bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of biosynthesis and release of crustacean hyperglycemic hormone isoforms in the X‐organ–sinus gland complex of the crayfish Orconectes limosus
Authors: Ollivaux C. , Soyez D.
Published: March 2003

The crustacean hyperglycemic hormone (CHH) is the major neuropeptide produced by the X‐organ–sinus gland neurosecretory system of the crayfish, Orconectes limosus. This hormone is synthesized by two different cell types, as two isomers (CHH and d‐Phe3‐CHH) which display different activities The aim of this report is to analyze and compare the synthetic and secretory activities of these specialized cells. In vitro pulse–chase incubations and time–course experiments were conducted on isolated X‐organ–sinus gland (XO–SG) complexes, followed by analysis of the labeled peptides. The different steps of the post‐translational processing of the CHH precursor, including proteolytic cleavage of the propeptide, C‐terminal amidation and N‐terminal pyroglutamylation were characterized and the kinetics of CHHs maturation were estimated in the different parts of the neuroendocrine complex. Furthermore, synthesis of CHHs in XO–SG complexes and release in incubation media were investigated using combined HPLC/immunoassay. Under basal conditions, i.e. without stimulation, similar dynamics for both isomers were found and results indicate that newly synthesized CHHs are preferentially released.
Quantitative analysis of Na+‐Ca2+ exchanger expression in guinea‐pig heart
Authors: McDonald Ruth L. , Colyer John , Harrison Simon M.
Published: March 2003

In previous studies, regional variations in the expression of the Na+‐Ca2+ exchanger (NCX) have been examined qualitatively in human heart using the C2C12 monoclonal antibody [Wang, J., Schwinger, R.H., Frank, K., Muller‐Ehmsen, J., Martin‐Vasallo, P., Pressley, T.A., Xiang, A., Erdmann, E. & McDonough, A.A. (1996) J. Clin. Invest.98, 1650–1658]. Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between atrial and ventricular tissue. NCX has been located in the general sarcolemma and t‐tubules of ventricular muscle and as t‐tubules are sparse in atrial tissue compared to ventricular tissue, it is surprising that NCX expression was found to be similar in both atria and ventricles [Wang et al. (1996)]. To reinvestigate this, we have used SDS/PAGE and a quantitative Western blotting technique to determine the pattern of expression of NCX in guinea‐pig heart in tissue samples from left atrium, right atrium, septum, left ventricle and right ventricle. NCX protein expression was 17.5 ± 3.9 pmol·mg−1 of protein in the left atrium and 29.2 ± 6.1 pmol·mg−1 of protein in the right atrium, which were both significantly lower (P < 0.05) than NCX expression in the septum, left ventricle and right ventricle (64.7 ± 15.2, 76.8 ± 19.5 and 69.4 ± 14.1 pmol·mg−1 of protein, respectively,n = 7). These differences in NCX expression may reflect variations in the cellular location of NCX protein in these regions. To study this, we used confocal immunofluorescence of single isolated myocytes to examine differences in the proportion of fluorescent staining on the general surface membrane compared with the interior of the cell (which presumably reflects a t‐tubular location). We found that the general membrane staining was 79.0 ± 1.2% in cells from the atria which was significantly higher (P < 0.001) than that seen in cells from the septum, left ventricle and right ventricle, with 48.1 ± 1.1%, 48.2 ± 1.8% and 45.6 ± 1.3%, respectively (n = 20). These results illustrate a similar pattern of NCX expression in guinea‐pig and human, with expression in atrial tissue significantly lower than in ventricular tissue. However, the cellular location of NCX differs regionally; in atrial tissue, the majority of the NCX protein is located in the general sarcolemma whereas in ventricular and septal tissue, approximately 50% of NCX protein is located within the cell (presumably at the level of the t‐tubules).
18O‐Labelling pattern of okadaic acid from H218O in dinoflagellate Prorocentrum lima elucidated by tandem mass spectrometry
Authors: Izumikawa Miho , Murata Michio , Tachibana Kazuo , Fujita Tsuyoshi , Naoki Hideo
Published: March 2003

Okadaic acid is a metabolite of the unicellular algae dinoflagellate. Its biosynthesis has attracted considerable attention since the skeletal structure was shown to be synthesized via an unprecedented route. However, its relevant intermediates or enzymes are unknown. In the course of our previous investigations on the oxygen source of okadaic acid by tandem mass spectrometry (CID MS/MS), we determined the level of 18O incorporation for each oxygen site from 18O2 and [18O2]acetate. In the present study, we examined H218O‐labelling patterns of okadaic acid from dinoflagellates in comparison with salinomycin from actinomycetes and has provided intriguing information regarding biosynthesis. Unexpectedly, oxygen atoms originating from acetate were not labelled from H218O; this can not be accounted for by the usual metabolic route where acetyl‐CoA is biosynthesized via pyruvate. Similar experiments for salinomycin revealed that all of its oxygen atoms derived from acetate or propionate were labelled by H218O. Another interesting feature is that two oxygen sites were derived from both O2 and H2O while the others were labelled only from O2. These results imply that an oxidation mechanism other than those in actinomycetes polyethers may be involved in the biosynthesis of okadaic acid.
Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene
Authors: Alape‐Girón Alberto , Flores‐Díaz Marietta , Guillouard Isabelle , Naylor Claire E. , Titball Richard W. , Rucavado Alexandra , Lomonte Bruno , Basak Ajit K. , Gutiérrez José M. , Cole Stewart T. , Thelestam Monica
Published: March 2003

Clostridium perfringens phospholipase C (PLC), also called α‐toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered α‐toxin variants harboring single amino‐acid substitutions in three loops of its C‐terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane‐interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103‐fold lower than that of the wild‐type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11–73% of the hemolytic activity and exhibited a cytotoxic potency 102‐ to 105‐fold lower than that of the wild‐type toxin. The results demonstrated that the sphingomyelinase activity and the C‐terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild‐type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.
Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana
Authors: Papenbrock Jutta , Schmidt Ahlert
Published: March 2003

Sulfurtransferases transfer a sulfane atom from a donor substrate to a thiophilic acceptor molecule. Recently a sulfurtransferase specific for the substrate 3‐mercaptopyruvate was isolated from Arabidopsis thaliana[Papenbrock, J. & Schmidt, A. (2000) Eur. J. Biochem. 267, 145–154]. In this study a second sulfurtransferase from Arabidopsis was characterized and compared to the enzyme described previously. Sequences of the mature proteins had an identity of 77.7%. The plant sulfurtransferases formed a distinct group within the known eukaryotic sulfurtransferases. When Southern blots were hybridized with labelled cDNA fragments from each of the plant sulfurtransferases the same pattern of bands was obtained indicating the existence of only these two closely related sulfurtransferases. The new sulfurtransferase was expressed in Escherichia coli fused with an N‐terminal His6‐tag, purified and tested for enzyme activity. Like the first enzyme, the newly isolated protein preferred 3‐mercaptopyruvate to thiosulfate as substrate. The Km of both enzymes determined for 3‐mercaptopyruvate and cyanide were almost identical. As a result of database searches it became obvious that sulfurtransferase proteins from higher plants showed high similarities to small senescence‐ and stress‐induced proteins. To prove the involvement of sulfurtransferases in senescence‐associated processes 3‐mercaptopyruvate sulfurtransferase activity was determined in crude protein extracts from Arabidopsis plants of different ages. 3‐mercaptopyruvate sulfurtransferase activity and steady‐state RNA levels of sulfurtransferases increased with increasing age. However, steady‐state protein levels as measured by using an antibody against the sulfurtransferase protein expressed previously decreased. Putative roles of sulfurtransferases in senescence‐associated processes are discussed.
Novel polyunsaturated n‐alkenes in the marine diatom Rhizosolenia setigera
Authors: Damsté Jaap S. Sinninghe , Schouten Stefan , Rijpstra W. Irene C. , Hopmans Ellen C , Peletier Harry , Gieskes Winfried W. C. , Geenevasen Jan A. J.
Published: March 2003

Four previously unknown n‐C25 and n‐C27 heptaenes of the marine diatom Rhizosolenia setigera were isolated and identified using NMR spectroscopy. They possess six methylene interrupted (Z)‐double bonds starting at C‐3 and an additional terminal or n‐2 (Z)‐double bond. Structural and stable carbon isotopic evidence suggests that these polyenes are biosynthesized by chain elongation of the C22:6n‐3 fatty acid, followed by decarboxylation and introduction of double bonds at specific positions.
Purification and cloning of pierisin‐2, an apoptosis‐inducing protein from the cabbage butterfly, Pieris brassicae
Authors: Matsushima‐Hibiya Yuko , Watanabe Masahiko , Kono Takuo , Kanazawa Takashi , Koyama Kotaro , Sugimura Takashi , Wakabayashi Keiji
Published: March 2003

The cabbage butterfly Pieris rapae contains a strong apoptosis‐inducing substance, pierisin, against human cancer cell lines, which is thought to act via ADP‐ribosylation. Here we report the purification and cloning of an apoptosis‐inducing substance, designated as pierisin‐2, from another cabbage butterfly, Pieris brassicae. Pierisin‐2 was purified from pupae by sequential chromatography and its cytotoxic and apoptosis‐inducing activities to various cancer cells were similar to those of pierisin, designated as pierisin‐1, from P. rapae. cDNA cloning of pierisin‐2 was performed on the basis of the partial amino‐acid sequence. The nucleotide sequence indicated that the cDNA encodes an 850‐amino‐acid protein with a calculated molecular mass of 97 986. The deduced amino‐acid sequence of pierisin‐2 was 91% identical with that of pierisin‐1. In vitro expressed protein in the reticulocyte lysate exhibited apoptosis‐inducing activities against human gastric carcinoma TMK‐1 and cervical carcinoma HeLa cells, similar to the purified native pierisin‐2 from the pupae. Pierisin‐2 shows regional sequence similarities with certain ADP‐ribosylating toxins such as the A‐subunit of cholera toxin. The results from site‐directed mutagenesis at Glu165, a conserved residue among ADP‐ribosylating enzymes necessary for NAD binding, and from experiments with ADP‐ribosylating enzyme inhibitors suggested that pierisin‐2 could be considered as an ADP‐ribosylating toxin like pierisin‐1.
Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28
Authors: Stohwasser Ralf , Salzmann Ulrike , Giesebrecht Jan , Kloetzel Peter‐Michael , Holzhütter Hermann‐Georg
Published: March 2003

The activation kinetics of constitutive and IFNγ‐stimulated 20S proteasomes obtained with homomeric (recPA28α, recPA28β) and heteromeric (recPA28αβ) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling.

The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28α/RecP28β/RecP28α+ RecP28β) exhibit biphasic characteristics which can be attributed to a low‐level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28αβ complex is about two orders of magnitude higher than towards the homomeric PA28α and PA28β complexes.

Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin‐like, trypsin‐like and peptidylglutamyl‐peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.
Ether lipids in the cell membrane of Mycoplasma fermentans
Authors: Wagner Frauke , Rottem Shlomo , Held Heinz‐Dieter , Uhlig Stefan , Zähringer Ulrich
Published: March 2003

Two new ether lipids, 1‐O‐alkyl/alkenyl‐2‐O‐acyl‐glycero‐3‐phosphocholine and its lyso form, 1‐O‐alkyl/alkenyl‐glycero‐3‐phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC‐MS, MALDI‐TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1′ (plasmalogen‐type lipid) or 9′ in a ratio ≈ 1 : 1. This is the first report of the presence of alkyl and vinyl (alk‐1′‐enyl) ether lipids in the cell membrane of aerobically grown mycoplasmas. Lipids of this type have been found in some Gram‐positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2‐O‐acetylated lyso form lipid can mimic platelet‐activating factor activity in isolated perfused and ventilated rat lungs.
Comparison of the substrate specificity of the human T‐cell leukemia virus and human immunodeficiency virus proteinases
Authors: Tözsér József , Zahuczky Gábor , Bagossi Péter , Louis John M. , Copeland Terry D. , Oroszlan Stephen , Harrison Robert W. , Weber Irene T.
Published: March 2003

Human T‐cell leukemia virus type‐1 (HTLV‐1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV‐1) PR inhibitors, the proteinase (PR) of HTLV‐1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV‐1 PR was characterized and compared to that of HIV‐1 PR. Two sets of substrates were used that contained single amino‐acid substitutions in peptides representing naturally occurring cleavage sites in HIV‐1 and HTLV‐1. The original HIV‐1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV‐1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV‐1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV‐1 and HIV‐1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV‐1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV‐1 substrate and on analysis of the modeled complex of HTLV‐1 PR with substrate, the substrate binding site of the HTLV‐1 PR appeared to be more extended than that of HIV‐1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV‐1 is evolutionarily optimized for rapid hydrolysis.
Phospholipases A2 from Callosellasma rhodostoma venom gland
Authors: Tsai Inn‐Ho , Wang Ying‐Ming , Au Lo‐Chun , Ko Tzu‐Ping , Chen Yi‐Hsuan , Chu Yi‐Fang
Published: March 2003

Callosellasma rhodostoma (Malayan pitviper) is a monotypic Asian pitviper of medical importance. Three acidic phospholipases A2 (PLA2s) and one basic PLA2‐homolog were purified from its venom while 10 cDNAs encoding distinct PLA2s were cloned from venom glands of a Thailand specimen of this species. Complete amino‐acid sequences of the purified PLA2s were successfully deduced from their cDNA sequences. Among the six un‐translated PLA2 cDNAs, two apparently result from recombination of its Lys49‐PLA2 gene with its Asp49‐PLA2 genes. The acidic PLA2s inhibit platelet‐aggregation, while the noncatalytic PLA2‐homolog induces local edema. This basic PLA2‐homolog contains both Asp49 and other, unusual substitutions unique for the venom Lys49‐PLA2 subtype (e.g. Leu5, Trp6, Asn28 and Arg34). Three‐dimensional modelling of the basic protein revealed a heparin‐binding region, and an abnormal calcium‐binding pocket, which may explain its low catalytic activity. Oxidation of up to six of its Met residues or coinjection with heparin reduced its edema‐inducing activity but methylation of its active site His48 did not. The distinct Arg/Lys‐rich and Met‐rich region at positions 10–36 of the PLA2 homolog presumably are involved in its heparin‐binding and the cell membrane‐interference leading to edema and myotoxicity.
Genomic organization of the human phosphodiesterase PDE11A gene
Authors: Yuasa Keizo , Kanoh Yutaka , Okumura Katsuzumi , Omori Kenji
Published: March 2003

PDE11A is a dual‐substrate, cAMP and cGMP, cyclic nucleotide phosphodiesterase (PDE). Presently four unique variants carrying distinct GAF sequences in the N‐terminal region have been identified. While human PDE11A3 and PDE11A4 are known to be specifically expressed in testis and prostate, respectively, PDE11A1 was mainly detected in skeletal muscle. The human PDE11A gene was investigated and revealed to span > 300 kb, contain 23 exons and be mapped on chromosome 2q31. The transcription start sites of PDE11A1, PDE11A3 and PDE11A4 were determined, and the promoter sequences were revealed. Although 5′ flanking genomic regions of PDE11A1 and PDE11A3 had a consensus TATA motif, that of PDE11A4 was a TATA‐less but contained CCAAT box and Sp1‐binding sequence. Interestingly, we found that the exon 2 sequence for N‐terminal region of PDE11A3 encoded an N‐terminal sequence of the cytochrome c pseudogene in an alternate reading frame, and that C‐terminal region of the cytochrome c pseudogene in intron 2 was disrupted by the insertion of Alu repetitive sequence. Furthermore, we examined the exon–intron organization of the PDE2A gene and compared the exon organization among GAF‐PDE family. The exon organization of the PDE11A catalytic domain was very similar to those of PDE5A and PDE6B. However, other GAF‐PDEs, PDE2A and PDE10A, displayed different exon organization from PDE11A although these three PDEs are similar in their amino‐acid sequences to each other. The findings suggested that PDE11A has a common ancestral gene with PDE5A and PDE6s, whereas PDE2A and PDE10A are generated separately from these three GAF‐PDEs.
Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology
Authors: Eriksson Tove L. J. , Rasool Omid , Huecas Sonia , Whitley Paul , Crameri Reto , Appenzeller Ulrich , Gafvelin Guro , van Hage‐Hamsten Marianne
Published: March 2003

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L. destructor‐sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and express new allergens from L. destructor and determine their recognition frequency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor‐sensitized individuals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C‐terminal His6‐tagged proteins. Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L. destructor‐sensitized subjects, respectively.
Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor
Authors: Rochel Natachu , Tocchini‐Valentini Guiseppe , Egea Pascal F. , Juntunen Kari , Garnier Jean‐Marie , Vihko Pirkko , Moras Dino
Published: March 2003

Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N‐terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild‐type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X‐ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild‐type is not well ordered.
A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale
Authors: Xu Feng , Golightly Elizabeth J. , Fuglsang Claus C. , Schneider Palle , Duke Kyle R. , Lam Loi , Christensen Søren , Brown Kimberly M. , Jørgensen Christel T. , Brown Stephen H.
Published: March 2003

A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (≤ 25% identity or 65% similarity) to other known flavin‐containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino‐acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono‐, oligo‐, or polymeric saccharides, and transfer their electrons to O2 or other acceptors. When d‐glucose served as electron‐donating substrate, an activity of 2 s−1 was observed at pH 5.5 and 23 °C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.
Expression and activity of ADAMTS‐5 in synovium
Authors: Vankemmelbeke Mireille N. , Holen Ingunn , Wilson Anthony G. , Ilic Mirna Z. , Handley Christopher J. , Kelner Gregory S. , Clark Melody , Liu Changlu , Maki Richard A. , Burnett David , Buttle David J.
Published: March 2003

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase‐1 (ADAMTS‐4) and aggrecanase‐2 (ADAMTS‐5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS‐5 from human arthritic and bovine synovium. ADAMTS‐5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin‐1α or all‐trans‐retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS‐5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte‐derived aggrecanases, and the activity was inhibited by heparin. ADAMTS‐5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium‐derived ADAMTS‐5 may play a role in cartilage aggrecan breakdown is discussed.
Overexpression of the Golgi‐localized enzyme α‐mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl‐N‐acetylchitobiose to tetramannosyl‐N‐acetylchitobiose in the N‐glycan‐processing pathway
Authors: Oh‐eda Masayoshi , Nakagawa Hiroaki , Akama Tomoya O. , Lowitz Kevin , Misago Masahiro , Moremen Kelley W. , Fukuda Michiko N.
Published: March 2003

Golgi α‐mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn1M5Gn2 to Gn1M3Gn2 during biosynthesis of N‐glycans. Previously, we isolated a cDNA encoding a protein homologous to α‐mannosidase II and designated it α‐mannosidase IIx. Here, we show by immunocytochemistry that α‐mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with α‐mannosidase II, α‐mannosidase IIx colocalizes with α‐mannosidase II in COS cells. A protein A fusion of the catalytic domain of α‐mannosidase IIx hydrolyzes a synthetic substrate, 4‐umbelliferyl‐α‐d‐mannoside, and this activity is inhibited by swainsonine. [3H]glucosamine‐labeled Chinese hamster ovary cells overexpressing α‐mannosidase IIx show a reduction of M6Gn2 and an accumulation of M4Gn2. Structural analysis identified M4Gn2 to be Manα1→6(Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc. The results suggest that α‐mannosidase IIx hydrolyzes two peripheral Manα1→6 and Manα1→3 residues from [(Manα1→6)(Manα1→3)Manα1→6](Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc, during N‐glycan processing.
A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin
Authors: Francky Andrej , Francky Bojana Mozetič , Štrukelj Borut , Gruden Kristina , Ritonja Anka , Križaj Igor , Kregar Igor , Pain Roger H. , Pungerčar Jože
Published: March 2003

Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active‐site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem.261, 746–752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide–enzyme interaction, along with residues nearer the N‐terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.
4‐Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB
Authors: Kamerbeek Nanne M. , Moonen Mariëlle J. H. , van der Ven Jos G. M. , van Berkel Willem J. H. , Fraaije Marco W. , Janssen Dick B.
Published: March 2003

A novel flavoprotein that catalyses the NADPH‐dependent oxidation of 4‐hydroxyacetophenone to 4‐hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4‐hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of ≈ 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4‐hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer–Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino‐acid protein with a deduced mass of 71 884 Da. Except for an N‐terminal extension of ≈ 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer–Villiger monooxygenases: cyclohexanone monooxygenase (27–33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site‐directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
Computer modelling and experimental evidence for two steady states in the photosynthetic Calvin cycle
Authors: Poolman Mark G. , Ölçer Hülya , Lloyd Julie C. , Raines Christine A. , Fell David A.
Published: March 2003

We present observations of photosynthetic carbon dioxide assimilation, and leaf starch content from genetically modified tobacco (Nicotiana tabacum) plants in which the activity of the Calvin cycle enzyme, sedoheptulose‐1,7‐bisphosphatase, is reduced by an antisense construct. The measurements were made on leaves of varying ages and used to calculate the flux control coefficients of sedoheptulose‐1,7‐bisphosphatase over photosynthetic assimilation and starch synthesis. These calculations suggest that control coefficients for both are negative in young leaves, and positive in mature leaves. This behaviour is compared to control coefficients obtained from a detailed computer model of the Calvin cycle. The comparison demonstrates that the experimental observations are consistent with bistable behaviour exhibited by the model, and provides the first experimental evidence that such behaviour in the Calvin cycle occurs in vivo as well as in silico.
BJ46a, a snake venom metalloproteinase inhibitor
Authors: Valente Richard H. , Dragulev Bojan , Perales Jonas , Fox Jay W. , Domont Gilberto B.
Published: March 2003

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl‐Sepharose and C4‐reversed phase chromatographies, resulted in the isolation of the anti‐hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P‐I) and jararhagin (P‐III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single‐chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI‐TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322‐amino‐acid protein containing four putative N‐glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.
Genetic and biochemical characterization of a short‐chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus
Authors: van der Oost John , Voorhorst Wilfried G. B. , Kengen Servé W. M. , Geerling Ans C. M. , Wittenhorst Vincent , Gueguen Yannick , de Vos Willem M.
Published: March 2003

The gene encoding a short‐chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the β‐glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26‐kDa subunits. AdhA is an NADPH‐dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2‐pentanol (46 U·mg−1) and pyruvaldehyde (32 U·mg−1) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 °C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.
The two‐component regulatory system BacRS is associated with bacitracin ‘self‐resistance’ of Bacillus licheniformis ATCC 10716
Authors: Neumüller Andrea M. , Konz Dirk , Marahiel Mohamed A.
Published: March 2003

Bacitracin is a peptide antibiotic produced by several Bacillus licheniformis strains that is most active against other Gram‐positive microorganisms, but not against the producer strain itself. Recently, heterologous expression of the bacitracin resistance mediating BcrABC transporter in Bacillus subtilis and Escherichia coli was described. In this study we could determine that the transporter encoding bcrABC genes are localized about 3 kb downstream of the 44‐kb bacitracin biosynthetic operon bacABC. Between the bac operon and the bcrABC genes two orfs, designated bacR and bacS, were identified. They code for proteins with high homology to regulator and sensor proteins of two‐component systems. A disruption mutant of the bacRS genes was constructed. While the mutant displayed no effects on the bacitracin production it exhibited highly increased bacitracin sensitivity compared to the wild‐type strain. Western blot analysis of the expression of BcrA, the ATP‐binding cassette of the transporter, showed in the wild‐type a moderate BcrA induction in late stationary cells that accumulate bacitracin, whereas in the bacRS mutant cells the BcrA expression was constitutive. A comparison of bacitracin stressed and nonstressed wild‐type cells in Western blot analysis revealed increasing amounts of BcrA and a decrease in BacR in the stressed cells. From these findings we infer that BacR acts as a negative regulator for controlling the expression of the bcrABC transporter genes.
Isolation and characterization of complex I, rotenone‐sensitive NADH:ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei
Authors: Fang Jing , Wang Yudong , Beattie Diana S.
Published: March 2003

Additional characterization of complex I, rotenone‐sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone‐sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl‐β‐d‐maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second‐dimensional Tricine–SDS/PAGE including the 51 kDa, PSST and TYKY subunits.
Dimethylsulfoxide promotes K+‐independent activity of pyruvate kinase and the acquisition of the active catalytic conformation
Authors: Ramírez‐Silva Leticia , T. Ferreira Sérgio , Nowak Thomas , Tuena de Gómez‐Puyou Marietta , Gómez‐Puyou Armando
Published: March 2003

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232–239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+‐independent activity [Ramírez‐Silva, L., Tuena de Gómez‐Puyou, M., & Gómez‐Puyou, A. (1993) Biochemistry32, 5332–5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 µmol·min−1·mg−1 in 40% dimethylsulfoxide, compared with 280 and 0.023 µmol· min1·mg−1 observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg2+, phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30–40% dimethylsulfoxide coincided with that of the enzyme–Mg2+–phosphenolpyruvate–K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase–Mn2+–(CH3)4N+ complex in 30–40% dimethylsulfoxide were similar to those of the pyruvate kinase–Mn2+–K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30–40% dimethylsulfoxide induces its active conformation.
Proteolytic processing of a human salivary proline‐rich protein precursor by proprotein convertases
Authors: Chan Maggie , Bennick Anders
Published: March 2003

Salivary proline‐rich proteins (PRPs) are synthesized as precursors that are cleaved before secretion giving rise to glycosylated PRPs which have lubricating function and basic PRPs which are potent precipitators of dietary tannins. The putative cleavage sites in the precursors for basic and glycosylated PRPs all conform to the sequence RSXR↓S (X can be A, S or P) in agreement with the recognition sequence (RXXR↓) for various proprotein convertases. PRB4S, a proprotein giving rise to a basic PRP (IB‐5) as well as a glycosylated PRP (II‐1) was synthesized by in vitro transcription‐translation. It was cleaved by furin at RSAR↓S(173–178) giving rise to two proteins II‐1 and IB‐5. Similarly another precursor with the sequence RSAR↓S(173–178) was also cleaved by furin. This together with previous results show that in vitro furin can cleave all RSXR↓S sequences in the proproteins that give rise to glycosylated and basic PRPs. To demonstrate cellular cleavage, a human submandibular cell line (HSG) was transfected with a vector encoding PRB4S. This resulted in secretion of II‐1 and IB‐5. The degree of cleavage was enhanced by coexpressing furin and PRB4S. No cleavage occurred if the cells expressed a mutant PRB4S, R177Q, where the furin cleavage site had been destroyed. Cleavage was also inhibited if a furin inhibitor was coexpressed with PRB4S. Incubating the cells at 20 °C which blocks exit of proteins from the trans‐Golgi network demonstrated that cleavage occurs before exit of the proteins from this network. These results show that furin may be responsible for in vivo cleavage of PRP precursors. Transfecting furin‐deficient RPE.40 cells with a vector encoding PRB4S also led to secretion of II‐1 and IB‐5 showing that convertases other than furin can also cleave PRB4S in tissue culture.
Characterization of a new brain‐specific isoform of the EWS oncoprotein
Authors: Melot Thomas , Dauphinot Luce , Sévenet Nicolas , Radvanyi François , Delattre Olivier
Published: March 2003

EWS and related TAFII68 and TLS/FUS genes are fused with different genes encoding transcription factors in various human cancers. The products of these genes have the ability to bind RNA and have been shown to be part of splicing and transcription complexes. We show that the EWS, TAFII68 and TLS/FUS proteins are expressed to various levels in all adult murine tissues. We characterize a new isoform of EWS that is specifically expressed in the central nervous system, in both mice and humans. It is shown to be related to a splice variant which includes a new 18‐bp exon, termed 4′, between exon 4 and 5. The detection of this isoform in spontaneously differentiating SH‐SY5Y neuroblastoma cells and in nerve growth factor‐induced PC12 cells further links this isoform to neural differentiation. RT‐PCR experiments indicate that the level of expression of the brain‐specific EWS isoform is stable during brain development whereas that of the ubiquitous EWS isoform decreases during this period. The two isoforms show a parallel decrease in expression after birth. The 4′ exon is not detected in tumour‐specific EWS fusion transcripts, suggesting that its presence may impair their oncogenic properties. Interestingly, sequences of the 4′ exon and flanking regions show remarkable similarities to that of the neural‐specific c‐src exon, suggesting common mechanisms for the alternative splicing of these exons. The phylogenetic conservation and relationship to neural differentiation strongly suggests an important functional role for this exon.
Characterization and mutagenesis of the recombinant N‐acetylneuraminate lyase from Clostridium perfringens
Authors: Krüger Dorothea , Schauer Roland , Traving Christina
Published: March 2003

The N‐acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His‐tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography. The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65–70 °C. In kinetic studies the lyase exhibits a Km of 3.2 mm for Neu5Ac and a Vmax of 27.5 U·mg−1. To clarify the functional role of some putative active site residues, site‐directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate. Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding. In this respect the carboxy group of aspartate 187 seems to be particularly important. Based on the results of these studies, a model of the reaction mechanism is discussed.