bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C‐terminal part of the Streptococcus pneumoniae Era GTPase
Authors: Hang Julie Qi , Meier Timothy I. , Zhao Genshi
Published: August 2003

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA‐ and membrane‐binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C‐terminal part of S. pneumoniae Era was systematically deleted while the N‐terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C‐terminal deletion of 9 or 19 amino‐acid residues did not affect 16S rRNA‐binding activity while further deletions of the C‐terminus (29–114 amino‐acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino‐acid residues between ≈ 22–83 from the C‐terminus, is required for 16S rRNA‐binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C‐terminus retained membrane‐binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane‐binding. Thus, these results indicate for the first time that the regions critical for the membrane‐ and 16S rRNA‐binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3′ terminus complemented an Escherishia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA‐binding activity and that part of the KH domain is also required for membrane‐binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.
Amphipathic α helical antimicrobial peptides.
Authors: Giangaspero Anna , Sandri Luca , Tossi Alessandro
Published: August 2003

Antimicrobial peptides (AMPs) that assume an amphipathic α helical structure are widespread in nature. Their activity depends on several parameters including the sequence, size, degree of structure formation, cationicity, hydrophobicity and amphipathicity. The analysis of numerous natural AMPs provided representative values for these parameters and led to a sequence template with which to generate potent artificial lead AMPs. Sequences were then varied in a rational manner, using both natural and nonproteinogenic amino acids, to probe the individual roles of each parameter in modulating biological activity. A high cationicity combined with a stabilized amphipathic α helical structure conferred enhanced cidal activity towards all the cell types considered, and was a requirement for Gram‐positive bacteria and fungi. An elevated helicity also correlated with increased hemolytic activity. The structural requirements for activity against several Gram‐negative bacteria were instead considerably less stringent, so that it persisted in peptides in which formation of a helical structure and/or amphipathicity were impeded. Either a reduced charge or a reduced hydrophobicity resulted in generally inactive peptides. These observations, combined with the kinetics of bacterial membrane permeabilization and time‐killing are discussed in terms of currently accepted models of action for this type of peptide. The simple guidelines obtained in this study allowed the design of highly active shortened AMPs and may be generally useful in the development of this type of peptides as anti‐infective agents.
NAD degradation and regulation of CD38 expression by human monocytes/macrophages
Authors: Pfister Martin , Ogilvie Adaling , da Silva Christina P. , Grahnert Andreas , Guse Andreas H. , Hauschildt Sunna
Published: August 2003

In recent years, evidence has accumulated that NAD+ serves as a precursor of metabolites that are involved in a number of regulatory processes. In this work we show that extracellularly added NAD+ was rapidly degraded by intact human monocytes to nicotinamide and ADP‐ribose. Besides these main products, minor amounts of AMP, ADP and cADP‐ribose were formed. Expression of CD38, which has been identified as NAD+‐glycohydrolase (EC 3.2.2.6) degrading NAD+ into nicotinamide and ADP‐ribose, was determined on freshly isolated human monocytes by flow cytometry and RT‐PCR. Upon ligation with anti‐CD38 mAb, CD38 underwent internalization, shedding and new expression. As monocytes possess an intracellular CD38 pool, it could serve as a source for newly expressed CD38. Differentiation of monocytes to macrophages resulted in down‐regulation of surface expression of CD38. This decrease correlates with a reduction in NADase activity, indicating that the amount of functional active CD38 molecules decrease during differentiation. As CD38 mRNA was found to be diminished in macrophages, regulation of the gene product seems to occur at the level of transcription or mRNA stability.
Caveolin‐1 negatively regulates SR‐BI mediated selective uptake of high‐density lipoprotein‐derived cholesteryl ester
Authors: Matveev Sergey , Uittenbogaard Annette , van der Westhuyzen Deneys , Smart Eric J.
Published: August 2003

The class B, type I scavenger receptor (SR‐BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR‐BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin‐1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin‐1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester‐induced differentiated THP‐1 cells. The goal of the present study was to determine if the expression of caveolin‐1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J‐774 cell lines that stably expressed caveolin‐1. Transfection with caveolin‐1 cDNA did not alter the amount of 125I‐labeled HDL that associated with the cells, although selective uptake of HDL [3H]cholesteryl ether was decreased by approximately 50%. The amount of [3H]cholesterol effluxed to HDL was not affected by caveolin‐1. To directly address whether caveolin‐1 inhibits SR‐BI‐dependent selective cholesteryl ester uptake, we overexpressed caveolin‐1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR‐BI. Caveolin‐1 inhibited the selective uptake of HDL [3H]cholesteryl ether by 50–60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR‐BI to demonstrate that the increase in selective [3H]cholesteryl ether uptake previously seen in differentiated THP‐1 cells was independent of SR‐BI. Finally, we used β‐cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR‐BI‐dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin‐1 is a novel negative regulator of SR‐BI‐dependent selective cholesteryl ester uptake.
Regioselective desulfation of sulfated l‐fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus.
Authors: Daniel Régis , Berteau Olivier , Chevolot Lionel , Varenne Anne , Gareil P. , Goasdoue Nicole
Published: August 2003

The study of the structural bases of the biological properties of algal fucoidan (Ascophyllum nodosum) led us to look for enzymes able to modify this sulfated polysaccharide. In this context, we found a sulfoesterase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on fucoidan. This sulfoesterase activity was shown by capillary electrophoresis and 13C–1H NMR (500 MHz) analysis of the enzymatic hydrolysis of the fucoidan, of fucoidan oligosaccharides and of sulfated fucose isomers. We report the exhaustive list of all proton and carbon chemical shifts for each of the three isomers of sulfated‐l‐fucose (including of their α/β anomers), i.e. the 2‐O‐, 3‐O‐ and 4‐O‐sulfated fucose, which have been useful reference values for the assignments of NMR spectra of fucoidan oligosaccharides upon enzymatic desulfation. Our results demonstrated a high regioselectivity for this sulfoesterase, which hydrolyzes only the sulfate group at the 2‐O position of the fucopyranoside. Therefore, this sulfoesterase is a helpful tool in the structure–activity study of the fucoidan, as the literature data suggest that the 2‐O‐sulfation level play a central role in the biological properties of the polysaccharide.
Breakdown of Na+/K+‐exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+‐ATPase activity.
Authors: Beaugé Luis
Published: August 2003

The reactivity towards Na+ and K+ of Na+/K+‐ATPase phosphoenzymes formed from ATP and Pi during Na+‐ATPase turnover and that obtained from Pi in the absence of ATP, Na+ and K+ was studied. The phosphoenzyme formed from Pi in the absence of cycling and with no Na+ or K+ in the medium showed a biphasic time‐dependent breakdown. The fast component, 96% of the total EP, had a decay rate of about 4 s−1 in K+‐free 130 mm Na+, and was 40% inhibited by 20 mm K+. The slow component, about 0.14 s−1, was K+ insensitive. Values for the time‐dependent breakdown of the phosphoenzymes obtained from ATP and from Pi during Na+‐ATPase activity were indistinguishable from each other. In K+‐free medium containing 130 mm Na+, the decays followed a single exponential with a rate constant of 0.45 s−1. The addition of 20 mm K+ markedly increased the decays and made them biphasic. The fast components had a rate of ≈ 220 s‐1 and accounted for 92–93% of the total phosphoenzyme. The slow components decayed at a rate of about 47–53 s−1. A second group of experiments examined the reactivity towards Na+ of the E2P forms obtained with ATP and Pi when the enzyme was cycling. In both cases, the rate of dephosphorylation was a biphasic function of [Na+]: inhibition at low [Na+], with a minimum at about 5 mm Na+, followed by recovery at higher [Na+]. Although qualitatively similar, the phosphoenzyme formed from Pi showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na+‐ATPase turnover share the same intermediates.
Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line
Authors: Jahnel Ricarda , Dreger Mathias , Gillen Clemens , Bender Olaf , Kurreck Jens , Hucho Ferdinand
Published: August 2003

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG‐derived cell line F‐11 (N18TG2 mouse neuroblastoma × rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N‐glycosylation sites, the predicted transient receptor potential channel‐like transmembrane folding proposes N604 is localized extracellularly. We used site‐directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross‐linking with dimethyladipimidate yielded almost exclusively dimers.

Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F‐11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti‐VR1 immunoreactivity in neurite‐like structures of F‐11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion‐derived F‐11 cells provide a powerful experimental system for the study of VR1 biochemistry.
Unfolding and refolding studies of frutalin, a tetrameric d‐galactose binding lectin
Authors: Campana Patricia T. , Moraes Derminda I. , Monteiro‐Moreira Ana C. O. , Beltramini Leila M.
Published: August 2003

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ·mol−1 and 12.34 kJ·mol−1, in the presence and absence of d‐galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size‐exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation‐condensation mechanism, whereby the sugar‐binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three‐dimensional structures, spontaneously assemble to form tetramers.
Role of a highly conserved YPITP motif in 2‐oxoacid:ferredoxin oxidoreductase.
Authors: Fukuda Eriko , Kino Hiroyasu , Matsuzawa Hiroshi , Wakagi Takayoshi
Published: August 2003

2‐Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A‐dependent oxidative decarboxylation of pyruvate and 2‐oxoglutarate, a cognate Zn‐7Fe‐ferredoxin serving as an electron acceptor. It comprises two subunits, a (632 amino acids) and b (305 amino acids). To further elucidate its structure and function, we constructed a gene expression system. The wild‐type recombinant enzyme was indistinguishable from the natural one in every criterion investigated. A series of variants was constructed to elucidate the role of the YPITP‐motif (residues 253–257) in subunit a, which is conserved universally in the 2‐oxoacid:ferredoxin oxidoreductase (OFOR) family. Single amino‐acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity. T256, the hydroxyl group of which has been proposed to be essential for binding of the 2‐oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2‐oxoglutarate being ≈ 33% and ≈ 51%, respectively, as compared with that of the wild‐type enzyme. Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2‐oxoacid was only slightly affected. Thus, the YPITP‐motif is essential for the turnover of the reaction rather than the affinity toward 2‐oxoacid.
Isolation and functional analysis of a cDNA encoding a myrcene synthase from holm oak (Quercus ilex L.)
Authors: Fischbach Robert J. , Zimmer Wolfgang , Schnitzler Jörg‐Peter
Published: August 2003

An 859‐bp cDNA segment of a terpene synthase gene was amplified by PCR from the evergreen sclerophyllous holm oak (Quercus ilex L.) using heterologous primers for conserved regions of terpene synthase genes (TPS) in dicotyledonous plants. Based on the sequence of this segment, homologous primers were designed for amplification by RACE‐PCR of a cDNA segment carrying the monoterpene synthase gene myrS. The gene encodes a protein of 597 amino acids including an N‐terminal putative plastid transit peptide. The gene without the segment encoding the transit peptide was cloned by PCR into a bacterial expression vector. Expression in Escherichia coli yielded an active monoterpene synthase, which converted geranyl diphosphate (GDP) predominantly into the acyclic monoterpene myrcene and to a very small extent into cyclic monoterpenes. Sequence comparison with previously cloned monoterpene synthases revealed that the myrcene synthase from Q. ilex belongs to the TPSb subfamily.
Fidelity and spatio‐temporal control in MAP kinase (ERKs) signalling
Authors: Pouysségur Jacques , Lenormand Philippe
Published: August 2003

The mitogen activated protein (MAP) kinase module: (Raf →MEK→ERKs) is central to the control of cell growth, cell differentiation and cell survival. The fidelity of signalling and the spatio‐temporal activation are key determinants in generating precise biological responses. The fidelity is ensured by scaffold proteins – protein kinase ‘insulators’ – and by specific docking sites. The duration and the intensity of the response are in part controlled by the compartmentalization of the signalling molecules. Growth factors promote rapid nuclear translocation and persistent activation of p42/p44 MAP kinases, respectively and ERK2/ERK1, during the entire G1 period with an extinction during the S‐phase. These features are exquisitely controlled by the temporal induction of the MAP kinase phosphatases, MKP1–3. MKP1 and 2 induction is strictly controlled by the activation of the MAP kinase module providing evidence for an auto‐regulatory mechanism. This negative regulatory loop is further enhanced by the capacity of p42/p44 MAPK to phosphorylate MKP1 and 2. This action reduces the degradation rate of MKPs through the ubiquitin–proteasomal system. Whereas the two upstream kinases of the module (Raf and MEK) remain cytoplasmic, ERKs (anchored to MEK in the cytoplasm of resting cells) rapidly translocate to the nucleus upon mitogenic stimulation. This latter process is rapid, reversible and controlled by the strict activation of the MAPK cascade. Following long‐term MAPK stimulation, p42/p44 MAPKs progressively accumulate in the nucleus in an inactive form. Therefore we propose that the nucleus represents a site for ERK action, sequestration and signal termination. With the generation of knockdown mice for each of the ERK isoforms, we will illustrate that besides controlling cell proliferation the ERK cascade also controls cell differentiation and cell behaviour.
Characterization of the serotoninergic system in the C57BL/6 mouse skin
Authors: Slominski Andrzej , Pisarchik Alexander , Semak Igor , Sweatman Trevor , Wortsman Jacobo
Published: August 2003

We showed expression of the tryptophan hydroxylase gene and of tryptophan hydroxylase protein immunoreactivity in mouse skin and skin cells. Extracts from skin and melanocyte samples acetylated serotonin to N‐acetylserotonin and tryptamine to N‐acetyltryptamine. A different enzyme from arylalkylamine N‐acetyltransferase mediated this reaction, as this gene was defective in the C57BL6 mouse, coding predominantly for a protein without enzymatic activity. Serotonin (but not tryptamine) acetylation varied according to hair cycle phase and anatomic location. Serotonin was also metabolized to 5‐hydroxytryptophol and 5‐hydroxyindole acetic acid, probably through stepwise transformation catalyzed by monoamine oxidase, aldehyde dehydrogenase and aldehyde reductase. Activity of the melatonin‐forming enzyme hydroxyindole‐O‐methyltransferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain). In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N‐acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain.
An Escherichia coli twin‐arginine signal peptide switches between helical and unstructured conformations depending on the hydrophobicity of the environment
Authors: San Miguel Miguel , Marrington Rachel , Rodger P. Mark , Rodger Alison , Robinson Colin
Published: August 2003

The Tat system catalyzes the transport of folded globular proteins across the bacterial plasma membrane and the chloroplast thylakoid. It recognizes cleavable signal peptides containing a critical twin‐arginine motif but little is known of the overall structure of these peptides. In this report, we have analyzed the secondary structure of the SufI signal peptide, together with those of two nonfunctional variants in which the region around the twin‐arginine, RRQFI, is replaced by KKQFI or RRQAA. Circular dichroism studies show that the SufI peptide exists as an unstructured peptide in aqueous solvent with essentially no stable secondary structure. In membrane‐mimetic environments such as SDS micelles or water/trifluoroethanol, however, the peptide adopts a structure containing up to about 40% α‐helical content. Secondary structure predictions and molecular modelling programs strongly suggest that the helical region begins at, or close to, the twin‐arginine motif. Studies on the thermal stability of the helix demonstrate a sharp transition between the unstructured and helical states, suggesting that the peptide exists in one of two distinct states. The two nonfunctional peptides exhibit almost identical spectra and properties to the wild‐type SufI peptide, indicating that it is the arginine sidechains, and not their contribution to the helical structure, that are critical in this class of peptide.
Mapping the functional domain of the prion protein
Authors: Cui Taian , Daniels Maki , Wong Boon Seng , Li Ruliang , Sy Man‐Sun , Sassoon Judyth , Brown David R.
Published: August 2003

Prion diseases such as Creutzfeldt–Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu‐binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu‐binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.
Glucose sensing in the intestinal epithelium
Authors: Dyer Jane , Vayro Steven , King Timothy P. , Shirazi‐Beechey Soraya P.
Published: August 2003

Dietary sugars regulate expression of the intestinal Na+/glucose cotransporter, SGLT1, in many species. Using sheep intestine as a model, we showed that lumenal monosaccharides, both metabolisable and nonmetabolisable, regulate SGLT1 expression. This regulation occurs not only at the level of transcription, but also at the post‐transcriptional level. Introduction of d‐glucose and some d‐glucose analogues into ruminant sheep intestine resulted in > 50‐fold enhancement of SGLT1 expression. We aimed to determine if transport of sugar into the enterocytes is required for SGLT1 induction, and delineate the signal‐transduction pathways involved.

A membrane impermeable d‐glucose analogue, di(glucos‐6‐yl)poly(ethylene glycol) 600, was synthesized and infused into the intestines of ruminant sheep. SGLT1 expression was determined using transport studies, Northern and Western blotting, and immunohistochemistry. An intestinal cell line, STC‐1, was used to investigate the signalling pathways.

Intestinal infusion with di(glucos‐6‐yl)poly(ethylene glycol) 600 led to induction of functional SGLT1, but the compound did not inhibit Na+/glucose transport into intestinal brush‐border membrane vesicles. Studies using cells showed that increased medium glucose up‐regulated SGLT1 abundance and SGLT1 promoter activity, and increased intracellular cAMP levels. Glucose‐induced activation of the SGLT1 promoter was mimicked by the protein kinase A (PKA) agonist, 8Br‐cAMP, and was inhibited by H‐89, a PKA inhibitor. Pertussis toxin, a G‐protein (Gi)‐specific inhibitor, enhanced SGLT1 protein abundance to levels observed in response to glucose or 8Br‐cAMP.

We conclude that lumenal glucose is sensed by a glucose sensor, distinct from SGLT1, residing on the external face of the lumenal membrane. The glucose sensor initiates a signalling pathway, involving a G‐protein‐coupled receptor linked to a cAMP–PKA pathway resulting in enhancement of SGLT1 expression.
Regulation of Cyr61/CCN1 gene expression through RhoA GTPase and p38MAPK signaling pathways
Authors: Han Ji‐Soo , Macarak Edward , Rosenbloom Joel , Chung Kwang Chul , Chaqour Brahim
Published: August 2003

Cysteine‐rich protein 61 (Cyr61/CCN1) is an angiogenic factor and a member of a family of growth factor‐inducible immediate‐early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr61/CCN1gene activation in smooth muscle cells. Treatment of these cells with sphingosine 1‐phosphate (S1P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr61/CCN1 gene at both the mRNA and protein levels. Cyr61/CCN1 mRNA stability was not altered but the transcription rate of the Cyr61/CCN1 gene was increased fivefold in isolated nuclei from S1P‐stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936‐bp promoter fragment of the human Cyr61/CCN1 gene is functional and induces a reporter gene activity in S1P‐treated cells. Using a combination of cis‐element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP‐1 site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP‐1, were responsible for the promoter activity in S1P‐stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr61/CCN1 gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr61/CCN1 promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK1. These findings demonstrate the transcriptional regulation of the Cyr61/CCN1 gene and provide clues to the signaling molecules and transcription factors involved in such regulation.
Pherokine‐2 and ‐3
Authors: Sabatier Laurence , Jouanguy Emmanuelle , Dostert Catherine , Zachary Daniel , Dimarcq Jean‐Luc , Bulet Philippe , Imler Jean‐Luc
Published: August 2003

Drosophila is a powerful model system to study the regulatory and effector mechanisms of innate immunity. To identify molecules induced in the course of viral infection in this insect, we have developed a model based on intrathoracic injection of the picorna‐like Drosophila C virus (DCV). We have used MALDI‐TOF mass spectrometry to compare the hemolymph of DCV infected flies and control flies. By contrast with the strong humoral response triggered by injection of bacteria or fungal spores, we have identified only one molecule induced in the hemolymph of virus infected flies. This molecule, pherokine‐2 (Phk‐2), is related to OS‐D/A10 (Phk‐1), which was previously characterized as a putative odor/pheromone binding protein specifically expressed in antennae. The virus‐induced molecule is also similar to the product of the gene CG9358 (Phk‐3), which is induced by septic injury. Both Phk‐2 and Phk‐3 are strongly expressed during metamorphosis, suggesting that they may participate in tissue‐remodeling.
Purification and properties of a new S‐adenosyl‐l‐ methionine:flavonoid 4′‐O‐methyltransferase from carnation (Dianthus caryophyllus L.)
Authors: Curir Paolo , Lanzotti Virginia , Dolci Marcello , Dolci Paola , Pasini Carlo , Tollin Gordon
Published: August 2003

A new enzyme, S‐adenosyl‐l‐methionine:flavonoid 4′‐O‐methyltransferase (EC 2.1.1.‐) (F 4′‐OMT), has been purified 1 399‐fold from the tissues of carnation (Dianthus caryophyllus L). The enzyme, with a molecular mass of 43–45 kDa and a pI of 4.15, specifically methylates the hydroxy substituent in 4′‐position of the flavones, flavanones and isoflavones in the presence of S‐adenosyl‐l‐methionine. A high affinity for the flavone kaempferol was observed (Km = 1.7 µm; Vmax = 95.2 µmol·min−1·mg−1), while other 4′‐hydroxylated flavonoids proved likewise to be suitable substrates. Enzyme activity had no apparent Mg++ requirement but was inhibited by SH‐group reagents. The optimum pH value for F 4′‐OMT activity was found to be around neutrality. Kinetic analysis of the enzyme bi‐substrate reaction indicates a Ping‐Pong mechanism and excludes the formation of a ternary complex. The F 4′‐OMT activity was increased, in both in vitro and in vivo carnation tissues, by the inoculation with Fusarium oxysporum f. sp. dianthi. The enzyme did not display activity towards hydroxycinnamic acid derivatives, some of which are involved, as methylated monolignols, in lignin biosynthesis; the role of this enzyme could be therefore mainly defensive, rather than structural, although its precise function still needs to be ascertained.
Subunit sequences of the 4 × 6‐mer hemocyanin from the golden orb‐web spider, Nephila inaurata
Authors: Averdam Anne , Markl Jürgen , Burmester Thorsten
Published: August 2003

The transport of oxygen in the hemolymph of many arthropod and mollusc species is mediated by large copper‐proteins that are referred to as hemocyanins. Arthropod hemocyanins are composed of hexamers and oligomers of hexamers. Arachnid hemocyanins usually form 4 × 6‐mers consisting of seven distinct subunit types (termed a–g), although in some spider taxa deviations from this standard scheme have been observed. Applying immunological and electrophoretic methods, six distinct hemocyanin subunits were identified in the red‐legged golden orb‐web spider Nephila inaurata madagascariensis (Araneae: Tetragnathidae). The complete cDNA sequences of six subunits were obtained that corresponded to a‐, b‐, d‐, e‐, f‐ and g‐type subunits. No evidence for a c‐type subunit was found in this species. The inclusion of the N. inaurata hemocyanins in a multiple alignment of the arthropod hemocyanins and the application of the Bayesian method of phylogenetic inference allow, for the first time, a solid reconstruction of the intramolecular evolution of the chelicerate hemocyanin subunits. The branch leading to subunit a diverged first, followed by the common branch of the dimer‐forming b and c subunits, while subunits d and f, as well as subunits e and g form common branches. Assuming a clock‐like evolution of the chelicerate hemocyanins, a timescale for the evolution of the Chelicerata was obtained that agrees with the fossil record.
Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes
Authors: Kukulski Filip , Komoszyński Michal
Published: August 2003

We purified to homogeneity and characterized NTPDase1 and NTPDase2 from porcine brain cortex synaptosomes. SDS/PAGE and immunoblotting with antibodies specific to these enzymes revealed a molecular mass estimated at 72 kDa for NTPDase1 and 66 for NTPDase2. Both enzymes exhibited kinetic properties typical for all members of the NTPDase family, e.g. low substrate specificity for tri‐ and diphosphonucleosides, divalent cations dependency and insensitivity towards ATPase inhibitors. The calculated Km value for NTPDase1 in respect to ATP as a substrate (97 μm) was three times lower in comparison to analogous values for NTPDase2 (270 μm). Additionally, NTPDase1 had a three times higher Kcat/Km coefficient than NTPDase2 (860 and 833 μmol product·s−1, respectively). We have also demonstrated that in spite of differences in the affinity of ATP for both hydrolases, these enzymes have similar molecular activity. Taken together, these results indicate that NTPDase1 would terminate P2 receptor‐mediated signal transmission whereas activity of NTPDase2 may contribute to decreasing high (toxic) concentrations of ATP and/or to production of another signal molecule, ADP.
Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow‐spotted longicorn beetle, Psacothea hilaris
Authors: Sugimura Masahiro , Watanabe Hirofumi , Lo Nathan , Saito Hitoshi
Published: August 2003

A cellulase (endo‐β‐1,4‐glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow‐spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello‐oligosaccharide (average degree of polymerization 34) and soluble cello‐oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min−1·(mg protein)−1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello‐oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min−1·(mg protein)−1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N‐terminal amino‐acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975‐bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino‐acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.
Structural characterization of the lipopolysaccharide O‐polysaccharide antigen produced by Flavobacterium columnare ATCC 43622
Authors: MacLean Leann L. , Perry Malcolm B. , Crump Elizabeth M. , Kay William W.
Published: August 2003

The structure of the antigenic O‐chain polysaccharide of Flavobacterium columnare ATCC 43622, a Gram‐negative bacterium that causes columnaris disease in warm water fish, was determined by high‐field 1D and 2D NMR techniques, MS, and chemical analyses. The O‐chain was shown to be an unbranched linear polymer of a trisaccharide repeating unit composed of 2‐acetamido‐2‐deoxy‐d‐glucuronic acid (d‐GlcNAcA), 2‐acetamidino‐2,6‐dideoxy‐l‐galactose (l‐FucNAm) and 2‐acetamido‐2,6‐dideoxy‐d‐xylo‐hexos‐4‐ulose (d‐Sug) (1 : 1 : 1), having the structure:

image
The Survival of Sympathetic Neurons Promoted by Potassium Depolarization, but Not by Cyclic AMP, Requires Phosphatidylinositol 3‐Kinase and Akt
Authors: Crowder Robert J. , Freeman Robert S.
Published: August 2003

Abstract : Phosphatidylinositol (PI) 3‐kinase and Akt protein kinase mediate trophic factor‐dependent survival in certain neurons. However, a role for these enzymes in neuronal survival promoted by other agents is unclear. We have tested PI 3‐kinase and Akt for their role in survival promoted by membrane‐depolarizing concentrations of extracellular potassium and the cell‐permeable cyclic AMP analogue 8‐(4‐chlorophenylthio)cyclic AMP (cpt‐cAMP). Depolarization of sympathetic neurons resulted in an increase in the activities of both PI 3‐kinase and Akt. In addition, the PI 3‐kinase inhibitor LY294002 was a potent inducer of cell death in depolarized neurons. Stimulation with cpt‐cAMP resulted in relatively small increases in PI 3‐kinase and Akt activities, and neurons maintained with cpt‐cAMP were more resistant to LY294002‐induced death than were depolarized neurons. Expression of either dominant‐negative PI 3‐kinase or dominant‐negative Akt blocked survival promoted by depolarization but not by cpt‐cAMP. These results indicate that a PI 3‐kinase/Akt pathway is required for survival of sympathetic neurons mediated by depolarization but not by cpt‐cAMP. Thus, the survival of sympathetic neurons can be maintained through PI 3‐kinase/Akt‐dependent and‐independent pathways.
Potential Use of Uniola paniculata Rhizome Fragments for Dune Restoration
Authors: Miller Deborah L. , Yager Lisa , Thetford Mack , Schneider Mica
Published: August 2003

Abstract Uniola paniculata (sea oats) rhizomes uprooted by hurricanes and deposited as wrack could be salvaged and replanted in dune restoration. To test this unexplored technique, percent tiller emergence was observed for 4 years from U. paniculata rhizomes replanted after submersion in seawater; air exposure of 0, 1, 3, 5, 7, and 11 days; freshwater rinse; and reburial in pots (watered) or on the beach (with or without supplemental water). In addition, U. paniculata rhizomes uprooted by Hurricane Georges were experimentally planted, and effects of soil salinity and moisture on emergence were tested in the greenhouse. Tiller emergence declined with increasing length of air exposure and decreasing size of rhizome. Tiller survival was enhanced by rainfall, rinsing with salt or fresh water during exposure and immediately after planting or supplemental beach watering. Although emergence was not reduced by soil salinity of 1,800 μS/cm, emergence was reduced by soil salinity of 5,800 μS/cm. Across the 4 years of the study tiller emergence from treated rhizomes varied considerably. U. paniculata rhizomes lost bud viability after 3–5 days of beach exposure, unless fresh water from rainfall, wet burlap storage, or watering was applied within 3 days. Bud viability was extended through 11 days when supplied with water. Thus, reburial within 3–11 days after a storm is a viable restoration technique.
The Restoration Gene Pool Concept: Beyond the Native Versus Non‐Native Debate
Authors: Jones T. A.
Published: August 2003

AbstractRestoration practitioners have long been faced with a dichotomous choice of native versus introduced plant material confounded by a general lack of consensus concerning what constitutes being native. The “restoration gene pool” concept assigns plant materials to one of four restoration gene pools (primary to quaternary) in order of declining genetic correspondence to the target population. Adaptation is decoupled from genetic identity because they often do not correspond, particularly if ecosystem function of the disturbed site has been altered. Because use of plant material with highest genetic identity, that is, the primary restoration gene pool, may not be ultimately successful, material of higher order pools may be substituted. This decision can be made individually for each plant species in the restored plant community in the scientific context that ecosystem management demands. The restoration gene pool concept provides a place for cultivars of native species and noninvasive introduced plant material when use of native‐site material is not feasible. The use of metapopulation polycrosses or composites and multiple‐origin polycrosses or composites is encouraged as appropriate. The restoration gene pool concept can be implemented as a hierarchical decision‐support tool within the larger context of planning seedings.
Restoring Forbs for Sage Grouse Habitat: Fire, Microsites, and Establishment Methods
Authors: Wirth Troy A. , Pyke David A.
Published: August 2003

Abstract The decline and range reduction of sage grouse populations are primarily due to permanent loss and degradation of sagebrush–grassland habitat. Several studies have shown that sage grouse productivity may be limited by the availability of certain preferred highly nutritious forb species that have also declined within sagebrush ecosystems of the Intermountain West, U.S.A. The purpose of this study was to determine the suitability of three species of forbs for revegetation projects where improving sage grouse habitat is a goal. Species suitability was determined by evaluating the emergence, survival, and reproduction of Crepis modocensis, C. occidentalis, and Astragalus purshii in response to method of establishment (seeding or transplanting), site preparation treatment (burned or unburned), and microsite (mound or interspace) in an Artemisia tridentata ssp. wyomingensis vegetation association in south central Oregon. For seeded plants A. purshii had the lowest emergence (8%) of all three species. Both seeded Crepis species had similar overall emergence (38%). Significantly more Crepis seedlings emerged from shrub mounds in unburned areas (50%) than in any other fire‐by‐microsite treatment (33 to 36%). Approximately 10% more Crepis seedlings survived in mounds compared with interspaces. Nearly twice as many emerging Crepis seedlings survived in the burned areas as opposed to unburned areas (p < 0.01). This resulted in more plant establishment in burned mounds despite higher emergence in unburned mounds. Astragalus purshii seedlings also survived better in burned areas (p = 0.06) but had no differential response to microsite. Fire enhanced survival of both Crepis and A. purshii transplants (p = 0.08 and p = 0.001). We believe additional research is needed to improve A. purshii emergence before it will become an effective plant for restoring sage grouse habitat. Conversely, we conclude that these Crepis species provide a viable revegetation option for improving sage grouse habitat in south central Oregon.
Burning and Grazing Management in a California Grassland: Growth, Mortality, and Recruitment of Nassella pulchra
Authors: Dyer Andrew R.
Published: August 2003

Abstract Annual grasslands in California are often managed with seasonal grazing and prescribed burning on the assumption that such practices have long‐term benefits for native species. Mature native perennial bunchgrasses, particularly Nassella pulchra (purple needlegrass), are often the focal species, although very little is known about responses at different life history stages. Thus, important questions remain about long‐term population dynamics of both mature plants and seedling recruitment. In plots receiving repeated grazing and burning events over 7 years, mortality of mature plants was threefold higher on mounds than on intermounds and likely reflected increased competition intensity associated with increased resource availability in deeper soil. Burning and grazing treatments had strong positive effects on basal area of mature N. pulchra. However, plants in grazed plots that were not burned contained considerable standing dead biomass. Topographic location strongly influenced growth as intermound plants grew relatively more than mound plants, but the effects on growth of burning and grazing did not vary with topographic location. In mapped plots N. pulchra recruitment was very low, and overall density dropped an average of 31%. However, a significant time‐by‐burning effect indicated that survival was significantly higher in burned plots. After 7 years of repeated treatments, effects of burning and grazing management on mature N. pulchra were positive but not for all phenological stages. Understanding long‐term influence of management on bunchgrass populations may not be easy to determine because short‐term results may not reflect long‐term responses and some life cycle dynamics may be observed only over very long periods.
A Test of Two Annual Cover Crops for Controlling Phalaris arundinacea Invasion in Restored Sedge Meadow Wetlands
Authors: Perry Laura G. , Galatowitsch Susan M.
Published: August 2003

Abstract Rapid establishment by aggressive plants such as Phalaris arundinacea (reed canarygrass) often interferes with sedge meadow establishment in restored prairie pothole wetlands in the mid‐continental United States. Introducing a cover crop during community establishment might suppress P. arundinacea invasion in restored prairie potholes by reducing resource availability. We evaluated two potential cover crops, Echinochloa crusgalli (barnyardgrass) and Polygonum lapathifolium (nodding smartweed), for suppressing P. arundinacea invasion in an experimental wetland using replacement series competition experiments. Further, we assessed the effects of E. crusgalli and P. lapathifolium on sedge meadow establishment by sowing Carex hystericina, a common wetland sedge, as a third species at a constant density in the replacement experiments. Echinochloa crusgalli, compared with no cover crop, reduced P. arundinacea biomass by more than 1,000 g/m2 (65%) after two growing seasons. Polygonum lapathifolium did not affect P. arundinacea biomass. Dense E. crusgalli canopies in the first year and thick E. crusgalli thatch in the second year substantially reduced light availability for P. arundinacea establishment. Echinochloa crusgalli also reduced C. hystericina biomass by more than 1,800 g/m2 (99%) after two growing seasons. Carex hystericina biomass was similar in plots sown with E. crusgalli to P. arundinacea monocultures. Neither E. crusgalli nor P. lapathifolium is likely to improve sedge meadow restoration success. These trends were not sensitive to initial sowing density or elevation above water level. Without methods to suppress P. arundinacea invasions, sedge meadow restorations may often fail. Thorough site preparation to remove P. arundinacea propagule sources before restoration is essential.
Propagation of a Long‐Lived and Threatened Prairie Plant, Lupinus sulphureus ssp. kincaidii
Authors: Severns Paul M.
Published: August 2003

Abstract Willamette Valley upland prairie in western Oregon, U.S.A. has been severely degraded and fragmented in the past 150 years after European settlement, resulting in vast population reductions of endemic species. Icaricia icarioides fenderi (Fender's blue butterfly) and Lupinus sulphureus ssp. kincaidii (Kincaid's lupine) are federally listed as Endangered and Threatened Species, respectively. Both are Willamette Valley upland prairie endemics, and Kincaid's lupine is the primary host plant for the Fender's blue butterfly. Attempts to grow Kincaid's lupine have been partially successful in a greenhouse situation; however, propagating plants from field‐sown seed can be tenuous and plant establishment is unpredictable. Kincaid's lupine seeds were planted in the fall 1997 at two different upland prairie sites, and the cohort was followed through the summer 2000. Based on cohort tables the most vulnerable life stages to mortality are the germinant stage and the first growing year. Mechanical scarification of Kincaid's lupine seeds yielded no significant differences in survivorship, maternal function, plant size, and the percentage of seeds germinated compared with unscarified seeds. Differential seed source performance detected at one planting site suggests that underlying differences in population genetics may affect Kincaid's lupine vigor, fitness, and establishment. Future restoration projects for Kincaid's lupine should focus on upland prairie sites with naturally occurring lupine populations because local ecological conditions are favorable for lupine establishment. Moreover, the addition of new individuals to small Kincaid's lupine colonies will buffer against the effects of inbreeding depression and increase the site carrying capacity for Fender's blue butterfly.