bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mechanisms underlying SHP‐1 gene expression
Authors: Tsui Hing Wo , Hasselblatt Kathleen , Martin Alberto , Mok Samuel Chi‐ho , Tsui Florence Wing Ling
Published: March 2003

SHP‐1, a protein‐tyrosine phosphatase with two src‐homology 2 domains, is expressed predominantly in hematopoietic and epithelial cells and has been implicated in numerous signaling pathways as a negative regulator. Two promoters direct the expression of human and murine SHP‐1, and two types of transcripts (I) and (II) SHP‐1, are initiated from each of these promoters. The cDNA sequences of (I)SHP‐1 and (II)SHP‐1 are identical except in the 5′ untranslated region and in the first few coding nucleotides. In this report, we show that promoter usage is similar in mouse and human hematopoietic cells, but different in epithelial cells. In human epithelial cells, only (I)SHP‐1 transcripts were expressed. In addition, 4β‐phorbol 12‐myristate 13‐acetate up‐regulates human (I)SHP‐1 transcript expression in SKOV3 cells (an ovarian cancer cell line). Indirect evidence suggests that nuclear factor‐κB might play a role in this induction. We also show that a 12‐bp repeat in the distal SHP‐1 promoter, which directs (I)SHP‐1 expression, is of functional relevance as deletion of one copy of this E‐box‐containing 12‐bp repeat resulted in a significant decrease in promoter activity. Electrophoretic mobility shift assays and supershift experiments showed that the upstream stimulatory factors USF1 and USF2 hetero‐dimerize and interact with this 12 bp repeat. Our results suggest that USFs which have antiproliferative functions might regulate the expression of SHP‐1, which itself is predominantly a negative growth regulator.
Transglutaminase‐mediated polyamination of vasoactive intestinal peptide (VIP) Gln16 residue modulates VIP/PACAP receptor activity
Authors: De Maria Salvatore , Metafora Salvatore , Metafora Vittoria , Morelli Francesco , Robberecht Patrick , Waelbroeck Magalì , Stiuso Paola , De Rosa Alfredo , Cozzolino Anna , Esposito Carla , Facchiano Angelo , Cartenì Maria
Published: March 2003

Previous data showing an increase of receptor binding activity of [R16]VIP, a vasoactive intestinal peptide (VIP) structural analogue containing arginine at the position 16 of its amino acid sequence, have pointed out the importance of a positive charge at this site. Here, the functional characterization of three VIP polyaminated adducts (VIPDap, VIPSpd, and VIPSpm), obtained by a transglutaminase‐catalysed reaction between the VIP Gln16 residue and 1,3‐diaminopropane (Dap), spermidine (Spd), or spermine (Spm), is reported. Appropriate binding assays and adenylate cyclase enzymatic determinations have shown that these VIP adducts act as structural VIP agonists, both in vitro and in vivo. In particular, their IC50 and EC50 values of human and rat VIP/pituitary adenylate cyclase activating peptide (PACAP)1 and VIP/PACAP2 receptors indicate that VIPDap is a VIP agonist, with an affinity and a potency higher than that of VIP, while VIPSpd and VIPSpm are also agonists but with affinities lower than that of VIP. These findings suggest that the difference in adduct agonist activity reflects the differences in the positive charge and carbon chain length of the polyamine covalently linked with the VIP Gln16 residue. In addition, the data obtained strongly suggest that the length of polyamine carbon chain could be critical for the interaction of the agonist with its receptor, even though possible hydrophobic interaction cannot be ruled out. In vivo experiments on murine J774 macrophage cell cultures have shown the ability of these compounds to stimulate the inducible nitric oxide synthase activity at the transcriptional level.
Balanced expression of single subunits in a multisubunit protein, achieved by cell fusion of individual transfectants
Authors: Norderhaug Lars , Johansen Finn‐Eirik , Sandlie Inger
Published: March 2003

To establish stable cell lines that produce recombinant multisubunit proteins, it is usually necessary to cotransfect cells with several independent gene constructs. Here, we show that a stepwise fusion of individually transfected cells, results in a fused cell‐line that secretes a complete multisubunit protein. Functional expression of recombinant multisubunit proteins may require a defined expression ratio between each protein subunit. The cell‐fusion technology described allows a predefined expression level of each subunit. Using SIgA as a model protein we demonstrate that the majority of the fused cells inherit the molar expression ratio of the parental transfected cells. These results indicate that simplified screening of clones expressing the expected subunit ratios may be possible using the cell‐fusion technology. This technology may therefore be an alternative to generic transfection methods for the establishment of cells that produce multiprotein complexes such as antibodies, receptors, ion channels and other multisubunit proteins.
Ligand‐induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle
Authors: Lezzi Markus , Bergman Thomas , Henrich Vincent C. , Vögtli Martin , Frömel Christina , Grebe Marco , Przibilla Sabina , Margarethe Spindler‐Barth
Published: March 2003

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR‐orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C‐terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose‐dependent manner. This was shown in vivo by a yeast two‐hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.
Fluorescent analogs of UDP‐glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile
Authors: Bhattacharyya Sudeep , Kerzmann Amy , Feig Andrew L.
Published: March 2003

Uridine‐5′‐diphospho‐1‐α‐d‐glucose (UDP‐Glc) is a common substrate used by glucosyltransferases, including certain bacterial toxins such as Toxins A and B from Clostridium difficile. Fluorescent analogs of UDP‐Glc have been prepared for use in our studies of the clostridial toxins. These compounds are related to the methylanthraniloyl‐ATP compounds commonly used to probe the chemistry of ATP‐dependent enzymes. The reaction of excess methylisatoic anhydride with UDP‐Glc in aqueous solution yields primarily the 2′ and 3′ isomers of methylanthraniloyl‐UDP‐Glc (MUG). As the 2′ and 3′ isomers readily interconvert, this isomeric mixture was copurified by HPLC away from the other isomeric products, and was characterized by a combination of NMR, fluorescence and mass spectrometric methods. TcdA binds MUG competitively with respect to UDP‐Glc with an affinity of 15 ± 2 µm in the absence of Mg2+. There is currently no evidence that the fluorescent substrate analog is turned over by the toxin in either glucosyltransferase or glucosylhydrolase reactions. Using a competition assay, the affinity of UDP‐Glc was determined to be 45±10 µm in the absence of Mg2+. The binding of UDP‐Glc and Mg2+ are highly coupled with Mg2+ affinities in the range of 90–600 µm, depending on the experimental conditions. These results imply that one of the significant roles of the metal ion might be to stabilize the enzyme–substrate complex prior to initiation of the transferase chemistry.
A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken α7 nicotinic acetylcholine receptors
Authors: Lay Poh Siew , Mourier Gilles , Thai Robert , Armugam Arunmozhiarasi , Molgó Jordi , Servent Denis , Jeyaseelan Kandiah , Ménez André
Published: March 2003

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx‐5, has been synthesized by solid‐phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx‐5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (Kd = 180 nm) with the muscular‐type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 µm) with the neuronal α7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve–muscle preparations revealed no blocking activity of sWntx‐5 at µm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.
A peptide derived from cyclin‐dependent kinase activator (p35) specifically inhibits Cdk5 activity and phosphorylation of tau protein in transfected cells
Authors: Zheng Ya‐li , Li Bing‐Sheng , Amin Niranjana D. , Albers Wayne , Pant Harish C.
Published: March 2003

Cyclin‐dependent kinase‐5 (Cdk5) is a serine/threonine kinase activated by its neuron‐specific activator, p35, or its truncated form, p25. It has been proposed that the deregulation of Cdk5 activity by association with p25 in human brain tissue disrupts the neuronal cytoskeleton and may be involved in neurodegenerative diseases such as Alzheimer's disease. In this study, we demonstrate that a short peptide (amino acid residues 154–279; Cdk5 inhibitory peptide; CIP), derived from p35, specifically inhibits Cdk5 activity invitro and in HEK293 cells cotransfected with the peptideand Cdk5/p25, but had no effect on endogenous cdc2 kinaseactivity. Moreover, we demonstrate that the phosphorylation of tau in HEK293 cells, cotransfected with Cdk5/p25 and CIP, is effectively reduced. These results suggest that CIP specifically inhibits both Cdk5/p25 complex activity and the tau hyperphosphorylation induced by Cdk5/p25. The elucidation of the molecular basis of p25 activation and CIP inhibition of Cdk5 activity may provide insight into mechanisms underlying the pathology of Alzheimer's disease and contribute to therapeutic strategies.
A chromatin‐associated protein from pea seeds preferentially binds histones H3 and H4
Authors: Castillo Josefa , Zúñiga Ángel , Franco Luis , Rodrigo M. Isabel
Published: March 2003

Pisum sativum p16 is a protein present in the chromatin of ungerminated embryonic axes. The purification of p16 and the isolation of a cDNA clone of psp54, the gene encoding its precursor have been recently reported [Castillo, J., Rodrigo, M. I., Márquez, J. A., Zúñiga, A and Franco, L. (2000) Eur. J. Biochem.267, 2156–2165]. In the present paper, we present data showing that p16 is a nuclear protein. First, after subcellular fractionation, p16 was clearly found in nuclei, although the protein is also present in other organelles. Immunocytochemical methods also confirm the above results. p16 seems to be firmly anchored to chromatin, as only extensive DNase I digestion of nuclei allows its release. Far Western and pull‐down experiments demonstrate a strong in vitro interaction between p16 and histones, especially H3 and H4, suggesting that p16 is tethered to chromatin through histones. Because the psp54 gene is specifically expressed during the late development of seed, the role of p16 might be related to the changes that occur in chromatin during the processes of seed maturation and germination.
Cellular retinol‐binding protein type II (CRBPII) in adult zebrafish (Danio rerio)
Authors: Cameron Marianne C. , Denovan‐Wright Eileen M. , Sharma Mukesh K. , Wright Jonathan M.
Published: March 2003

We have determined the nucleotide sequence of a zebrafish cDNA clone that codes for a cellular retinol‐binding protein type II (CRBPII). Radiation hybrid mapping revealed that the zebrafish and human CRBPII genes are located in syntenic groups. In situ hybridization and emulsion autoradiography localized the CRBPII mRNA to the intestine and the liver of adult zebrafish. CRBPII and intestinal fatty acid binding protein (I‐FABP) mRNA was colocalized to the same regions along the anterior‐posterior gradient of the zebrafish intestine. Similarly, CRBPII and I‐FABP mRNA are colocalized in mammalian and chicken intestine. CRBPII mRNA, but not I‐FABP mRNA, was detected in adult zebrafish liver which is in contrast to mammals where liver CRBPII mRNA levels are high during development but rapidly decrease to very low or undetectable levels following birth. CRBPII and I‐FABP gene expression appears therefore to be co‐ordinately regulated in the zebrafish intestine as has been suggested for mammals and chicken, but CRBPII gene expression is markedly different in the liver of adult zebrafish compared to the livers of mammals. As such, retinol metabolism in zebrafish may differ from that of mammals and require continued production of CRBPII in adult liver. The primary sequence of the coding regions of fish and mammalian CRBPII genes, their relative chromosomal location in syntenic groups and possibly portions of the control regions involved in regulation of CRBPII gene expression in the intestine appear therefore to have been conserved for more than 400 million years.
Barley α‐amylase Met53 situated at the high‐affinity subsite −2 belongs to a substrate binding motif in the β→α loop 2 of the catalytic (β/α)8‐barrel and is critical for activity and substrate specificity
Authors: Mori Haruhide , Bak‐Jensen Kristian Sass , Svensson Birte
Published: March 2003

Met53 in barley α‐amylase 1 (AMY1) is situated at the high‐affinity subsite −2. While Met53 is unique to plant α‐amylases, the adjacent Tyr52 stacks onto substrate at subsite −1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in β→α loop 2 of the catalytic (β/α)8‐barrel and site‐directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild‐type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2‐chloro‐4‐nitrophenyl β‐d‐maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5–0.01 and 1.7–0.02% of wild‐type, respectively. Km increased up to 20‐fold for these soluble substrates and the attack on glucosidic linkages in 4‐nitrophenyl α‐d‐maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite −2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild‐type released 4‐nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation – promoted probably by slow substrate hydrolysis – to produce up to maltoundecaose from PNPG6.
Subsite mapping of the binding region of α‐amylases with a computer program
Authors: Gyémánt Gyöngyi , Hovánszki György , Kandra Lili
Published: March 2003

A computer program has been evaluated for subsite map calculations of depolymerases. The program runs in windows and uses the experimentally determined bond cleavage frequencies (BCFs) for determination of the number of subsites, the position of the catalytic site and for calculation of subsite binding energies. The apparent free energy values were optimized by minimization of the differences of the measured and calculated BCF data. The program called suma (SUbsite Mapping of α‐Amylases) is freely available for research and educational purposes via the Internet (E‐mail: gyemant@tigris.klte.hu).

The advantages of this program are demonstrated through α‐amylases of different origin, e.g. porcine pancreatic α‐amylase (PPA) studied in our laboratory, in addition to barley and rice α‐amylases published in the literature.

Results confirm the popular ‘five subsite model’ for PPA with three glycone and two aglycone binding sites. Calculations for barley α‐amylase justify the ‘6 + 2 + (1) model’ prediction. The binding area of barley α‐amylase is composed of six glycone, two aglycone binding sites followed by a barrier subsite at the reducing end of the binding site. Calculations for rice α‐amylase represent an entirely new map with a ‘(1) + 2 + 5 model’, where ‘(1)‘ is a barrier subsite at the nonreducing end of the binding site and there are two glycone and five aglycone binding sites. The rice model may be reminiscent of the action of the bacterial maltogenic amylase, that is, suggesting an exo‐mechanism for this enzyme.
Biphasic reductive unfolding of ribonuclease A is temperature dependent
Authors: Yan Yong‐Bin , Zhang Ri‐Qing , Zhou Hai‐Meng
Published: March 2003

The kinetics of the reversible thermal unfolding, irreversible thermal unfolding, and reductive unfolding processes of bovine pancreatic ribonuclease A (RNase A) were investigated in NaCl/Pi solutions. Image parameters including Shannon entropy, Hamming distance, mutual information and correlation coefficient were used in the analysis of the CD and 1D NMR spectra. The irreversible thermal unfolding transition of RNase A was not a cooperative process, pretransitional structure changes occur before the main thermal denaturation. Different dithiothreitol (dithiothreitolred) concentration dependencies were observed between 303 and 313 K during denaturation induced by a small amount of reductive reagent. The protein selectively follows a major unfolding kinetics pathway with the selectivity can be altered by temperature and reductive reagent concentration. Two possible explanations of the selectivity mechanism were discussed.
The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal‐recognition particle in Escherichia coli
Authors: Adams Hendrik , Scotti Pier A. , de Cock Hans , Luirink Joen , Tommassen Jan
Published: March 2003

Signal sequences often contain α‐helix‐destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer‐membrane protein PhoE, the glycine residue at position −10 (Gly−10) is thought to be responsible for the break in the α‐helix. Previously, we showed that substitution of Gly−10 by α‐helix‐promoting residues (Ala, Cys or Leu) reduced the proton‐motive force dependency of the translocation of the precursor, but the actual role of the helix breaker remained obscure. Here, we considered the possibility that extension of the α‐helical structure in the signal sequence resulting from the Gly−10 substitutions affects the targeting pathway of the precursor. Indeed, the mutations resulted in reduced dependency on SecB for targeting in vivo. In vitro cross‐linking experiments revealed that the G‐10L and G‐10C mutant PhoE precursors had a dramatically increased affinity for P48, one of the constituents of the signal‐recognition particle (SRP). Furthermore, in vitro cross‐linking experiments revealed that the G‐10L mutant protein is routed to the SecYEG translocon via the SRP pathway, the targeting pathway that is exploited by integral inner‐membrane proteins. Together, these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby, together with the hydrophobicity of the signal sequence, a determinant of the targeting pathway.
Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coli
Authors: Adams Hendrik , Scotti Pier A. , Luirink Joen , Tommassen Jan
Published: March 2003

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564–5571], we showed that the precursor of outer‐membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position −10 in the signal sequence (G‐10L) is targeted to the SecYEG translocon via the signal‐recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G‐10L mutant precursor, which is normally exported in a wild‐type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross‐linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K‐accessibility experiments in pulse‐labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral‐membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.
Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution
Authors: Katavic Vesna , Mietkiewska Elzbieta , Barton Dennis L. , Giblin E. Michael , Reed Darwin W. , Taylor David C.
Published: March 2003

Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose‐inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.
The C‐terminal region of ammodytoxins is important but not sufficient for neurotoxicity
Authors: Prijatelj Petra , Križaj Igor , Kralj Bogdan , Gubenšek Franc , Pungerčar Jože
Published: March 2003

Ammodytoxins (Atxs) are presynaptically acting snake venom phospholipase A2 (PLA2) toxins the molecular mechanism of whose neurotoxicity is not completely understood. Two chimeric PLA2s were prepared by replacing the C‐terminal part of a nontoxic venom PLA2, ammodytin I2, with that of AtxA(K108N). The chimeras were not toxic, but were able to bind strongly to an Atxs‐specific neuronal receptor, R25. They also showed an increased affinity for calmodulin, a recently identified high‐affinity binding protein for Atxs, whereas affinity for a neuronal M‐type PLA2 receptor remained largely unchanged. The results show that the C‐terminal region of Atxs, which is known to be involved in neurotoxicity, is critical for their interaction with specific binding proteins, but that some other part of the molecule also contributes to toxicity.
Structural analysis of Francisella tularensis lipopolysaccharide
Authors: Vinogradov Evgeny , Perry Malcolm B. , Conlan J. Wayne
Published: March 2003

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a β‐GlcN‐(1–6)‐GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O‐1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O‐3 and O‐4 of β‐GlcN, and no substituent at O‐4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure:

where Δ‐GalNA‐(1–3)‐β‐QuiNAc represents a modified fragment of the O‐chain repeating unit. Two shorter oligosaccharides lacking the O‐chain fragment were also identified. A minor amount of the disaccharide β‐GlcN‐(1–6)‐α‐GlcN‐1‐P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end.

The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2‐N‐(3‐O‐acyl4‐acyl2)‐β‐GlcN‐(1–6)‐2‐N‐acyl1−3‐O‐acyl3‐GlcN where acyl1, acyl2 and acyl3 are 3‐hydroxyhexadecanoic or 3‐hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH‐1 of the reducing end glucosamine, and OH‐3 and OH‐4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.
Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes
Authors: Persson Egon , Olsen Ole H.
Published: March 2003

The most active factor VIIa (FVIIa) variants identified to date carry concurrent substitutions at positions 158, 296 and 298 with the intention of generating a thrombin‐mimicking motif, optionally combined with additional replacements within the protease domain [Persson, E., Kjalke, M. & Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA98, 13583–13588]. Here we have characterized variants of FVIIa mutated at one or two of these positions to assess the relative importance of the individual replacements. The E296V and M298Q mutations gave an increased intrinsic amidolytic activity (about two‐ and 3.5‐fold, respectively) compared with wild‐type FVIIa. An additive effect was observed upon their combination, resulting in the amidolytic activity of E296V/M298Q‐FVIIa being close to that of the triple mutant. The level of amidolytic activity of a variant was correlated with the rate of inhibition by antithrombin (AT). Compared with wild‐type FVIIa, the Ca2+ dependence of the intrinsic amidolytic activity was significantly attenuated upon introduction of the E296V mutation, but the effect was most pronounced in the triple mutant. Enhancement of the proteolytic activity requires substitution of Gln for Met298. The simultaneous presence of the V158D, E296V and M298Q mutations gives the highest intrinsic activity and is essential to achieve a dramatically higher relative increase in the proteolytic activity than that in the amidolytic activity. The N‐terminal Ile153 is most efficiently buried in V158D/E296V/M298Q‐FVIIa, but is less available for chemical modification also in the presence of the E296V or M298Q mutation alone. In summary, E296V and M298Q enhance the amidolytic activity and facilitate salt bridge formation between the N‐terminus and Asp343, E296V reduces the Ca2+ dependence, M298Q is required for increased factor X (FX) activation, and the simultaneous presence of the V158D, E296V and M298Q mutations gives the most profound effect on all these parameters.
Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis
Authors: Ågren Jyrki J. , Ravandi Amir , Kuksis Arnis , Steiner George
Published: March 2003

Triacylglycerols secreted by liver and carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases. These enzymes have been shown to have positional and fatty acid specificity in vitro. If there were specificity in basal lipolysis in vivo, triacylglycerol compositions of circulating and newly secreted VLDL would be different. To study this we compared the composition of normal fasting VLDL triacylglycerol of Wistar rats to that obtained after blocking lipolysis by Triton WR1339, which increased plasma VLDL triacylglycerol concentration about 4.7‐fold in 2 h. Analyses of molecular species of sn‐1,2‐ and sn‐2,3‐diacylglycerol moieties and stereospecific triacylglycerol analysis revealed major differences between the groups in the VLDL triacylglycerol composition. In nontreated rats, the proportion of 16:0 was higher and that of 18:2n‐6 lower in the sn‐1 position. The proportion of 14:0 was lower in all positions and that of 18:0 was lower in the sn‐1 and sn‐3 positions in nontreated rats whereas the proportions of 20:4n‐6, 20:5n‐3, 22:5n‐3 and 22:6n‐3 were higher in the sn‐1 and lower in the sn‐2 position. These results suggest that the fatty acid of the sn‐1 position is the most decisive factor in determining the sensitivity for hydrolysis of the triacylglycerol. In addition, triacylglycerol species with highly unsaturated fatty acids in the sn‐2 position also favoured hydrolysis. The in vivo substrate specificity followed only partly that obtained in in vitro studies indicating that the nature of molecular association of fatty acids in natural triacylglycerol affects its susceptibility to lipolysis. To conclude, our results indicate that preferential basal lipolysis leads to major structural differences between circulating and newly secreted VLDL triacylglycerol. These differences extend beyond those anticipated from analysis of total fatty acids and constitute a previously unrecognized feature of VLDL triacylglycerol metabolism.
Characterization of the lectin from females of Phlebotomus duboscqi sand flies
Authors: Volf Petr , Skarupová Sona , Man Petr
Published: March 2003

Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti‐lectin immunoglobulins. Carbohydrate‐binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d‐glucosamine, d‐galactosamine and d‐mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44‐kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI‐TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.
Molecular characterization of artemin and ferritin from Artemia franciscana
Authors: Chen Tao , Amons Reinout , Clegg James S. , Warner Alden H. , MacRae Thomas H.
Published: March 2003

Embryos of the brine shrimp, Artemia franciscana, exhibit remarkable resistance to physiological stress, which is temporally correlated with the presence of two proteins, one a small heat shock/α‐crystallin protein termed p26 and the other called artemin, of unknown function. Artemin was sequenced previously by Edman degradation, and its relationship to ferritin, an iron storage protein, established. The isolation from an Artemia expressed sequence tag library of artemin and ferritin cDNAs extends this work. Artemin cDNA was found to contain an ORF of 693 nucleotides, and its deduced amino‐acid sequence, except for the initiator methionine, was identical with that determined previously. Ferritin cDNA is 725 bp in length with an ORF of 516 nucleotides. Artemin amino‐acid residues 32–185 are most similar to ferritin, but artemin is enriched in cysteines. The abundance of cysteines and their intramolecular spatial distribution suggest that artemin protects embryos against oxidative damage and/or that its function is redox regulated. The conserved regions in artemin and ferritin monomers are structurally similar to one another and both proteins assemble into oligomers. However, modeling of the quaternary structure indicated that artemin multimers lack the central space used for metal storage that characterizes ferritin oligomers, implying different roles for this protein. Probing of Northern blots revealed two artemin transcripts, one of 3.5 kb and another of 2.2 kb. These transcripts decreased in parallel and had almost disappeared by 16 h of development. The ferritin transcript of 0.8 kb increased slightly during reinitiation of development, then declined, and was almost completely gone by 16 h. Clearly, the loss of artemin and ferritin during embryo development is due to transcriptional regulation and proteolytic degradation of the proteins.
Antioxidant protein 2 prevents methemoglobin formation in erythrocyte hemolysates
Authors: Stuhlmeier Karl M. , Kao Janet J. , Wallbrandt Pia , Lindberg Maria , Hammarström Barbro , Broell Hans , Paigen Beverly
Published: March 2003

Antioxidant protein 2 (AOP2) is a member of a family of thiol‐specific antioxidants, recently renamed peroxiredoxins, that evolved as part of an elaborate system to counteract and control detrimental effects of oxygen radicals. AOP2 is found in endothelial cells, erythrocytes, monocytes, T and B cells, but not in granulocytes. AOP2 was found solely in the cytoplasm and was not associated with the nuclear or membrane fractions; neither was it detectable in plasma. Further experiments focused on the function of AOP2 in erythrocytes where it is closely associated with the hemoglobin complex, particularly with the heme. An investigation of the mechanism of this interaction demonstrated that the conserved cysteine‐47 in AOP2 seems to play a role in AOP2‐heme interactions. Recombinant AOP2 prevented induced as well as noninduced methemoglobin formation in erythrocyte hemolysates, indicating its antioxidant properties. We conclude that AOP2 is part of a sophisticated system developed to protect and support erythrocytes in their many physiological functions.
Plasmodium falciparum merozoite surface protein 1
Authors: Hoessli Daniel C. , Poincelet Monique , Gupta Ramneek , Ilangumaran Subburaj , Nasir‐ud‐Din
Published: March 2003

In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP‐1) bears O‐GlcNAc modifications predominantly in β‐anomeric configuration, in both the C‐ and N‐terminal portions of the protein. Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI‐anchored C‐terminal fragments of MSP‐1 are recovered in Triton X‐100 resistant, low‐density membrane fractions. Our results suggest that O‐GlcNAc‐modified MSP‐1 N‐terminal fragments tend to localize within the parasitophorous vacuolar membrane while GPI‐anchored MSP‐1 C‐terminal fragments associate with low‐density, Triton X‐100 resistant membrane domains (rafts), redistribute in the parasitized erythrocyte and are eventually shed as membrane vesicles that also contain the endogenous, GPI‐linked CD59.
Bovine tryptases
Authors: Gambacurta Alessandra , Fiorucci Laura , Basili Paolo , Erba Fulvio , Amoresano Angela , Ascoli Franca
Published: March 2003

A complementary DNA encoding a new bovine tryptase isoform (here named BLT) was cloned and sequenced from lung tissue. Analysis of sequence indicates the presence of a 26‐amino acid prepro‐sequence and a 245 amino acid catalytic domain. It contains six different residues when compared with the previously characterized tryptase from bovine liver capsule (BLCT), with the most significant difference residing at the primary specificity S1 pocket. In BLT, the canonical residues Asp‐Ser are present at positions 188–189, while in BLCT these positions are occupied by residues Asn‐Phe. This finding was confirmed by mass fingerprinting of the peptide mixture obtained upon in‐gel tryptic digestion of BLT. Analysis by gel filtration of the purified protein shows that BLT is probably tetrameric, similar to the previously identified tryptases from other species, with monomer migrating as 35–40 kDa multiple bands in SDS/PAGE. As expected, the catalytic abilities of the two bovine tryptases are different. The specificity constant values (kcat/Km) assayed with model substrates are 10‐ to 60‐fold higher in the case of BLT. The tissue‐specific expression of the two tryptases was evaluated at the RNA level by analysis of their different restriction patterns. In lung, only BLT was found to be expressed, while in liver capsule only BLCT is present. Both isoforms are distributed in similar amounts in heart and spleen. Analysis of the two gene sequences reveals the presence of several recognition sequences in the promoter regions and suggest a role for hormones in governing the mechanism of tissue expression of bovine tryptases.
Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis
Authors: Migita Catharina T. , Zhang Xuhong , Yoshida Tadashi
Published: March 2003

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho‐1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric‐biliverdin IXα in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH‐cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH‐cytochrome P450 reductase is greatly retarded after the oxy‐heme complex formation. The optical absorption spectra of the heme‐enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue‐shifted and relatively strong CT bands of the high‐spin component in the ferric form spectrum. The heme‐enzyme complex shows the acid‐base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase‐1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform‐1 is different from that of rat heme oxygenase isoform‐1, rather resembling that of bacterial heme oxygenase, Hmu O.
Deciphering metabolic networks
Authors: Fiehn Oliver , Weckwerth Wolfram
Published: March 2003

All higher organisms divide major biochemical steps into different cellular compartments and often use tissue‐specific division of metabolism for the same purpose. Such spatial resolution is accompanied with temporal changes of metabolite synthesis in response to environmental stimuli or developmental needs. Although analyses of primary and secondary gene products, i.e. transcripts, proteins, and metabolites, regularly do not cope with this spatial and temporal resolution, these gene products are often observed to be highly coregulated forming complex networks. Methods to study such networks are reviewed with respect to data acquisition, network statistics, and biochemical interpretation.
Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts
Authors: Shimaoka Taise , Miyake Chikahiro , Yokota Akiho
Published: March 2003

Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi‐uni‐uni‐uni‐ping‐pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The Km value for DHA and the summed Km value for GSH were determined to be 53 ± 12 µm and 2.2 ± 1.0 mm, respectively, with a turnover rate of 490 ± 40 s−1. Incubation of 10 µm DHAR with 1 mm DHA and 10 µm GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH–enzyme adduct by 2‐mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site‐directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.
Purification and characterization of the nuclear ribonuclease P of Aspergillus nidulans
Authors: Han Sang Jun , Lee Byeong Jae , Kang Hyen Sam
Published: March 2003

In Aspergillus nidulans, the nuclear ribonuclease P was separated from its mitochondrial counterpart by Q‐Sepharose chromatography, and a precursor‐tRNAHis processing assay system was used to discriminate nuclear ribonuclease P activity from the mitochondrial counterpart. The nuclear ribonuclease P was purified to near homogeneity from whole‐cell extracts. A 2150‐fold purification with a yield of 2.3 % was achieved by five types of chromatography including tRNA affinity chromatography and glycerol gradient velocity sedimentation. This enzyme, which had a molecular mass of 580 kDa determined by both glycerol‐gradient sedimentation analysis and gel‐permeation chromatography, appeared to be composed of seven polypeptides and an RNA molecule. Seven polypeptides, with masses of 125, 85, 45, 33, 30, 21, 19 kDa, were consistently copurified with nuclear ribonuclease P activity through MonoS and tRNA affinity chromatography and in a glycerol gradient. As judged by a micrococcal‐nuclease‐sensitivity assay, nuclear ribonuclease P required an RNA component for its activity, as do other ribonuclease Ps. Analysis of the radiolabeled 5′ end of RNAs copurified with nuclear ribonuclease P implied that RNA molecules in the purified nuclear ribonuclease P originated from a common RNA molecule, the putative RNA molecule of nuclear ribonuclease P. Comparison of the two ribonuclease Ps in A. nidulans showed that the protein and RNA components of the nuclear ribonuclease P were different from those of the mitochondrial counterpart.
Multiple polypeptides immunologically related to β‐poly( L‐malate) hydrolase (polymalatase) in the plasmodium of the slime mold Physarum polycephalum
Authors: Karl Michael , Holler Eggehard
Published: March 2003

Plasmodia of Physarum polycephalum contain large amounts of the cell‐type‐specific polyanion β‐poly(
L‐malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68‐kDa form giving rise to a 54‐kDa form when deglycosylated, and the 97‐kDa form giving rise to an 88‐kDa polypeptide that was indistinguishable from an 88‐kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two‐dimensional non‐denaturing in the first dimension and SDS‐denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200‐kDa ’precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular‐mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of β‐poly(
L‐malate) from the nucleus to the culture medium.
A substrate‐induced change in the stereospecificity of the serine‐hydroxymethyltransferase‐catalysed exchange of the α‐protons of amino acids
Authors: Fitzpatrick Teresa B. , Malthouse J. Paul G.
Published: March 2003

NMR has been used to study the catalysis of the hydrogen−deuterium exchange of the α‐protons of amino acids by serine hydroxymethyltransferase ( EC2.1.2.1) from Escherichia coli.13C‐NMR was used to follow the exchange of the α‐protons of [2‐13C]glycine. The enzyme‐catalysed first‐order exchange rate of the pro‐2S proton of glycine was ≈7000 times more efficient than that of the pro‐2R proton of glycine at both pH 7.0 and 7.8. 1H‐NMR was used to follow the hydrogen−deuterium exchange rates of the α‐protons of
L‐ and
D‐2‐amino derivatives of butyric, pentanoic and hexanoic acids at pH 7.8. Increasing the size of the R‐group leads to a progressive change in the stereospecificity of the exchange reaction from the pro‐2S proton of glycine to the 2R proton of
L‐amino acids. The stereospecificity for the α‐protons of
L‐amino acids increased as the size of the R‐group increased. With glycine, removal of tetrahydrofolate led to a large decrease in the stereospecificity of the exchange reaction but did not affect the exchange rates of the α‐protons of any of the larger amino acids studied. We show that the Schiff base formed between
L‐2‐aminohexanoic acid (
L‐norleucine) and pyridoxal 5′‐phosphate binds at a different site from the Schiff base between glycine and pyridoxal 5′‐phosphate. The molecular basis of these results is discussed.