bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C
Authors: Benen Jacques A. E. , Kester Harry C. M. , Visser Jaap
Published: March 2003

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3–7).

 Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 µkat·mg–1, 36.5 µkat·mg–1 and 415 nkat·mg–1 for endopolygalacturonases I, II and C, respectively. Km values were < 0.15 mg·mL–1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond‐cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes.

 Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non‐esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase‐generated Δ4,5‐unsaturated oligogalacturonates of degree of polymerization 4–8. Whereas endopolygalacturonase I showed a strong preference for generating the Δ4,5‐unsaturated dimer, with endopolygalacturonase II the Δ4,5‐unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the Δ4,5‐unsaturated dimer and trimer were observed, although in different ratios.
Cloning and expression of the human p8, a nuclear protein with mitogenic activity
Authors: Vasseur Sophie , Mallo Gustavo Vidal , Fiedler Fritz , Bödeker Hans , Cánepa Eduardo , Moreno Silvia , Iovanna Juan Lucio
Published: March 2003

We have previously identified a new rat mRNA, provisionally named p8, which showed a strong, but transient, induction in the pancreas in response to acute pancreatitis. We report here the cloning and sequencing of the human p8 cDNA. The human p8 polypeptide is 82 aminoacids long and shows an overall identity of 74% with the rat counterpart. The complete structure of the p8gene was also established. The p8 gene comprises three exons separated by two introns and the gene was mapped to chromosome 16. Analysis of the p8 primary structure suggested the presence of a bipartite motif of nuclear targeting, indicating that p8 may function within the nucleus. This presumption has been confirmed by transfection of COS‐7 cells with the p8 cDNA followed by immunostaining with specific antibodies. p8 mRNA expression is induced in some, but not all, cells by serum starvation and ceramide. To analyze the p8 function, we stably transfected HeLa cells with a p8 expression plasmid, and measured their growth and their sensitivity to apoptosis. Response to TNFα and staurosporine‐induced apoptosis was not modified by p8 expression. However, cells expressing p8 grew significantly more rapidly.
Localization of neutral N‐linked carbohydrate chains in pig zona pellucida glycoprotein ZPC
Authors: Yonezawa Naoto , Fukui Norio , Kudo Katsuyasu , Nakano Minoru
Published: March 2003

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays important roles in fertilization and consists of three glycoproteins; ZPA, ZPB and ZPC. In pig, neutral complex‐type N‐linked chains obtained from a ZPB/ZPC mixture possess sperm‐binding activity. We have recently reported that among neutral N‐linked chains triantennary and tetraantennary chains have a sperm‐binding activity stronger than that of diantennary chains. Triantennary and tetraantennary chains are localized at the second of the three N‐glycosylation sites of ZPB. In this study, we focused on the localization of neutral N‐linked chains in ZPC. ZPB and ZPC can not be separated from each other unless the acidic N‐acetyllactosamine regions of their carbohydrate chains are removed by endo‐β‐galactosidase digestion. A large part of the acidic N‐linked chains becomes neutral by the digestion, but the main neutral N‐linked chains are not susceptible to the enzyme. N‐glycanase digestion indicated that ZPC has three N‐glycosylation sites. Three glycopeptides each containing one of the N‐glycosylation sites were obtained by tryptic digestion of ZPC and the N‐glycosylation sites were revealed as Asn124, Asn146 and Asn271. The carbohydrate structures of the neutral N‐linked chains from each glycopeptide were characterized by two‐dimensional sugar mapping analysis taking into consideration the structures of the main, intact neutral N‐linked chains of ZPB/ZPC mixture reported previously. Triantennary and tetraantennary chains were found mainly at Asn271 of ZPC, whereas diantennary chains were present at all three N‐glycosylation sites. Thus, ZPC has tri‐antennary and tetra‐antennary chains as well as ZPB, but the localization of the chains is different from that in ZPB.
Stimulation of P‐glycoprotein‐mediated drug transport by prazosin and progesterone
Authors: Shapiro Adam B. , Fox Kelly , Lam Ping , Ling Victor
Published: March 2003

P‐glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad‐specificity, ATP‐dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug‐binding sites of P‐glycoprotein involved in transport [Shapiro, A.B. & Ling, V. (1997) Eur. J. Biochem.250, 130–137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non‐P‐glycoprotein‐specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug‐binding site on P‐glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P‐glycoprotein by [125I]iodoarylazidoprazosin [Safa, A.R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry33, 256–265] and is likely not to be capable of drug transport.
Pertussis toxin‐sensitive and insensitive intracellular signalling pathways in undifferentiated 3T3‐L1 cells stimulated by insulin converge with phosphatidylinositol 3‐kinase upstream of the Ras mitogen‐activated protein kinase cascade
Authors: Uehara Takashi , Tokumitsu Yukiko , Nomura Yasuyuki
Published: March 2003

We have previously reported that pertussis toxin (PTX)‐sensitive GTP binding protein (G‐protein) and phosphatidylinositol 3‐kinase (PI 3‐K) are involved in adipocyte differentiation of 3T3‐L1 cells induced by insulin/dexamethasone/methylisobutyl xanthine. The aim of this study was to examine the effect of PTX on the tyrosine kinase cascade stimulated by insulin acting through insulin‐like growth factor‐I (IGF‐I) receptors in undifferentiated 3T3‐L1 cells. A high level of mitogen‐activated protein kinase (MAPK) activation was sustained for up to 4 h after insulin treatment, and mobility shifted and tyrosine phosphorylated MAPK was also detected. MAPK kinase activity measured by the incorporation of 32P into kinase‐negative recombinant MAPK was enhanced by insulin treatment. We previously discovered that insulin activates Ras and that this is mediated by wortmannin‐sensitive PI 3‐K. Tyrosine‐phosphorylation of IRS‐1 and Shc also occurred in response to insulin. Subsequently, we investigated the effects of PTX on the activation of these proteins by insulin. Interestingly, treating 3T3‐L1 cells with PTX attenuates the activation by insulin of both the Ras‐MAPK cascade and PI 3‐K. In contrast, neither tyrosine‐phosphorylation of IRS‐1 and Shc nor the interaction between IRS‐1 and PI 3‐K is sensitive to PTX. However, activation of the Ras‐MAPK cascade and tyrosine‐phosphorylation of Shc by epidermal growth factor are insensitive to PTX. These results indicate that there is another pathway which regulates PI 3‐K and Ras‐MAPK, independent of the pathway mediated by IGF‐I receptor kinase. These findings suggest that in 3T3‐L1 fibroblasts, PTX‐sensitive G‐proteins cross‐talk with the Ras‐MAPK pathway via PI 3‐K by insulin acting via IGF‐I receptors.
Molecular cloning of human chondromodulin‐I, a cartilage‐derived growth modulating factor, and its expression in Chinese hamster ovary cells
Authors: Hiraki Yuji , Mitsui Kaori , Endo Naoto , Takahashi Kazuhiro , Hayami Tadashi , Inoue Hiroyuki , Shukunami Chisa , Tokunaga Kunihiko , Kono Takaharu , Yamada Mitsunori , Takahashi Hideaki E. , Kondo Jun
Published: March 2003

Bovine chondromodulin‐I (ChM‐I) purified from fetal cartilage stimulated the matrix synthesis of chondrocytes, and inhibited the growth of vascular endothelial cells in vitro. The human counterpart of this bovine growth regulating factor has not been identified. We report here the cloning of human ChM‐I precursor cDNA and its functional expression in Chinese hamster ovary (CHO) cells. We first identified a genomic DNA fragment which encoded the N‐terminus of the ChM‐I precursor, and then isolated human ChM‐I cDNA from chondrosarcoma tissue by PCR. The deduced amino acid sequence revealed that mature human ChM‐I consists of 120 amino acids. In total, 16 amino acid residues were substituted in the human sequence, compared to the bovine counterpart. Almost of all the substitutions were found in the N‐terminal hydrophilic domain. In the C‐terminal hydrophobic domain (from Phe42 to Val120), the amino acid sequence was identical except for Tyr90, indicating a functional significance of the domain. Northern blotting and in situ hybridization indicated a specific expression of ChM‐I mRNA in cartilage. We also successfully determined the cartilage‐specific localization of ChM‐I protein, using a specific antibody against recombinant human ChM‐I. Multiple transfection of the precursor cDNA into CHO cells enabled us to isolate the mature form of human ChM‐I from the culture supernatant. Purified recombinant human ChM‐I stimulated proteoglycan synthesis in cultured chondrocytes. In contrast, it inhibited the tube morphogenesis of cultured vascular endothelial cells in vitro and angiogenesis in chick chorioallantoic membrane in vivo.
Biosynthesis of ansatrienin (mycotrienin) and naphthomycin
Authors: Chen Shuo , von Bamberg Daniel , Hale Valerie , Breuer Michael , Hardt Birgit , Müller Rolf , Floss Heinz G. , Reynolds Kevin A. , Leistner Eckhard
Published: March 2003

The polyketide chains of the two ansamycin antibiotics, ansatrienin (mycotrienin) and naphthomycin produced by Streptomyces collinus are assembled using 3‐amino‐5‐hydroxybenzoic acid (AHBA) as a starter unit. The gene encoding AHBA synthase, an enzyme which catalyzes the final step of AHBA biosynthesis in the recently discovered aminoshikimate pathway, has been used to identify two separate antibiotic biosynthetic gene clusters in S. collinus. In one of these clusters, analysis of approximately 20 kb of contiguous sequence has revealed both a cluster of six genes presumed to play a role in the AHBA pathway and the beginning of a polyketide synthase (PKS) gene containing an acyl ACP ligase domain. This domain is likely responsible for loading AHBA onto the PKS. This gene cluster also contains chcA, encoding the enzyme 1‐cyclohexenylcarbonyl CoA reductase, which is essential for the biosynthesis of the cyclohexanecarboxylic acid moiety of ansatrienin from shikimic acid, and a peptide synthetase. This gene cluster thus seems to control the biosynthesis of ansatrienin, which contains a side chain of N‐cyclohexanecarbonyl‐d‐alanine esterified to the macrocyclic lactam backbone. In the putative naphthomycin biosynthetic gene cluster approximately 13 kb of contiguous sequence has revealed a second set of the genes required for AHBA biosynthesis. In addition the end of a polyketide synthase and a gene putatively involved in termination of the chain extension process, formation of an intramolecular amide bond between the AHBA nitrogen and the carboxyl group of the fully extended polyketide chain, have been identified. Thus, despite commonality in biosynthesis, the ansatrienin and naphthomycin biosynthetic gene clusters show clear organizational differences and carry separate sets of genes for AHBA biosynthesis.
Expression of 3β‐hydroxysteroid dehydrogenase type I and type VI isoforms in the mouse testis during development
Authors: Baker Paul J. , Sha Jiaho A. , McBride Martin W. , Peng Lihong , Payne Anita H. , O’Shaughnessy Peter J.
Published: March 2003

Six isoforms of the enzyme 3β‐hydroxysteroid dehydrogenase (3βHSD) have been identified in the mouse, each the product of a distinct gene. Two of these isoforms (type I and type VI) are detectable in the adult testis but changes in their expression during development are unknown. In this study we have examined changes in testicular expression and localization of mRNA encoding the type I and type VI isoforms of 3βHSD. Total 3βHSD (type I plus type VI) mRNA was measured by reverse transcription‐polymerase chain reaction and showed a peak of expression at day 5 after birth followed by a decline and then a further rise after day 10 that continued up to adulthood. When each isoform was measured individually it was clear that the type I isoform was expressed at all ages from embryonic day 13 to adulthood. In contrast, the type VI isoform was only expressed at significant levels during fetal life on embryonic day 13 and then not again until after day 10 postnatally. Expression of the type VI isoform mRNA increased markedly after day 10 so that by adulthood it was the predominant 3βHSD isoform present in the testis. Closer examination of the timing of type VI expression showed that the isoform mRNA was first detectable at a significant level on day 11. In‐situ hybridization confirmed that the type I isoform is the only one expressed in the fetal/neonatal animal and showed that expression was limited to the interstitial tissue. In the adult, both type I and type VI expression was within the interstitial tissue. The timing of 3βHSD type VI mRNA expression suggests, strongly, that this isoform is expressed only by adult‐type Leydig cells in the mouse testis and that this development starts shortly before day 11. The limited expression of the type VI isoform means that it will be a useful marker in studies of adult Leydig cell development.
Identification of chURP, a nuclear calmodulin‐binding protein related to hnRNP‐U
Authors: Lodge Anthony P. , Walsh Adrian , McNamee Christine J. , Moss Diana J.
Published: March 2003

In a screen for myosin‐like proteins in embryonic chicken brain, we have identified a novel nuclear protein structurally related to hnRNP‐U (heterogeneous nuclear ribonuclear protein U). We have called this protein chURP, for chicken U‐related protein. In this screen, chURP was immunoreactive with two myosin antibodies and, in common with the unconventional myosins, bound calmodulin in vitro in both the presence and absence of calcium ions. Determination of 757 amino acids of the chURP sequence revealed that it shares 41% amino acid identity with human and rat hnRNP‐U, although chURP and hnRNP‐U appear not to be orthologous proteins. ChURP is ubiquitously expressed in the nuclei of all chick tissues and, as one of a growing number of calmodulin‐binding proteins to be identified in the nucleus, further highlights the potential of calmodulin as a regulator of nuclear metabolism.
Two‐dimensional electrophoresis of Malassezia allergens for atopic dermatitis and isolation of Mal f 4 homologs with mitochondrial malate dehydrogenase
Authors: Onishi Yoshimi , Kuroda Masanobu , Yasueda Hiroshi , Saito Akemi , Sono‐Koyama Eiko , Tunasawa Susumu , Hashida‐Okado Takashi , Yagihara Tomoko , Uchida Katsuhisa , Yamaguchi Hideyo , Akiyama Kazuo , Kato Ikunoshin , Takesako Kazutoh
Published: March 2003

The yeast Malassezia furfur is a natural inhabitant of the human skin microflora that induces an allergic reaction in atopic dermatitis. To identify allergens of M. furfur, we separated a crude preparation of M. furfur antigens as discrete spots by 2‐D PAGE and detected IgE‐binding proteins using sera of atopic dermatitis patients. We identified the known allergens, Mal f 2 and Mal f 3, and determined N‐terminal amino acid sequences of six new IgE‐binding proteins including Mal f 4. The cDNA and genomic DNA encoding Mal f 4 were cloned and sequenced. The gene was mitochondrial malate dehydrogenase and encoded Mal f 4 composed of 315 amino acids and a signal sequence of 27 amino acids. We purified Mal f 4, which had a molecular mass of 35 kDa from a membrane fraction of a lysate of cultured cells. Thirty of 36 M. furfur‐allergic atopic dermatitis patients (83.3%) had elevated serum levels of IgE to purified Mal f 4, indicating that Mal f 4 is a major allergen. There was a significant correlation of the Phadebas RAST unit values of Mal f 4 and the crude antigen, but not between Mal f 4 and the known allergen Mal f 2.
Structure of the O‐specific polysaccharide of a serologically separate strain Proteus penneri 2 from a new proposed serogroup O66
Authors: Arbatsky Nikolay P. , Shashkov Alexander S. , Toukach Filip V. , Moll Hermann , Zych Krystyna , Knirel Yuriy A. , Zähringer Ulrich , Sidorczyk Zygmunt
Published: March 2003

O‐specific polysaccharide chain of Proteus penneri strain 2 lipopolysaccharide was studied by full and partial acid hydrolysis, Smith degradation, methylation analysis, and NMR spectroscopy, including two‐dimensional rotating‐frame NOE spectroscopy (ROESY) and 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments. Together with d‐glucose and 2‐acetamido‐2‐deoxy‐d‐glucose, the polysaccharide was found to contain two rarely occurring sugars, 6‐deoxy‐l‐talose (L‐6dTal) and 2,3‐diacetamido‐2,3,6‐trideoxy‐l‐mannose (L‐RhaNAc3NAc), and the following structure of a non‐stoichiometrically O‐acetylated tetrasaccharide repeating unit was established:

The O‐specific polysaccharide studied has a unique composition and structure and, accordingly, P. penneri 2 is serologically separate among Proteus strains. Therefore, we propose for P. penneri 2 a new Proteus O‐serogroup O66 where this strain is at present the single representative.
N‐Benzoyl‐l‐tyrosyl‐p‐aminobenzoic acid hydrolase β (human meprinβ)
Authors: Pischitzis Anastassios , Hahn Dagmar , Leuenberger Boris , Sterchi Erwin E.
Published: March 2003

N‐Benzoyl‐l‐tyrosyl‐p‐aminobenzoic acid hydrolase or human meprin (PPH) is a brush‐border membrane enzyme of small intestinal epithelial cells. It is a type I integral membrane protein composed of two disulphide‐bridged subunits (α and β). PPH and its homologous counterparts in rodents belong to the astacin family of zinc‐metalloendopeptidases. Although the amino‐acid sequence of the β subunits is 80–90% identical in these three species, processing is different. Expression of PPHβ in simian virus 40‐transformed African green monkey kidney cells (COS‐1) and Madin Darby canine kidney (MDCK) cells results in its cell surface localization and secretion, whereas mouse meprinβ is only found at the plasma membrane. To investigate proteolytic processing of PPHβ and to identify the cleavage site, different C‐terminal domains of wild‐type PPHβ were exchanged with the homologous domains of mouse meprinβ. We identified a 13‐amino‐acid sequence (QIQLTPAPSVQDL) necessary for cleavage and subsequent secretion of PPHβ. Using brefeldin A, the site of processing was identified as being after passage through the Golgi compartment. Proteolytic processing of PPHβ thus provides a means for secretion of αβ heterodimers.
A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus
Authors: Ziegler Rolf , Willingham Leslie A. , Engler Diane L. , Tolman Kenneth J. , Bellows David , Van der Horst Dick J. , Yepiz‐Plascencia Gloria M. , Law John H.
Published: March 2003

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin‐III.
Mixed reconstitution of mutated subunits of HIV‐1 reverse transcriptase coexpressed in Escherichia coli – two tags tie it up
Authors: Maier Gottfried , Dietrich Ursula , Panhans Barbara , Schröder Britta , Rübsamen‐Waigmann Helga , Cellai Luciano , Hermann Thomas , Heumann Hermann
Published: March 2003

The active form of HIV‐1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C‐terminal proteolytic cleavage of p66. A well‐known problem of p66 recombinant expression is partial cleavage of a 15‐kDa peptide from the C‐terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N‐terminal fusion protein of glutathione S‐transferase (GST) with p51 and a C‐terminally His‐tagged p66, respectively. The two‐plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione‐agarose, phenyl‐superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility‐shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility‐shift assays. This indicates the p51 subunit has an active role in DNA binding
Mode of action of chitin deacetylase from Mucor rouxii on N‐acetylchitooligosaccharides
Authors: Tsigos Iason , Zydowicz Nathalie , Martinou Aggeliki , Domard Alain , Bouriotis Vassilis
Published: March 2003

The mode of action of chitin deacetylase from the fungus Mucor rouxii on N‐acetylchitooligosaccharides with a degree of polymerization 1–7 has been elucidated.

 Identification of the sequence of chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo‐glycosidases in conjunction with HPLC. The results were further verified by 1H‐NMR spectroscopy.

 It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate chitin oligomers with a degree of polymerization lower than three. Tetra‐N‐acetylchitotetraose and penta‐N‐acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri‐N‐acetylchitotriose, hexa‐N‐acetylchitohexaose and hepta‐N‐acetylchitoheptaose the reducing‐end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing‐end residue of all chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion.

 The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.
Ecdysone‐regulation of synthesis and processing of Fat Body Protein 1, the larval serum protein receptor of Drosophila melanogaster
Authors: Burmester Thorsten , Antoniewski Christophe , Lepesant Jean‐Antoine
Published: March 2003

At the end of the third larval instar of Drosophila melanogaster, larval serum proteins 1 and 2 (LSP‐1 and ‐2) are taken up by cells of the fat body. Here, we show that the product of the ecdysteroid‐inducible gene Fbp‐1 (Fat Body Protein 1) is the receptor that binds LSP‐1. Transcription and translation of Fbp‐1 is stage‐specifically restricted to the end of the third larval instar, starting around 99 h after egg laying. Expression of Fbp‐1 is induced by a low level of 20‐hydroxy‐ecdysone (≥ 10−7 m). After translation, the FBP‐1 protein is thought to be proteolytically cleaved in three subsequent steps. The final cleavage step is delayed by 6 h and relies on a higher concentration of ecdysone (≥ 10−5 m). Therefore, 20‐hydroxy‐ecdysone regulates Fbp‐1 expression and function at two different levels. To the best of our knowledge, this study is the first to date to demonstrate two distinct functions for different concentrations of a steroid hormone on a single biochemical process.
Assembly of heteromeric connexons in guinea‐pig liver en route to the Golgi apparatus, plasma membrane and gap junctions
Authors: Diez Juan A. , Ahmad Shoeb , Evans W. Howard
Published: March 2003

Guinea‐pig liver gap junctions are constructed from approximately equal amounts of connexins 26 and 32. The assembly of these connexins into connexon hemichannels and gap junctions was studied using antibodies specific to each connexin. Intracellular membranes were shown to contain low amounts of connexin 26 relative to connexin 32 in contrast to the equal connexin ratios detected in lateral plasma membranes and gap junctions. Assembly of gap junctions requires oligomerization of connexins into connexons that may be homomeric or heteromeric. Immunoprecipitation using antibodies to connexins 26 and 32 showed that liver gap junctions were heteromeric. A chemical cross‐linking procedure showed that connexons solubilized from guinea‐pig liver gap junctions were constructed of hexameric assemblies of connexin subunits. The intracellular site of oligomerization of connexins was investigated by velocity sedimentation in sucrose–detergent gradients. Oligomers of connexins 26 and 32 were extensively present in Golgi membranes and oligomeric intermediates, especially of connexin 26, were detected in the endoplasmic reticulum–Golgi intermediate subcellular fraction. Two intracellular trafficking pathways that may account for the delivery of connexin 26 to the plasma membrane and explain the heteromeric nature of liver gap junctions are discussed.
A novel isoform of the low molecular weight phosphotyrosine phosphatase, LMPTP‐C, arising from alternative mRNA splicing
Authors: Tailor Pankaj , Gilman Jennifer , Williams Scott , Mustelin Tomas
Published: March 2003

The low molecular weight protein tyrosine phosphatase (LMPTP) is an 18‐kDa enzyme that it distantly related to other protein tyrosine phosphatases. The single gene for LMPTP is known to undergo an alternative splicing event in which exon 3 or exon 4 is excised, resulting in two isoforms termed A and B; the latter is more mobile in SDS/PAGE. In this paper we report the existence of a third isoform, which we call C, in which both exons 3 and 4 are lacking. We find the resulting mRNA to be ubiquitously expressed at levels that exceed those of the mRNAs for isoforms A and B. This mRNA was reverse‐transcribed, cloned and sequenced, confirming the direct splicing of exon 2 to exon 5. In‐vitro transcription and translation of the cDNA for the novel isoform resulted in the expected 16 kDa protein. This protein was also detected in Jurkat T cells using an antipeptide antiserum. LMPTP‐C immunoprecipitated from transfected cells, as well as bacterially produced recombinant LMPTP‐C, lacked phosphatase activity. Unlike LMPTP‐B, LMPTP‐C was not phosphorylated on tyrosine when coexpressed with Lck despite the presence of the two acceptor tyrosines. Finally, whereas c‐fos induction by platelet‐derived growth factor was inhibited by LMPTP‐B, LMPTP‐C augmented it. These results suggest that the lack of the 38‐amino acid fragment encoded by exon 3 or 4 results in a protein product with a different three‐dimensional folding, that lacks a functional catalytic pocket and that may function as a natural antagonist of isoforms A and B.
Polyproline binding is an essential function of human profilin in yeast
Authors: Ostrander Darin B. , Ernst Eileen G. , Lavoie Thomas B. , Gorman Jessica A.
Published: March 2003

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5‐bisphosphate (PIP2) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline‐binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1Δ. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline‐binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.
Molecular cloning and tissue expression of an insect farnesyl diphosphate synthase
Authors: Castillo‐Gracia Manuela , Couillaud Franck
Published: March 2003

The enzyme farnesyl‐diphosphate synthase (FPS, EC2.5.1.1/EC2.5.1.10), which has been shown to play a key role in isoprenoid biosynthesis, catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and di‐methylallyl diphosphate. Insects do not synthesize cholesterol de novo, rather farnesyl diphosphate leads to the formation of nonsterol isoprenoids, which are essential for insect development and reproduction. In this paper, we describe the characterization of one FPS from the moth Agrotis ipsilon, the first insect FPS to be reported. An homologous probe was obtained through a nested PCR strategy using degenerate primers designed from the conserved domains of FPS from other organisms. The complete cDNA clone was isolated by PCR screening of a brain cDNA library by using homologous primers deduced from the probe. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 412 amino acids (Mr = 47 170), which shares regions similar to the FPS of other organisms, but exhibits singularities such as an extra N‐terminal extension of ≈ 70 amino acid residues. Using an RNase protection assay, a protected fragment corresponding to the region encoding the FPS catalytic site was found in brain, ovary, fat body and corpora allata samples, but not in muscle. FPS is overexpressed in the corpora allata, the endocrine gland that produces the juvenile hormones. These hormones are specific to insects and play a crucial role in regulating insect physiology.
Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria
Authors: Angeli Sergio , Ceron Francesca , Scaloni Andrea , Monti Maria , Monteforti Gaia , Minnocci Antonio , Petacchi Ruggero , Pelosi Paolo
Published: March 2003

Soluble low‐molecular‐mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40–50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29–Cys38 and Cys57–Cys60) and indicated the absence of any additional post‐translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2‐sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.
Spectroscopic characterization of the spinach Lhcb4 protein (CP29), a minor light‐harvesting complex of photosystem II
Authors: Pascal Andy , Gradinaru Claudiu , Wacker Ulrich , Peterman Erwin , Calkoen Florentine , Irrgang Klaus‐Dieter , Horton Peter , Renger Gernot , van Grondelle Rienk , Robert Bruno , van Amerongen Herbert
Published: March 2003

A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b‐binding protein CP29 (or the Lhcb4 protein) from spinach, prepared by a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.‐E. (1989) Biochim. Biophys. Acta977, 301–308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment‐binding protein shows similarities to other related light‐harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcb5). It is also, in the main, similar to other preparations of CP29, although some significant differences are discussed. In common with CP26, the protein binds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem.36, 12984–12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.
The relevance of N‐linked glycosylation to the binding of a ligand to guanylate cyclase C
Authors: Hasegawa Makoto , Hidaka Yuji , Wada Akihiro , Hirayama Toshiya , Shimonishi Yasutsugu
Published: March 2003

The role of carbohydrate moieties at the N‐linked glycosylation sites of guanylate cyclase C (GC‐C), a receptor protein for guanylin, uroguanylin and heat‐stable enterotoxin, in ligand binding and structural stability was examined using site‐directed mutagenesis of the putative N‐linked glycosylation sites in the extracellular domain (ECD) of porcine GC‐C. For this purpose, eight mutant proteins of ECD (N9A, N20A, N56A, N172A, N261A, N284A, N334A and N379A) and six mutant proteins of the complete GC‐C (N9A, S11A, N172A, T174A, N379A and T381A) were prepared, in which Ala replaced Asn, Ser and Thr at the N‐linked glycosylation consensus sites. All the mutant proteins showed a ligand‐binding affinity (Kd) similar to those of the wild‐type proteins, although the deletion of a carbohydrate moiety at each of the N‐linked glycosylation sites affected the ligand‐binding ability of ECD or GC‐C to some degree. However, the mutant proteins of ECD (N379A) and GC‐C (N379A and T381A) showed considerably decreased binding ability in the context of maximum capacity (Bmax) to a ligand, despite the fact that the expression levels of these mutant proteins were nearly the same as the wild‐type proteins. Moreover, the mutant protein of ECD (N379A) was considerably less stable to a denaturant. These results clearly indicate a crucial role for the carbohydrate moiety at N379, which is located near the transmembrane region, in structural stability, the ability to bind to a ligand and the cyclase catalytic activity of GC‐C, and provide a route for the elucidation of the mechanism of the interaction between GC‐C and a ligand.
Deregulation of the signaling pathways controlling urokinase production
Authors: Aguirre Ghiso Julio A. , Alonso Daniel F. , Farías Eduardo F. , Gomez Daniel E. , Bal de Kier Joffè Elisa
Published: March 2003

We review the evidence in support of the notion that, upon experimental oncogenic transformation or in spontaneous human cancers, mitogenesis and expression of urokinase (uPA) and its receptor (uPAR) are activated through common signaling complexes and pathways. It is well documented that uPA, uPAR or metalloproteinases (MMPs) are overexpressed in tumor cells of mesenchymal or epithelial origin and these molecules are required for tumor invasion and metastasis. Furthermore, oncogenic stimuli, which may render the transformed cells tumorigenic and metastatic in vivo, activate, in a constitutive fashion, the extracellular‐regulated kinases (Erk 1 and 2) classical mitogenic pathway and others such as the NH2‐Jun‐kinase (Jnk). Cells from human tumors or oncogene‐transformed cells overexpress uPA and uPAR, and also show a sustained activation of the above‐mentioned signaling modules. In this paper we show that the classical mitogenic pathway involving Ras‐Erk, PKC‐Erk or Rac‐JNK, among others, is activated by growth factors or endogenously by oncogenes, and constitutively activates uPA and uPAR expression. All the data obtained from human tumors or experimental systems, incorporated into a general model, indicate that oncogenic stimuli lead to the constitutive activation of mitogenesis and uPA and its receptor expression, through the activation of the same classical and nonclassical signaling complexes and pathways that regulate cell proliferation. We also discuss contrasting points of view. For instance, what governs the differential regulation of mitogenesis and the signal that leads to protease overexpression in a way that allows normal cells during physiological events to respond to growth factors, and proliferate without overexpressing extracellular matrix (ECM) proteases? Or how can cells remodel their microenvironment without proliferating? What restrains benign tumors from overexpressing tumor‐associated proteases when they certainly have the mitogenic signal fully activated? This may occur by the differential regulation of transcriptional programs and recent reports reviewed in this paper may provide an insight into how this occurs at the signaling and transcriptional levels.
Structural maintenance of chromosomes (SMC) proteins
Authors: Strunnikov Alexander V. , Jessberger Rolf
Published: March 2003

The evolutionarily‐conserved eukaryotic SMC (structural maintenance of chromosomes) proteins are ubiquitous chromosomal components in prokaryotes and eukaryotes. The eukaryotic SMC proteins form two kind of heterodimers: the SMC1/SMC3 and the SMC2/SMC4 types. These heterodimers constitute an essential part of higher order complexes, which are involved in chromatin and DNA dynamics. The two most prominent and best‐characterized complexes are cohesin and condensin, necessary for sister chromatid cohesion and chromosome condensation. Here we discuss these functions together with additional roles in gene dosage compensation and DNA recombination.
A role for the immunoglobulin‐like domain of the human IL‐6 receptor
Authors: Vollmer Petra , Oppmann Birgit , Voltz Nicole , Fischer Martina , Rose‐John Stefan
Published: March 2003

Interleukin (IL)‐6, IL‐11 and cililary neurotrophic factor (CNTF) belong to the same family of hematopoietic and neurotrophic cytokines. Their receptor complexes contain a cytokine‐binding α receptor and the common glycoprotein (gp)130 subunit for signal transduction. The extracellular parts of the α‐receptor subunits consist of a membrane‐proximal cytokine‐binding domain and an N‐terminal immunoglobulin (Ig)‐like domain with unknown function. We examined the role of the Ig‐like domain of IL‐6R by constructing deletion mutants lacking the Ig domain (IL‐6RΔIg and soluble IL‐6RΔIg). IL‐6RΔIg was shed as effectively as wild‐type IL‐6R from transfected COS‐7 cells upon 4β‐phorbol 12‐myristate 13‐acetate (PMA) treatment, whereas nonstimulated shedding of IL‐6RΔIg was not observed. The shed sIL‐6RΔIg from PMA‐treated cells, as well as the transmembrane IL‐6RΔIg, had the same biological activity as wild‐type sIL‐6R, as measured by the induction of haptoglobin secretion in HepG2‐IL‐6 cells and IL‐6‐dependent proliferation of IL‐6RΔIg transfected BAF/gp130 cells. In COS‐7 cells transfected with IL‐6RΔIg or soluble IL‐6RΔIg cDNA, transport of the deletion mutants through the secretory pathway appeared to be delayed because a sizeable proportion of the mutants was detected as an endo‐β‐N‐acetylglucosaminidase‐sensitive intermediate, suggesting that transport and processing of the ΔIg mutants on the secretory pathway were impaired. These experiments suggest that the Ig‐like domain of the IL‐6R is important for intracellular transport of IL‐6R through the secretory pathway. Furthermore, the Ig‐like domain is necessary for noninduced shedding of the IL‐6R, whereas it has no function in PKC‐dependent shedding of the IL‐6R.
Structural and membrane‐binding properties of saposin D
Authors: Tatti Massimo , Salvioli Rosa , Ciaffoni Fiorella , Pucci Piero , Andolfo Annapaola , Amoresano Angela , Vaccaro Anna Maria
Published: March 2003

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane‐binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine‐containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues.

 The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5–Cys78, Cys8–Cys72 and Cys36–Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes.

 The analysis of the sugar moiety of saposin D revealed that the single N‐glycosylation site present in the molecule is mainly modified by high‐mannose‐type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid‐binding site.

 The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid‐containing membranes.
Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis
Authors: Charest Marie‐Claude , Rhainds David , Falstrault Louise , Matzouranis Tony , Brissette Louise
Published: March 2003

Low density lipoprotein (LDL) can follow either a holoparticle uptake pathway, initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl esters (CE) selectively to HepG2 cells. Although high density lipoprotein‐CE selective uptake has been shown to be linked to cell cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL‐CE selective uptake on hepatic cell cholesterol homeostasis. In order to define the role of the LDL‐CE selective uptake pathway in hepatic cell cholesterol homeostasis, we used a cellular model that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2‐all cells). The addition of a specific antibody anti‐LDLr (IgG‐C7) reduces LDL protein degradation (LDLr activity) to 7%. This cellular model therefore reflects, above all, LDL‐CE selective uptake activity when incubated with LDL. The inactivation of LDLr reduces LDL–protein association by 78% and LDL–CE association by only 43%. The LDL‐CE selective uptake was not reduced by the inactivation of LDLr. The activities of the various enzymes involved in cell cholesterol homeostasis were measured in normal and LDLr‐deficient cells during incubation in the absence or presence of LDL as a cholesterol source. Essentially, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase (ACAT) activities responded to LDL in LDLr‐deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroquine abolished the effect measured on ACAT activity in the presence of LDL, suggesting that CE of LDL, but not free cholesterol, maintains cell cholesterol homeostasis. Thus, in HepG2 cells, when LDLr function is virtually abolished, LDL‐CE selective uptake is coupled to cell cholesterol homeostasis.