bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenylalanyl‐tRNA Synthetase from Yeast and Its Discrimination of 19 Amino Acids in Aminoacylation of tRNAPhe‐C‐C‐A and tRNAPhe‐C‐C‐A(3′NH2)
Authors: Freist Wolfgang , Sternbach Hans , Cramer Friedrich
Published: July 2004

For discrimination between phenylalanine and 18 other naturally occurring non‐cognate amino acids by the class II aminoacyl‐tRNA synthetase specific for phenylalanine, discrimination factors, D, of 190–6300 have been determined from kcat and Km values. Generally, phenylalanyl‐tRNA synthetase is more specific than the class II enzymes specific for Lys and Thr, but works with lower accuracy than the class I enzymes specific for IIe, Tyr, and Arg.

In aminoacylation of tRNA Phe‐C‐C‐A(3′NH2) discrimination factors D1 vary between 80–1610. Pre‐transfer proof‐reading factors II1 are in the range 2.3–74, post‐transfer proof‐reading factors II2 in the range 1.0–4.6, showing that pre‐transfer proof‐reading is the main correction step, post‐transfer proof‐reading is less effective or negligible.

Initial discrimination factors (I1 and I2) caused by differences in Gibbs free energies of binding between phenylalanine and non‐cognate amino acids have been calculated assuming a two‐step binding process. Factors I1 can be related to hydrophobic‐interaction forces depending on accessible surface areas of the amino acids, factors I2 scatter about a low mean value and do not show any relation to amino acid structures or surfaces, indicating less checking of amino acid side chains in the putative second binding step.
Characterization and Isolation of Enzymes that Hydrolyze Short‐Chain acyl‐CoA in Rat‐Liver Mitochondria
Authors: Svensson L. Thomas , Kilpeläinen Seppo H. , Hiltunen J. Kalervo , Alexson Stefan E. H.
Published: July 2004

In this study we investigated the presence of short‐chain acyl‐CoA hydrolases in rat liver mitochondria. One acetyl‐CoA‐hydrolyzing enzyme with a molecular mass of about 48 kDa was purified to apparent homogeneity as judged by SDS/PAGE. Immunoprecipitation experiments with antibodies raised to the purified protein showed that this enzyme corresponds to a minor portion of the total mitochondrial acetyl‐CoA hydrolase activity, most (about 90%) of the total activity being due to an enzyme which was labile and required Triton X‐100 for its stability. Neither of these acetyl‐CoA‐hydrolyzing enzymes appeared to be induced by treatment of rats with di(2‐ethylhexyl)phthalate, a peroxisome proliferator which mediates induction of several cytosolic and mitochondrial long‐chain acyl‐CoA thioesterases. In addition, an enzyme that hydrolyzed acetoacetyl‐CoA was partially purified; it was induced about 3.5‐fold by di(2‐ethylhexyl)phthalate treatment. In conclusion, these results demonstrate that rat liver mitochondria contain several enzymes capable of hydrolyzing short‐chain acyl‐CoA, indicating that regulation of the metabolism of short‐chain acyl‐CoAs and formation of ketone bodies, could be complex.
Activation of Signaling Pathways in HL60 Cells and Human Neutrophils by Farnesylthiosalicylate
Authors: Tisch Daphna , Halpern Matya , Marciano Daniela , Kloog Yoel , Aviram Irit
Published: July 2004

Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl‐sulfoxide‐differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (≤20 μM) dose‐dependently elevated cytosolic Ca2+ concentrations, suggesting phospholipase‐C‐mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47‐phox and p67‐phox to the membrane. The calcium signal, inositol‐trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussis‐toxin‐sensitive and pertussis‐toxin‐resistant elements. In HL60 cells, farnesylthiosalicylate (≤20 μM) did not activate NADPH oxidase but dose‐dependently augmented PMA‐elicited activity of the enzyme. This effect was resistant to pertussis‐toxin treatment. In vitro augmentation of PKC‐mediated phosphorylation of histone and cytosolic p47‐phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine‐docking sites on cellular signaling elements.
Purification and Characterization of a Proline‐Rich Antibacterial Peptide, with Sequence Similarity to Bactenecin‐7, from the Haemocytes of the Shore Crab, Carcinus Maenas
Authors: Schnapp Denni , Kemp Graham D. , Smith Valerie J.
Published: July 2004

Antibacterial peptides are important for non‐specific host defence in many animals. They have been extensively characterized from mammals, amphibians, insects and chelicerates but have not so far been found in crustaceans. Here we report the presence of several constitutive antibacterial proteins, active against both gram‐positive and gram‐negative bacteria, in the haemocytes of the shore crab, Carcinus maenas. These proteins have molecular masses of >70 kDa, ≈45 kDa, ≈14 kDa and 6.5 kDa. The 6.5 kDa peptide has been purified to homogeneity by Sep Pak C18 extraction, gel filtration and reverse‐phase HPLC. Partial N‐terminal sequence analysis further shows that it is proline rich and shares more than 60% identity in a 28‐amino‐acid overlap with the mature form of bactenecin 7, an antimicrobial peptide from bovine neutrophils which belongs to the cathelicidin family of mammalian peptide antibiotics.
Structural Analysis of the O‐Antigenic Polysaccharide from the Enteropathogenic Escherichia coli O125
Authors: Kjellberg Alexandra , Urbina Felipe , Weintraub Andrej , Widmalm Göran
Published: July 2004

The structure of the polysaccharide part of the lipopolysaccharide obtained from the enteropathogenic Escherichia coli O125 has been investigated. Methylation analysis, 1H‐NMR spectroscopy and 13C‐NMR spectroscopy revealed that the polysaccharide is composed of repeating hexasaccharide units. Smith degradation of the native O‐polysaccharide resulted in a polysaccharide with four sugar residues in the repeating unit. Information on the sequence of the native O‐polysaccharide and the Smith‐degraded product was obtained by two‐dimensional‐NMR techniques, namely heteronuclear‐mutiple‐bond‐connectivity and NOESY experiments. The structure of the repeating unit of the O‐polysaccharide of E. coli strain O125, which has two adjacent branch‐point residues, is

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Identification of Differentially Expressed Genes During Hepatocytes Development and Characterization of their Prenatal Hormonal Induction
Authors: Cohen Hannah , Trus Michael , Benvenisty Nissim , Reshef Lea
Published: July 2004

Upon birth, the liver acquires new functions as a result of the initiation of expression of key enzymes. One example is the initiation of gluconeogenesis which depends on the induced appearance of phosphoenolpyruvate carboxykinase (P‐pyruvate‐CK) at birth. To characterize other genes that undergo such regulation, a differential screening was performed on a cDNA library from well‐differentiated hepatoma cells. The pattern of tissue‐specific and developmental‐specific expression was determined for seven genes. Three clones, out of which two encode for the known genes alcohol dehydrogenase class I (ADH) and phenylalanine 4‐monooxygenase (PAH) and a new gene (clone 116‐3), exhibited a pattern of expression similar to that of the P‐pyruvate‐CK gene, i.e. their expression was liver and kidney specific and induced in the liver upon birth. Determination of the sequence of clone 116‐3 revealed that it belonged to the UDP‐glucuronosyltransferases type 2 (UGT2) family and thus was named UGT2B‐rH4. To examine whether expression of the various genes could be prematurely induced by hormones in the fetal liver, either high levels of CAMP or low levels of insulin were induced in utero. The results demonstrated that cAMP induced a marked expression only of the genes for P‐pyruvate‐CK and ADH but not of those for PAH or UGT2B‐rH4, while insulin deficiency induced premature expression of all four genes. We suggest that a set of genes whose expression is specifically induced in the liver upon birth can be prematurely induced by the hormones in utero.
Lipopolysaccharide Activates Transcription of the Heme Oxygenase Gene in Mouse M1 Cells Through Oxidative Activation of Nuclear Factor κB
Authors: Kurata Shun‐Ichi , Matsumoto Midori , Tsuji Yoshitaka , Nakajima Hiroshi
Published: July 2004

It has been known for a long time that heme oxygenase (HO) is a key enzyme in heme catabolism, and it was found to act as an oxidative‐stress protein to produce carbon monoxide, which has similar actions to those of nitrogen monoxide. We examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during treatment with lipopolysaccharide (LPS; an oxidative reagent). Since the promoter region of this gene in human cells contains a 12‐O ‐tetradecanoyl‐phorbol‐13‐acetate(TPA)‐responsive element (TRE) and a nuclear‐factor‐κB–responsive element, HO mRNA expression might be regulated by an oxidative activation pathway. We investigated activation of the HO gene after treatment of M1 cells with LPS. Upon treatment with LPS, H2O2 was produced, the nuclear proto‐oncogenes fos and jun were activated, then the HO gene was activated. The extent of transcriptional activation of the fos, jun and HO genes in M1 cells treated with LPS was strongly reduced by a scavenger of oxygen radicals (N ‐acetyl‐l‐cysteine), but a specific inhibitor of protein kinase C only reduced transcriptional activation by 10–20%. These results suggest that LPS may be an oxidative reagent. Some oxidative reagents (e.g., H2O2 are strong activators of NF‐κB, and therefore we treated M1 cells with H2O2. Essentially the same extends of transcriptional activation of the fos, jun and HO genes were observed as those observed after LPS treatment. Super‐shift assays with DNA that contained the TRE motif revealed that the Fos and Jun proteins from nuclei of M1 cells treated with LPS and H2O2, bound weakly to the TRE motif, and, in assays with DNA that contained the NF‐κB motif, nuclear protein from M1 cells treated with H2O2, or LPS bound strongly to the NF‐κB motif. These results strongly suggest that the HO gene in M1 cells is mainly activated by LPS through oxidative activation of NF‐κB due to production of H2O2.
NMR Studies of the Escherichia coli Trp Repressor ·trpRs Operator Complex
Authors: Evans Paul D. , Jaseja Mahesh , Jeeves Mark , Hyde Eva I.
Published: July 2004

To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H‐NMR spectroscopy. We report here the 1H‐NMR chemical shifts of a 20‐bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37‐kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.
Regulation of Leukotriene‐Biosynthetic Enzymes During Differentiation of Myelocytic HL‐60 Cells to Eosinophilic or Neutrophilic Cells
Authors: Scoggan Kylie A. , Nicholson Donald W. , Ford‐Hutchinson Anthony W.
Published: July 2004

Leukotrienes (LTs) are potent mediators of bronchial inflammation and are predominantly produced by myeloid cells. As myelocytic cells differentiate towards either eosinophils or neutrophils, the profile of leukotrienes they produce upon stimulation diverges. Eosinophils produce mainly cysteinyl leukotrienes whereas neutrophils predominantly synthesize 5(S),12(R)‐dihydroxy‐6,8,10,14‐eicosatetraenoic acid (LTB4). The mechanism by which this change in leukotriene composition occurs is unknown. In this study, we investigated the control of leukotriene biosynthetic enzymes during myeloid cell differentiation. Western‐blot analyses of myelocytic leukemia cell lines, HL‐60#7 and HL‐60, differentiated towards eosinophilic or neutrophilic cell types, respectively, demonstrated that as myelocytic cells differentiate towards eosinophils or neutrophils, the protein levels of cytosolic phospholipase A2 (cPLA2) remain constant, whereas 5‐lipoxygenase and 5‐lipoxygenase‐activating protein (FLAP) levels are simultaneously elevated. As myelocytic cells become more eosinophil‐like, 5(S)‐hydroxy‐6(R)‐S ‐glutathionyl‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acid (LTC4) synthase activity and expression of both the protein and messenger RNA in the cells are dramatically increased (approximately 75‐fold), while the LTC4 synthase level and activity in neutrophil‐like cells remain constant at very low levels. In contrast, in neutrophilic cells, the amount of 5,6‐oxido‐7,9,11,14‐eicosatetraenoic acid (LTA4) hydrolase was elevated approximately 100‐fold greater than the increase in LTA4 hydrolase from eosinophilic cells. These results indicate that as a myeloid cell differentiates towards a granulocyte, similar mechanisms of regulation may be applied to the leukotriene biosynthetic pathway up to the point at which the pathway diverges. At the stage in the leukotriene biosynthetic pathway where LTA4 may be converted to either LTC4 or to LTB4, specific regulators of transcription may become activated as a myelocytic cell differentiates, thereby causing increased LTA4 hydrolase or LTC4 synthase expression.
2,4‐Dioxygenases Catalyzing N‐Heterocyclic‐Ring Cleavage and Formation of Carbon Monoxide
Authors: Bauer Iris , Max Nicole , Fetzner Susanne , Lingens Franz
Published: July 2004

1H‐3‐Hydroxy‐4‐oxoquinaldine 2,4‐dioxygenase (MeQDO) was purified from quinaldine‐grown Arthrobacter sp. Rü61a. It was enriched 59‐fold in a yield of 22%, and its properties were compared with 1H‐3‐hydroxy‐4‐oxoquinoline 2,4‐dioxygenase (QDO) purified from Pseudomonas putida 33/1. The enzyme‐catalyzed conversions were performed in an (8O)O2/(16O)O2, atmosphere. Two oxygen atoms of either (18O)O2 or (16O)O2 were incorporated at C2 and C4 of the respective substrates, indicating that these unusual enzymes, which catalyze the cleavage of two carbon‐carbon bonds concomitant with CO formation, indeed are 2,4‐dioxygenases. Both enzymes are small monomeric proteins of 32 kDa (MeQDO) and 30 kDa (QDO). The apparent Km values of MeQDO for 1H‐3‐hydroxy‐4‐oxoquinaldine and QDO for 1H‐3‐hydroxy‐4‐oxoquinoline were 30 μM and 24 μM, respectively. In both 2,4‐dioxygenases, there was no spectral evidence for the presence of a chromophoric cofactor. EPR analyses of MeQDO did not reveal any signal that could be assigned to an organic radical species or to a metal, and X‐ray fluorescence spectrometry of both enzymes did not show any metal present in stoichiometric amounts. Ethylxanthate, metal‐chelating agents (tiron, α,α′‐bipyridyl, 8‐hydroxyquinoline, o‐phenanthroline, EDTA, diphenylthiocarbazone, diethyldithiocarbamate), reagents that modify sulfhydryl groups (iodoacetamide, N‐ethylmaleimide, p‐hydroxymercuribenzoate), and reducing agents (sodium dithionite, dithiothreitol, mercaptoethanol) either did not affect 2,4‐dioxygenolytic activities at all or inhibited at high concentrations only. With respect to the supposed lack of any cofactor and with respect to the inhibitors of dioxygenolytic activities, MeQDO and QDO resemble aci‐reductone oxidase (CO‐forming) from Klebsiella pneumoniae, which catalyzes 1,3‐dioxygenolytic cleavage of 1,2‐dihydroxy‐3‐keto‐S‐methylthiopentene anion (Wray, J. W. & Abeles, R. H. (1993) J. Biol. Chem. 268, 21466–21469; Wray, J. W. & Abeles, R. H. (1995) J. Biol. Chem. 270, 3147–3153). 1H‐3‐Hydroxy‐4‐oxoquinaldine and 1H‐3‐hydroxy‐4‐oxoquinoline were reactive towards molecular oxygen in the presence of the base catalyst potassium‐tert.‐butoxide in the aprotic solvent N,N‐dimethylformamide. Base‐catalyzed oxidation, yielding the same products as the enzyme‐catalyzed conversions, provides a non‐enzymic model reaction for 2,4‐dioxygenolytic release of CO from 1H‐3‐hydroxy‐4‐oxoquinaldine and 1H‐3‐hydroxy‐4‐oxoquinoline.
A Positive Regulatory Sequence of Hepatitis B Viral Small X Promoter
Authors: Choi Cheol Yong , Park Geon Tae , Rho Hyune Mo
Published: July 2004

Hepatitis B viral X protein (HBx) and small X proteins (HBSx) are known to transactivate promoters for RNA polymerase II and RNA polymerase III. Small X promoter has been mapped in the 5′‐distal half of the X open reading frame. A 5′‐serial deletion analysis showed that there was a positive regulatory sequence for the efficient transcription of the small X promoter. Two cellular proteins of 110 kDa (p110) and 33 kDa (p33) bound at the 3′ and 5′ regions of the regulatory sequence, respectively. Mutation of p33‐binding and p110‐binding sites led to diminution and elevation, respectively, of activation properties of the positive element, suggesting that p33 participates in the transactivation and that p110 has an inhibitory effect on the function of p33. This possibility was further supported by the result demonstrating that in vitro phosphorylation of p110 reduced its target DNA‐binding capability.
Purification, Characterization and Subcellular Localization of a Type‐1 Ribosome‐Inactivating Protein from the Sarcocarp of Cucurbita pepo
Authors: Yoshinari Shigeo , Yokota Sadaki , Sawamoto Hiroshi , Koresawa Shigehiro , Tamura Minoru , Endo Yaeta
Published: July 2004

The flesh of the fruit of Cucurbita pepo contains a type‐1 ribosome‐inactivating protein (RIP), which we named pepocin. Pepocin was purified to apparent homogeneity by acid fractionation, ion‐exchange chromatography and adsorption chromatography. The protein was found to have a molecular mass of 26 kDa and a PI of about 9.9. It does not contain glycosidic linkages. The protein inhibits protein synthesis in a rabbit‐reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 15.4 pM, and depurinates 28S rRNA in the ribosomes of the lysate in a manner identical to that of ricin A‐chain and other RIP. The enzyme is also active on wheat‐germ ribosomes and on Escherichia coli ribosomes. The sequence of the N‐terminal 20 amino acids of the protein reveals a close relationship to other RIP. Immunoelectron‐microscopic localization of pepocin in the sarcocarp shows that the protein is predominantly localized in intercellular spaces. In addition, the immunolocalized signals are observed in leaf intercellular spaces.
Molecular Characterization and Expression Analysis ofManduca sextaAllatotropin
Authors: Taylor Phillip A. , Bhatt Tejal R. , Horodyski Frank M.
Published: July 2004

Juvenile hormones (JH) are a class of regulatory sesquiterpenoids that control metamorphosis in immature insects and reproduction in adult insects. The regulation of JH synthesis by the corpora allata (CA), a pair of endocrine glands with nervous connections to the brain, is achieved by a complex interplay of stimulatory and inhibitory factors mediated in part by the brain. The neuropeptide, allatotropin (Mas AT), was recently isolated and sequenced from the brain of the tobacco hornwormManduca sexta.Mas AT is a 13‐residue amidated peptide that activates JH synthesis in adult, but not larval, lepidopteran CA. A 23‐nucleotide degenerate oligonucleotide was designed based on the peptide sequence and was used to isolate the Mas AT genomic clone. The Mas AT gene is expressed as three mRNAs which differ from one another by alternative splicing. These mRNAs are predicted to encode three distinct prohormones, each containing Mas AT. A restriction fragment from the genomic clone was then used to isolate the cDNA clone.In situhybridization and immunohistochemistry studies show that Mas AT is expressed in both the central and enteric nervous systems. Cells expressing Mas AT were identified in the brain, frontal ganglion and terminal ganglion.
Activation and Enzyme Characteristics of a DNA‐Restrained Phosphatase in Chromatin‐Associated Complexes
Authors: Loeffler Harald , Spiess Eberhard , Juodka Benediktas , Stammer Hermann , Werner Dieter
Published: July 2004

DNA‐bound polypeptide complexes composed of several non‐histone polypeptides that resisted harsh DNA deproteinization procedures were characterized. The three major polypeptides of these complexes have molecular masses of 62, 52, and 40 kDa. They constitute supramolecular structures that reside on isolated DNA in dense clusters. The supramolecular complexes were released from DNA as globular 12.8±0.8‐nm particles; these particles were gradually disassembled to form smaller supramolecular structures. The DNA‐bound complexes comprise of an encrypted adenosinetriphosphatase/phosphatase activity, which is a minor but intrinsic component of the complexes. The enzyme remained inactive as long as the complexes were bound to DNA. However, the enzyme was activated concomitantly with the progression of DNA digestion, which indicated that DNA was involved in the downregulation of the enzyme. The inactive DNA‐restrained complex could not be restored in vitro, which indicated its non‐trivial nature. Once released from DNA, the enzyme was inactivated over a period of several hours. However, in the DNA‐associated complexes its potential to become activated during DNA digestion was conserved for several months. In the activated state, the enzyme showed an optimum activity at pH 9.5, was stimulated by Mg2+, inhibited by vanadate and EDTA, but was not significantly inhibited by okadaic acid. The active enzyme, which consists of two subunits of 56 kDa and 59 kDa, can be released from the supramolecular structures by agarose gel electrophoresis. A regulatory mechanism therefore exists for the downregulation of this phosphatase by DNA.
Unfolding Kinetics of Bovine Trypsinogen
Authors: Otlewski Jacek , Sywula Agnieszka , Kolasinski Marcin , Krowarsch Daniel
Published: July 2004

The unfolding kinetics of bovine trypsinogen were studied by a fluorescence‐detected stopped‐flow technique at pH 5.8. Trypsinogen unfolding appeared to be a rather complex reaction. Two phases, fast (with a time constant in the millisecond range) and slow, were detected in the range 2–7 M guanidium chloride (GdmC1). The natural logarithm of the rate constant of the slow phase exhibited strong dependence on [GdmCl], changing from hundreds of seconds at low denaturant concentration to about 20 ms at 7 M GdmC1. The curvature of this dependence further suggests a complex mechanism of unfolding. Generally, similar kinetics were observed for the trypsinogen · Ca complex. Small differences could be noticed, however, for the fast phase. In agreement, Ca2+ influenced only this stage of the reaction. Analysis of the dependence of the time constant of the fast phase on [CaCl2] indicates that at 4 M GdmC1, trypsinogen · Ca unfolds about sixfold slower than free zymogen, and that native trypsinogen at 4 M GdmCl still exhibits high affinity for Ca2+. Limited data on trypsin unfolding show virtually an identical dependence of the slow phase on [GdmC1]; the fast phase, however, was not observed. Moreover, in the 3–4.5 M GdmCl range, a separate phase was detected. It is postulated that this phase is a manifestation of the activation‐domain unfolding. The Eyring plots for the fast phase of trypsinogen and trypsinogen · Ca unfolding are linear, indicating little change in heat capacity for this stage of reaction. The slow step of unfolding, however, shows significant curvature, which indicates a substantial increase in heat capacity.
Soyacystatin, a Novel Cysteine Proteinase Inhibitor in Soybean, is Distinct in Protein Structure and Gene Organization from Other Cystatins of Animal and Plant Origin
Authors: Misaka Takumi , Kuroda Masaharu , Iwabuchi Kyoko , Abe Keiko , Arai Soichi
Published: July 2004

Cystatins, cysteine proteinase inhibitors, deserve note because of their regulatory and protective functions in plant tissues. We isolated both genomic DNA and cDNA clones from soybean that encode a cystatin consisting of 245 amino acid residues (soyacystatin). It is, while basically similar in sequence to known cystatins that are generally in the range of 12–15 kDa, characterized by having extremely large extension sequences in both its amino and carboxyl termini. The genomic DNA encoding soyacystatin is also unique in that it consists of four exons with three introns in its coding regions. The mRNA for soyacystatin is distinctly expressed in soybean seeds 2 weeks after flowering. Soyacystatin purified from mature soybean seeds had a molecular mass of about 26 kDa on SDS/PAGE which suggests that it contains the extension sequences. Papain‐inhibition experiments demonstrate that this endogenous soyacystatin has almost the same inhibitory activity as that of its deletion mutant (102 amino acid residues) recombinantly produced by truncation of the amino and carboxyl terminal extensions, indicating that the occurrence of the extensions does not affect the cystatin activity. Immunohistochemical experiments reveal that soyacystatin is expressed nearly uniformly in the cotyledons. These results also suggest the possible occurrence of a cysteine proteinase as the target enzyme of soyacystatin.
Photoaffinity Labelling of a DNA‐Binding Site on the Globular Domain of Histone H5
Authors: Goytisolo Fermín A. , Packman Leonard C. , Thomas Jean O.
Published: July 2004

We have labelled a DNA‐binding site on the globular domain of histone H5 (GH5) by ultraviolet‐activated cross‐linking of a self‐complementary 5‐bromodeoxyuridine (5BrU)‐substituted oligonucleotide with the sequence 5′‐AGCGA5BrUATCGCT‐3′. Cross‐linking was to His62, mainly to the protein back‐bone. This observation provides further support for the mode of binding of GH5 to DNA proposed on the basis of the similarity between the X‐ray crystal structure of GH5 and the DNA‐bound structures of catabolite activator protein and hepatic nuclear factor 3y [Ramakrishnan, V. (1994) Curr. Opin. Struct. Biol. 4, 44–50].
Nuclear Factor k B (NF‐k B), Nuclear Factor Interleukin‐6 (NFIL‐6 or C/EBPβ) and Nuclear Factor Interleukin‐6β (NFIL6‐β or C/EBPδ) are Not Sufficient to Activate the Endogenous Interleukin‐6 Gene in the Human Breast Carcinoma Cell Line MCF‐7
Authors: Faggioli Laura , Costanzo Chiara , Merola Marcello , Bianchini Ercolina , Furia Adriana , Carsana Antonella , Palmieri Marta
Published: July 2004

A comparative study of the molecular mechanism of interleukin‐6 (IL‐6) gene induction on two breast‐carcinoma‐derived cell lines has been performed. MDA‐MB‐231 cells produce constitutive detectable levels of both secreted 1L‐6 and mRNA which, as expected. are dramatically enhanced following induction by either IL‐1β or tumor necrosis factor‐α (TNF‐α). The levels of both secreted IL‐6 and IL‐6 mRNA are significantly higher in response to IL‐1β in spite of the fact that stimulation by TNF‐α alone enhances the half life of IL‐6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL‐6 in these cells. In contrast, MCF‐7 cells fail to produce detectable IL‐6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF‐κB, NFIL6 and NFIL6β, which have been described to be sufficient to activate the IL‐6 gene in other cell systems, shows a similar pattern of expression in both MCF‐7 and MDA‐MB‐231 cells. Furthermore, transfection of a recombinant plasmid carrying the IL‐6 promoter linked to a luciferase reporter gene shows that both cell lines are able to drive IL‐1β or TNF‐α activation of this construction in a very similar manner. Finally, when MCF‐7 cells were treated with IL‐1β or TNF‐α in the presence of cycloheximide, transcription of IL‐6 mRNA from the endogenous IL‐6 gene was observed. These data suggest that a mechanism of IL‐6 gene repression is active in MCF‐7 cells.
Extremely Thermophilic and Thermostable 5′‐Methylthioadenosine Phosphorylase from the Archaeon Sulfolobus solfataricus
Authors: Cacciapuoti Giovanna , Porcelli Marina , Bertoldo Costanzo , Fusco Stefania , De Rosa Mario , Zappia Vincenzo
Published: July 2004

A gene encoding an extremely thermophilic and thermostable 5′‐methylthioadenosine phosphorylase was cloned from the archaeon Sulfolobus solfataricus. Two degenerate oligodeoxyribonucleotide probes synthesized on the basis of the N‐terminal amino acid sequence of the protein were used to screen a genomic library of S. solfataricus cloned into the pGEM7Zf(+) vector. The DNA fragment of 2118 bp containing the 5′‐methylthioadenosine phosphorylase gene was sequenced. The open reading frame comprises 711 nucleotides, which includes the stop codon, and encodes a protein of 236 residues whose molecular mass is in good agreement with the value determined by gel filtration for the purified enzyme. The N‐ and C‐terminal sequences of the protein and the sequences of the peptides prepared by cyanogen bromide cleavage exactly match with the corresponding sequences deduced from the gene, thus confirming the identity of the 5′‐methylthioadenosine phosphorylase gene. Typical archaebacterial regulatory sites were identified in the flanking regions and a potential Shine‐Dalgarno‐like sequence was recognized around the ATG initiation codon. The deduced amino acid sequence showed 32% identity and 30% identity with Escherichia coli purine‐nucleoside phosphorylase and with E. coli uridine phosphorylase, respectively. Evolutionary and structural implications of this similarity are discussed.
Primary Structure of Stallion Seminal Plasma Protein HSP‐7, a Zona‐Pellucida‐Binding Protein of the Spermadhesin Family
Authors: Reinert Markus , Calvete Juan J. , Sanz Libia , Mann Karlheinz , Töpfer‐Petersen Edda
Published: July 2004

The primary structure of HSP‐7, a 14‐kDa protein isolated from stallion seminal plasma, has been determined. HSP‐7 belongs to the spermadhesin protein family, shares 98% sequence identity with the boar seminal plasma protein AWN, and, like its boar homolog, displays zona‐pellucida‐binding activity. Despite these conserved structural and functional features, the equine and porcine spermadhesins differ in their topography on spermatozoa.
A Sec1‐Related Vesicle‐Transport Protein that is Expressed Predominantly in Epithelial Cells
Authors: Riento Kirsi , Jäntti Jussi , Jansson Sanna , Hielm Sebastian , Lehtonen Eero , Ehnholm Christian , Keränen Sirkka , Olkkonen Vesa M.
Published: July 2004

Sec1‐related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Sec1‐related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc‐18–2/Munc‐18b/muSec1 protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associatesin vitrowith syntaxins 1A, 2, and 3.In situhybridization analysis of embryonic mouse tissues revealed prominent expression of the munc‐18‐2 mRNA in the epithelia of several tissues. Cell‐fractionation studies demonstrated that the majority of Munc‐18‐2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc‐18‐2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS‐1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin 1A, it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc‐18‐2 as a predominantly epithelial vesicle‐transport protein with a polarized distribution and provided novelin vivoevidence for the association of Sec1‐related proteins with members of the syntaxin family.
Pregnenolone‐7β‐Hydroxylating Activities of Yeast‐expressed Mouse Cytochrome P450‐1A1 and Mouse‐tissue Microsomes
Authors: Doostzadehi Jaleh , Urban Philippe , Pompon Denis , Morfin Robert
Published: July 2004

In many tissues from different species, pregnenoIone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7α position by a cytochrome P450 (P450)‐containing microsomal enzyme com‐plex. In addition, 7‐hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7β‐hydroxypregnenolone and 7β‐hydroxy‐DHEA was not supported by formal identifica‐tion, and the P450 responsible for 7α‐hydroxylation and 7β‐hydroxylation of pregnenolone and DHEA have not been identified. Based on results of analyses by crystallization to constant specific activity and gas chromatographylmass spectrometry, we report that mouse‐liver and mouse‐brain microsomes carried out 7β‐hydroxylation of pregnenolone and DHEA, and that yeast‐expressed mouse cytochrome P450‐1Al (P450 1Al) transformed pregnenolone into 7β‐hydroxypregnenolone (Km= 25.1 ± 0.4 μM, turnover number = 979 ± 30pmol. min−1± nmol−1 mouse P450 1Al). Neither 7‐hydroxy derivatives of DHEA nor 7a‐hydroxypregnenolone was produced by P450 1Al. The presence of P450 1Al in liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1 Al by β‐naphthoflavone resulted in increased 7β‐hydroxylation of pregnenolone in liver microsomes. Studies of the brain‐microsome 7β‐hydroxylating enzyme with pregnenolone or DHEA gave Km of 5.0 μM and 4.9 μM, respectively, and Vmax of 4.5 pmol. min−1± mg−1 and 6.1 pmol ± min−1± mg−1 respectively, and showed the absence of cross‐inhibitions between the two steroids. These findings indicate that, in addition to unidentified P450, P450 1Al is involved in 7β‐hydroxylation of pregnenolone and may contribute in part to the production of the 7‐hydroxylated steroids necessary for activation of immune defenses.
HlyA Hemolysin of Vibrio Cholerae O1 Biotype El Tor
Authors: Menzl Karin , Maier Elke , Chakraborty Trinad , Benz Roland
Published: July 2004

Hemolysin (HlyA) was concentrated from supernatants of different Vibrio cholerae O1 biotype El Tor strains by ammonium sulfate precipitation. The concentration of the toxin in the supernatants and in the precipitates was quantified using its hemolytic activity. The toxin formed a high molecular‐mass band (about 220 kDa) on SDS/PAGE while the toxin monomer had a molecular mass of 60 kDa when it was heated. The addition of the El Tor hemolysin oligomers, but not that of the monomers, to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion‐permeable channels, which had long lifetimes at small voltages. The hemolysin channel had a single‐channel conductance of 350 pS in 1 M KC1. These results defined hemolysin (HlyA) from V. cholerae as a channel‐forming component with properties similar to other cytolytic toxins. The long lifetime of the channel suggested that the channel‐forming oligomer did not show a rapid association/dissociation reaction. At voltages larger than 50 mV, the hemolysin channel was voltage dependent in an asymmetric fashion dependent on the side of its addition. The single‐channel conductance of the hemolysin (HlyA) from V. cholerae O1 biotype El Tor channel was a linear function of the bulk aqueous conductance, which suggested that the toxin forms aqueous channels with an estimated minimum diameter of about 0.7 nm. The hemolysin channel of V. cholerae was found to be moderately anion‐selective. The pore‐forming properties of hemolysin (HlyA) from V. cholerae O1 biotype El Tor were compared with those of aerolysin of Aeromonas sobria and α‐toxin from Staphylococcus aureus. All these cytolytic toxins must probably oligomerize for activity in biological and artificial membranes and form anion‐selective channels.
Calcium Influx is a Determining Factor of Calpain Activation and Microparticle Formation in Platelets
Authors: Pasquet Jean‐Max , Dachary‐Prigent Jeanne , Nurden Alan T.
Published: July 2004

We have related the release of procoagulant microparticles from platelets to calcium movement and the activation of the Ca2+‐dependent protease calpain. The effects of the Ca2+‐ATPase inhibitors thapsigargin, cyclopiazonic acid and 2,5‐di‐(t‐butyl)‐1,4‐benzohydroquinone were compared with those of the Ca2+ ionophore A23187. Whereas all three Ca2+‐ATPase inhibitors induced aminophospholipid exposure on platelets, only thapsigargin and cyclopiazonic acid promoted microparticle formation and only when strong Ca2+ influx, calpain activation and proteolysis of cytoskeletal proteins occurred concomitantly. Preincubation with dibutylbenzohydroquinone inhibited the responses to thapsigargin and cyclopiazonic acid but not to A23187. When platelets were suspended in a Ca2+‐free medium, calpain activation and microparticle formation were not observed, even with maximum mobilisation of internal Ca2+ stores by A23187. Incubation of fluo‐3–loaded platelets with A23187 in 0.1 mM EGTA followed by the sequential addition of 25 μM Ca2+ increments to the medium showed that calpain activation occurred when the intraplatelet [Ca2+] reached 3–8 μM. To assess the physiologic significance of these results, the subpopu‐lation of platelets that expressed procoagulant activity after stimulation by a thrombin/collagen mixture was isolated by means of annexin‐V–coupled magnetic beads. Subsequent western blotting experiments confirmed that this subpopulation contained activated calpain. Overall, our results provide evidence that microparticle formation and calpain activation require an elevated intraplatelet [Ca2+] that is brought about by influx across the plasma membrane.
Lipid‐Binding Properties of Synthetic Peptide Fragments of Human Apolipoprotein A‐II
Authors: Benetollo Claire , Lambert Gilles , Talussot Corinne , Vanloo Berlinda , Van Cauteren Tom , Rouy Didier , Dubois Hervé , Baert Johan , Kalopissis Athina , Denèfle Patrice , Chambaz Jean , Brasseur Robert , Rosseneu Maryvonne
Published: July 2004

Human apolipoprotein A‐II (apo A‐II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid‐binding properties of this apolipoprotein. The conformation and lipid‐binding properties of these peptides, either as single‐helix or as two‐helix peptides, were investigated by turbidity, fluorescence, electron‐microscopy and circular‐dichroism measurements, and are compared in this article. The lipid affinity of shorter C‐terminal segments of apo A‐II was compared with those of the single‐helix or two‐helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo‐A‐II‐(13–48)‐peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo‐A‐II‐(13–30)‐(33–48)‐peptide, is predicted to form a long uninterrupted helix.

The single helices of apo A‐II could not form stable complexes with phospholipids, and the helix‐turn‐helix segment spanning residues 13–48 was not active either. The apo‐A‐II‐(37–77)‐peptide and the apo‐A‐II‐(40–73)‐peptide could form complexes with lipids, which appear as discoidal particles by negative‐staining electron microscopy. The shortest C‐terminal domain of apo A‐II, able to associate with lipids to form stable complexes was the apo‐A‐II‐(40–73)‐peptide, which consisted of the C‐terminal helix, a β‐turn and part of the preceding helix. The shorter apo‐A‐II‐(49–77)‐peptide, and the helical apo‐A‐II‐(13–30)‐(33–48)‐peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid.

These data suggest that the C‐terminal pair of helices of apo A‐II, which is the most hydrophobic pair, is responsible for the lipid‐binding properties of the entire protein. The N‐terminal pair of helices of apo A‐II at residues 13–48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A‐II is thus less pronounced than in apo A‐I or apo A‐IV. The N‐terminal and C‐terminal domains of apo A‐II appear to behave as two distinct entities with regard to lipid‐protein association.
Further Characterization of the Peroxisomal 3‐Hydroxyacyl‐Coa Dehydrogenases from Rat Liver
Authors: Dieuaide‐Noubhani Martine , Novikov Dmitry , Baumgart Eveline , Vanhooren Johannes C. T. , Fransen Marc , Goethals Marc , Vandekerckhove Joël , Van Veldhoven Paul P. , Mannaerts Guy P.
Published: July 2004

Recently, we purified five 3‐hydroxyacyl‐CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I–V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (l‐ or d‐hydroxyacyl‐CoA) stereospecificity and (de)hydratase measurements with the different 3‐hydroxyacyl‐COA stereoisomers of straight‐chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I–V and molecular cloning of enzyme III revealed the following picture.

Rat liver peroxisomes contain two multifunctional β‐oxidation proteins: (a) multifunctional protein 1 (the classical multifunctional protein; MFP‐1) displaying 2‐enoyl‐CoA hydratase, l–3‐hydroxyacyl‐CoA dehydrogenase and Δ3, Δ2‐enoyl‐CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP‐2) displaying 2‐enoyl‐CoA hydratase and d‐3‐hydroxyacyl‐CoA dehydrogenase activity (enzyme III).

Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring β‐oxidation intermediates, MFP‐1 and MFP‐2 appear to be involved in the β‐oxidation of fatty acids and bile acids intermediates, respectively.

The deduced amino acid sequence of the cloned MFP‐2 cDNA is highly similar to that of the recently described porcine endometrial estradiol 17β‐dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, H. H. & Jungblut, P. W. (1994) Eur. J. Biochem. 222, 221–227]. In agreement, MFP‐2 also displayed estradiol 17β‐dehydrogenase activity, indicating that MFP‐2 and the steroid dehydrogenase are identical enzymes.

MFP‐2 is partially cleaved, most probably in vivo, in a estradiol 17β‐dehydrogenasel/d‐3‐hydroxyacyl‐CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP‐1 (enzyme IV) is artefactually cleaved during purification giving rise to 3‐hydroxyacyl‐CoA dehydrogenase V. 3‐Hydroxyacyl‐CoA dehydrogenase II is a mitochondrial contaminant similar to porcine and murine mitochondrial 3‐hydroxyacyl‐CoA dehydrogenase.
Biosynthesis of Acylpeptidolactones of the Daptomycin Type
Authors: Wessels Petra , Von Döhren Hans , Kleinkauf Horst
Published: July 2004

A21978C and A54145 are antibacterial 13‐residue peptides with a medium‐chain‐acylated amino terminus and a 10‐residue lactone ring; they are produced by strains of Streptomyces roseosporus and Streptomyces fradiae, respectively. The structural differences in their peptide chains, which include amino acid replacements and modifications (L‐Glu2→L‐Asn, L‐Asn(OH)3→L‐Asp, Sar5→Gly, L‐Ala6→L‐Orn, L‐Lys8→D‐Ala, L‐Asp(OMe)9→L‐Asp, L‐Asn11→D‐Ser, and L‐IIe13→L‐Kyn; Sar = sarcosine; L‐Orn =L‐ornithine, L‐Kyn =L‐kynurenine), reside in the multienzymatic templates directing their biosynthesis. We have examined the peptide synthetases employing immunodetection and substrate activation detected by the amino‐acid‐dependent ATP‐PP1‐exchange reaction. Two different antibodies specific for actinomycin synthetase 2 and a peptide sequence characteristic of acyL‐CoA‐synthetases/peptide synthetases were applied. For the A21978 system two peptide synthetases of 670 and 240 kDa were detected, together with two similar proteins of 630 and 440 kDa occurring in varying amounts. The latter are suggested to be degradation products of an unstable multienzyme. Activation of L‐Asp, L‐Thr, Gly, L‐Orn, L‐Ala and L‐Ser were assigned to the high‐molecular‐mass components of 670, 630 and 440 kDa. The 240‐kDa protein was purified to homogeneity and shown to catalyse activation of L‐kynurenine. The A54145 system consisted of three peptide synthetases of 690, 590 and 250 kDa. Activations of L‐Asn, L‐Thr and Gly were found. The 250‐kDa synthetase was capable of activating isoleucine and valine. Both systems thus show a comparable organisation; implications for the modular construction of their peptide synthetases are discussed.
The Human IgE Germline Promoter is Regulated By Interleukin‐4, Interleukin‐13, Interferon‐α and Interferon‐γ Via an Interferon‐γ‐Activated Site and Its Flanking Regions
Authors: Ezernieks Juris , Schnarr Bernd , Metz Kurt , Duschl Albert
Published: July 2004

Class switching to IgE is preceded by the appearance of ɛ germline transcripts, which are induced by Interleukin‐4 (IL‐4) and by IL‐13. A 51‐bp fragment of the human ɛ germline promoter conferred in reporter gene assays with the erythroleukemic cell line TF‐1 upregulation of transcription by IL‐4 or IL‐13, and repression by interferon‐α (IFN‐α) and IFN‐γ. A central IFN‐γ activated sequence within the 51‐bp fragment was sufficient for transcriptional regulation by the cytokines in the absence of its normal flanking regions. In contrast, deletion of either upstream or downstream sequences abolished repression by IFN‐α or INF‐γ, but not upregulation by IL‐4 or IL‐13. IL‐4 stimulated reporter gene transcription required more than ten times higher concentrations than cell proliferation or tyrosine phosphorylation of the IL‐4 receptor.
Transient‐State Kinetic Evidence Against a Channelled Transfer of Glyceraldehyde 3‐Phosphate from Aldolase to Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: Arias Wilma Martínez , Pettersson Gösta
Published: July 2004

Theoretical and experimental evidence is presented which leads to a rejection of the proposal that the transient‐state kinetics of the coupled two‐enzyme reaction involving aldolase and glyceraldehyde‐3‐phosphate dehydrogenase are indicative of a channelled transfer of glyceraldehyde 3‐phosphate from the producing enzyme to the consuming one.
Refolding Pathway and Association Intermediates of Glutamate Dehydrogenase from the Hyperthermophile Pyrococcus furiosus
Authors: Consalvi Valerio , Chiaraluce Roberta , Millevoi Stefania , Pasquo Alessandra , Vecchini Paola , Chiancone Emilia , Scandurra Roberto
Published: July 2004

The denaturation and renaturation processes of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been investigated using guanidinium chloride as denaturant. The enzyme is highly stable and the transition midpoint for guanidinium chloride denaturation is 6.1 M. The recovery of enzyme structure occurs after dilution of the denaturant at 20°C through the formation of structured monomers. Concentration of the structured monomers leads to the formation of higher association states with a tertiary structure different from that of the native enzyme. Activity is observed only in the presence of the hexamers, although a heating step at 70°C is required to fully reactivate the hexamer formed at 20 °C. The refolding process and the intermediate(s) were studied by activity assay, spectroscopic methods, size‐exclusion chromatography, and ultracentrifugation analysis.