein possesses DNA‐binding activity as well as biochemical properties similar to those reported for the 30‐kDa rat liver form of nuclear factor 1 (NF1‐L). Results from both gel mobility supershift assays and Western‐blot analyses, performed in combination with a polyclonal antibody directed against the DNA‐binding domain of NF1‐L, indicated that rat liver nuclear factor 1 might indeed correspond to one of the transcription factors interacting with the rat growth‐hormone proximal silencer element. Further experiments using gel mobility shift assays also indicated that, as for NF1‐L, multiple proteins among the 52–66‐kDa CTF/NF‐I isoforms from human HeLa cells also possess the ability to bind the rat growth‐hormone silencer.
Probing olfactory receptors with sequence‐specific antibodies
Authors: KRIEGER Jürgen , SCHLEICHER Sabine , STROTMANN Jörg , WANNER Ina , BOEKHOFF Ingrid , RAMING Klaus , DE GEUS Pieter , BREER Heinz
Published: March 2005

Molecular cloning has revealed the structure of several putative odorant receptors. Chemically synthesized peptides, that correspond to a predicted extracellular domain of the encoded proteins, were employed to generate receptor‐specific antibodies. Immunohistological approaches as well as Western‐blot analysis confirmed the specificity of the antipeptide sera. Furthermore, deglycosylation experiments explained the observed discrepancy between the molecular mass of odorant receptors, as determined by SDS/PAGE and Western‐blot analysis of ciliary proteins (Mr 50000), and the predicted protein size based on the deduced primary structure from cloned receptor genes (Mr 30000–35000). Receptor proteins become phosphorylated upon odorant stimulation of olfactory cilia preparations; this was demonstrated by immunoprecipitation experiments employing the sequence‐directed, receptor‐specific antibodies. Functional assays revealed that the receptor‐specific antibodies significantly attenuate second messenger signalling elicited by inositol 1,4,5‐trisphosphate‐inducing odorants, whereas activation of the cAMP cascade by appropriate odorants was not affected. These observation indicate that the sequence‐specific antibodies not only recognize odorant receptors, but also discriminate between receptor subtypes coupling to different second‐messenger pathways.
Norepinephrine, unlike ATP, induces all‐or‐none increase in cytosolic calcium in thyroid cells
Authors: BIZZARRI Cinzia , CORDA Daniela
Published: March 2005

The mechanism of action of norepinephrine and ATP has been analyzed in single FRTL5 cells (a normal thyroid cell line), loaded with the fluorescent Ca2+ probe Fura2. ATP increased the cytosolic Ca2+ in an apparently concentration‐dependent manner with a maximal effect at 10 μM (413±26% over basal levels of 135±7 nM). In contrast, the norepinephrine‐induced increase (198±5% over basal) was concentration independent in individual cells, the minimal effective concentration being 1 nM. However, the number of cells responding to norepinephrine was concentration dependent. The ATP‐induced Ca2+ rise was biphasic, consisting of a rapid rise (2–4 s, 252±15%), resembling the effect of norepinephrine, followed by a slower and longer component, which reached a plateau in 0.5–2 min. The second component appeared to be related to the opening of a channel, since it required extracellular Ca2+ and was abolished by SC38249, an inhibitor of the second‐messenger‐operated and voltage‐operated channels. Moreover, it was inhibited by 4β‐phorbol 12‐myristate 13‐acetate, suggesting that protein kinase C might be involved in the modulation of this Ca2+ channel.
The bilin‐binding protein of Pieris brassicae
Authors: SCHMIDT Frank S. , SKERRA Arne
Published: March 2005

The bilin‐binding protein (BBP) is a blue pigment protein which is abundant in the butterfly Pieris brassicae. In an attempt to clarify the physiological role of this member of the lipocalin family of proteins, its complete cDNA was cloned and expression of the BBP gene in P. brassicae was investigated. It was found that synthesis of the BBP mRNA is highly regulated during the insect's ontogenesis. In larvae after the third ecdysis as well as in pupae and adults, large amounts of the mRNA are present. Each of these stages itself displays a distinct time course of mRNA synthesis. In addition, BBP is expressed tissue specifically, with the fat body being the major source of this secretory protein in the larvae. Hence, the expression pattern of BBP in this organism is markedly different from the closely related pigment protein insecticyanin in Manduca sexta. Finally, the bacterial expression of BBP in a functional state was established as a basis for the future analysis of its ligand‐binding functions by protein engineering.
Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase
Authors: AMEIS Detlev , MERKEL Martin , ECKERSKORN Christoph , GRETEN Heiner
Published: March 2005

Lysosomal acid lipase (LAL) is a hydrolase essential for the intracellular degradation of cholesteryl esters and triacylglycerols. This report describes a multi‐step procedure for the purification of LAL from human liver. After solubilization with non‐ionic detergent, acid hydrolase activity was purified 17000‐fold to apparent homogeneity by sequential chromatography on Concanavalin A Sepharose, carboxymethyl‐cellulose, phenyl Superose, Mono S cation exchange and Superose 12 gel‐filtration columns. This procedure yielded two silver‐staining protein bands of 56 kDa and 41 kDa on SDS/PAGE. Size‐exclusion chromatography of the 41‐kDa protein indicated that the enzyme was catalytically competent as a monomer of approximately 38 kDa. When assayed in the presence of cholesteryl oleate or trioleoylglycerol, purified acid lipase had Vmax values of 4390 nmol fatty acid · min−1· mg protein and 4756 nmol fatty acid · min−1· mg protein−1, and apparent Km values of 0.142 mM and 0.138 mM, respectively. The purified enzyme was most active at low pH (4.5–5.0) and required non‐ionic detergent and ethylene glycol for optimal stability. Incubation of the 41‐kDa acid lipase with endoglucosaminidase H reduced the molecular mass by 4–6 kDa, demonstrating Asn‐linked glycosylation with high‐mannose oligosaccharides. Deglycosylation did not affect enzymic activity, indicating that carbohydrates are not required for LAL activity. Based on partial peptide sequence, an oligonucleotide was synthesized and utilized to isolate LAL cDNA clones from a human liver cDNA library. A full‐length LAL cDNA contained 2626 nucleotides and coded for a predicted protein of 372 amino acids, preceded by a 27 residue hydrophobic signal peptide. Hepatic LAL differed from fibroblast acid lipase at the N‐terminus and revealed extensive similarities with human gastric lipase and rat lingual lipase, confirming a gene family of acid lipases. Northern hybridization using the complete LAL cDNA as a radiolabeled probe indicated striking differences in mRNA expression among human tissues. LAL mRNA was most abundant in brain, lung, kidney and mammary gland. Placenta and HeLa cells expressed intermediate amounts of LAL mRNA, while RNA extracted from liver and heart showed low levels of expression.
Effects of protons on the thrombin‐fibrinogen interaction
Authors: PICOZZI Matilde , LANDOLFI Raffaele , DE CRISTOFARO Raimondo
Published: March 2005

Amidase activity of human α‐thrombin toward the synthetic substrate Tosyl‐Gly‐Pro‐Arg‐NH‐Ph and fibrinogen has been studied as a function of pH at t= 25°C, under steady‐state conditions. A viscosity‐perturbation method allowed us to compute the equilibrium binding constant along with the rate constants for the acylation and deacylation reactions. The ionization constants for the groups affecting binding and hydrolysis of the synthetic substrate were measured by application of linkage thermodynamics principles. The binding of the synthetic substrate is controlled by two ionizable groups having pKa values of 7.5 and 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. These two groups were found to control the acylation process as well.

Thrombin‐fibrinogen interaction has been studied by measurements of steady‐state hydrolysis of the synthetic substrate Phe‐pipecolyl‐Arg‐NH‐Ph in the presence of fibrinogen, used as a competitive inhibitor. This method allowed us to measure the Km of thrombin‐fibrinogen interaction. The values of Km computed at different solution viscosities were used in order to calculate the equilibrium dissociation constant and both k2/k3 and k2/k‐1 ratios. The same residues that were found to control binding of Tosyl‐Gly‐Pro‐Arg‐NH‐Ph to α‐thrombin, do modulate binding of fibrinogen as well. These residues shift their pKa values upon the formation of the Michaelis adduct from 7.5 to 5.7 and from 8.7 to 9.7, respectively. Furthermore the ratio kcat/Km as a function of pH has been obtained by HPLC measurements of fibrinopeptides release. The kcat/Km values along with the ratio k2/k‐1, derived from viscometric experiments, allowed us to calculate the forward‐rate constant, k+1, for the thrombin‐fibrinogen interaction. The association process was found to depend on pH, namely in the alkaline region. The results for Tosyl‐Gly‐Pro‐Arg‐NH‐Ph and fibrinogen are compared and discussed on the basis of the structural elements which differentiate the interactions of these substrates with human α‐thrombin.
Differential secretion of α1‐acid glycoprotein occurs in the Golgi complex of isolated rat hepatocytes
Authors: POÜS Christian , GUIBOURDENCHE Jean , DRECHOU Anne , DURAND Geneviève
Published: March 2005

Using weakly basic amines, we investigated the step at which the secretion kinetics of concanavalin‐A‐retained and nonretained α1‐acid glycoprotein glycoforms diverge in isolated rat hepatocytes. Both chloroquine and primaquine, whose action on protein secretion is targeted to terminal domains of the Golgi apparatus, cancelled the kinetic difference without influencing carbohydrate chain sialylation. To test for a possible interaction of α1‐acid glycoprotein with Golgi membranes, we also permeabilized control and primaquine‐treated hepatocytes, as well as purified Golgi preparations, with saponin. In each case, we found that α1‐acid glycoprotein was associated with Golgi membranes, the association being more marked in primaquine‐treated cells than in control cells. Membrane‐bound α1‐acid glycoprotein appeared to be preferentially retained on concanavalin A. Such retention could account for the divergent secretion kinetics of α1‐acid glycoprotein glycoforms.
Identification of a ligand‐binding site of the human endothelin‐A receptor and specific regions required for ligand selectivity
Authors: ADACHI Miki , FURUICHI Yasuhiro , MIYAMOTO Chikara
Published: March 2005

To investigate the ligand‐binding site of the human endothelin‐A‐receptor subtype (ETA), we have produced various chimeric and mutated receptors in chinese hamster ovary cells. The substitution of Lys140 with Ile located in the C‐terminus of the second transmembrane region caused a 13‐fold reduction in affinity for endothelin‐1 (ET‐1) and 3.6‐fold lower Bmax than those values for the original receptor. Correspondingly, the mutated ETA receptor with the Lys140→Ile substitution failed to induce an increase in the intracellular calcium concentration in the presence of 1 nM ET‐1. Thus, the Lys140 in the ETA receptor is important in ligand binding. ETA and ETB receptors posses the ET isopeptides selective and non‐selective binding activities, respectively. Displacement experiments and the binding of 125I‐ET‐3 to various chimera receptors demonstrated that both the third and fourth extracellular regions, including the flanking transmenbrane regions, are responsible for the ligand‐binding selectivity of the ETA receptor.
Solution structure of seminal plasmin in the presence of micelles
Authors: MAURER Till , RÜTERJANS Heinz
Published: March 2005

The 47‐amino‐acid peptide seminal plasmin maintains a random‐coil characteristic feature when in aqueous solution, as can be shown by CD and NMR measurements. However, upon binding to a hydrophobic/hydrophilic interface, an increase in the content of secondary structure is observed. This change in conformation can also be induced with the addition of a detergent. The secondary structure segments thus formed were assigned by two‐dimensional NMR. The results indicate that hydrophobic interactions are a driving force in the folding of seminal plasmin. The interaction of seminal plasmin with detergent micelles could serve as a model for the binding of this interesting protein with the acrosome membrane of sperm cells.
H2: heterodisulfide oxidoreductase complex from Methanobacterium thermoautotrophicum
Authors: SETZKE Edgar , HEDDERICH Reiner , HEIDEN Stefanie , THAUER Rudolf K.
Published: March 2005

The reduction of the heterodisulfide (CoM‐S‐S‐HTP) of coenzyme M (H‐S‐CoM) and N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP) with H2 is an energy‐conserving step in most methanogenic Archaea. In this study, we show that in Methanobacterium thermoautotrophicum (strain Marburg) this reaction is catalyzed by a stable H2: heterodisulfide oxidoreductase complex of F420‐non‐reducing hydrogenase and heterodisulfide reductase. This complex, which was loosely associated with the cytoplasmic membrane, was purified 17‐fold with 80% yield to apparent homogeneity. The purified complex was composed of six different subunits of apparent molecular masses 80, 51, 41, 36, 21 and 17 kDa, and 1 mol complex, with apparent molecular mass 250 kDa, contained approximately 0.6 mol nickel, 0.9 mol FAD, 26 mol non‐heme iron and 22 mol acid‐labile sulfur. In 25 nM Chaps, the complex partially dissociated into two subcomplexes. The first subcomplex was was composed of the 51‐, 41‐ and 17‐kDa subunits; 1 mol trimer contained 0.7 mol nickel, 10 mol non‐heme iron and 9 mol acid‐labile sulfur and exhibited F420‐non‐reducing hydrogenase activity. The other subcomplex was composed of the 80‐, 36‐ and 21‐kDa subunits; 1 mol trimer contained 0.8 mol FAD, 22 mol non‐heme iron and 15 mol acid‐labile sulfur and exhibited heterodisulfide‐reductase activity. The stimulatory effects of potassium phosphate, a membrane component, uracil derivatives and coenzyme F430 on the H2:heterodisulfide‐oxidoreductase activity of the purified complex are described.
Protein organization in the matrix of leaf peroxisomes
Authors: HEUPEL Ralf , HELDT Hans Walter
Published: March 2005

This report is an investigation on how the compartmentation of peroxisomal metabolism, involved in the photorespiratory cycle, is accomplished. With isolated peroxisomes from spinach leaves the conversion of serine to glycerate, as coupled to the conversion of glycolate to glycine, was measured. Not only with intact but also with osmotically shocked peroxisomes, which had retained the aggregated state of the peroxisomal matrix but lost the integrity of the boundary membrane, the rates of glycerate synthesis were as high as required for the photorespiratory cycle in vivo. With both intact and shocked peroxisomes the intermediates glyoxylate and hydroxypyruvate did not equilibrate with the medium. It appears from these results that the apparent compartmentation of peroxisomal metabolism is not due to the function of the boundary membrane but to the organization of peroxisomal enzymes in multi‐enzyme complexes. When glycolate was added to peroxisomes without transamination partners, glyoxylate was released from the peroxisomes while the peroxisomal matrix partially disintegrated. With solubilized peroxisomes a partial reconstitution of functional enzyme complexes was achieved by the addition of poly(ethylene glycol). The function of the apparently very stable peroxisomal multi‐enzyme complexes in protecting the cells from the toxic intermediates H2O2 and glyoxylate is discussed.
Light‐activated cleavage of DNA by cobalt‐bleomycin
Authors: NIGHTINGALE Karl P. , FOX Keith R.
Published: March 2005

We have studied the light‐activated cleavage of DNA by cobalt‐bleomycin using a series of synthetic DNA fragments containing (AT)n and (GC)n. This cleavage reaction requires high concentrations of the antibiotic and appears to be a stoichiometric process rather than a catalytic process. We find that, in common with the iron‐complex, cobalt‐bleomycin can cleave at ApT steps within regions of alternating AT residues; ApT steps within other sequences including (AAT)n· (ATT)n are not good substrates for cobalt‐bleomycin cleavage. Some repetitive regions display an alternating pattern of cleavage products, revealing the preferred arrangement of ligand molecules along a saturated DNA lattice. A similar repetitive pattern is found for diethylpyrocarbonate modification and hydroxyl‐radical cleavage. Although cleavage of ApT and GpC proceeds at equivalent rates, the data suggest that bleomycin binds more tightly to the latter. Adenine residues on the 3′ side of both GpC‐cleavage and ApT‐cleavage sites are rendered more reactive to diethylpyrocarbonate, consistent with a ligand‐induced alteration in local DNA structure. The cobalt‐bleomycin‐binding site consists of not more than four base pairs, and may be as small as three base pairs.
Characterization of the lipopolysaccharide of Moraxella catarrhalis
Authors: MASOUD Hussein , PERRY Malcolm B. , RICHARDS James C.
Published: March 2005

The lipopolysaccharide of Moraxella catarrhalis serotype A (ATCC 25238) was found to consist of a short‐chain oligosaccharide attached to a lipid A moiety. Composition and NMR analyses showed the oligosaccharide component in O‐deacylated LPS to be composed of d‐glucose, d‐galactose, 2‐acetamido‐2‐deoxy‐d‐glucose and 3‐deoxy‐d‐manno‐octulosonic acid in the molar ratio of 5:2:1:2. In addition, the lipid A region contained phosphate, d‐glucosamine, ethanolamine, 3‐hydroxydodecanoic acid, dodecanoic acid and decanoic acid. The lipid A was examined in detail by high‐field NMR spectroscopy and mass spectrometry. It was found to consist of a β‐1,6‐d‐glucosamine disaccharide backbone esterified at C4′ by a phosphomonoester and glycosidically at C1 by diphosphoethanolamine or phosphomonoester. The amide group of the reducing and non‐reducing glucosamine residues were acylated by 3‐dodecanoyloxydodecanoic acid and 3‐decanoyloxydodecanoic acid, respectively. The hydroxyl group at C3 and C3′ were acylated by 3‐decanoyloxydodecanoic acid and 3‐hydroxydodecanoic acid respectively, while the hydroxyl groups at C4 and C6′ were unsubstituted.
Increased thermal aggregation of proteins in ATP‐depleted mammalian cells
Authors: Van NGUYEN Trung , BENSAUDE Olivier
Published: March 2005

In an attempt to understand the influence of the intracellular environment on protein stability, the thermal denaturation of various reporter proteins was examined within cultured mammalian cells. Loss of solubility and of enzymatic activities were taken as indicators of thermal denaturation. Photinus pyralis luciferase, Escherichia coliβ‐galactosidase, the 70‐kDa constitutive heat‐shock proteins and the 68‐kDa dsRNA‐dependent protein kinase are found mostly in the supernatant fractions of centrifuged lysates from control unshocked mammalian cells. However, when cells are lysed after heat shock, a proportion of the reporter molecules is found to be aggregated to the nuclear pellets. This insolubilization does not affect all cellular proteins; many of them remain unaffected by heat shock.

The heat‐induced insolubilization of all four reporter proteins is markedly enhanced when the intracellular ATP concentration is drastically decreased after inhibition of both oxidative phosphorylation and glycolysis. Although ATP molecules bind to luciferase and protect it from thermal inactivation in vitro, the consequences of strong ATP depletion on luciferase thermal stability within the cells are found to be much greater than expected from in vitro data. The 70‐kDa constitutive heat‐shock proteins and the 68‐kDa protein kinase are ATP‐binding proteins but ATP depletion also considerably increases the aggregation of β‐galactosidase to the nuclear pellets, although this enzyme is not known to be an ATP‐binding molecule.

Insolubilization of all four reporter proteins occurs in ATP‐depleted cells even at normal growing temperatures (37°C). Protein denaturation may be enhanced either by the aggregation and disappearance of the intracellular ‘free’ chaperones or by the trapping of unfolded protein molecules on chaperones; the chaperone/unfolded protein complexes could not dissociate in the absence of ATP.

Enhanced protein denaturation due to ATP depletion is proposed to account for the greater heat sensitivity of ATP‐depleted cells and for the ability of mitochondrial uncouplers to trigger a heat‐shock response in some cells.
The upstream region of the gene for the pathogenesis‐related protein 1 a from tobacco responds to environmental as well as to developmental signals in transgenic plants
Authors: GRÜNER Rose , PFITZNER Ursula M.
Published: March 2005

Pathogenesis‐related proteins (PR proteins) are a heterogeneous group of proteins which are induced in plants by diverse stimuli, e.g. PR proteins are elicited by pathogen attack in the course of the hypersensitive defense reaction of the plant. To examine the regulation of these genes, the 5′‐flanking region of the tobacco (Nicotiana tabacum cv. Wisconsin 38) PR‐1 a gene up to position–1533 was isolated from genomic DNA by the polymerase chain reaction. Two chimeric gene constructs containing 1533 bp and 906 bp, respectively, of the PR‐1 a upstream region fused to the GUS reporter gene were stably integrated into the tobacco genome. All primary transformants exhibited induced expression of the reporter gene after infection of the plants with tobacco mosaic virus or treatment with acetylsalicylic acid. In addition, similar expression of the reporter gene was observed in leaves of adult transgenic plants without any prior inductive treatments. To study this phenomenon in more detail, the F1 progeny of independent transgenic lines were monitored during the ontogeny of the plants. In normally developing tobacco plants, strong GUS activities were typically detected approximately 12 weeks after germination in the lowest leaves of vegetative plants. When successive leaves of individual plants were tested during the following weeks, a clear gradient of reporter gene activity had developed in the green leaves including the sepals from the bottom to the top of the plants. In all cases analyzed, this gradient of reporter gene expression was strictly parallelled by the expression of the endogenous PR‐1 proteins. These results suggest that the acidic PR‐1 proteins from tobacco fulfill a role during the later stages of plant development and that the PR‐1 a upstream region –906 to –335 contains positive regulatory elements for both environmental and developmental signals.
Cloning, chromosomal location and functional expression of the human voltage‐dependent calcium‐channel β3 subunit
Authors: COLLIN Thibault , LORY Philippe , TAVIAUX Sylvie , COURTIEU Christophe , GUILBAULT Pierre , BERTA Philippe , NARGEOT Joël
Published: March 2005

A novel human‐voltage‐dependent‐calcium‐channel (VDCC) β subunit was isolated from a 9‐week‐old human total‐embryo cDNA library. Of the four genes encoding β‐subunit isoforms that have been identified in animal species, this isoform shares strong similarity with the rat and rabbit β3‐related gene product and is referred to here as Hβ3 subunit. The Hβ3 isoform is the second β subunit identified in human. Its open reading frame encodes a 482‐amino‐acid protein with a predicted molecular mass of 54.571 kDa. The Hβ3 mRNA is expressed mostly in brain, smooth muscle and ovary. The gene for the human Hβ3 was specifically localized on chromosome 12q13. The cloned Hβ3 subunit was further expressed in Xenopus oocytes to demonstrate its ability to modulate VDCC activity.
Structure and expression of a gene coding for thermostable α‐glucosidase with a broad substrate specificity from Bacillus sp. SAM1606
Authors: NAKAO Masahiro , NAKAYAMA Toru , KAKUDO Akemi , INOHARA Misa , HARADA Masami , OMURA Fumihiko , SHIBANO Yuji
Published: March 2005

We cloned an α‐glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5′‐noncoding region as well as expression of the α‐glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12000‐fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the α‐glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the α‐glucosidase from N‐terminal serine was calculated to be 68886Da. Sequence analysis revealed the SAM1606 α‐glucosidase showed extremely high sequence identity (62–65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo‐1,6‐glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80–82%) among these three enzymes. However, the substrate specificity of the SAM1606 α‐glucosidase was significantly different from those of the oligo‐1,6‐glucosidases. The thermostability of these three α‐glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at β turns and coils in the enzymes.
Transcriptional regulatory regions for expression of the rat pyruvate kinase M gene
Authors: WANG Ziyuan , TAKENAKA Masaru , IMAI Enyu , YAMADA Kazuya , TANAKA Takehiko , NOGUCHI Tamio
Published: March 2005

To study the regulatory mechanism of pyruvate kinase M gene transcription, we analyzed its chromatin structure and cis‐acting DNA regions. Two DNase‐I‐hypersensitive sites were detected in dRLh‐84 hepatoma cells, but not in hepatocytes, which coincides with expression of the M gene in the two types of cells. These sites, designated HS2 and HS1, were located around the major transcription start site and about 2.9 kb downstream from this site, respectively. A transient chloramphenicol acetyltransferase expression assay indicated that the region around HS1 did not show any activity, whereas the upstream region up to ‐457 had promoter activity in hepatoma cells. Most of this activity was lost by a 5′‐deletion from ‐286 to ‐225. Further analysis identified a cluster of three cis‐acting regions from ‐279 to ‐216, which are named boxes A, B and C. These regions did not have any independent effect, but the inclusion of all regions were synergistic. These regions were not active in hepatocytes, suggesting that they have cell‐type specificity. A gel mobility shift assay indicated that unidentified, but distinct, nuclear proteins bound to the three boxes. These results suggest that transcriptional regulation of the M gene involves alteration of chromatin structure and binding of proteins to three cis‐acting elements.
The oligosaccharides of the Fe(III)‐Zn(II) purple acid phosphatase of the red kidney bean
Authors: STAHL Bernd , KLABUNDE Thomas , WITZEL Herbert , KREBS Bernt , STEUP Martin , KARAS Michael , HILLENKAMP Franz
Published: March 2005

Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110‐kDa glycoprotein related to the mammalian purple acid phosphatases with a two‐metal cluster at the active site contains five oligosaccharide side chains/monomer. The N‐linked glycan structures were characterized by selective enzymic degradation in combination with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). The purified protein was cleaved by cyanogen bromide. One 30‐kDa large methionine‐free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an α1–3 fucose attached to the asparagine‐linked N‐acetylglucosamine prevented removal of the glycan by peptide N‐glycosidase F; peptide N‐glycosidase A removed all carbohydrates from the peptides.

To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex‐type xylose‐containing structure with four of the five having an additional fucose.
Pore formation in artificial membranes by the secreted hemolysins of Proteus vulgaris and Morganella morganii
Authors: BENZ Roland , HARDIE Kim R. , HUGHES Colin
Published: March 2005

Lipid‐bilayer experiments were performed with the related hemolysins from Proteus vulgaris and Morganella morganii (HlyA). The addition of the toxins to the aqueous phase bathing lipid‐bilayer membranes composed of different lipids resulted in the formation of transient ion‐permeable channels. Membranes formed of pure lipids were rather inactive targets for the hemolysins as compared with lipid mixtures such as asolectin. The channels had several different substates. The major open state had single‐channel conductances of 500 pS in 0.15 M KCl at small transmembrane voltages. Experiments with differents with different salts suggested that the hemolysin‐induced channels of P. vulgaris and M. morganii were exclusively cation selective at neutral pH, caused by negative charges localized at the channel mouth. The mobility sequence of the cations within the channels was similar if not identical to their mobility sequence in the aqueous phase. The single‐channel data were consistent with wide, water‐filled channels with estimated minimal diameters of about 1 nm since the large organic cation Tris+ can permeate the channels without any detectable interaction with its interior. Pore‐forming properties of these hemolysins were compared with those of HlyA of Escherichia coli. All these toxins share common features, oligomerize probably to form pores in lipid‐bilayer membranes and form channels with similar properties which suggests that their structures are more or less identical.
NMR and circular dichroic studies of the solution structure of conformationally constrained antigenic peptides
Authors: PRÊCHEUR Bénédicte , BOSSUS Marc , GRAS‐MASSE Hélène , QUINIOU Eric , TARTAR André , CRAESCU Constantin T.
Published: March 2005

Circular dichroic and nuclear magnetic resonance spectroscopies were used to evaluate the conformational properties in solution of a series of 20‐amino‐acid peptides derived from the primary structure of an antigen from Echinococcus granulosus. The linear peptide corresponding to the sequence 93–112 in the antigen was found to populate in a significant proportion the α‐helix conformational state. In the presence of 2,2,2‐trifluoroethanol, a cosolvent known to stabilize peptide secondary structure, the helical population, estimated from circular dichroic spectra, increases up to 60–70%. Two‐dimensional nuclear magnetic resonance studies under these conditions showed that the segment K96–K108 meets all the criteria of an α‐helix at 281 K and 298 K. Three different variants were synthesized with the same or similar primary structure but containing a lactam‐bridged (>) side chain: D107>K110, D97>K100 and K94>E98. Generally, the observed helical content in these variants was lower than in the parent molecule and the stability of the helical conformation decreased in the order D107, K110, K94, E98, D97, K100. Analysis of chemical shift and nuclear Overhauser enhancement data suggested that the lactam rings induce significant distortions of the local features of helix secondary structure. The possible factors of helix destabilization induced by lactam bridges, observed in the studied peptides are discussed in relation to the stabilizing effect of ion pairs in model compounds.
Elucidation of the structure of SA‐FF22, a lanthionine‐containing antibacterial peptide produced by Streptococcus pyogenes strain FF22
Authors: JACK Ralph W. , CARNE Alan , METZGER Jörg , STEFANOVIĆ Stefan , SAHL Hans‐Georg , JUNG Günther , TAGG John
Published: March 2005

The antibacterial peptide SA‐FF22, produced by the pathogen Streptococcus pyogenes strain FF22 was purified and features of its primary and secondary structure were characterised. Mass spectrometry demonstrated the pure peptide had a mass of 2794Da while, amino acid analysis revealed the presence of the unusual, thioether amino acids lanthionine and 3‐methyllanthionine; thus SA‐FF22 is a member of the group of antibacterial polypeptides termed lantibiotics. Furthermore, amino acid sequencing showed a unique sequence which was blocked at position 23 by a residue of the unsaturated amino acid 2,3‐didehydrobutyrine. Carboxypeptidase‐Y digestion could be used to demonstrate that serine occupies the C‐terminal position only after complete oxidation of the thioether amino acid bridges, suggesting that the three‐dimensional structure of the native peptide may prevent access of the enzyme to the C‐terminus. Fragmentation of the native peptide with a variety of proteolytic enzymes failed to yield a peptide containing less than all three of the cross‐linked lanthionine and methyllanthionine residues and demonstrated that all three thioether bridges overlapped. Analysis of the circular dichroism of SA‐FF22 in various concentrations of 2,2,2‐trifluoroethanol in water, SDS micelles and in the presence of artificial phospholipid vesicles suggested that there is significant change in its secondary structure from aqueous to lipophilic environments.
Two human melanoma cell‐line variants with enhanced in vivo tumor growth and metastatic capacity do not express the β3 integrin subunit
Authors: BOUKERCHE Habib , BENCHAIBI Miloud , BERTHIER‐VERGNES Odile , LIZARD Gerard , BAILLY Maryse , BAILLY Mireille , McGREGOR John L.
Published: March 2005

The αvβ3 integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express αvβ3. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of αvβ3 expression. Established human melanoma cell lines (M3Da., M4Beu.) were isolated from an achromic skin metastasis of lymph nodes. Two stable variants (7GP, T1P26), derived from a poorly metastatic M4Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow‐cytometry analysis shows an absence of the β3 integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, T1P26) compared to M3Da. and M4Beu. cell lines which express a relatively high number of β3 subunits. The expression levels of the integrin subunits β1, β5, β6 and αv were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of β3 in 7GP or T1P26 cell lines and its presence in M3Da. and M4Beu. Moreover, similar levels of αv transcript were present in the four melanoma cell lines. The functional effect of the absence of β3 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and T1P26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M3Da. or M4Beu. cell lines. The β3 integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M4Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of β3 or αvβ3 integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the αvβ3 integrin complex.
Triple‐helix formation interferes with the transcription and hinged DNA structure of the interferon‐inducible 6–16 gene promoter
Authors: ROY Christian
Published: March 2005

The interferon responsive element (IRE) of the 6–16 gene lies within two 39‐bp elements in tandem. A purine‐rich oligodeoxynucleotide, oligo(dN), was found to be able to pair with the purinerich strand of the IRE in an antiparallel orientation which led to triple‐helix formation with Mg2+ being necessary for triplex stability. Footprinting analysis confirmed these results. The interaction between the IRE and the oligo(dN) was reversible and had a Kd equal to 20 nM. The two repeats of the 6–16 gene IRE can form a hinged DNA structure through pairing of their purine‐rich regions; exonuclease III experiments support this model. The hybrid DNA structure leads to a parallel pairing of the purine strands of the 6–16 gene IRE and this conformation was shown to be destabilized by triplex formation. When co‐transfected with a reporter gene whose promoter was under the control of the 6–16 gene IRE, the triple‐helix‐forming oligo(dN)s inhibit the interferon‐induced stimulation of the reporter gene with complete inhibition being obtained with 1 μM oligo(dN) at the time of transfection. When added to the cell culture medium after transfection, the concentrations of oligo(dN) needed to obtain 50% inhibition of the interferon effect on gene transcription must be 50–100 times higher. Besides the existence of a peculiar structure for the 6–16 gene IRE, the possibility of interfering with gene expression by means of oligo(dN)s is demonstrated.
Molecular characterisation of the enolase gene from the human malaria parasite Plasmodium falciparum Evidence for ancestry within a photosynthetic lineage
Authors: READ Martin , HICKS Karen E. , SIMS Paul F. G. , HYDE John E.
Published: March 2005

We have isolated and characterised the gene encoding the glycolytic enzyme enolase (2‐phospho‐d‐glycerate hydrolase) from the human malaria parasite Plasmodium falciparum. This was achieved using a combination of cDNA sequencing and inverse‐PCR techniques. The gene maps to chromosome 10 of the parasite. We have also mapped two further glycolytic enzyme genes, glyceraldehyde‐3‐phosphate dehydrogenase and triose‐phosphate isomerase, to chromosome 14. The enolase gene encodes a protein of 446 amino acids (48.7 kDa), and all amino acid residues implicated in substrate/cofactor binding and catalysis are conserved in the malarial enolase molecule. The predicted protein sequence displays approximately 60–70% identity to enolase molecules of other eukaryotes, the closest relationship with its homologues seen amongst the seven fully described glycolytic pathway enzymes of P. falciparum. Of particular significance in this well conserved molecule is a characteristic 5‐amino‐acid insertion sequence that is identical in position and virtually identical in primary structure to that which is otherwise found uniquely in plant enolase proteins. This pentapeptide, together with other features of the plasmodial sequence, points to a common ancestry with photosynthetic organisms at the level of a protein‐encoding nuclear gene, thus extending earlier analyses of nuclear small‐subunit ribosomal RNA genes, and of an extrachromosomal circular 35‐kb DNA element found in P. falciparum, which have also indicated such a relationship.
Disruption of the gene encoding the NADH‐binding subunit of NADH: ubiquinone oxidoreductase in Neurospora crassa
Authors: FECKE Wolfgang , SLED Vladimir D. , OHNISHI Tomoko , WEISS Hanns
Published: March 2005

In this study, the gene of the 51‐kDa NADH‐binding subunit of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) in Neurospora crassa was inactivated by homologous replacement with a defective gene copy. The resulting mutant, nuo51, lacks the 51‐kDa subunit and shows no complex I activity but still grows at one third of the wild‐type growth rate. The enzyme activity of the alternative NADH:ubiquinone oxidoreductase(s) is increased twofold while the activities of the other mitochondrial respiratory enzymes are normal. Complex I is almost completely assembled except for the NADH‐binding subunit and still possesses three out of the four EPR‐detectable iron‐sulphur clusters. Since the deleted subunit contains the sequence motif for one tetranuclear iron‐sulphur cluster, the missing cluster N‐3 is considered to be bound to this subunit.