bluuebunny/crossref_metadata_2025 huggingface dataset:
Different mechanisms of protection against apoptosis by valproate and Li+
Authors: Mora Alfonso , González‐Polo Rosa A. , Fuentes José M. , Soler Germán , Centeno Francisco
Published: March 2003

Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mm). Because the protection was unaffected by MK801 (N‐methyl‐d‐aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low‐K+‐induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3‐kinase inhibitor). These results suggest a role for phosphatidylinositol 3‐kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched‐chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low‐K+‐induced apoptosis by acting in the phosphatidylinositol 3‐kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.
Characterization of two bifunctional Arabdopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl‐tRNA synthetase and threonyl‐tRNA synthetase by alternative use of two in‐frame AUGs
Authors: Souciet Ginette , Menand Benoît , Ovesna Jaroslava , Cosset Anne , Dietrich André , Wintz Henri
Published: March 2003

We characterized two Arabidopsis thaliana cDNAs coding for class I valyl‐tRNA synthetase and class II threonyl‐tRNA synthetase. The proteins display characteristics of cytosolic enzymes, yet possess an N‐terminal extension relative to their prokaryotic homologs. The proximal part of the N‐terminal extension is a mitochondrial‐targeting signal. Through transient expression of GFP fusions in tobacco cells, we demonstrated that both genes encode the cytosolic and mitochondrial forms of the enzymes by alternative use of two in‐frame initiation codons. A long, mitochondrial form of the enzyme is translated from a first initiation codon at reduced levels because of a poor sequence context and a shorter, cytosolic form is translated from a second in‐phase AUG, which is in a better context for translation initiation. Primer extension experiments revealed several transcript ends mapping upstream of the first AUG and between the two AUGs. Distal to the mitochondrial transit peptide both valyl‐tRNA synthetase and threonyl tRNA synthetase possess an NH2‐appended domain compared with their prokaryotic counterparts. This domain’s amphiphilic helix is conserved between yeast and A. thaliana valyl‐tRNA synthetase, suggesting an important role in translation. Based on the high structural similarities between yeast and A. thaliana valyl‐tRNA synthetase, we propose that the acquisition of bifunctionality of valyl‐tRNA synthetase predates the divergence of these two organisms.
Analysis of lissencephaly‐causing LIS1 mutations
Authors: Sapir T. , Eisenstein M. , Burgess H. A. , Horesh D. , Cahana A. , Aoki J. , Hattori M. , Arai H. , Inoue K. , Reiner O.
Published: March 2003

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly‐causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Δ301–334; deletion of exon 4, which results in deleted amino acids Δ40–64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. We studied the consequences of the point mutation, deletion mutation and one of the reported truncations. In order to study LIS1 structure function, we introduced an additional point mutation and other truncations in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of our results, we suggest that all truncation mutations and lissencephaly‐causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein.
Atlantic salmon (Salmo salar L.) skin contains a novel kininogen and another cysteine proteinase inhibitor
Authors: Ylönen Anne , Rinne Ari , Herttuainen Jukka , Bøgwald Jarl , Järvinen Mikko , Kalkkinen Nisse
Published: March 2003

We describe the purification and characterization of two novel cysteine proteinase inhibitors found in Atlantic salmon skin. One of these, salmon kininogen, has a molecular mass of 52 kDa as determined by matrix‐assisted laser desorption/ionization time‐of‐flight MS, is multiply charged with pI values of 4.0, 4.2 and 4.6 and shows homology to kininogens including the bradykinin motif. The other, salarin, has a molecular weight of 43 kDa, a pI of 5.1 and shows weak homology to cysteine proteinases. Both proteins are N‐ and O‐glycosylated and inhibit papain and ficin but not trypsin.
Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl‐tRNA synthetase
Authors: Madore Eric , Florentz Catherine , Giegé Richard , Sekine Shun‐ichi , Yokoyama Shigeyuki , Lapointe Jacques
Published: March 2003

Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild‐type E. coli contains five modified nucleosides: Ψ13, mnm5s2U34, m2A37, T54 and Ψ55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Ψ, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D‐ and T‐arms and in the variable region. The lack of a 2‐thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520‐fold the specificity of E. coli glutamyl‐tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl‐tRNA synthetase for the recognition of glutamate and ATP.
Molecular characterization of a novel nucleolar protein in starfish oocytes which is phosphorylated before and during oocyte maturation
Authors: Nakajima Hiromi , Matoba Koji , Matsumoto Yoshihiro , Hongo Tomokatsu , Kiritaka Keiko , Sugino Hiroyuki , Nagamatsu Yasunori , Hamaguchi Yukihisa , Ikegami Susumu
Published: March 2003

In response to 1‐methyladenine, a maturation‐inducing substance, starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown through activation of p34cdc2‐cyclin B, which results in the dispersal of the nucleolus. Little information has been elucidated thus far on nucleolar proteins that are phosphorylated by p34cdc2‐cyclin B during meiotic maturation. Here, we describe a novel nucleolar protein of the starfish Asterina pectinifera oocyte, which is designated ANO39 and which is phosphorylated during meiotic maturation. A full‐length ANO39 cDNA of 2106 base pairs encodes a polypeptide of 346 amino acids having a calculated Mr of 39 005. The amount of ANO39 is kept nearly constant during oocyte maturation and embryogenesis up to the midgastrula stage. The transcript encoding ANO39 was present in growing oocytes but not in full‐grown ones, as evidenced by Northern blot hybridization. Ser145 is specifically phosphorylated when ANO39 is incubated in vitro with purified starfish p34cdc2‐cyclin B. This phosphorylation site corresponds to that is phosphorylated during meiotic maturation in vivo. Immunoblot analysis using phosphoserine145‐specific antibody as a probe revealed that some populations of ANO39 of the immature oocytes at the G2 stage have been already phosphorylated on Ser145 and Ser145 is maximally phosphorylated during meiotic maturation.
Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic cell cycle
Authors: Tokumoto Mika , Horiguchi Ryo , Nagahama Yoshitaka , Ishikawa Katsutoshi , Tokumoto Toshinobu
Published: March 2003

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2‐phase) and mature (in M‐phase) oocytes, and compared its subunits by immunoblotting. At least two protein bands, at 30 kDa (detected by GC3β antibody) and 62 kDa (detected by 1‐4D5 antibody), differed between 26S proteasomes. A monoclonal antibody, GC3β cross‐reacted with two bands in the 26S proteasome from immature oocytes, however, the upper band was absent in the 26S proteasome from mature oocytes. The 62‐kDa protein band detected by 1‐4D5 antibody was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these proteins were isolated by an immunoscreening method using the monoclonal antibodies. The 30‐kDa protein was an α4 subunit, which is one of the α‐subunit group of the 20S proteasome, and the 62‐kDa protein was a homologue of CCTε, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the α4 subunit of goldfish 20S proteasome, α4_ca, is phosphorylated in G2‐phase and dephosphorylated in M‐phase. A binding assay using a recombinant goldfish CCTε revealed that unmodified CCTε interacts with the 26S proteasome.

Fertilization triggers a transition from meiotic metaphase to mitotic interphase. During fertilization, a GC3β cross‐reacting upper band reappeared. The 62‐kDa band dissociated from the 26S proteasome. As a result, the 26S proteasome changed to an immature type from a mature type during fertilization. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.
Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase
Authors: Martínez‐Chantar María L. , Pajares María A.
Published: March 2003

Rat liver methionine adenosyltransferase incorporated 8 mol ofN‐ethylmaleimide per mol of subunit upon denaturation in the presence of 8 murea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re‐examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N‐terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver‐specific enzyme.
Formation of sarcoglycan complex with differentiation in cultured myocytes
Authors: Noguchi Satoru , Wakabayashi Eriko , Imamura Michihiro , Yoshida Mikiharu , Ozawa Eijiro
Published: March 2003

The sarcoglycan complex consists of four transmembrane protein subunits. Mutation of any one of the genes encoding these four subunits causes complete loss or marked decrease in expression of the whole complex, resulting in the phenotype of Duchenne‐like autosomal recessive muscular dystrophy, termed sarcoglycanopathy. As the basis for understanding this process, we examined how the sarcoglycan complex is formed and associates with other proteins during myogenic differentiation, using a myogenic cell line. Accumulation of the sarcoglycan subunits and formation of the sarcoglycan complex were accomplished with myotube formation. In protein transport inhibition experiments with blefeldin A, we found that the sarcoglycan complex is formed in the endoplasmic reticulum and then associates with the dystroglycan complex and sarcospan en route from the Golgi apparatus to the cell surface. In early myotubes, limited kinds of incomplete sarcoglycan complexes were observed. Their analyses would provide information on the possible patterns of formation of the sarcoglycan complex.
Characterization of an α‐macroglobulin‐like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata
Authors: Kopáček Petr , Weise Christoph , Saravanan Thangamani , Vítová Kateřina , Grubhoffer Libor
Published: March 2003

We report the identification of the first representative of the α‐2‐macroglobulin family identified in terrestrial invertebrates.

An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B.

The N‐terminal amino‐acid sequence of subunit A is similar to the N‐terminus of invertebrate α‐2‐macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the α‐2‐macroglobulin family, and the protein is therefore referred to as tick α‐macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high‐molecular‐weight substrate azocoll in a manner similar to that of bovine α‐2‐macroglobulin. This effect is abolished by pre‐treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active‐site inhibition by soybean trypsin inhibitor.

We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N‐terminus of subunit B and the putative ‘bait region’ (a segment of α‐2‐macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post‐translational processing.
Isolation and characterization of a self‐sufficient one‐domain protein
Authors: Gruber Christine , Stürzenbaum Stephen , Gehrig Peter , Sack Ragna , Hunziker Peter , Berger Burkhard , Dallinger Reinhard
Published: March 2003

Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd‐MT isolated from the compost‐dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino‐acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino‐acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N‐terminal site and at the linker region between the two putative metal‐binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys‐motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self‐sufficient one‐domain MT which is stable in vitro. The isolation of the single‐domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post‐translational processing of the translated gene product to yield functional one‐domain MT moieties.
In vitro folding, purification and characterization of Escherichia coli outer membrane protease OmpT
Authors: Kramer R. A. , Zandwijken Davika , Egmond Maarten R. , Dekker Niek
Published: March 2003

OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n‐dodecyl‐N,N‐dimethyl‐1‐ammonio‐3‐propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217–Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz‐Ala‐Arg‐Arg‐Ala‐Tyr(NO2)‐NH2 (where Abz is o‐aminobenzoyl and Tyr(NO2) is 3‐nitrotyrosine) enabled the determination of the kinetic parameters. The wild‐type enzyme has an affinity Km of 0.4 µm for the substrate and a turnover number kcat of 40 s−1. The Km and kcat for the double variant were 1.1 µm and 1.6 s−1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of β‐strand conformation, and on that basis a β‐barrel topology model is proposed.
Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture
Authors: Marc Nathalie , Galisteo Milagros , Lagadic‐Gossmann Dominique , Fautrel Alain , Joannard Florence , Guillouzo André , Corcos Laurent
Published: March 2003

Phenobarbital (PB) has long been known as an inducer of drug‐metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB‐treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP‐dependent pathway, as treatment with cAMP‐dependent protein kinase (PKA) activators (10 µm dibutyryl‐cAMP and 50 µm forskolin) dramatically inhibited PB‐induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP‐dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB‐induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura‐2 fluorescence ratio in murine hepatocytes showed that long‐term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA‐AM) nearly completely abolished PB‐induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin‐dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.
Sulfakinin neuropeptides in a crustacean
Authors: Johnsen Anders H. , Duve Hanne , Davey Marlene , Hall Mike , Thorpe Alan
Published: March 2003

The sulfakinin (SK) family of neuropeptides are characterized by a C‐terminal octapeptide sequence that begins with two acidic residues (most commonly DD), and ends with YGHMRF‐NH2, usually with the tyrosyl residue sulfated. So far, sulfakinins have only been identified in insects and the present study was initiated to investigate if the family is more widely distributed within the arthropods. Purification of an extract of the central nervous system of the giant tiger prawn Penaeus monodon has revealed three novel members of the sulfakinin peptide family. One of the peptides, Pem SKI, has the sequence <QFDEY(SO3H)GHMRF‐NH2, where <Q denotes a pyroglutamic acid residue, and is for all criteria typical of insect sulfakinins, several of which also have an N‐terminal pyroglutamic acid. Tyrosyl O‐sulfation was verified by mass spectrometry. The two other peptides have a hitherto unknown L/M variation at position three from the C‐terminus. One of these, Pem SKII, has a particularly glycine‐rich N‐terminus, AGGSGGVGGEYDDYGHLRF‐NH2. The other, Pem SKIII, is a truncated form of Pem SKII, with the sequence VGGEYDDYGHLRF‐NH2. Mass spectrometry of the latter two peptides indicated that only one of the two tyrosyl residues is sulfated. By analogy, it is suggested that the sulfation is located at the residue in position six from the C‐terminus. A small amount of a nonsulfated variant of Pem SKII was also present in the extract. Immunocytochemical studies with sulfakinin antisera show a sparse neuronal distribution pattern, similar to that of insects. A prominent pair of large (≈ 25 μm) cells and 6–8 pairs of smaller (≈ 10 μm) cells are present in the protocerebrum. The larger cells have prominent neurites that give rise to varicosities in the centre of the brain. Their axons exit the brain via the circumoesophageal connectives and continue along the intersegmental connectives. Each of the thoracic and abdominal ganglia has sulfakinin‐immunoreactive arborizations as a result of branching from the intersegmental nerves. This distribution pattern strongly suggests a role in neurotransmission or neuromodulation, although it remains to be elucidated what the exact role(s) is. However, on account of the conservation of peptide structure during the evolutionary period spanning the insect/crustacean lineage, especially between Pem SKI and insect sulfakinins, it may be assumed that the sulfakinins have a significant physiological role.
A switch in the direction of the effect of insulin on the partitioning of hepatic fatty acids for the formation of secreted triacylglycerol occurs in vivo, as predicted from studies with perfused livers
Authors: Rennie Susan M. , Park Byun‐Sung , Zammit Victor A.
Published: March 2003

The direct effects of insulin on hepatic triacylglycerol secretion are important because they may determine the degree of postprandial hyperlipidaemia, a known risk factor for the development of atherosclerotic lesions. Previous work from this laboratory, conducted on isolated perfused rat livers [Zammit, V.A., Lankester, D.J., Brown, A.M. & Park, B.S. (1999) Eur. J. Biochem.263, 859–864], has indicated that the effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state of the donor animals. In this paper, we demonstrate that a switch in the direction of insulin action on hepatic partitioning of fatty acyl moieties towards triacylglycerol secretion also occurs in vivo between the fed, normoinsulinaemic state and the fasted or severely insulin‐deficient states. The partitioning of fatty acids in the liver of awake, unstressed rats was studied using selective labelling of hepatic fatty acids during hyperinsulinaemic‐euglycaemic clamps achieved through the use of hepatocyte‐targeted liposome‐encapsulated insulin preparations. The data show that, whereas in the fed, normoinsulinaemic state, insulinization of the liver raises the proportion of fatty acids directed towards secreted triacylglycerol, in the fasted or insulin‐deficient states, insulin inhibits the partitioning of acyl moieties into secreted triacylglycerol. These data show that observations on the direction of insulin action on hepatic triacylglycerol secretion obtained using isolated perfused rat livers are reflected in the effects of the hormone on hepatic fatty acid partitioning in vivo. They offer an explanation for the positive relationship between chronic hyperinsulinaemia, hepatic VLDL‐triacylglycerol secretion and hypertriglyceridaemia observed previously in insulin‐resistant states.
Alkaline phosphatase from the Antarctic strain TAB5
Authors: Rina Maria , Pozidis Charalambos , Mavromatis Konstantinos , Tzanodaskalaki Maria , Kokkinidis Michael , Bouriotis Vassilis
Published: March 2003

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology‐based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino‐acid sequence can be interpreted in terms of potential psychrophilic adaptations.

The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity.

The use of this heat‐labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.
Molecular mode of interaction of plant amine oxidase with the mechanism‐based inhibitor 2‐butyne‐1,4‐diamine
Authors: Frébort Ivo , Šebela Marek , Svendsen Ib , Hirota Shun , Endo Masaaki , Yamauchi Osamu , Bellelli Andrea , Lemr Karel , Peč Pavel
Published: March 2003

2‐Butyne‐1,4‐diamine (DABI) is a mechanism‐based inhibitor of copper‐containing plant amine oxidases; the number of turnovers that leads to enzyme inactivation is ≈ 20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295 nm and gives coloured derivatives with p‐dimethylaminobenzaldehyde and p‐dimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the p‐dimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stopped‐flow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4‐amino‐2‐butynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.
Mouse Hsp25, a small heat shock protein
Authors: Lindner Robyn A. , Carver John A. , Ehrnsperger Monika , Buchner Johannes , Esposito Gennaro , Behlke Joachim , Lutsch Gudrun , Kotlyarov Alexey , Gaestel Matthias
Published: March 2003

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C‐terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild‐type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild‐type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize α‐lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild‐type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C‐terminal extension of Hsp25 leads to induction of extra C‐terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol‐reduced α‐lactalbumin by 1H NMR spectroscopy indicates that the C‐terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C‐terminal extension in mammalian sHsps is required for full chaperone activity.
Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells
Authors: Hubbard Michael J. , McHugh Nicola J. , Carne Diana L.
Published: March 2003

Recently we cloned and described ERp29, a novel 29‐kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze–thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C‐terminal tetrapeptide (KEEL motif) confers effective ER‐retention in enamel cells. ERp29 behaved as a single species (≈ 40 kDa) during size‐exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up‐regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory‐protein synthesis. Unlike other reticuloplasmins, ERp29 was down‐regulated during enamel mineralization and thereby dissociated from a calcium‐handling role. Tissue‐specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein‐processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.
Sequencing and analysis of the Methylococcus capsulatus (Bath) soluble methane monooxygenase genes
Authors: Coufal David E. , Blazyk Jessica L. , Whittington Douglas A. , Wu Wayne W. , Rosenzweig Amy C. , Lippard Stephen J.
Published: March 2003

The soluble methane monooxygenase (sMMO) hydroxylase is a prototypical member of the class of proteins with non‐heme carboxylate‐bridged diiron sites. The sMMO subclass of enzyme systems has several distinguishing characteristics, including the ability to catalyze hydroxylation or epoxidation chemistry, a multisubunit hydroxylase containing diiron centers in its α subunits, and the requirement of a coupling protein for optimal activity. Sequence homology alignment of known members of the sMMO family was performed in an effort to identify protein regions giving rise to these unique features. DNA sequencing of the Methylococcus capsulatus (Bath) sMMO genes confirmed previously identified sequencing errors and corrected two additional errors, each of which was confirmed by at least one independent method. Alignments of homologous proteins from sMMO, phenol hydroxylase, toluene 2‐, 3‐, and 4‐monooxygenases, and alkene monooxygenase systems revealed an interesting set of absolutely conserved amino‐acid residues, including previously unidentified residues located outside the diiron active site of the hydroxylase. By mapping these residues on to the M. capsulatus (Bath) sMMO hydroxylase crystal structure, functional and structural roles were proposed for the conserved regions. Analysis of the active site showed a highly conserved hydrogen‐bonding network on one side of the diiron cluster but little homology on the opposite side, where substrates are presumed to bind. It is suggested that conserved residues on the hydroxylase surface may be important for protein–protein interactions with the reductase and coupling ancillary proteins and/or serve as part of an electron‐transfer pathway. A possible way by which binding of the coupling protein at the surface of the hydroxylase might transfer information to the diiron active site at the interior is proposed.
The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids
Authors: Jiang Weiping , Gupta Dwijendra , Gallagher Dan , Davis Scott , Bhavanandan Veer P.
Published: March 2003

We previously elucidated five distinct protein domains (I–V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of ≈ 55 tandem repeats of 329 amino acids and that domains III–V are encoded by a 58.4‐kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2.2–q2.3. The amino‐acid sequence of six tandem repeats (two full and four partial) were found to have only 92–94% identities. We propose that the variability in the amino‐acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68–94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81‐amino‐acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N‐terminal and C‐terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.
Design syntheses and mitochondrial complex I inhibitory activity of novel acetogenin mimics
Authors: Kuwabara Kaoru , Takada Motoyuki , Iwata Jun , Tatsumoto Kaorin , Sakamoto Kimitoshi , Iwamura Hajime , Miyoshi Hideto
Published: March 2003

Some natural acetogenins are the most potent inhibitors of mitochondrial complex I. These compounds are characterized by two functional units [i.e. hydroxylated tetrahydrofuran (THF) and α,β‐unsaturated γ‐lactone ring moieties] separated by a long alkyl spacer. To elucidate which structural factors of acetogenins, including their active conformation, are crucial for the potent inhibitory activity we synthesized a novel bis‐acetogenin and its analogues possessing two γ‐lactone rings connected to bis‐THF rings by flexible alkyl spacers. The inhibitory potency of the bis‐acetogenin with bovine heart mitochondrial complex I was identical to that of bullatacin, one of the most potent natural acetogenins. This result indicated that one molecule of the bis‐acetogenin does not work as two reactive inhibitors, suggesting that a γ‐lactone and the THF ring moieties act in a cooperative manner on the enzyme. In support of this, either of the two ring moieties synthesized individually showed no or very weak inhibitory effects. Moreover, combined use of the two ring moieties at various molar ratios exhibited no synergistic enhancement of the inhibitory potency. These observations indicate that both functional units work efficiently only when they are directly linked by a flexible alkyl spacer. Therefore, some specific conformation of the spacer must be important for optimal positioning of the two units in the enzyme. Furthermore, the α,β‐unsaturated γ‐lactone, the 4‐OH group in the spacer region, the long alkyl tail attached to the THF unit and the stereochemistry surrounding the hydroxylated bis‐THF rings were not crucial for the activity, although these are the most common structural features of natural acetogenins. The present study provided useful guiding principles not only for simplification of complicated acetogenin structure, but also for further wide structural modifications of these molecules.
Cancer‐associated alternative usage of multiple promoters of human GalCer sulfotransferase gene
Authors: Tsuda Masayuki , Egashira Masayuki , Niikawa Norio , Wada Yoshinao , Honke Koichi
Published: March 2003

The galactosylceramide sulfotransferase (cerebroside sulfotransferase, CST) (EC 2.8.2.11) gene is highly expressed in human renal cancer cells. To elucidate the regulatory mechanism of its gene expression, we have determined the genomic organization of the human CST gene. The gene comprises at least four exons and spans about 20 kb. The coding region is located in exons 3 and 4. To determine the transcription initiation sites, 5′‐rapid amplification of cDNA ends analysis was performed using mRNA obtained from four human renal cancer cell lines, SMKT‐R1–R4, and normal human renal proximal tubular cells. We found four transcription initiation sites and alternative usage of six exons corresponding to the 5′‐untranslated region in cancer cells. On the other hand, the only transcript beginning at exon 1a was observed in normal cells. Using reverse transcriptase‐PCR analysis, we confirmed that all of the exons 1a–d, especially exons 1c and 1d, are used as a transcription initiation site in cancer cells, whereas only exons 1a and 1b, mostly 1a, are utilized in normal cells. Analyzing the protein production from the mRNA variants with different 5′‐UTRs, we found that all the transcripts examined produced the identical proteins. These observations suggest that the aberrant usage of transcription initiation sites flanked with promoters/enhancers is involved in the cancer‐associated expression of the CST gene. Furthermore, this gene was assigned to human chromosome 22q12 by means of fluorescence in situ hybridization.
Polytomella spp. growth on ethananol
Authors: Atteia Ariane , van Lis Robert , Ramírez Jorge , González‐Halphen Diego
Published: March 2003

A defined medium with ethanol as sole carbon source was devised for growth of the colorless, unicellular alga Polytomella spp. Cell density on this carbon source was related to extracellular pH. An acidic pH was required for ethanol utilization; best yields were obtained at pH 3.7. Spectroscopic analysis of the cells showed that the concentration of cytochrome c per cell was 40% higher than at pH 6.0; the concentrations of cytochrome a606 (cytochrome c oxidase) and b566 (cytochrome bc1 complex) were the same. A soluble cytochrome c550 was purified from cells grown at pH 3.7 and characterized by peptide sequencing as the 12‐kDa cytochrome c550 of the mitochondrial respiratory chain. Immunoblots of total cell proteins showed higher accumulation of cytochrome c550 at pH 3.7 than at pH 6.0. RNA blot analysis gave clear evidence of the abundance of c550 transcript in cells grown at pH 3.7. The amount of mitochondrial proteins obtained from cells grown at pH 3.7 was twofold higher than that of cells grown at pH 6.0. Mitochondria isolated from both cell types readily oxidized succinate, malate or ethanol. The rates of oxygen uptake were 20–25% higher in mitochondria from cells grown at pH 3.7. Cyanide and antimycin A inhibited respiration with succinate up to 95% in both types of mitochondria. The participation of cytochrome c550 in mitochondrial electron transport from succinate to oxygen was shown by spectral measurements.
Characterization and molecular cloning of two different type 2 ribosome‐inactivating proteins from the monocotyledonous plant Polygonatum multiflorum
Authors: Van Damme Els J. M. , Hao Qiang , Charels Diana , Barre Annick , Rougé Pierre , Van Leuven Fred , Peumans Willy J.
Published: March 2003

Leaves of the monocotyledonous plant Polygonatum multiflorum L. (Solomon's seal) contain besides a monocot mannose‐binding lectin two galactose/N‐acetylgalactosamine (Gal/GalNAc)‐binding type 2 ribosome‐inactivating proteins (RIPs). Both RIPs were purified using a combination of classical protein purification techniques and affinity chromatography. Although both RIPs consist of protomers of 65 kDa, the P. multiflorum RIP monomer (PMRIPm) occurs as a monomer of approximately 60 kDa, whereas the tetramer (PMRIPt) is a tetramer of 240 kDa. Both RIPs exhibit similar RNA N‐glycosidase activity but differ in their specific agglutination activity and carbohydrate‐binding specificity, PMRIPt being a GalNAc‐specific lectin whereas PMRIPm is Gal/GalNAc‐specific. Toxicity tests indicated that both Polygonatum RIPs exhibit a very low cytotoxicity towards human and animal cells. Analysis of the genomic clones encoding both RIPs revealed a high degree of sequence similarity to other type 2 RIPs. Molecular modelling confirmed that both Polygonatum RIPs have a similar structure to ricin.
Proteomic analysis of the Escherichia coli outer membrane
Authors: Molloy Mark P. , Herbert Ben R. , Slade Martin B. , Rabilloud Thierry , Nouwens Amanda S. , Williams Keith L. , Gooley Andrew A.
Published: March 2003

Outer membrane proteins (OMPs) of Gram‐negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram‐negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two‐dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K‐12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS‐PROT release 37 and predicted to separate within the range of pH 4–7 and molecular mass 10–80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two‐dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.
Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl‐reactive agent
Authors: Rhee Hae Jin , Kim Geum‐Yi , Huh Jae Wan , Kim Seung‐Wook , Na Doe Sun
Published: March 2003

Annexin I (also called lipocortin 1) is a 37‐kDa member of the annexin family of proteins. It has been proposed to be involved in the regulation of cell growth and differentiation, apoptosis, and inflammation. Previously, we have reported that annexin I displays a chaperone‐like function (Kim, G.Y., Lee, H.B., Lee, S.O., Rhee, H.J. & Na, D.S. (1997) Biochem. Mol. Biol. Int.43, 521–528). To determine the possibility that annexin I is a stress protein, we examined whether expression of annexin I and annexin I mRNA increases in response to stresses in A549 and HeLa cells. Treatments of cells with heat, hydrogen peroxide or sodium arsenite resulted in (a) an increase in annexin I and annexin I mRNA and (b) translocation of annexin I from the cytoplasm to the nucleus and perinuclear region. The annexin I gene promoter region, cloned upstream of a reporter gene, was inducible in response to heat, hydrogen peroxide, and sodium arsenite. These results indicate that annexin I serves as a stress protein and annexins may constitute a new class of stress proteins.
Sulfhydryls of tubulin
Authors: Roychowdhury Manami , Sarkar Nabanita , Manna Tapas , Bhattacharyya Shankar , Sarkar Taradas , BasuSarkar Pampi , Roy Siddhartha , Bhattacharyya Bhabatarak
Published: March 2003

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three‐dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 °C leads to loss of self‐assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 °C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8‐anilino‐1‐naphthalenesulfonate (ANS) and bis‐ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.