bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and immunological characterization of group V allergen isoforms from velvet grass pollen (Holcus lanatus)
Authors: Schramm Gabriele , Bufe Albrecht , Petersen Arnd , Schlaak Max , Becker Wolf‐Meinhard
Published: March 2003

Group V grass pollen allergens belong to the major grass pollen allergens causing reactions of type I allergy. cDNAs coding for two isoforms of the group V allergen of velvet grass pollen (Holcus lanatus), a widespread grass species, were isolated from a cDNA library by hybridization with a 5′‐terminal reverse‐transcribed PCR‐derived cDNA probe. Amino acid sequences of the two isoforms, designated rHol l 5.01 and rHol l 5.02, revealed high similarity between them (78 % identity) and to group V allergens and their isoforms found in other grass species. Recombinant Hol l 5 isoforms were expressed in Escherichia coli and purified as fusion proteins. To compare their immunological reactivities with group‐V‐specific monoclonal antibodies and patients' IgE, immunoblotting, ELISA and histamine release assay were performed. Interestingly, monoclonal antibody Bo9, specific for group Vb isoforms of timothy grass, bound only to isoform rHol l 5.01, not to rHol l 5.02. On the other side, IgE reactivities of patients' sera revealed no differences between the two isoforms when investigated by immunoblotting and only slight differences when investigated by ELISA. In histamine release assay both isoforms released comparable amounts of histamine from basophils of four individual patients. Thus, the two group V isoforms of velvet grass pollen exhibit differential binding when tested with monoclonal antibodies, i.e. different structure of single epitopes, but negligible differences concerning overall IgE‐binding capacity and histamine‐releasing capacity.
3,5‐Diiodothyronine binds to subunit Va of cytochrome‐c oxidase and abolishes the allosteric inhibition of respiration by ATP
Authors: Arnold Susanne , Goglia Fernando , Kadenbach Bernhard
Published: March 2003

The short‐term effects of thyroid hormones, which do not occur via gene expression, were postulated to be based on interaction of diiodothyronines with mitochondria. We demonstrate specific binding of labelled 3,5‐diiodothyronine to subunit Va of cytochrome‐c oxidase from bovine heart. 3,5‐Diiodothyronine, and to a small extent triiodothyronine, but not thyroxine and thyronine, abolish the allosteric inhibition of ascorbate respiration of reconstituted cytochrome c oxidase by ATP [Arnold, S. & Kadenbach, B. (1997) Eur. J. Biochem. 249, 350−354]. This abolition of ATP‐inhibition by 3,5‐diiodothyronine is completely prevented by a monoclonal antibody to subunit Va. The results explain at the molecular level the short‐term action of thyroid hormones on basal metabolic rate.
Conformation of desmopressin, an analogue of the peptide hormone vasopressin, in aqueous solution as determined by NMR spectroscopy
Authors: Walse Björn , Kihlberg Jan , Drakenberg Torbjörn
Published: March 2003

Desmopressin (1‐desamino‐[ DArg8]vasopressin, is a synthetic analogue of the neurohypophyseal peptide hormone vasopressin which has high antidiuretic and antibleeding potency. The structure of desmopressin has been determined in aqueous solution by two‐dimensional NMR techniques and molecular dynamics simulations. Both standard and time‐averaged distance restraints were used in structure calculations because of the inherent flexibility in small peptides. 21 models calculated with standard restraints were compared with structures refined with time‐averaged distance restraints and were found to be good representatives of the conformational ensemble of desmopressin. The macrocyclic ring forms an inverse γ‐turn centered around Gln4. Residues 1 and 2, the disulphide bridge and the three‐residue acyclic tail were found to be flexible in solution. Residues 4−6 in the ensemble of calculated structures contain essentially the same backbone conformation as in the crystal structure of pressinoic acid, the cyclic moiety of vasopressin, whereas residues 2−6 superimpose on the NMR‐derived conformation of oxytocin bound to neurophysin. The results presented in this work suggest that, in addition to the differences in sequence between desmopressin and vasopressin, differences in conformational and dynamic properties between the two compounds explain their pharmacological differences.
Tryptophan mutants of troponin C from skeletal muscle
Authors: She Mingda , Dong Wen‐Ji , Umeda Patrick K. , Cheung Herbert C.
Published: March 2003

We have generated a series of chicken skeletal muscle troponin C mutants to study the conformation of the regulatory domain in the N‐terminal half of the molecule. These mutants each contained a single Trp at position 22 (helix A), 52 (linker of helices B and C), or 90 (central helix). Some of these mutants also contained additional mutations to introduce a single Cys at a desired position. The mutants were characterized by molecular graphics and CD and found to have a minimum of structural perturbations when compared with the native structure. They also retained the ability to regulate myofibrillar ATPase activity. The fluorescence of Trp22 was sensitive to Ca2+ binding only to the regulatory sites, whereas Trp52 and Trp90 responded to Ca2+ binding to both the regulatory and the Ca2+/Mg2+ sites. The tryptophan quantum yield (Q) of all Trp22‐containing mutants was very high (0.33) in the absence of bound Ca2+, compared to that of
L‐tryptophan in aqueous solution (0.14). Q decreased 25 % upon binding of Ca2+ to the regulatory sites. The quantum yields of Trp52 and Trp90 in apo mutants were close to 0.14. In the presence of bound Ca2+ at the regulatory sites, the quantum yield of Trp52 decreased 16 %, whereas that of Trp90 increased 25 %. Results from acrylamide quenching of the fluorescence of the three Trp residues indicated that Trp22 was the least exposed and Trp52 was the most exposed, consistent with other spectral data that Trp22 was in a relatively nonpolar environment and Trp52 was in a highly polar environment. The ability of Trp52 and Trp90 to sense Ca2+ binding to sites located at both domains suggests inter‐domain communication in the protein. These single Trp TnC mutants provide specific signals for probing Ca2+‐induced conformational changes in the regulatory domain.
Carbamate kinase from Enterococcus faecalis and Enterococcus faecium
Authors: Marina Alberto , Uriarte Matxalen , Barcelona Belén , Fresquet Vicente , Cervera Javier , Rubio Vicente
Published: March 2003

Carbamate kinase (CK) catalyzes the reversible reaction NH2COO− + ATP  NH2COOPO.es2−.rb.ei3.rb + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X‐ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate‐dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl‐phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high Km and the similar Km values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5′)pentaphospho(5′)adenosine (Ap5A) and Ap6A and, less powerfully, by Ap4A, whereas Ap3A is essentially non‐inhibitory. Thus, inhibition by Ap5A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap5A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis : (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C‐terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity.

Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N‐acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.
Priority paper Partially deglycosylated human choriogonadotropin, stabilized by intersubunit disulfide bonds, shows full bioactivity
Authors: Heikoop Judith C. , van den Boogaart Paul , de Leeuw Renato , Rose Ursula M. , Mulders John W. M. , Grootenhuis Peter D. J.
Published: March 2003

Several studies indicate that in human choriogonadotropin the N‐linked oligosaccharide at position 52 of the α‐subunit is important for bioactivity. We have generated choriogonadotropin mutants in which the α52 glycosylation site is removed and the α and β subunits are covalently linked by intersubunit disulfide bonds. These mutants display wild‐type receptor binding and bioactivity. Furthermore, we show that removal of the α52 sugar leads to instability of heterodimeric choriogonadotropin. Therefore, we conclude that the α52 oligosaccharide of choriogonadotropin is not involved in signal transduction, but in the stability of the heterodimer.
Biochemical characterization and localization of transglutaminase in wild‐type and cell‐death mutants of the nematode Caenorhabditis elegans
Authors: Mádi András , Punyiczki Mária , di Rao Massimo , Piacentini Mauro , Fésüs László
Published: March 2003

Transglutaminase activity was characterized in extracts of the nematode Caenorhabditis elegans using a microtiter plate method, and found to be Ca2+‐dependent, optimal at pH 8.0, and to be inhibited by EGTA, ammonia, iodoacetamide and GTP. Monoclonal and polyclonal antibodies raised against human tissue transglutaminase also inhibited the activity and detected a 61‐kDa protein from the worm lysate. Constitutive expression of the enzyme in the wild‐type intestinal cells was revealed by immunohistochemistry. Potential protein substrates for the enzyme were found in worm lysates using a biotin‐labelled amine substrate. There is a basal level of protein‐bound ε(γ‐glutamyl)lysine cross‐links, characteristic of transglutaminase activity, formed in situ in adult wild‐type animals. Developmental studies have revealed that the enzyme activity is highest in adult animals, and relatively higher in L1 larvae than in other larval stages. As compared to wild types, lower transglutaminase activity has been measured in lysates of ced3, ced‐4 and ced‐9 mutants. Cross‐link levels were also low in ced‐4 and ced‐9 mutants. By contrast, the crosslink content was high in several phagocytosis mutants. The highest concentration was found in the ced‐5; ced‐7 double phagocytosis mutants which carry an extra number of dead cells during their lifespan. In accordance with this finding, several transglutaminase‐immunopositive cells were found in both the embryos and in the head of these double phagocytosis mutants. The results suggest that a transglutaminase is involved in, or related to, the death program of cells in C. elegans and the expression and crosslinking activity of the enzyme may be perturbed in some ced mutants.
Oxygen exchange with water in heme‐oxo intermediates during H2O2‐driven oxygen incorporation in aromatic hydrocarbons catalyzed by microperoxidase‐8
Authors: Dorovska‐Taran Victoria , Posthumus Maarten A. , Boeren Sjef , Boersma Marelle G. , Teunis Cees J. , Rietjens Ivonne M. C. M. , Veeger Cees
Published: March 2003

The present paper describes the oxygen incorporation into naphthalene and anthracene by H2O2‐driven microperoxidase‐8, forming α‐naphthol and anthraquinone, respectively. Microperoxidase‐8 is a minienzyme containing a histidinyl‐coordinated Fe3+‐protoporphyrin IX cofactor covalently attached to an eight‐amino‐acid peptide.

Additional experiments were performed to investigate whether the reaction mechanism involved is like that of peroxidase and/or cytochrome P‐450. A reaction pathway like that of cytochrome P‐450 implies oxygen transfer to the substrate from the as yet uncharacterized iron‐oxo species formed in the reaction of the heme cofactor with H2O2. In contrast, a peroxidase‐type reaction chemistry involves reaction pathways proceeding by initial one‐electron oxidation of, or H‐abstraction from, the substrate, followed by incorporation of oxygen from sources other than the iron‐oxo species, i.e. from other than H2O2.

The results of the present study exclude Fenton‐type chemistry and prove that the minicatalyst is able to catalyze the oxygen incorporation by both peroxidase and cytochrome P‐450 types of reaction pathways, while exchange occurs between the high‐valency iron‐oxo species and H2O. The mechanistic implications of this exchange for cytochrome P‐450 are discussed.
Sulfhydryl reagent susceptibility in proteins with high sequence similarity
Authors: Garza‐Ramos Georgina , Cabrera Nallely , Saavedra‐Lira Emma , Tuena de Gómez‐Puyou Marietta , Ostoa‐Saloma Pedro , Pérez‐Montfort Ruy , Gómez‐Puyou Armando
Published: March 2003

The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68 %. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100‐fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8‐anilino‐1‐napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half‐maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accesibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.
Cloning and characterization of a cDNA coding for Astacus embryonic astacin, a member of the astacin family of metalloproteases from the crayfish Astacus astacus
Authors: Geier Gebhard , Zwilling Robert
Published: March 2003

The astacin family of zinc endopeptidases was named after the digestive enzyme astacin isolated from the crayfish Astacus astacus. Employing a reverse transcription/PCR strategy with degenerate oligonucleotide primers specific for two signature seqences of the astacin family, we have isolated a 1602‐bp cDNA from embryos of developing A. astacus eggs, which was designated Astacus embryonic astacin (AEA). This cDNA was found to code for an astacin‐like protease domain which accounts for the N‐terminal half of the predicted protein. The C‐terminal half mainly consists of two complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein 1 (CUB) domains. The metalloprotease domain displays an amino acid sequence identity of 42 % with astacin. A higher sequence similarity was found to astacin family members that act as hatching enzymes in different species, e.g. chorioallantoic membrane protein 1 (CAM‐1; from quail) and Xenopus hatching enzyme (formerly UVS.2), both of which show 54 % identity, and high and low choriolytic enzymes (HCE and LCE) from the teleost Oryzias latipes (52 % and 48 % identity, respectively). A relationship to astacin‐like hatching enzymes is further supported by a phylogenetic analysis of the protease domains. Expression of AEA mRNA in developing embryos was found to be restricted to unhatched juveniles (larvae) during the last 8 days before hatching. AEA transcripts could not be detected in various tissues of adult animals or in eggs and embryos from an earlier developmental stage. AEA expression starts about 8 days prior to hatching, followed by a strong (18‐fold) induction with a maximum at day 4 before hatching. Newly hatched juveniles were found not to express the AEA mRNA.
Secretion of tetrain, a Tetrahymena cysteine protease, as a mature enzyme and its identification as a member of the cathepsin L subfamily
Authors: Suzuki Kazu‐Michi , Hayashi Nakanobu , Hosoya Natsumi , Takahashi Tadao , Kosaka Toshikazu , Hosoya Hiroshi
Published: March 2003

A protease in the culture medium of Tetrahymena pyriformis was purified to homogeneity. The purified protease had an apparent molecular mass of 28 kDa on SDS/PAGE. The amino acid sequences of the N‐terminal and internal peptides of the protease showed complete identity with those of tetrain, an enzyme previously reported as a Tetrahymena cysteine protease but not characterized in detail. Two overlapping cDNA clones for tetrain were sequenced, and the nucleotide sequence predicts that these clones encode a 330‐amino acid protein composed of a 16‐residue N‐terminal signal sequence followed by a 103‐residue propeptide and a 211‐residue mature protease. The primary structure and enzymatic properties support the conclusion that tetrain belongs to the cathepsin L subfamily. Immunoblotting analyses showed that mature tetrain was found exclusively in the culture medium. Immunofluorescence microscopy demonstrated that tetrain was concentrated in or around the food vacuoles of cells in the late logarithmic phase, but the staining of food vacuoles was not obvious in the stationary phase. These results suggest that tetrain is synthesized at the logarithmic phase and is secreted into the culture medium as a mature form.
A pancreas‐specific glycosylated protein disulphide‐isomerase binds to misfolded proteins and peptides with an interaction inhibited by oestrogens
Authors: Klappa Peter , Stromer Thusnelda , Zimmermann Richard , Ruddock Lloyd W. , Freedman Robert B.
Published: March 2003

Using a cross‐linking approach, we have demonstrated that radiolabeled model peptides or misfolded proteins specifically interact in vitro with two different luminal proteins in a crude extract from sheep pancreas microsomes. One of the proteins was identified as protein disulphide‐isomerase (PDI), the other one was a related protein (PDIp). We have shown that PDIp was expressed exclusively in the pancreas. Interspecies conservation of PDIp was confirmed and, unlike other members of the PDI family, PDIp from various sources was found to be a glycoprotein. PDIp interacted with peptides and also a misfolded protein, but not with native proteins, suggesting that it might act as a molecular chaperone. The inital binding process was independent of the presence of Cys residues in the probed peptides. Certain oestrogens strongly inhibited the interaction between peptides and PDIp, with 17β‐oestradiol being the most potent inhibitor.
Transport of LDS‐751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine‐123‐selective site of P‐glycoprotein
Authors: Shapiro Adam B. , Ling Victor
Published: March 2003

P‐glycoprotein is an ATP‐dependent transporter of an extremely wide variety of lipophilic compounds. We showed previously [Shapiro, A. B. & Ling, V. (1997a) Eur. J. Biochem. 250, 130−137] that P‐glycoprotein contains two drug transporting sites, dubbed H (for Hoechst 33342‐selective) and R (for rhodamine‐123‐selective), that interact with positive cooperativity. The H site transports 2‐[2‐(4‐ethoxyphenyl)‐6‐benzimidazolyl]‐6‐(1‐methyl‐4‐piperazyl)benzimidazole (Hoechst 33342) from the cytoplasmic leaflet of the plasma membrane to the aqueous extracellular medium [Shapiro, A. B. & Ling, V. (1997b) Eur. J. Biochem. 250, 122−129]. The environment from which the R site transports its substrates is unknown. In this paper, we used the fluorescent DNA dye 2‐{4‐[4‐(dimethylamino)phenyl]‐1,3‐butadienyl}‐3‐ethylbenzothiazolium perchlorate (LDS‐751), a substrate of the R site, to address this issue.

LDS‐751 which, like Hoechst 33342, exhibits lipid‐dependent fluorescence and slow transleaflet diffusion, allowed us to use the same methodology that we used for the H site to study the location of the R site. As with Hoechst 33342, the specific initial rate of LDS‐751 transport by P‐glycoprotein‐rich, isolated plasma membrane vesicles from CHRB30 cells was directly proportional to the amount of membrane‐bound LDS‐751 and inversely proportional to the concentration of free, aqueous LDS‐751. This result demonstrates that the R site of P‐glycoprotein transports LDS‐751 out of the lipid membrane. The slight decrease, instead of an increase, in the initial rate of active transport of LDS‐751 with the amount of time elapsed for slow diffusion of LDS‐751 from the cytoplasmic leaflet to the extracellular leaflet indicates that the R site of P‐glycoprotein removes LDS‐751 from the cytoplasmic leaflet of the plasma membrane. Thus, both known drug‐transporting sites of P‐glycoprotein remove their substrates from the cytoplasmic leaflet. Since all of the P‐glycoprotein substrates we have examined so far are recognized by one or both of the two known drug‐transporting sites, these two sites in the cytoplasmic leaflet of the plasma membrane may be able to account for all substrate transport by P‐glycoprotein.
Analysis of the F‐actin binding fragments of vinculin using stopped‐flow and dynamic light‐scattering measurements
Authors: Goldmann Wolfgang H. , Guttenberg Zeno , Tang Jay X. , Kroy Klaus , Isenberg Gerhard , Ezzell Robert M.
Published: March 2003

Using amino acids 884−1066 and 884−1012 expressed from chicken vinculin as fusion proteins with schistosomal glutathione S‐transferase, we determined the binding kinetics of the protein fragments with F‐actin. We established by the stopped‐flow method a two‐step binding process : an initial rapid reaction followed by a slower process. The latter is attributed to F‐actin cross‐linking and/or bundling, which was previously detected by viscometry and electron microscopy [Johnson, R. P. & Craig, S. W. (1995) Nature 373, 261−264]. This is also supported by dynamic light‐scattering measurements, indicating dramatic changes in the internal actin filament dynamics, i.e. in bending undulations due to thermal noise. The similar size of the binding reaction for both fusion proteins with F‐actin indicates that the F‐actin binding site(s) on vinculin are located between residues 884−1012. No binding of pure glutathione S‐transferase or its fusion protein with vinculin peptide 1012−1066 with F‐actin was detected by either method.
cDNA cloning of the 43‐kDa latex allergen Hev b 7 with sequence similarity to patatins and its expression in the yeast Pichia pastoris
Authors: Sowka Slawomir , Wagner Stefan , Krebitz Monika , Arija‐Mad‐Arif Siti , Yusof Faridah , Kinaciyan Tamar , Brehler Randolf , Scheiner Otto , Breiteneder Heimo
Published: March 2003

IgE‐mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years. We purified natural Hev b 7, a 43‐kDa patatin‐like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences. A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library. The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa. The deduced amino acid sequence had 39−42 % identity to patatins from Solanum tuberosum. The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S. tuberosum, esterase activity. Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex‐sensitized allergic individuals. In contrast to patatins from S. tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N‐terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated. These results establish the 43‐kDa patatin‐like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins.
Expression, structure and chromosomal localization of the human cGMP‐binding cGMP‐specific phosphodiesterase PDE5A gene
Authors: Yanaka Noriyuki , Kotera Jun , Ohtsuka Akio , Akatsuka Hiroyuki , Imai Yuji , Michibata Hideo , Fujishige Kotomi , Kawai Eri , Takebayashi Shin‐Ichiro , Okumura Katsuzumi , Omori Kenji
Published: March 2003

cGMP‐binding, cGMP‐specific phosphodiesterase which is encoded by the PDE5A gene plays important roles in cardiovascular system, and is a significant target molecule of therapeutic agents. However, little is known about molecular characteristics of the human PDE5A gene. The 4.4‐kb cDNA encoding human PDE5A was isolated from lung and placenta cDNA libraries. The deduced amino acid sequence analysis demonstrated that N‐terminal amino acid sequence is dissimilar to that of rat PDE5A [Kotera, J., Yanaka, N., Fujishige, K., Imai, Y., Akatsuka, H., Ishizuka, T., Kawashima, K. & Omori, K. (1997) Eur. J. Biochem. 249, 434−442]. Human PDE5A mRNA is produced in high amounts in various tissues such as pancreas, skeletal muscle, placenta, heart, thyroid, adrenal cortex, testis, small intestine and stomach. In addition, the megakaryocyte‐like cell line Dami cells and two types of human vascular smooth muscle cells also produce the mRNA. Over 100‐kb chromosomal DNA corresponding to the human PDE5A gene was isolated and analyzed. The human PDE5A gene was revealed to contain 21 exons. Comparison of genomic organization with the rod photoreceptor phosphodiesterase β‐subunit gene (PDE6B), which is another kind of cGMP‐specific phosphodiesterase, has shown that the PDE5A and PDE6B genes are very similar in their relative exon−intron organization. In particular, the evolutionary relatedness of these genes was suggested in the catalytic domain. Furthermore, chromosomal location of the PDE5A gene was defined as being chromosome 4q26 by fluorescent in situ hybridization analysis.
ERp28, a human endoplasmic‐reticulum‐lumenal protein, is a member of the protein disulfide isomerase family but lacks a CXXC thioredoxin‐box motif
Authors: Ferrari David M. , Nguyen Van Phuc , Kratzin Hartmut D. , Söling Hans‐Dieter
Published: March 2003

We report on the isolation, sequence and a putative role of a human endoplasmic‐reticulum‐lumenal protein, ERp28. The protein has the C‐terminal retention signal KEEL and localizes to the endoplasmic reticulum (ER) as seen by subcellular fractionation and immunofluorescence studies. The protein has significant sequence similarity to members of the protein disulfide isomerase (PDI) family, although it lacks the thioredoxin box (CGHC) motif. We propose, on the basis of sequence analysis, a model of the domain structure of PDI, representing a significant extension of previously proposed models. Our results are in partial agreement with recently published NMR data [Kemmink, J., Darby, J., Dijkstra, K., Nilges, M. & Creighton, T. E. (1997) Curr. Biol. 7, 239−245] and indicate that PDI contains, in addition to the two thioredoxin folds described in previous models, two thioredoxin folds within the domains previously defined as b and b′. The thioredoxin domain of ERp28 shares a higher degree of similarity with the corresponding active and inactive domains of PDI than with other members of the PDI family, indicating that ERp28 developed from an ancient form of PDI or a PDI precursor. In contrast to Ig‐heavy‐chain‐binding protein, human ERp28 is not induced by metabolic stress (tunicamycin). In in vitro experiments, ERp28 and calnexin precipitate with overexpressed, wild‐type hepatitis B small surface antigen and with a mutated ER‐retained form. This indicates that ERp28, as calnexin, may be involved in the processing of secretory proteins within the ER.
Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis
Authors: Bouvier‐Navé Pierrette , Husselstein Tania , Benveniste Pierre
Published: March 2003

Two methyl transfers are involved in the biosynthesis of 24‐methyl and 24‐ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24‐methylene lophenol. About ten cDNA clones encoding S‐adenosyl‐
L‐methionine (AdoMet) sterol methyltransferases (SMTs) have been isolated so far from various plants. According to their deduced amino acid sequences, they were classified in two families, smt1 and smt2; in addition, smt2 cDNAs were shown to encode a 24‐methylene lophenol C241 methyltransferase [Bouvier‐Navé, P., Husselstein, T., Desprez, T. & Benveniste, P. (1997) Eur. J. Biochem. 246, 518−529]. We now report the comparison of two cDNAs isolated from Nicotiana tabacum, Ntsmt1‐1 which belongs to the first SMT cDNA family and Ntsmt2‐1 which belongs to the second. Both cDNAs were expressed in the yeast null mutant erg6, deficient in SMT. Whereas erg6 is devoid of 24‐alkyl sterols, erg6 Ntsmt1‐1 contained a majority of 24‐methylene sterols and erg6 Ntsmt2‐1, a majority of 24‐ethylidene sterols, indicating distinct functions for the expression products of these cDNAs. In the presence of AdoMet, delipidated microsomes from erg6 Ntsmt1‐1 efficiently converted cycloartenol into 24‐methylene cycloartanol, but did not produce any 24‐ethylidene lophenol upon incubation with 24‐methylene lophenol. This demonstrates that cDNA Ntsmt1‐1 (and most probably the other plant SMT cDNAs of the first family) encode(s) a cycloartenol C24 methyltransferase. In contrast, delipidated microsomes of erg6 Ntsmt2‐1 were shown to methylate preferentially 24‐methylene lophenol, as expected from an SMT encoded by an smt2 cDNA. In summary, among various cDNAs isolated from N. tabacum, one (Ntsmt1‐1) belongs to the first family of plant SMT cDNAs according to its deduced amino acid sequence and was shown to encode a cycloartenol C24 methyltransferase, whereas another (Ntsmt2‐1) belongs to the second family and was shown to encode a 24‐methylene lophenol C241 methyltransferase. Meanwhile, two cDNAs were isolated from Oriza sativa and shown to belong to smt1 and to smt2 families, respectively. These data disclose the coexistence, in a given plant species, of two distinct SMTs, each catalyzing one step of methylation in the sterol biosynthesis pathway.
Hypothermia enhances the biological activity of lipopolysaccharide by altering its fluidity state
Authors: Luhm Jürgen , Schromm Andra Beate , Seydel Ulrich , Brandenburg Klaus , Wellinghausen Nele , Riedel Eberhard , Schumann Ralf Reiner , Rink Lothar
Published: March 2003

Lipopolysaccharides (LPS, endotoxin) of Gram‐negative bacteria are among the main causes of sepsis and septic shock. In the present study, the influence of temperature on the biological activity of LPS was investigated. Lowering the temperature from 37 +C to 34.5 +C or to 30 +C significantly enhances in vitro tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β and IL‐6 release induced by different LPS chemotypes and heat‐inactivated Escherichia coli. This cytokine‐increasing effect of lowering the temperature is highly mediated by serum proteins, particularly by LPS‐binding protein (LBP) and low‐density lipoproteins (LDL). In contrast, cytokine production induced by the superantigen toxic shock syndrome toxin‐1 (TSST‐1) from Gram‐positive Staphyloccoccus aureus decreases by around 70 % at 30 +C as compared with 37 +C, corresponding to the expected effect of change in temperature and regardless of the presence of serum proteins. In order to explain the unexpected biological hypothermia effect with regard to LPS, the fluidity state of the lipid A portion of LPS as one important physico‐chemical property possibly involved was investigated. The fluidity, determined by fluorescence polarization measurements, was found to decrease with decreasing temperature. These data suggest that a low fluid LPS chemotype is biologically more active than a more fluid one (and vice versa). Statistical analysis of the results shows a strong correlation between cytokine secretion and fluidity state of a given LPS chemotype (0.71 < r < 0.89, all P < 0.01). As a clinical consequence, these data may be one possible explanation for the higher mortality rate of hypothermic Gram‐negative sepsis.
Subunits composition and allosteric control in Carcinus aestuarii hemocyanin
Authors: Dainese Enrico , Di Muro Paolo , Beltramini Mariano , Salvato Benedetto , Decker Heinz
Published: March 2003

Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca2+ : 24S accounting for 90 % of total hemocyanin and 16S accounting for 10 %. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component. The 5S component consists of three monomeric fractions named CaeSS1 (10 %), CaeSS2 (50 %) and CaeSS3 (40 %); the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 have been studied as far as their reassociation properties to form hexamers are concerned. We investigated the oxygen‐binding properties of the native form (24S), the mixture of the two 16S forms, the pH‐stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggregate. The analysis of O2‐binding data reveals that 24S‐Hc can be well described by the modified Monod Wyman and Changeaux‐model (nested MWC‐model), while the half‐molecules (16S) bind oxygen according to the simple MWC‐model. The two hexameric 16S within the dodecameric 24S hemocyanin can be regarded as nested allosteric units. They behave as being functionally coupled in the T‐states (tT and rT). In the R‐states (tR and rR) the two half‐molecules seem to be functionally uncoupled since they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.
Structural and serological studies on a new acidic O‐specific polysaccharide of Proteus vulgaris O32
Authors: Bartodziejska Beata , Shashkov Alexander S. , Babicka Dorota , Grachev Aleksei A. , Torzewska Agnieszka , Paramonov Nikolay A. , Chernyak Anatoly Y. , Rozalski Antoni , Knirel Yuriy A.
Published: March 2003

The following structure of the O‐specific polysaccharide chain (O‐antigen) of the Proteus vulgaris O32 lipopolysaccharide (LPS) was established by 1H‐NMR and 13C‐NMR spectroscopy, including two‐dimensional NOESY and H‐detected 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments :
←2)‐α‐
L‐RhapI‐(1←2)‐α‐
L‐RhapII‐(1←4)‐β‐
D‐GalpAI‐(1←3)‐β‐
D‐GlcpNAc‐(1←4)‐α‐
D‐GalpAII‐(1←.
In addition, an O‐acetyl group was detected, which, most probably, is located at position 3 of a part of RhapI residues.

Serological studies, using rabbit polyclonal anti‐(P. vulgaris O32) serum, homologous and heterologous Proteus O‐antigens and related artificial antigens, revealed the importance of an α‐
D‐GalA‐associated epitope in manifesting the immunospecificity of P. vulgaris O32 and substantiated serological relationships between the O‐antigen studied and those of some other Proteus strains.
Structure of the promoter and complete sequence of the gene coding for the rabbit translationally controlled tumor protein (TCTP) P23
Authors: Thiele Holger , Berger Mario , Lenzner Claudia , Kühn Hartmut , Thiele Bernd‐Joachim
Published: March 2003

We have isolated genomic recombinants containing the complete gene coding for the rabbit translationally controlled tumor protein (TCTP), also known as histamine‐releasing factor (HRF) P23. The gene is organized into five introns and six exons and its total length amounts to 3819 nucleotides. All intron/exon boundaries are in accordance with the GT/AG rule. Transcription of the gene generates two mRNAs of 843 and 1163 nucleotides differing in the length of their 3′‐untranslated regions. They are formed by alternative polyadenylation. The transcription initiation site has been determined by comparison of sequences of the gene and several processed TCTP pseudogenes. The full‐length 5′‐untranslated region comprises 116 nucleotides and starts with an oligopyrimidine tract important for translational regulation. Additionally 1.2 kb of the 5′‐flanking promoter region has been sequenced. The promoter contains a TATA box at −30 and potential binding sites for transcription factors such as stimulating protein 1 (Sp1), nuclear factor 1 (NF1), activator protein 1 (AP1), c‐Ets1, cAMP‐response element (CP2), myeloid‐specific zinc finger protein 1 (MZF1) and others. For functional analysis 5′‐flanking sequences up to −918 were fused to the chloramphenicol acetyltransferase (CAT) gene and tested using a rabbit aortic smooth‐muscle cell line by cell transfection and CAT assays. The results confirm that the analyzed gene is the actively transcribed TCTP gene.
Deletion of the PB‐loop in the LCM subunit does not affect phycobilisome assembly or energy transfer functions in the cyanobacterium Synechocystis sp. PCC6714
Authors: Ajlani Ghada , Vernotte Claudie
Published: March 2003

In cyanobacteria, light energy is mainly harvested for photosynthesis by the phycobilisome (PBS) : a large pigment‐protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides in order to optimize light‐energy absorbance and transfer to photosystem II. The core membrane linker subunit (LCM) is a fascinating multifunctional polypeptide that participates in the PBS structure, function and anchoring to the photosynthetic membrane. Sequence analysis has defined several domains within the LCM polypeptide. The C‐terminal portion contains two to four repeated domains that are similar to the conserved domains of linker polypeptides and are believed to play the same role. The N‐terminal portion is similar to phycobiliproteins (PB‐domain) and carries, like phycobiliproteins, a covalently linked phycobilin chromophore. This domain is interrupted by a so‐called PB‐loop insertion. The PB‐domain of the LCM is thus regarded as one of the core subunits, with its PB‐loop protruding towards the photosynthetic membrane. The PB‐loop was thought to be involved in the attachment of the PBS to the photosynthetic membrane. We generated an apcE gene (encoding LCM), in which we deleted the sequence encoding 54 amino acids of the PB‐loop domain. The modified gene was expressed in a Synechocystis PCC6714 strain in which the apcE gene had been inactivated. The truncated polypeptide was functionally equivalent to the wild‐type LCM; PBSs were assembled and functioned as in the wild‐type. The PB‐loop of the LCM seems thus dispensable for the PBS biogenesis and function.
Identification of the reaction products of (2′‐5′)oligoadenylate synthetase in the marine sponge
Authors: Kuusksalu Anne , Subbi Juhan , Pehk Tõnis , Reintamm Tõnu , Müller Werner E. G. , Kelve Merike
Published: March 2003

Previously we reported on the presence of a high (2′‐5′)oligoadenylate synthetase activity in the marine sponge Geodia cydonium [Kuusksalu, A., Pihlak, A., Müller, W. E. G. & Kelve, M. (1995) Eur. J. Biochem. 232, 351−357]. The presence of (2′‐5′)oligoadenylates [(2′‐5′)A] in crude sponge extract was shown by radioimmunoassay and by their HPLC comigration with authentic (2′‐5′)A oligomers. In addition, the sponge (2′‐5′)oligoadenylates displayed biological activity, as determined by inhibition studies of protein biosynthesis in rabbit reticulocyte lysate. In the present study individual (2′‐5′)oligoadenylates synthesized by sponge enzyme were separated by HPLC. The exact composition of every oligonucleotide peak eluted was determined by matrix‐assisted laser‐desorption‐ionization mass spectrometry (MALDI‐MS) analysis. The 2′‐5′ phosphodiester bond in oligoadenylates was verified by NMR analysis. Based on the high concentration of (2′‐5′)A oligomers in G. cydonium and their similarity with those found in mammals we propose that the (2′‐5′)A system is involved in a cytokine‐mediated pathway and/or in a protection system against viruses, present in the marine environment.
Characterisation of acyl binding by a plant lipid‐transfer protein
Authors: Zachowski Alain , Guerbette Françoise , Grosbois Michèle , Jolliot‐Croquin Alain , Kader Jean‐Claude
Published: March 2003

Maize lipid‐transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluorescent‐labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyrene moiety attached at the end, alone or in conjunction with an anthroyloxy analogue, indicated that LTP could bind two fatty acids although with a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing that the four S‐S bonds of LTP are essential for its lipid binding property. Other analogues used were 16‐carbon or 18‐carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is more important around the middle of the chain than at its extremities. Binding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond did not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduced the affinity. Fatty acyl‐CoA or lysoderivatives bound as well as the corresponding fatty acid.
Identification of an essential gene encoding a class‐V unconventional myosin in Drosophila melanogaster
Authors: MacIver Bryce , McCormack Angela , Slee Roger , Bownes Mary
Published: March 2003

Class‐V myosins are a unique type of myosin motor with roles in intracellular transport. The mouse dilute gene was the first member of this class to be cloned, with mutations resulting in lightening of the coat colour or neurogenic defects leading to early death. Further examples of class‐V myosins have been described in yeast, chicken and rat. Here, we report the cloning of the first class‐V myosin from Drosophila. We show that expression of this myosin is predominantly in the adult germ line and early embryo and that the transcript is localised in the oocyte during oogenesis. Genetic and in situ hybridisation experiments have determined that this gene is located in the 43C region. We have evidence that it maps to a mutation in this region with an embryonic lethal phenotype.
Degradation of the morpholino ring in the crystal structure of cyanomorpholinodoxorubicin complexed with d(CGATCG)
Authors: Ettorre Alessandro , Cirilli Maurizio , Ughetto Giovanni
Published: March 2003

The cyanomorpholino analogue of antitumor anthracycline doxorubicin possesses an intense potency and differs from the parent compound in cross‐resistance and other biological properties. The induction by cyanomorpholinodoxorubicin of both DNA cross‐links and strand scission suggests an altered mode of action relative to doxorubicin with a different DNA‐interacting capacity. We have co‐crystallized 3′‐(3‐cyano‐4‐morpholinyl)‐3′‐desaminodoxorubicin (CMD) with the DNA hexamer d(CGATCG) and have determined the crystal structure at 0.16‐nm resolution. The complex crystallizes in the space group P41212 and is similar to the previously reported anthracycline/DNA structures, with the drug intercalated at the CpG step, forming hydrogen bonds with the guanine residue. The structure reveals that the morpholino moiety has undergone a major rearrangement with loss of the cyano group and opening of the morpholino ring. The compound actually bound to DNA in the complex resembles N‐(2‐hydroxyethyl)doxorubicin, which was previously identified as a hydrolysis product of CMD. No DNA alkylation has been observed. However, the structure shows that the active site of the morpholino ring, after dissociation of the cyano group, lies in the minor groove in proximity of the A/T base pair. This may indicate that a C/G base pair next to the intercalation site, with NH2 group in the minor groove, is required for DNA alkylation.
Purification and cloning of the mitochondrial branched‐chain amino acid aminotransferase from sheep placenta
Authors: Faure Magali , Glomot Françoise , Bledsoe Randy , Hutson Susan , Papet Isabelle
Published: March 2003

This paper presents the first purification of the mitochondrial branched‐chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82–85%) than with the cytosolic isoenzymes (60%).

By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched‐chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched‐chain amino acid catabolism is specific to each species.
Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non‐native outer membrane proteins
Authors: De Cock Hans , Schäfer Ute , Potgeter Martin , Demel Rudy , Müller Matthias , Tommassen Jan
Published: March 2003

The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins. In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases. The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations. Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo. In addition, it interacts specifically with outer membrane proteins that are in their non‐native state. We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane.