bluuebunny/crossref_metadata_2025 huggingface dataset:
Response of native and denatured hen lysozyme to high pressure studied by 15N/1H NMR spectroscopy
Authors: Kamatari Yuji O. , Yamada Hiroaki , Akasaka Kazuyuki , Jones Jonathan A. , Dobson Christopher M. , Smith Lorna J.
Published: March 2003

High‐pressure 15N/1H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 m urea, over the pressure range 30–2000 bar. Most 1H and 15N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 ± 0.101 p.p.m. (1H) and 0.51 ± 0.36 p.p.m. (15N). The shifts indicate that the hydrogen bonds formed to carbonyl groups or water molecules by the backbone amides are, on average, shortened by ≈ 0.02 Å as a result of pressure. In native lysozyme, six residues in the β domain or at the α/β domain interface have anomalously large nonlinear 15N and 1H chemical‐shift changes. All these residues lie close to water‐containing cavities, suggesting that there are conformational changes involving these cavities, or the water molecules within them, at high pressure. The pressure‐induced 1H and 15N shifts for lysozyme denatured in 8 m urea are much more uniform than those for native lysozyme, with average backbone amide shifts of 0.081 ± 0.029 p.p.m. (1H) and 0.57 ± 0.14 p.p.m. (15N). The results show that overall there are no significant variations in the local conformational properties of denatured lysozyme with pressure, although larger shifts in the vicinity of a persistent hydrophobic cluster indicate that interactions in this part of the sequence may rearrange. NMR diffusion measurements demonstrate that the effective hydrodynamic radius of denatured lysozyme, and hence the global properties of the denatured ensemble, do not change detectably at high pressure.
Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor
Authors: Mouillet Jean‐François , Henrich Vincent C. , Lezzi Markus , Vögtli Martin
Published: March 2003

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N‐terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full‐length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentiallly controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand‐independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.
Trichosanthin interacts with acidic ribosomal proteins P0 and P1 and mitotic checkpoint protein MAD2B
Authors: Chan Siu‐Hong , Hung Fanny S.‐J. , Chan Denize S.‐B. , Shaw Pang‐Chui
Published: March 2003

Trichosanthin is a ribosome‐inactivating protein with multiple pharmacological properties. By a yeast two‐hybrid system, ribosomal phosphoproteins P0 and P1 and a putative mitotic checkpoint protein, MAD2B, were found to interact with an active‐site mutated trichosanthin (TCS). The interactions were verified by an in vitro binding assay of recombinant wild‐type TCS and target proteins. The interaction domain of P0 was mapped to amino acids 220–273, which had been previously reported to be involved in the interaction with P1 and P2 in yeast. Consistent with our previous finding that the last seven residues of TCS are not essential for an active conformation, the same deletion did not affect the interaction with P0. Our present study suggests that TCS may disrupt the binding of elongation factors to the P‐complex, in addition to the well‐known N‐glycosidase activity for ribosome inactivation.
Defects of type I procollagen metabolism correlated with decrease of prolidase activity in a case of lethal osteogenesis imperfecta
Authors: Galicka Anna , Wolczyñski Slawomir , Anchim Tomasz , Surazyñski Arkadiusz , Lesniewicz Ryszard , Palka Jerzy
Published: March 2003

We have studied the structure and metabolism of type I procollagen in a case of perinatal lethal osteogenesis imperfecta (OI) type II. Cultured skin fibroblasts from the proband synthesized both normal and abnormal forms of type I procollagen. Some abnormal, overmodified molecules were secreted by OI cells, although less efficiently than normal molecules from control cells. The OI fibroblasts accumulated large amounts of abnormal proα1(I) and proα2(I) chains intracellularly. The extracellular collagenolytic activity was decreased compared to control cells. Furthermore, OI cells produced less type I procollagen and demonstrated lower capacity to synthesize DNA than control cells. We have found that in contrast to prolinase activity, the activity of prolidase (an enzyme essential for collagen synthesis and cell growth) is also significantly reduced in OI cells. No differences were found in the amount of the enzyme protein recovered from both the OI and control cells. However, we found that expressions of β1 integrin and insulin‐like growth factor‐I receptor (receptors known to play an important role in up regulation of prolidase activity) were decreased in OI cells compared to control cells. The decrease in prolidase activity may provide an important mechanism of altered cell growth and collagen metabolism involved in producing the perinatal lethal form of the OI phenotype.
Growth factor‐stimulated protein synthesis is inhibited by sodium orthovanadate
Authors: Viñals Francesc , McKenzie Fergus R. , Pouysségur Jacques
Published: March 2003

The study of intracellular signaling pathways has been aided by the use of sodium orthovanadate, a cell‐permeable inhibitor of tyrosine phosphatases. However, long‐term addition of sodium orthovanadate is often cytotoxic. In this study we demonstrate that the growth factor‐mediated increase in the rate of protein synthesis was inhibited by sodium orthovanadate. This effect of sodium orthovanadate was dose‐dependent, with an IC50 of 40 µm and maximal inhibition obtained at 100 µm. As a consequence, the fetal bovine serum‐mediated induction of the immediate‐early genes, c‐Fos and MKP‐1, at the protein level was inhibited by orthovanadate. Orthovanadate’s ability to attenuate protein synthesis was partially reversible, and was no longer evident when the agent was added 6 h after addition of growth factor to cells. Analysis of several elements of signaling pathways which are known to regulate protein synthesis in a positive manner (p42/p44 MAPK, AKT and p70 S6K stimulation, and hyperphosphorylation of PHAS‐I) were not inhibited but rather were stimulated by orthovanadate. Thus, sodium orthovanadate is a potent inhibitor of growth factor‐stimulated protein synthesis independent of p42/p44 MAPK or PI3K‐p70 S6K activation.
Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum
Authors: Wittmann Christoph , Heinzle Elmar
Published: March 2003

In the present work, a novel comprehensive approach of 13C‐tracer studies with labeling measurements by MALDI‐TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI‐TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 µL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI‐TOF MS in tracer experiments either with 1‐13C glucose or with mixtures of 13C6/12C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose‐6‐phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI‐TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.
Activation of pro‐astacin
Authors: Möhrlen Frank , Baus Stefanie , Gruber Anja , Rackwitz Hans‐Richard , Schnölzer Martina , Vogt Günter , Zwilling Robert
Published: March 2003

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., Stöcker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys.337, 300–307]. To localize and follow the processing of pro‐astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro‐astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro‐astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro‐astacin, present in the cardiac stomach. We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro‐astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position −1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino‐acid residues in this position, in these cases also astacin‐specific cleavage would be possible.
Characterization of a functional serum response element in the Actin403 gene promoter from the crustacean Artemia franciscana
Authors: Casero Marie‐Carmen , Sastre Leandro
Published: March 2003

The serum response factor (SRF) activates expression of several genes in response to growth factors present in serum. SRF also regulates the expression of tissue‐specific genes, including those in vertebrate muscles. An SRF‐binding site (CArG box) present in the Artemia franciscana Actin403 promoter was shown to be necessary for transcriptional activity in cultured cells from Drosophila melanogaster and mammals. This DNA region bound mammalian and Drosophila SRFs in vitro and mediated transcriptional activation of the Actin403 promoter in response to serum, phorbol esters and lysophosphatidic acid in transfected cultured mammalian cells. Mutations in the CArG box greatly reduced promoter activity and stimulation by extracellular compounds.
Complete primary structure of rainbow trout type I collagen consisting of α1(I)α2(I)α3(I) heterotrimers
Authors: Saito Masataka , Takenouchi Yoshitaka , Kunisaki Naomichi , Kimura Shigeru
Published: March 2003

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be α1(I)α2(I)α3(I) and [α1(I)]2α2(I), respectively. The occurrence of α3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP‐13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 °C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N‐terminal and/or a part of triple‐helical domains of proα(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple‐helical domain of each proα(I) chain had 338 uninterrupted Gly‐X‐Y triplets consisting of 1014 amino acids and was unique in its high content of Gly‐Gly doublets. In particular, the bonyfish‐specific α(I) chain, proα3(I) was characterized by the small number of Gly‐Pro‐Pro triplets, 19, and the large number of Gly‐Gly doublets, 38, in the triple‐helical domain, compared to 23 and 22, respectively, for proα1(I). The small number of Gly‐Pro‐Pro and the large number of Gly‐Gly in proα3(I) was assumed to partially loosen the triple‐helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proα3(I) had diverged from proα1(I). This study is the first report of the complete primary structure of fish type I procollagen.
Mutation of residues in the coenzyme binding pocket of Dopa decarboxylase
Authors: Bertoldi Mariarita , Castellani Silvia , Voltattorni Carla Borri
Published: March 2003

Residues D271, H192, H302 and N300 of l‐3,4‐dihydroxyphenylalanine decarboxylase (DDC), a homodimeric pyridoxal 5′‐phosphate (PLP) enzyme, were mutated in order to acquire information on the catalytic mechanism. These residues are potential participants in catalysis because they belong to the common PLP‐binding structural motif of group I, II and III decarboxylases and other PLP enzymes, and because they are among the putative active‐site residues of structural modelled rat liver DDC. The spectroscopic features of the D271E, H192Q, H302Q and N300A mutants as well as their dissociation constants for PLP suggest that substitution of each of these residues causes alteration of the state of the bound coenzyme molecule and of the conformation of aromatic amino acids, possibly in the vicinity of the active site. This supports, but does not prove, the possibility that these residues are located in the coenzyme‐binding cleft. Interestingly, mutation of each residue generates an oxidative decarboxylase activity towards l‐3,4‐dihydroxyphenylalanine (l‐Dopa), not inherent in the wild‐type in aerobiosis, and reduces the nonoxidative decarboxylase activity of l‐Dopa from 3‐ to 390‐fold. The partition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 × 10−4 for N300A mutant to 946 × 10−4 for H302Q mutant. Unlike wild‐type enzyme, the mutants catalyse these two reactions to the same extent either in the presence or absence of O2. In addition, all four mutants exhibit an extremely low level of the oxidative deaminase activity towards serotonin with respect to wild‐type. All these findings demonstrate that although D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship among the integrity of the PLP cleft, the productive binding of O2 and the transition to a closed conformational state of DDC is discussed.
Possible involvement of the membrane‐bound form of peroxiredoxin 4 in acrosome formation during spermiogenesis of rats
Authors: Sasagawa Isoji , Matsuki Shingo , Suzuki Yasuhiro , Iuchi Yoshihito , Tohya Kazuo , Kimura Michio , Nakada Teruhiro , Fujii Junichi
Published: March 2003

Peroxiredoxins are novel peroxidases that exhibit divergent biological functions. The fourth member, Prx4, is synthesized with a signal sequence and, after processing, secreted as a 27‐kDa form in most tissues. A 31‐kDa Prx4 which corresponds to the unprocessed, membrane‐bound form is found only in testis. This study was undertaken in order to investigate the role of the membrane‐bound Prx4 during spermiogenesis in rats. The Prx4 transcript was observed in testes at 14 days of age, the age before puberty, and its level increased only slightly during aging. Only the secretable form of Prx4 was present before 30 days of age, but the membrane‐bound Prx4 became detectable in adult testes, although sum of the two forms appeared to be the same after 21 days old. While weak immunoreactivity to the Prx4 antibody was detected in spermatogenic cells for all ages, a strong immunoreactivity was observed in the elongating spermatid and residual body of adult testis. Prx4 was present in the lumen of the endoplasmic reticulum, Golgi bodies, and the perinuclear space in young rat testes. However, the localization of Prx4 was restricted to membranes of the acrosomal vesicle of the elongated spermatid and was not detected in spermatozoon. Once spermiogenesis is accomplished, vesicles containing the membrane‐bound Prx4 were released into the residual body, along with residual membranous components. Thus the conversion of the soluble form to the membrane‐bound form may have a role in the acrosome formation during vesicular reorganization during spermiogenesis.
Cell surface human α‐L‐fucosidase
Authors: Cordero Oscar J. , Merino Ana , de la Cadena María Páez , Bugía Begoña , Nogueira Montserrat , Viñuela Juan E. , Martínez‐Zorzano Vicenta S. , de Carlos Alejandro , Rodríguez‐Berrocal F. Javier
Published: March 2003

The acid α‐l‐fucosidase is usually found as a soluble component of lysosomes where fucoglycoconjugates are degraded. In the present investigation, we have demonstrated the existence of a cell surface protein with enzymatic α‐l‐fucosidase activity that crossreacts specifically with a rabbit anti‐(α‐l‐fucosidase) Ig. By different approaches, this α‐l‐fucosidase, which represents 10–20% of the total cellular fucosidase activity, was detected in all the tested human cells (hemopoietic, epithelial, mesenchymal). Two bands of ≈ 43–49 kDa were observed, although theoretical data support the possibility of having the same genetic origin that the known 50 to 55‐kDa Mrα‐l‐fucosidase. We speculate about an alternative traffic pathway for the plasma membrane α‐l‐fucosidase to work on the rapid turnover of glycoproteins.
The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60
Authors: Levy‐Rimler Galit , Viitanen Paul , Weiss Celeste , Sharkia Rajach , Greenberg Anat , Niv Adina , Lustig Ariel , Delarea Yacov , Azem Abdussalam
Published: March 2003

Mitochondrial chaperonins are necessary for the folding of newly imported and stress‐denatured mitochondrial proteins. The goal of this study was to investigate the structure and function of the mammalian mitochondrial chaperonin system. We present evidence that the 60 kDa chaperonin (mt‐cpn60) exists in solution in dynamic equilibrium between monomers, heptameric single rings and double‐ringed tetradecamers. In the presence of ATP and the 10 kDa cochaperonin (mt‐cpn10), the formation of a double ring is favored. ADP at very high concentrations does not inhibit malate dehydrogenase refolding or ATP hydrolysis by mt‐cpn60 in the presence of mt‐cpn10. We propose that the cis (mt‐cpn60)14·nuleotide·(mt‐cpn10)7 complex is not a stable species and does not bind ADP effectively at its trans binding site.
Transfection of the c‐erbB2/neugene upregulates the expression of sialyl Lewis X, α1,3‐fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell line
Authors: Liu Fei , Qi Hui‐Ling , Zhang Ying , Zhang Xia‐Ying , Chen Hui‐Li
Published: March 2003

The pCMV4 plasmid containing the cancer‐promoting gene, c‐erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the ‘neo’ selectable marker. Several clones showing stable expression of c‐erbB2/neuwere established and characterized by determination of c‐erbB2/neumRNA and its encoded protein p185. Expression of Lewis antigens and α1,3‐fucosyltransferases and the biological behavior of 7721 cells after c‐erbB2/neutransfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLexexpression on the surface of mock cells was high, whereas expression of SDLex, Lexand SLeawas absent or negligible. This is compatible with the abundant expression of α1,3‐fucosyltransferase VII, very low expression of αfucosyltransferase III/VI, and almost absent expression of α1,3‐fucosyltransferase IV in the mock cells. After transfection of c‐erbB2/neu, expression of SLexand α1,3‐fucosyltransferase VII were simultaneously elevated, but that of αfucosyltransferase III/VI was not altered. The expression of both SLexand α1,3‐fucosyltransferase VII correlated positively with the expression of c‐erbB2/neuin different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P‐selectin, as well as cell migration and invasion, were increased in c‐erbB2/neu‐transfected cells. These increases also correlated positively with the expression intensities of c‐erbB2/neu, SLexand α1,3‐fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLex(KM93) and SDLex(FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P‐selectin and cell migration and invasion. mAbs to SDLexand SLeadid not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of α1,3‐fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c‐erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of α1,3‐fucosyltransferase VII and its specific product, SLex, and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the c‐erbB2/neugene is proposed to be a metastasis‐promoting gene, and its effects are at least partially mediated by the increased expression of α1,3‐fucosyltransferase VII and SLex.
The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning
Authors: Rodina Elena V. , Vainonen Yuliya P. , Vorobyeva Nataliya N. , Kurilova Svetlana A. , Nazarova Tatjana I. , Avaeva Svetlana M.
Published: March 2003

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42→Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH‐independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition.
Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart
Authors: Shiraki Kentaro , Nishikori Shingo , Fujiwara Shinsuke , Hashimoto Hiroshi , Kai Yasushi , Takagi Masahiro , Imanaka Tadayuki
Published: March 2003

Comparison of the conformational stability of an O6‐methylguanine‐DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk‐MGMT), and its mesophilic counterpart C‐terminal Ada protein from Escherichia coli (Ec‐AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk‐MGMT unfolded at Tm = 98.6 °C, which was 54.8 °C higher than the unfolding temperature of Ec‐AdaC. The maximum free energy (ΔGmax) of the proteins were different; the value of Tk‐MGMT (42.9 kJ·mol−1 at 29.5 °C) was 2.6 times higher than that of Ec‐AdaC (16.6 kJ·mol−1 at 7.4 °C). The high conformational stability of Tk‐MGMT was attributed to a 1.6‐fold higher enthalpy value than that of Ec‐AdaC. In addition, the ΔGmax temperature of Tk‐MGMT was considerably higher (by 22.1 °C). The apparent heat capacity of denaturation (ΔCp) of Tk‐MGMT was 0.7‐fold lower than that of Ec‐AdaC. These three synergistic effects, increasing ΔGmax, shifted ΔG vs. temperature curve, and low ΔCp, give Tk‐MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk‐MGMT possessed higher stability than Ec‐AdaC in all conditions studied. These results indicate that the high stability of Tk‐MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.
Hormonal regulation of multiple promoters of the rat mitochondrial glycerol‐3‐phosphate dehydrogenase gene
Authors: Weitzel Joachim M. , Kutz Sabine , Radtke Christiane , Grott Stefan , Seitz Hans J.
Published: March 2003

Rat mitochondrial glycerol‐3‐phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue‐specific manner. Here, we demonstrate that thyroid hormone (3,5,3′‐tri‐iodo‐l‐thyronine) and steroid hormone but not the peroxisome proliferator clofibrate and retinoic acid stimulate the activation of the ubiquitous promoter B in a receptor‐dependent manner, whereas the more tissue‐restricted promoters A and C are not inducible by these hormones. Thyroid hormone action is mediated by a direct repeat +4 (DR+4) hormone‐response element as identified by deletion and mutation analyses of promoter B in transient transfection analyses. The DR+4 element was able to bind to an in vitro translated thyroid hormone receptor in band‐shift and supershift experiments. The hormone‐response element comaps with a recognition site for the transcription factor Sp1, suggesting complex regulation of this sequence element. Mutation of this Sp1‐recognition site reduces the basal promoter B activity dramatically in HepG2 and HEK293 cells in transient transfection and abolishes the binding of Sp1 in band‐shift experiments. As demonstrated by Western‐blot experiments, administration of tri‐iodothyronine to euthyroid rats increases hepatic mGPDH protein concentrations in vivo. As it has recently been reported that human mGPDH promoter B is not regulated by tri‐iodothyronine, this is the first example of a differentially tri‐iodothyronine‐regulated orthologous gene promoter in man and rat.
Estrogen induces a rapid secretion of amyloid β precursor protein via the mitogen‐activated protein kinase pathway
Authors: Manthey Dieter , Heck Stefanie , Engert Stefanie , Behl Christian
Published: March 2003

The female sex hormone estrogen (17β‐estradiol; E2) may function as a neurohormone and has multiple neuromodulatory functions in the brain. Its potent neuroprotective activities can be dependent and independent of estrogen receptors (ERs). In addition, E2 influences the processing of the amyloid β precursor protein (APP), one central step in the pathogenesis of Alzheimer's disease. Here, we show: (a) that physiological concentrations of E2 very rapidly cause an increased release of secreted nonamyloidogenic APP (sAPPα) in mouse hippocampal HT22 and human neuroblastoma SK‐N‐MC cells; and (b) that this effect is mediated through E2 via the phosphorylation of extracellular‐regulated kinase 1 and 2 (ERK1/2), prominent members of the mitogen‐activated protein kinase (MAPK) pathway. Furthermore, we show that the activation of MAPK‐signaling pathway and the enhancement of the sAPP release is independent of ERs and could be induced by E2 to a similar extent in neuronal cells either lacking or overexpressing a functional ER.
The molecular interaction of human salivary histatins with polyphenolic compounds
Authors: Wróblewski Karol , Muhandiram Ranjith , Chakrabartty Avi , Bennick Anders
Published: March 2003

Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan‐3‐ol ester related to condensed tannins (polymerized products of flavan‐3‐ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst–polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1‐mm dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.
Metabolic conversion as a pre‐receptor control mechanism for lipophilic hormones
Authors: Nobel Stefan , Abrahmsen Lars , Oppermann Udo
Published: March 2003

The majority of physiological effects mediated by steroids, retinoids and thyroids is accomplished by binding to members of the nuclear receptor superfamily of ligand activated transcription factors. The complex specific effects of lipid hormones depend not only on receptor expression, distribution and interactions, but also on the availability and metabolic conversion of the hormone itself. The cell‐specific metabolic activation of inactive hormone precursors introduces a further level of hormonal regulation, and constitutes an important concept in endocrinology. The metabolic reactions carried out are achieved by dehydrogenases/reductases, hydroxylases and other enzymes, acting on ligands of the steroid/thyroid/retinoic hormone receptor superfamily. The concept implies that these tissue‐ and cell‐specific metabolic conversions contribute to lipid hormone action, thus pointing to novel targets in drug development. All components of this signalling system, the hormone compounds, the receptor proteins, and modifying enzyme families originate from an early metazoan date, emphasizing the essential nature of all elements for development and diversification of vertebrate life.
Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect onCandida albicans
Authors: Kim Dae‐Hee , Lee Dong Gun , Kim Kil Lyong , Lee Younghoon
Published: March 2003

Tenecin  3 is a glycine‐rich, antifungal protein of 78 residues isolated from the insectTenebrio molitorlarva. As an initial step towards understanding the antifungal mechanism of tenecin  3, we examined how this protein interacts with the pathogenic fungusCandida albicansto exert its antifungal action. Tenecin  3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential ofC. albicansby the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin  3 is rapidly internalized into the cytoplasmic space in energy‐dependent and temperature‐dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin  3 into the cytoplasm ofC. albicansis mediated by a fungal cellular process. The internalized tenecin  3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization.
Dimerization, stability and electrostatic properties of porcine β‐lactoglobulin
Authors: Ugolini Raffaella , Ragona Laura , Silletti Erika , Fogolari Federico , Visschers Ronald W. , Alting Arno C. , Molinari Henriette
Published: March 2003

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico‐chemical characterization of porcine β‐lactoglobulin, purified from pooled milk: size‐exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine β‐lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer–dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess α helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine β‐lactoglobulin in the crystal structure. Interaction studies revealed that porcine β‐lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.
Focal adhesion kinase affects the sensitivity of human hepatocellular carcinoma cell line SMMC‐7721 to tumor necrosis factor‐α/cycloheximide‐induced apoptosis by regulating protein kinase B levels
Authors: Fang Yi , Wang Liying , Jin Jiawei , Zha Xiliang
Published: March 2003

Most cell lines are resistant to tumor necrosis factor‐α (TNF‐α) cytotoxicity and require cotreatment of TNF‐α with cycloheximide (Chx) to undergo apoptosis. Recently, the serine/threonine protein kinase, protein kinase B has been demonstrated to protect cells from apoptosis induced by TNF‐α. In this study, we have shown that the human hepatocellular carcinoma cell line, SMMC‐7721, was insensitive to TNF‐α cytotoxicity and underwent apoptosis quickly in the presence of TNF‐α and Chx. PKB levels decreased during TNF‐α/Chx‐induced apoptosis. No significant change in PKB levels was found in the presence of TNF‐α or Chx alone. It seemed that the level of PKB closely correlated with apoptosis. The protein level of focal adhesion kinase (FAK) was reduced by 66% by transfecting FAK antisense cDNA recombinant vector into SMMC‐7721 cells. We determined the apoptosis‐induced effect of TNF‐α/Chx on the FAK antisense cDNA transfectant cells. The results indicated that the percentage of apoptotic cells was enhanced at lower doses of TNF‐α (10, 20 or 50 U·mL−1) and decreased at a higher dose of TNF‐α (1000 U·mL−1) in the transfected cells as compared to the control. Correspondingly, in the FAK antisense cDNA transfectant cells treated with lower doses of TNF‐α in presence of 10 µg·mL−1 Chx, the PKB level was lower, but in the FAK antisense cDNA transfectants treated with higher doses of TNF‐α in presence of 10 µg·mL−1 Chx, the PKB level was higher. In response to TNF‐α alone, FAK antisense cDNA transfectants showed a decrease in the level of PKB. However, in the case of TNF‐α cotreated with wortmannin, a specific inhibitor of phosphatidylinositol 3‐kinase (PtdIns3K), the FAK antisense cDNA transfectants produced significantly less amounts of PKB than the control. It seemed that FAK could stimulate PKB levels through a pathway not involving PtdIns3K. These results suggest that FAK can affect the sensitivity of SMMC‐7721 cells to TNF‐α/Chx‐induced apoptosis in a biphasic manner by regulating PKB levels.
A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis
Authors: Ohshima Toshihisa , Nunoura‐Kominato Naoki , Kudome Tomomi , Sakuraba Haruhiko
Published: March 2003

A novel NADH‐dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH‐dependent glyoxylate reductase was purified ≈ 560‐fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of ≈ 37 kDa. The optimum pH and temperature for enzyme activity were ≈ 6.5 and 90 °C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 °C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was ≈ one‐quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mm, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36 807. The amino‐acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye‐affinity chromatography were needed.
Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) β‐mannanase gene
Authors: Xu Bingze , Sellos Daniel , Janson Jan‐Christer
Published: March 2003

Using PCR, cloning and sequencing techniques, a 1.1‐kb complementary DNA fragment encoding for a β‐mannanase (mannan endo‐1,4‐β‐mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several β‐mannanases in glycoside hydrolase family 5. The β‐mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The β‐mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiaeα‐factor signal sequence. The β‐mannanase was produced in a 14‐L fermenter with an expression level of 900 mg·L−1. The expression level is strongly affected by the induction temperature. A two‐step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure β‐mannanase. However, due to post‐translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulisβ‐mannanase was close to that of the wild‐type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel β‐mannanase. This is the first time a mollusc β‐mannanase has been characterized at the DNA level.
Brood Reduction in White Storks Mediated through Asymmetries in Plasma Testosterone Concentrations in Chicks
Authors: Sasvári Lajos , Hegyi Zoltán , Péczely Péter
Published: March 2003

We hypothesized that increasing chick plasma testosterone concentrations, transmitted from the mothers via their eggs, enhances survival of their offspring and that the fitness of the young, depending on the maternal hormones, is influenced by parental quality. To test our hypotheses we distinguished the broods of white storks Ciconia ciconia L. where chicks died and those where all chicks survived. We analysed the plasma testosterone concentrations in the chicks, the ability of the chicks to be first to receive food and the mass of chicks before fledging in relation to their hatching order and recorded the body mass of parents and food mass delivered by them.

Female storks used the asymmetries in testosterone concentrations within a brood to control brood size and adjusted the number of young hatched to match the parental ability to rear offspring. Females of poor condition altered the testosterone concentrations to produce large differences between the chicks: The first‐hatched chicks, which had high plasma testosterone levels, responded faster to the feeding parent and received more food than did their younger siblings. One or two later‐hatched chicks, which had lower testosterone levels, died in these broods. Females in good condition produced small differences in testosterone concentrations between the chicks and all chicks survived in their brood. Chicks that were raised by the females of poor condition in reduced broods were heavier than chicks that were raised by females of good condition in broods where all chicks survived.

We suggest that the control of brood size by testosterone concentration, transmitted by the mother to the chicks, is a hormonal means of condition‐dependent reproductive strategy in the white stork.
Reproductive Conflict and Division of Labor inEutetramorium mocquerysi, a Myrmicine Ant Without Morphologically Distinct Female Reproductives
Authors: Heinze Jürgen , Hölldobler Bert , Alpert Gary
Published: March 2003

The myrmicine antEutetramorium mocquerysiEmery from Madagascar exhibits a unique social organization. All female individuals are similar in size and appearance; female reproductives with a distinct external morphology do not exist. Based on ovarian anatomy, however, two major types of females can be distinguished: females with six ovarioles and a spermatheca, which can mate and produce diploid offspring, and females with only two ovarioles, which lack a spermatheca but can lay unfertilized eggs. Individuals with three to five ovarioles are rare. Anatomical differences are not correlated with different roles. Both types of females were observed foraging, tending brood, and laying eggs. However, only females with six ovarioles and a spermatheca were the reproductively and socially most dominant individuals. Nestmate antagonism, which for the first time is demonstrated for an ant species belonging neither to the Ponerinae nor the Formicoxenini, consists of biting, antennation bouts, and ritualized dominance postures. In two colonies, removal of the dominant individual resulted in the destruction of all larvae and pupae.
Aggressiveness in Two Male Morphs of the Bulb Mite Rhizoglyphus robini
Authors: Radwan Jacek , Czyż Magorzata , Konior Magdalena , Kolodziejczyk Maria
Published: March 2003

We studied differences in aggressiveness between two male morphs of the bulb mite Rhizoglyphus robini (Acari: Acaridae). Heteromorphic males have a thickened and sharply terminating third pair of legs, whereas homeomorphic males have unmodified legs. In another genus (Caloglyphus) of the family Acaridae, modified legs are used during fights to stab (often mortally) other males, but fights have not so far been observed in the genus Rhizoglyphus.

In this study we showed that in Rhizoglyphus robini males fight in a way similar to that previously described for the genus Caloglyphus. The presence of females increased the duration of aggressive interactions and caused earlier initiation, but fights were also observed in the absence of females. Pairs of heteromorphic males spent more time on aggressive interactions than did mixed pairs of heteromorphs and homeomorphs or pairs of homeomorphic males. Although the proportion of pairs involved in aggressive interactions did not differ between these three morph combinations, clasping of the opponent with legs, which is a typical behaviour of killer Caloglyphus males that precedes stabbing, occurred more often between pairs of heteromorphs than between mixed pairs and pairs of homeomorphs. This is consistent with our other finding that mortality attributable to male aggression was only recorded in pairs of males containing at least one heteromorphic male. We conclude that the modified legs of heteromorphic R. robini males function as a weapon in aggressive interactions between males and that, as previously described for the genus Caloglyphus, male morphs differ in aggressiveness.