bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of a cDNA encoding the porcine type XVII collagen noncollagenous 16 A domain and localization of the domain to the upper part of porcine skin basement membrane zone
Authors: XU LUTING , OLIVRY THIERRY , CHAN LAWRENCE S.
Published: June 2004

Abstract   Bullous pemphigoid is an autoimmune blistering human skin disease mediated by immunoglobulin (Ig)G autoantibodies targeting skin basement membrane component type XVII collagen, a transmembrane protein. Also designated BP180 and BPAG2, type XVII collagen is an extracellular matrix element essential for the connection between the epidermis and the underlying dermis. In addition to being a target antigen in the human disease bullous pemphigoid, type XVII collagen is also targeted by autoantibodies of canine, feline, equine and porcine patients suffering from a similar blistering skin disease. Previously, enzyme‐linked imunosorbent assay and Western blot analyses have shown that autoantibodies from pigs affected with bullous pemphigoid recognize the human NC16A domain of type XVII collagen. To facilitate the development of porcine model of bullous pemphigoid, we isolated cDNA encoding the porcine type XVII collagen NC16A domain using a reverse transcription–polymerase chain reaction technique. The amino acids deduced from the NC16A cDNA showed 61% identity with the sequence of human NC16A. An antibody generated against a 20‐amino acid peptide within the porcine NC16A localized the NC16A epitope to the upper part of porcine skin basement membrane zone. Our data provide further information of the porcine bullous pemphigoid target antigen and may help investigators for their further studies of this disease.
Up‐regulation of cytokeratin expression in canine distemper virus‐infected canine footpad epidermis
Authors: GRÖNE ANDREA , DOHERR MARCUS G. , ZURBRIGGEN ANDREAS
Published: June 2004

Abstract Cytokeratin expression was assessed in footpad epidermis from dogs using immunohistochemistry. Four groups of dogs were studied: dogs with experimentally induced distemper and with canine distemper virus (CDV) in footpad epidermis (group 1,n = 7); dogs with experimentally induced distemper and without CDV in footpad epidermis (group 2,n = 4); inoculated dogs without distemper and without CDV in the footpad epidermis (group 3,n = 8), and noninoculated dogs without distemper (group 4,n = 2). No increase in thickness of the footpad epidermis was present in any of these groups. Sections of metacarpal or metatarsal pads were stained for cytokeratin (CK)14 (proliferation‐associated), CK10 (correlated with early differentiation), and for involucrin (associated with terminal differentiation). CK14 was present in basal keratinocytes of all groups, but staining intensity decreased towards the corneal layer in groups 2–4, but not in group 1. CK10 was present in the spinous and granular layer of all groups, but staining of the granular layer was much stronger in group 1. Involucrin was present in the granular layer of footpads of group 1 and only in the upper part of this layer in groups 2–4. The results demonstrate increased staining intensity and/or wider distribution within the footpad epidermis in group 1 dogs when compared to the other groups. This was interpreted as up‐regulation in expression of these proteins. These findings suggest that presence of CDV antigen and mRNA in footpad epidermis was associated with an increase in expression of CK14, CK10 and involucrin. The potential role of this up‐regulation in cytokeratin expression in the development of CDV‐induced digital hyperkeratosis remains speculative at the moment and requires further studies.
Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients
Authors: Gebara E. C. E. , Pannuti C. , Faria C. M. , Chehter L. , Mayer M. P. A. , Lima L. A. P. A.
Published: June 2004

Helicobacter pylori is an important gastrointestinal pathogen associated with gastritis, peptic ulcers, and an increased risk of gastric carcinoma. The oral cavity has been indicated as a possible H. pylori reservoir, and may therefore be involved in the reinfection of the stomach which sometimes follows treatment of H. pylori infection. The objective of the present study was to evaluate the prevalence of H. pylori as detected by polymerase chain reaction (PCR) in the oral cavity of periodontitis patients testing positive for this bacterium in the stomach. Thirty adult patients with alterations of the superior digestive tract, testing urease positive after endoscopy and biopsy, were selected. A full‐mouth periodontal examination was performed in every patient and the subjects were allocated to two groups: gingivitis (15 patients) and chronic periodontitis (15 patients). Plaque and saliva samples collected from each patient were stored in 0.5 ml of TE buffer. DNA was extracted from the samples by the boiling method and was evaluated for the presence of H. pylori using the PCR method. JW 22/23 primers were used. The DNA of ATCC H. pylori 43629 (positive control) and water (negative control) were used for controlling the reactions. Of the 30 evaluated patients, 13 (43.3%) harbored H. pylori in the mouth. The bacterium was not found on the dorsum of the tongue of any patient, but was found in saliva in three patients (10%), in the supragingival plaque in six patients (20%), and in the subgingival plaque in eight patients (26.6%). The presence of H. pylori was similar in the gingivitis and chronic periodontitis groups. In conclusion, a high percentage of patients harbored H. pylori in their mouth. The bacterium was detected in saliva, supragingival and subgingival plaque, suggesting that these sites may be considered reservoirs for H. pylori in urease‐positive patients.
Photosynthesis in the water‐stressed C4grassSetaria sphacelatais mainly limited by stomata with both rapidly and slowly imposed water deficits
Authors: Da Silva Jorge Marques , Arrabaça Maria Celeste
Published: June 2004

A comparison of the effects of a rapid and a slowly imposed water deficit on photosynthesis was performed inSetaria sphacelatavar.splendida(Stapf) Clayton, a C4NADP‐ME grass. Gas exchange was measured in rapidly and slowly dehydrated adult leaves either under atmospheric CO2partial pressure with an infrared gas analyser or under saturating CO2partial pressure with a leaf disc oxygen electrode. These measurements were used to calculate stomatal and non‐stomatal limitations to photosynthesis. These were further investigated using modulated chlorophyllafluorescence measurements and photosynthetic pigment quantification. The decrease of net photosynthesis, leaf conductance and water use efficiency was more pronounced under rapid stress than in slow stress. However, photosynthesis is always mainly limited by stomata in both types of stress, albeit the contribution of non‐stomatal limitations increases at severe water deficits in slow stress experiments. The substomatal CO2partial pressure significantly increased in both types of stress, suggesting an increased resistance due to an internal barrier to CO2diffusion. Physical alterations in the structure of the intercellular spaces due to leaf shrinkage may account for these results. The maximal photochemical efficiency of photosystem II (PSII) was remarkably resistant to stress, as theFv/Fmratio decreased only at severe water deficit. On the contrary, the effective photochemical efficiency of PSII (ΔF/F′m) measured under high actinic light decreased linearly in both types of stress, although in a more pronounced way under rapid stress. A similar variation in photochemical quenching suggests that the decrease of ΔF/F′mis mainly due to the closure of PSII reaction centres. The non‐photochemical quenching did not change significantly except under severe dehydration indicating that the energization state of thylakoids remained stable under stress. The decrease observed in photosynthetic pigments may be an adaptation to stress rather than a limiting factor to photosynthesis. Results suggests that, although intrinsic mesophyll metabolic inhibitions occur, stomatal limitation to CO2diffusion is the main reason for the decrease in photosynthesis.
Drought‐induced spikelet sterility is associated with an inefficient antioxidant defence in rice panicles
Authors: Selote Devarshi S. , Khanna‐Chopra Renu
Published: June 2004

Water stress‐induced spikelet sterility limits rice production under upland conditions. The causes of spikelet sterility under drought stress are poorly understood. In this study the role of antioxidant defence management in drought‐induced spikelet sterility was investigated in two rice (Oryza sativa) genotypes differing in drought resistance. Drought‐resistant N22 genotype showed less water stress‐induced spikelet sterility when compared to the susceptible N118 genotype under upland conditions. The N22 panicles maintained higher RWC and turgor potential and lower H2O2 levels across the developmental stages under water stress than that of N118 panicles. Drought‐induced enhancement in superoxide dismutase (SOD, EC 1.15.1.1) activity coupled with higher ascorbate (AsA), glutathione (GSH) content and enhanced ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities resulted in lower H2O2 levels in N22 panicles. In contrast, insufficient enhancement in SOD, APX and GR activities resulted in relatively higher H2O2 levels under water stress in N118 panicles. The N22 panicles exhibited a higher number of SOD and APX isozymes in comparison with N118 panicles that might provide better reactive oxygen species scavenging. Hence it is concluded that well‐equipped antioxidant defence plays an important role in minimizing water stress‐induced spikelet sterility in upland rice.
Specific hypomethylation of DNA is induced by heavy metals in white clover and industrial hemp
Authors: Aina Roberta , Sgorbati Sergio , Santagostino Angela , Labra Massimo , Ghiani Alessandra , Citterio Sandra
Published: June 2004

The present study was to assess the effect of heavy metal stress on the DNA methylation of a metal‐sensitive plant, Trifolium repens L. and of a metal‐tolerant plant, Cannabis sativa L. The changes in the level of 5‐methylcytosine (5mC) in the root DNA of plants grown on soils contaminated with different concentrations of Ni2+, Cd2+ and Cr6+ compared with that of untreated plants, were determined by immunolabelling with a monoclonal antibody, using the Slot‐Blot technique. Results showed that DNA of hemp control plants was about three times more methylated than clover DNA, for the same amount of root DNA. Heavy metal treatments induced a global dose‐dependent decrease of 5mC content, both in hemp and clover, ranging from 20 to 40%. Changes in methylation pattern of 5′‐CCGG‐3′ containing sequences were investigated by methylation‐sensitive amplification polymorphism (MSAP) technique. Control plants of the same species showed a very similar pattern, suggesting that, in normal condition, methylation involves precise sites. Heavy metals induced DNA methylation changes mainly related to hypomethylation events. These variations were not randomly directed but involved specific DNA sequences, since the detected polymorphisms were the same in all the plants analysed for each treatment.
Cloning and Characterization of the Arabidopsis Thaliana SQS1 Gene Encoding Squalene Synthase
Authors: Kribii Rachida , Arró Montserrat , Del Arco Ana , González Victor , Balcells Lluís , Delourme Didier , Ferrer Albert , Karst Francis , Boronat Albert
Published: June 2004

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A full‐length Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR‐based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQS1 and SQS2) which are organized in a tandem array. SQS1 and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQS1 gene product, SQS1. RNA blot analysis has shown that the 1.6‐kb SQS1 mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQS1 isoform is unable to complement the SQS‐defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C‐terminal residues of the Arabidopsis enzyme by the 111 C‐terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using [3H]farnesyl‐P2, and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQS1 or chimeric Arabidopsis/S. pombe SQS derivatives indicated that the C‐terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.
Characterization of a 200‐kDa Diatom Protein that is Specifically Associated with a Silica‐Based Substructure of the Cell Wall
Authors: Kröger Nils , Lehmann Gerhard , Rachel Reinhard , Sumper Manfred
Published: June 2004

The cell wall of a diatom is made up of a silica‐based scaffold and organic macromolecules. Proteins located in the cell wall are believed to control morphogenesis of the species‐specific silica structures of the scaffold. However, data that correlate distinct silica elements and specific proteins within the diatom cell wall have not been reported. Here, the cell wall protein HEP200 (200‐kDa HF‐extractable protein) from the diatom Cylindrotheca fusiformis is identified and characterized. HEP200 is tightly associated with a substructure of the silica scaffold. It is a member of a new protein family, of which two more members are identified. Each member displays the same bipartite structure. The N‐terminal part consists of a variable number of a repeated sequence motif (PSCD domain), whereas the C‐terminal part is unique. Immunolocalization experiments revealed the arrangement of different proteins within the cell wall. Frustulins, a previously described group of glycoproteins, constitute the outer coat of the cell wall and exhibit a ubiquitous distribution. In contrast, HEP200 is specifically located at a subset of about six silica strips in intact cell walls, shielded by frustulins. This study therefore identifies a diatom cell wall protein (HEP200) that is associated with a distinct substructure of the silica scaffold.
Extraction of Hoechst 33342 from the Cytoplasmic Leaflet of the Plasma Membrane by P‐Glycoprotein
Authors: Shapiro Adam B. , Ling Victor
Published: June 2004

P‐glycoprotein is an ATP‐dependent plasma membrane multidrug transporter of broad specificity. A common chemical property of its substrates is that all are lipophilic. Using Hoechst 33342 as the substrate, we have previously shown that P‐glycoprotein extracts the substrate directly from the lipid bilayer [Shapiro, A. B., Corder, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 115–121]. In this paper, we determined the leaflet of the plasma membrane from which P‐glycoprotein extracts Hoechst 33342.

The initial rate of Hoechst 33342 transport upon ATP addition to P‐glycoprotein‐rich inside‐out plasma membrane vesicles decreased slightly with the amount of time previously elapsed for slow diffusion of Hoechst 33342 to the extracellular leaflet. This result is consistent with transport from the cytoplasmic leaflet. Fluorescence resonance energy transfer from donor Hoechst 33342 to acceptor 2‐[6‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]hexanoyl‐sn‐glycero‐3‐phosphocholine (Nbd‐C6‐HPC) in the cytoplasmic leaflet was used to monitor the amount of Hoechst 33342 in the cytoplasmic leaflet versus time. The initial rate of decrease of the energy‐transfer‐related Nbd‐C6‐HPC fluorescence after ATP addition exceeded that of the Hoechst 33342 fluorescence and continued to decrease after decrease of the Hoechst 33342 fluorescence had ceased. These effects were consistent with transport of Hoechst 33342 from the cytoplasmic leaflet to the aqueous interior of the vesicles, followed by rebinding to the extracellular leaflet. This demonstrates that P‐glycoprotein transports drugs from the cytoplasmic leaflet of the plasma membrane directly to the aqueous extracellular medium. This finding has implications for efforts to localize the drug‐binding site(s) within P‐glycoprotein.
Further Insights into the Mechanism of Action of Methylmalonyl‐CoA Mutase by Electron Paramagnetic Resonance Studies
Authors: Abend Andreas , Illich Valentin , Rétey Jànos
Published: June 2004

Novel analogues of methylmalonyl‐CoA and succinyl‐CoA have been prepared and used for mechanistic investigations on the coenzyme‐B12‐dependent methylmalonyl‐CoA mutase. 1‐Carboxyethyl‐CoA (1) and 2‐carboxyethyl‐CoA (2) as well as their sulphoxides (3 and 4) were moderately good inhibitors with Ki values 4–20 times higher than the Km for succinyl‐CoA. 2‐Carboxyethyl‐CoA (2) and its sulphoxide 4 induced EPR signals when bound to the enzyme–coenzyme‐B12 complex. The EPR spectrum of 2 and its sulphoxide 4 differed very much from those induced by the other substrates. In the case of 2 the EPR spectrum of the holoenzyme/inhibitor complex showed the presence of an organic radical coupled to cobal(II)amin. The same experiment with 4 leads to the formation of enzyme‐bound cobal(II)amin with no detectable organic radical. The analogues 1 and 3 exhibited higher Ki values and did not induce EPR signals binding to the enzyme‐coenzyme‐B12 complex.

Formyl‐CoA and acrylate inhibited the enzyme synergistically but were unable to induce EPR signals and to form the product. Ethylmalonyl‐CoA, known as a poor substrate, induced a similar but less intense EPR signal than the natural substrate methylmalonyl‐CoA. The results are discussed in terms of the mechanism of the methylmalonyl‐CoA mutase reaction.
Polyadenylation‐Mediated Translational Regulation of Maternal P450(llβ) mRNA in Frog Oocytes
Authors: Takemori Hiroshi , Halder Sunil Krishna , Nonaka Yasuki , Fujii Tomomi , Ohta Miho , Hatano Osamu , Okamoto Mitsuhiro
Published: June 2004

Northern blot analysis of bullfrog tissues using a cDNA probe of cytochrome P450(11β) showed that a large amount of message was present in the ovary as well as in the adrenal tissue. Two kinds of mRNA of different sizes were found in the ovary. Sequence determination of the two cDNAs and analysis by reverse‐transcription polymerase chain reaction indicated that the protein encoded by the larger mRNA was identical to the adrenal enzyme, while the protein encoded by the smaller had a truncated sequence lacking an extension peptide necessary for the protein transport to the mitochondria. The mRNAs were present in the oocytes but not in the follicular cells, and their content in an oocyte varied little during its maturation. Immunoblot analyses of the mitochondrial fraction of oocytes failed to demonstrate the presence of P450(11β) protein. In contrast the eggs were found to contain a large amount of enzymatically active protein. Interestingly the mRNA has a cis‐element called cytoplasmic polyadenylation element at its 3′ untranslated region. When poly(A) tails of the message prepared from eggs and oocytes were examined by RNase H digestion or reverse‐transcription polymerase chain reaction, those of eggs were about 150 nucleotides longer than those of oocytes. These results suggest that translation of the message is stimulated during the oocyte maturation as a result of enhanced polyadenylation at its 3′‐end. Finally a finding is presented that progesterone was converted to 11β‐hydroxyprogesterone by the frog P450(11β), implying that the enzyme expressed in eggs may control a level of progesterone which is needed to initiate the oocyte maturation.
Isolation of a Rat Histidase cDNA Sequence and Expression in Escherichia coli— Evidence of Extrahepatic/Epidermal Distribution
Authors: Sano Hirofumi , Tada Toyohiro , Moriyama Akihiko , Ogawa Hisamitsu , Asai Kiyofumi , Kawai Yoko , Hodgson Mark Emory , Kato Taiji , Wada Yoshiro , Suchi Mariko
Published: June 2004

Histidase (histidine ammonia‐lyase) is a cytosolic enzyme responsible for catalyzing the non‐oxidative deamination of histidine to urocanic acid. Full‐length cDNAs encoding rat histidase have been isolated from a λZAP liver cDNA library using a partial cDNA fragment obtained by PCR. Whereas the initial description of the rat histidase 3′ untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28‐bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA. Dideoxynucleotide chain‐termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5′ and 3′ untranslated sequences, respectively. A selected rat histidase cDNA clone was introduced into the pET‐16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli. After purification by nickel‐chelation chromatography, recombinant histidine‐tagged protein was employed to raise anti‐(rat histidase) immunoglobulin in a Japanese white rabbit. The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins. Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin. Among other histidase‐positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex. Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I). Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.
Rescue of a Mutant G‐Protein by Substrate‐Assisted Catalysis
Authors: Zor Tsaffrir , Bar‐Yaacov Margalith , Elgavish Sharona , Shaanan Boaz , Selinger Zvi
Published: June 2004

Signaling by guanine‐nucleotide‐binding proteins (G‐proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G‐proteins remains controversial. We have employed substrate‐assisted catalysis as a novel approach to study catalysis by G‐proteins. In these studies, we have used diaminobenzophenone‐phosphonoamidate‐GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase‐deficient [Leu227]Gsα mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild‐type enzyme at the same rate. On the other hand, modification of Gsα by cholera toxin, which catalyses ADP‐ribosylation of Arg201 of Gsα, decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]Gsα mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild‐type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate‐limiting step of the GTPase reaction.
Crystallographic Analysis of Human Immunodeficiency Virus 1 Protease with an Analog of the Conserved CA‐p2 Substrate
Authors: Weber Irene T. , Wu Jin , Adomat Jill , Harrison Robert W. , Kimmel Alan R. , Wondrak Ewald M. , Louis John M.
Published: June 2004

Human immunodeficiency virus type 1 (HIV‐1) protease hydrolysis of the Gag CA‐p2 cleavage site is crucial for virion maturation and is optimal at acidic pH. To understand the processing of the CA‐p2 site, we have determined the structure of HIV‐1 protease complexed with an analog of the CA‐p2 site, the reduced peptide inhibitor Arg‐Val‐Leu‐r‐Phe‐Glu‐Ala‐Ahx‐NH2 [r denotes the reduced peptide bond and Ahx 2‐aminohexanoic acid (norleucine), respectively]. The crystal structure was refined to an R‐factor of 0.17 at 0.21‐nm resolution. The crystals have nearly the same lattice as related complexes in P212121, which have twofold disordered inhibitor, but are in space group P21, and the asymmetric unit contains two dimers of HIV‐1 protease related by 1800 rotation. An approximate non‐crystallographic symmetry has replaced the exact crystal symmetry resulting in well‐ordered inhibitor structure. Each protease dimer binds one ordered inhibitor molecule, but in opposite orientations. The interactions of the inhibitor with the two dimers are very similar for the central P2 Val to P2' Glu residues, but show more variation for the distal P3 Arg and P4' Ahx residues. Importantly, the carboxylate oxygens of Glu at P2' in the inhibitor are within hydrogen‐bonding distance of a carboxylate oxygen of Asp30 of the protease suggesting that the two side chains share a proton. This interaction suggests that the enzyme‐substrate complex is additionally stabilized at lower pH. The importance of this interaction is emphasized by the absence of polymorphisms of Asp30 in the protease and variants of P2' Glu in the critical CA‐p2 cleavage site.
The 40‐kDa Component of the Phagocyte NADPH Oxidase (p40phox) is Phosphorylated during Activation in Differentiated HL60 Cells
Authors: Fuchs Alexandra , Bouin Anne‐Pascale , Rabilloud Thierry , Vignais Pierre V.
Published: June 2004

The superoxide‐generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane‐bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67phox, p47phox and p40phox which translocate to the membrane upon activation. Known mechanisms underlying the translocation of these proteins include polyphosphorylation of p47phox and specific Src homology 3/polyproline motif interactions. In this study, through two‐dimensionnal electrophoresis and immunoprecipitation experiments, we show using dimethylsulfoxide‐differentiated HL60 promyelocytes that p40phox is in a basal phosphorylated state in resting cells and undergoes further phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol myristate acetate or by the formyl peptide fMet‐Leu‐Phe‐Lys. Moreover, the extent of phosphorylation is strongly correlated with the level of superoxide production. Typically, in cells transiently activated by fMet‐Leu‐Phe‐Lys, onset of superoxide production coincides with the appearance of new phosphorylated species of p40phox and, at. the end of the respiratory burst, dephosphorylation of p40phox is observed. In vitro assays show that the kinase(s) involved in the phosphorylation of p40phox differ from those which participate in the phosphorylation of p47phox. This suggests that, in the cell, the phosphorylation of p40phox and of p47phox are under the control of two different kinase pathways.
Metabolic Processing of Gangliosides by Human Fibroblasts in Culture — Formation and Recycling of Separate Pools of Sphingosine
Authors: Chigorno Vanna , Riva Carmen , Valsecchi Manuela , Nicolini Marco , Brocca Paola , Sonnino Sandra
Published: June 2004

Human cultured fibroblasts were fed with GM3 ganglioside species isotopically labeled at C3 of C18‐sphingosine, or at C3 of C18‐sphinganine, or at the sialic acid acetyl group, and with C18‐sphingosine and C18‐sphinganine, both labeled at Cl. After a lipid pulse the cells were subjected until 7‐day chase; measurements were then made of the radioactive products resulting from the administered long‐chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions.

The GM3 species differing in the long‐chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic C18‐sphingosine and C18‐sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded.

Results obtained by administering ganglioside species of GM3 containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive GM3 highdensity microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the GD3 species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the GD3 species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of GD3. Administration of the ganglioside species of GM3 containing sphinganine or free sphinganine to fibroblasts yielded the GD3 species containing mainly very long‐chain fatty acids and sphingosine.

These results show the possible existence of a pool of ganglioside‐derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway.
Isolation and Identification of Multiple Neuropeptides of the Allatostatin Superfamily in the Shore Crab Carcinus Maenas
Authors: Duve Hanne , Johnsen Anders H. , Maestro Jose‐Luis , Scott Alan G. , Jaros Peter P. , Thorpe Alan
Published: June 2004

20 neuropeptides belonging to the allatostatin superfamily were isolated from extracts of cerebral and thoracic ganglia of the shore crab Carcinus maenas. They were purified by HPLC, monitored by radioimmunoassay and identified by mass spectrometry and amino acid sequencing. The allatostatins are characterised by a common C‐terminal pentapeptide sequence ‐YXFGL‐NH2. Previously such peptides have only been reported from insects. In insects the variable post‐tyrosyl residue is restricted to Ala, Asn, Asp, Gly or Ser. In C. maenas, however, there are only two types; thirteen of the peptides having a post‐tyrosyl Ala and the other seven, a post‐tyrosyl Ser. The crab peptides include the shortest allatostatins so far identified (YAFGL‐NH2 and YSFGL‐NH2) as well as the longest, a 27‐residue peptide. The total of 20 peptides exceeds the highest number of allatostatins found in any of the insects investigated so far (14 in Periplaneta americana). It is of interest that, despite their clear homology, none of the peptides of C. maenas is identical to any of the more than 50 known insect allatostatins. The crab allatostatins show evidence of gene duplication and mutation that has resulted in several sub‐groups with close structural similarities. For example, there are four heptapeptides with the common C‐terminus ‐PYAFGL‐NH2 that differ only at the N‐terminal residue, which is either Glu, Asp, Asn or Ser. Other motifs, variously extended at the N‐terminus, include ‐GPY(A/S)FGL‐NH2 (three peptides), ‐DMY(A/S)FGL‐NH2 (three peptides), and ‐GQY(A/S)FGL‐NH2 (two peptides). Unique among the allatostatin superfamily, one of the crab peptides has a Tyr for Phe substitution at position three from the C‐terminus (GGPYSYGL‐NH2). Immunocytochemistry has provided clues to the functions of the allatostatins in crustaceans by showing their widespread presence in the central and stomatogastric nervous systems.
Conformational Features and Thermal Stability of Bovine Seminal Plasma Protein PDC‐109 Oligomers and Phosphorylcholine‐Bound Complexes
Authors: Gasset María , Saiz José L. , Laynez Josél , Sanz Libia , Gentzel Marc , Töpfer‐Petersen Edda , Calvete Juan J.
Published: June 2004

At ejaculation, PDC‐109, the major heparin‐binding protein of bull seminal plasma, binds to the phosphorylcholine group of sperm lipids and modulates capacitation promoted by glycosaminoglycans during sperm residence in the female genital tract. Combination of size‐exclusion chromatography, analytical ultracentrifugation, circular dichroism, Fourier‐transform infrared spectroscopy, and differential scanning calorimetry has allowed us to biophysically characterize PDC‐109 and its interaction with phosphorylcholine. PDC‐109 can be regarded as a polydisperse molecule whose aggregation state can be modulated by the solute composition of its solution environment. Dissociation of PDC‐109 oligomers occurs upon increasing the concentration of either NaCl, EDTA, CaCl2, or phosphorylcholine, suggesting that both ionic and hydrophobic interactions are responsible for the aggregation tendency of PDC‐109 monomers. Dissociation processes are accompanied by exposure of peptide bonds to the solvent, changes in the environment of tyrosine and tryptophan residues, and a slight increase in the turn content at the expense of non‐regular structure. Analysis of the heat‐induced denaturation of PDC‐109 oligomers revealed two melting transitions at about 36 °C (irreversible) and 55 °C (partially reversible) characterized by calorimetric enthalpy changes of 42 kJ/mol and 217 kJ/mol, respectively. These transitions could be assigned to the dissociation of oligomers and to the cooperative unfolding of PDC‐109 monomers, respectively. The modulation of the aggregation state of PDC‐109 by its molecular environment and by phosphorylcholine binding suggests possible mechanisms for capacitation mediated by the seminal plasma protein.
Structure of a Hydroxymycolic Acid Potentially Involved in the Synthesis of Oxygenated Mycolic Acids of the Mycobacterium Tuberculosis Complex
Authors: Quémard Annaïk , Lanéelle Marie‐Antoinette , Marrakchi Hédia , Prome Danielle , Dubnau Eugenie , Daffé Mamadou
Published: June 2004

Mycolic acids are believed to play a crucial role in the architecture of the mycobacterial envelope. However, very few steps of their biosynthetic pathway have yet been elucidated. We previously isolated [Dubnau, E., Lanéelle, M. A., Soares, S., Bénichou, A., Vaz, T., Promé, D., Promé, J. C., Daffé, M. & Quémard, A. (1997) Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto and hydroxy‐mycolic acids, Mol. Microbiol. 23, 313–322] a gene cluster from Mycobacterium bovis BCG, cmaA‐D, which confers upon M. smegmatis the ability to synthesize cyclopropanated ketomycolic acid, and a new type of mycolic acid which is hydroxylated. A meticulous analysis of all the mycolic like fatty acids of M. bovis BCG and M. tuberculosis showed that these organisms produce small amounts of the hydroxymycolic acid. The structure of this molecule, determined by NMR spectroscopy, mass spectrometry and stereochemical studies, strongly suggests that there is a direct biosynthetic relationship between the keto‐and the hydroxyl‐mycolic acids.
RNA‐unwinding and RNA‐folding Activities of RNA Helicase II/Gu — Two Activities in Separate Domains of the Same Protein
Authors: Valdez Benigno C. , Henning Dale , Perumal Karthika , Busch Harris
Published: June 2004

The human RNA helicase II/Gu protein (RH‐TI/Gu) is a member of the D‐E‐A‐D box protein family. It is a unique enzyme, which possesses an ATP‐dependent RNA‐unwinding activity and has an RNA‐folding activity that introduces an intramolecular secondary structure in single‐stranded RNA. This report shows that these two enzymatic activities are distinct. ATP[S], GTP and low concentrations of ATP enhance the RNA‐folding activity of RH‐II/Gu but not the RNA‐helicase activity. High concentrations of ATP are required for the helicase activity but are inhibitory to the RNA‐folding activity. Mg2 is required for the helicase activity but not for the RNA‐folding reaction. Affinity‐purified anti‐(RH‐II/Gu) polyclonal Ig inhibit the RNA‐unwinding activity but not the folding activity. Mutations of the DEVD sequence, which corresponds to the DEAD box, and the SAT motif enhanced RNA‐folding activity of RH‐II/Gu but completely inhibited the RNA‐helicase activity. A mutant that lacks the COOH‐terminal 76 amino acid residues, including the four FRGQR repeats, had unwinding activity but did not catalyze the folding of a single‐stranded RNA. The two enzymatic activities of RH‐II/Gu reside in distinct domains. Amino acids 1–650 are active in the RNA‐unwinding reaction but lack RNA‐folding activity. Amino acids 646–801 fold single‐stranded RNA but lack helicase activity. This report shows distinct RNA‐unwinding and RNA‐folding activities residing in separate domains within the same protein.
Characterization and Evolution of a Gene Encoding a Trimeresums flavoviridis Serum Protein that Inhibits Basic Phospholipase A2 Isozymes in the Snake's Venom
Authors: Nobuhisa Ikuo , Inamasu Satoshi , Nakai Makoto , Tatsui Akira , Mimori Tomohiro , Ogawa Tomohisa , Shimohigashi Yasuyuki , Fukumaki Yasuyuki , Hattori Shosaku , Kihara Hiroshi , Ohno Motonori
Published: June 2004

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were ractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I–V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI‐IV and PLI‐V correspond to PLI‐A and PLI‐B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate‐recognition domain of C‐type lectins. PLI‐I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA‐B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA,s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI‐I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI‐I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI‐I was isolated. It is 9.6‐kb long and consists of five exons and four introns. Comparison of the exonintron structure of the PLI‐I gene with those of genes encoding urokinaseype‐plasminogen‐activator receptor (uPAR), Ly‐6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI‐I gene belongs to the uPAR, Ly‐6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.
Biochemical and Immunological Characterization of Recombinant Allergen Lol p 1
Authors: Tamborini Elena , Faccini Silvia , Lidholm Jonas , Svensson Margareta , Brandazza Anna , Longhi Renato , Groenlund Hans , Sidoli Alessandro , Arosio Paolo
Published: June 2004

Pollen from perennial rye grass (Lolium perenne), a major cause of type‐I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des(H6–240)‐Lol p 1, des‐(1–88)‐Lol p 1 and des‐(133–189)‐Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17–18‐residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.
The C‐Terminal Domain V of Perlecan Promotes β1 Integrin‐Mediated Cell Adhesion, Binds Heparin, Nidogen and Fibulin‐2 and Can be Modified by Glycosaminoglycans
Authors: Brown Judith C. , Sasaki Takako , Göhring Walter , Yamada Yoshihiko , Timpl Rupert
Published: June 2004

Domain V of the major basement‐membrane proteoglycan perlecan, a domain which consists of three laminin type G (LG) and four epidermal‐growth‐factor‐like (EG) modules, was obtained in recombinant form by transfecting embryonic kidney cells with an episomal expression vector. A major 90‐kDa fragment V was obtained together with fragments Va (74 kDa) and Vb (26 kDa) which were generated by endogenous proteolysis in front of the most C‐terminal LG module. All three fragments bound to a heparin affinity column and could be displaced at a moderate (0.2 M) NaCl concentration. Rotary‐shadowing electron microscopy demonstrated a three‐globule structure for fragment V. Fragment V also showed a strong immunological cross‐reaction with tissue‐derived perlecan, indicating that it was folded into a native structure. A further, larger fragment, Vc, was apparently substituted with heparan sulphate and/or chondroitin sulphate chains and failed to bind to heparin. Fragment V but not fragment Vc promoted a distinct adhesion of several cell lines and this could be blocked by antibodies against the integrin β1 chain. This domain may, however, represent only one of several cell‐adhesive sites of perlecan. The recombinant perlecan fragment V bound in surface plasmon resonance assays to fibulin‐2, laminin‐nidogen complex, nidogen and two nidogen fragments. This indicated two different nidogen‐binding epitopes on perlecan domain V with about a 10‐fold difference in their affinities (Kd= 0.05–0.2 μM and about 2 μM). Perlecan domain V therefore seems to participate in the supramolecular assembly and cell connections of basement membranes.
Cross‐Linking of Chloroplast F0F1‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor
Authors: Schulenberg Birte , Wellmer Frank , Lill Holger , Junge Wolfgang , Engelbrecht Siegfried
Published: June 2004

Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6 → Ser mutant of subunit ɛ of spinach chloroplast F0F1 ATP synthase. Wild‐type and engineered ɛ were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1 lacking the ɛ subunit [F1(‐ɛ)]. Cys‐containing ɛ variants were modified with a sulfhydryl‐reactive photolabile cross‐linker. Photocross‐linking of ɛ to F1(‐ɛ) yielded the same SDS gel pattern of cross‐link products independent of the presence or absence of Mg2+ ADP, phosphate and Mg2+ ATP. ɛ (wild type) [Ser6, Cys28]ɛ and [Ser6,Cys41]ɛ were cross‐linked with subunit γ. With chloroplast F0F1 the same cross‐link pattern was obtained, except for one extra cross‐link, probably between [Ser6, Cys28]ɛ and F0 subunit III. [Ser6, Cys17]ɛ and [Ser6, Cys85]ɛ did not produce cross‐links. Cross‐linking of ɛ, [Ser6,Cys28]ɛ, [Ser6,Cys41]ɛ to γ in soluble chloroplast F1 impaired the ability of ɛ to inhibit Ca2+‐ATPase activity. The Mg2+‐ATPase activity of soluble F1 (measured in the presence of 30% MeOH) was not affected by cross‐linking ɛ with γ. Functional reconstitution of photophosphorylation in F1‐depleted thylakoids was observed with F1 in which γ was cross‐linked to [Ser6,Cys28]ɛ or [Ser6,Cys41]ɛ but not with wild‐type ɛ. In view of the intersubunit rotation of γ relative to (αβ)3, which is driven by ATP hydrolysis, γ and ɛ would seem to act concertedly as parts of the ‘rotor’ relative to the ‘stator’ (αβ)3.
Transferrin
Authors: Pakdaman Rowchanak , El Hage Chahine Jean‐Michel
Published: June 2004

The interaction of apolactoferrin with hydrogen carbonate (bicarbonate) has been investigated in the pH range 6.5–9.2. In the absence of bicarbonate apolactoferrin loses a single proton with pK1a of 8.10. This proton loss is independent of the interaction with the synergistic anion. The C‐site of apolactoferrin interacts with bicarbonate with a very low affinity (K−1c= 3.2 M−1). This process is accompanied by a proton loss, which is probably provided by the bicarbonate in interaction with the protein. This proton loss can possibly be the result of a shift in the proton dissociation constant, pKa, of the bicarbonate/carbonate acid/base equilibrium, which would decrease from pKa, 10.35 to pK2a 6.90 in the bicarbonate‐lactoferrin adduct. The N‐site of the protein interacts with bicarbonate with an extremely low affinity, which excludes the presence of the N‐site‐synergistic anion adduct in neutral physiological media. Contrary to serum transferrin, the concentration of the apolactoferrin in interaction with bicarbonate is pH dependent. Between pH 7.4 and pH 9 with [HCO−3] about 20 mM, the concentration of the serum transferrin‐bicarbonate adduct is always about 30%, whereas that of the apolactoferrin‐synergistic anion adduct varies from 25% at pH 7.5 to 90% at pH 9. This implies that, despite an affinity for bicarbonate two orders of magnitude lower than that of serum transferrin, lactoferrin interacts better with the synergistic anion. This can be explained by the possible interaction of lactoferrin with carbonate in neutral media, whereas transferrin only interacts with bicarbonate.
Purification and Characterization of two Isoforms of Isopentenyl‐Diphosphate Isomerase from Elicitor‐Treated Cinchona Robusta Cells
Authors: Ramos‐Valdivlv Ana C. , Van Der Heijden Robert , Verpoorte Robert , Camara Bilal
Published: June 2004

In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl‐diphosphate isomerase (isopentenyl‐POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl‐POP and dimethylallyl diphosphate (dimethylallyl‐POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells. From elicitor‐treated C. robusta cells, two isoforms of isopentenyl‐POP isomerase have been purified to apparent homogeneity in four chromatographic steps. The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with ATm values for isopentenyl‐POP of 5.1 μM and 1.0 μM, respectively. Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5–2 mM. Isoform I was most active in the presence of 0.5–1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+. A pH optimum of 7–7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (K, 96 μM for isoform I) and the transition state analogue 2‐(dimethylamino)ethyl diphosphate. Rechromatography of purified isoforms did not indicate any interconversion of both forms. Western blot analysis, using antibodies raised against isopentenyl‐POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C. robusta cells. Isoform II was specifically induced by elicitation, non‐treated cells contained low activity of this isoform. The possible role of isopentenyl‐POP isomerase in the biosynthesis of anthraquinones is discussed.