Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

Identification of transmembrane domains that regulate spatial arrangements and activity of prokineticin receptor 2 dimers.

The chemokine prokineticin 2 (PK2) activates its cognate G protein-coupled receptor (GPCR) PKR2 to elicit various downstream signaling pathways involved in diverse biological processes. Many GPCRs undergo dimerization that can modulate a number of functions including membrane delivery and signal transduction. The aim of this study was to elucidate the interface of PKR2 protomers within dimers by analyzing the ability of PKR2 transmembrane (TM) deletion mutants to associate with wild type (WT) PKR2 in yeast using co-immunoprecipitation and mammalian cells using bioluminescence resonance energy transfer. Deletion of TMs 5-7 resulted in a lack of detectable association with WT PKR2, but could associate with a truncated mutant lacking TMs 6-7 (TM1-5). Interestingly, TM1-5 modulated the distance, or organization, between protomers and positively regulated Gαs signaling and surface expression of WT PKR2. We propose that PKR2 protomers form type II dimers involving TMs 4 and 5, with a role for TM5 in modulation of PKR2 function.

Pharmacological activity of a Bv8 analogue modified in position 24.

BACKGROUND AND PURPOSE: The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK(1) and PK(2) , in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation. EXPERIMENTAL APPROACH: We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK(1) and PK(2) receptors in cell preparations and in affecting nociceptive thresholds in rodents. KEY RESULTS: A-24 preferentially bound to PK(2) receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased ß-endorphin levels in mouse brain. CONCLUSIONS AND IMPLICATIONS: A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK(1) receptors and by activating the central opioid system and the descending pain control pathway through brain PK(2) receptors.

Alpha-defensin increase in peripheral blood mononuclear cells from patients with hepatitis C virus chronic infection.

The alpha-defensin genes promoter regions contain a putative nuclear factors of activated T cells (NFAT)-binding site and it is known that hepatitis C virus (HCV) core protein activates the interleukin (IL)-2 gene transcription through the NFAT pathway. The aims of this study were to investigate if HCV affects the alpha-defensin expression in peripheral human mononuclear cells (PBMCs) and to evaluate the existence of a correlation between alpha-defensins and liver damage in patients with chronic hepatitis C. Ninety patients with chronic hepatitis C, 30 with chronic hepatitis B and 25 healthy controls were enrolled. Alpha-defensins were identified and quantified in PBMCs by mass spectrometry, enzyme-linked immunosorbent assay, antibacterial activity and mRNA levels. PBMCs from three patients and controls were stimulated with HCV core protein, hepatitis B virus core antigen and the alpha-defensin mRNAs level was quantified. We found that HCV core protein activates in vitro the alpha-defensin transcription. Alpha-defensin levels in patients with chronic hepatitis C (mean +/- SD = 1.103 +/- 0.765 ng/10(6) cells), chronic hepatitis B (0.53 +/- 0.15) and healthy controls (0.217 +/- 0.09) resulted significantly different (P < 0.001). In patients with chronic hepatitis C, the alpha-defensin levels and antibacterial activity correlate with the liver fibrosis. Our data suggest that HCV induces alpha-defensin expression. The high linear correlation of alpha-defensin levels with advancing fibrosis makes the measure of these peptides a reliable marker to evaluate fibrosis stage.

Short-term comparative outcomes associated with the use of GP IIb/IIIa antagonists in patients undergoing coronary intervention.

BACKGROUND: Platelet glycoprotein (GP) IIb/IIIa antagonists reduce the occurrence of death, myocardial infarction (MI) and urgent revascularization among patients undergoing percutaneous coronary intervention (PCI). Despite a similar mechanism of platelet inhibition, the three currently approved agents vary widely in cost. PURPOSE: The purpose of this prospectively designed, retrospective analysis was to determine clinical outcomes for patients receiving abciximab, tirofiban or eptifibatide as adjunctive therapy during PCI at a single center. We hypothesized that there would be no difference in outcomes during hospitalization following PCI in patients receiving tirofiban or eptifibatide compared with those patients who received abciximab. Outcomes examined included in-hospital mortality, hemorrhagic procedural complications, need for recatheterization, peak creatine kinase following intervention and length of hospital stay (LOS). RESULTS: Two hundred and sixty seven consecutive patients in whom GP IIb/IIIa antagonist therapy was initiated in the catheterization laboratory for PCI were analyzed. Abciximab-treated patients were more likely to be undergoing primary (p<0.001) and rescue (p=0.022) PCI and to have received fibrinolytic therapy (p=0.013) when compared to patients receiving tirofiban or eptifibatide. There were no significant differences between abciximab- and non abciximab-treated patients in either the primary PCI or non primary PCI groups in any of the studied endpoints. In patients undergoing primary PCI, abciximab-treated patients when compared with non abciximab-treated patients exhibited a trend toward an increase in hospital LOS (7.8+/-7.0 d vs 6.2+/-3.9, p=0.19) and in the frequency of hemorrhagic complications (22.1% vs 5.3%, p=0.11). In patients not receiving fibrinolytic therapy, abciximab-treated patients experienced a trend toward increased hemorrhagic complications following PCI when compared to non abciximab-treated patients (10.2% vs 6.0%, p=0.28). Complications distant from the vascular access site comprised 62.5% of hemorrhagic complications in the abciximab-treated group, but only 20% of the complications in the non-abciximab treated population (p<0.001). These data suggest no differences in acute outcomes between groups of patients receiving abciximab or other approved GP IIb/IIIa antagonists highlighting a potential significant cost saving. These data will require interpretation following the publication of comparative trials.

Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system.

High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.

Utilization management and information technology: adapting to the new era.

Overuse of clinical laboratory services has been written about for many years (1), but remedies that easily could be implemented and effective over the long term have been in short supply. This issue has been acute for CLS, Inc., which is wholly owned by a managed care organization (HIP Health Plan of New York). Because CLS is assessing the possibility of acquiring a new laboratory information system, we reviewed past reports on approaches to utilization management (UM) and considered how developments in information technology (IT) may affect the latter. We feel there is a distinct possibility for implementation of UM in real time, and we propose this as a new paradigm whose realization has implications for choice of IT and for how clinical laboratories operate in the future.

Involvement of Rel factors in the expression of antimicrobial peptide genes in amphibia.

Genes coding for antimicrobial peptides in amphibia reveal a remarkably high number of structural motifs for response elements, previously identified in the genes of insect antimicrobial peptides and in those of the mammalian acute phase response. This study focuses on the functional analysis of the bombinin gene promoter in a Drosophila blood cell line, and the identification of kappaB-binding factors in skin secretions of the frog Bombina orientalis. Transfection experiments demonstrated that the bombinin gene promoter was activated in a lipopolysaccharide-dependent manner, and that insect Rel factors target specific sequences in the amphibian gene promoter. After bathing frogs in bacteria, their skin secretions contained kappaB-specific binding complexes, indicating that Rel factors are crucial components in the response against gram-negative bacteria in this species. These results suggest that a common ancestral control mechanism governs the expression of the first line host-defence from insects to vertebrates.

Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7.

The structure of a gene coding for bombinin-like peptides (BLP) in Bombina orientalis was determined. It comprises two exons separated by a 1337 bp intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for BLP-7 and a bombinin H-type peptide (GH-2). The promoter region contains putative recognition sites for nuclear factors, such as NF-IL6 and NF-kappaB. The analysis of the structure of this gene, compared with that of the previously reported BLP-3 gene sequence, suggests the occurrence of a gene duplication event, rather than an alternative splicing mechanism, which leads to the generation of both inter- and intra-families variability in this class of cytolytic peptides. Furthermore, chromosome walking analysis indicates that this gene family is not densely clustered.

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis.

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.

Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity.

The properties of recombinant staphylokinase (SakSTAR) expressed in Pichia pastoris cells have been determined. The single consensus N-linked oligosaccharide linkage site in SakSTAR (at Asn28 of the mature protein) was occupied in approximately 50% of the expressed protein with high-mannose-type oligosaccharides. The majority of these glycans ranged in polymerization state from Man8GlcNAc2 to Man14GlcNAc2, with the predominant species being Man10GlcNAc2 and Man11GlcNAc2. Glycosylated SakSTAR (SakSTARg) did not differ from its aglycosyl form in its aggregation state in solution, its thermal denaturation properties, its ability to form a complex with human plasmin (hPm), the amidolytic properties of the respective SakSTAR-hPm complexes, or its ability to liberate the amino-terminal decapeptide required for formation of a functional SakSTAR-hPm plasminogen activator complex. However, this latter complex with SakSTARg showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same complex with the aglycosyl form of SakSTAR. We conclude that glycosylation at Asn28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but it is detrimental to the ability of the SakSTAR-hPm complex to serve as a hPg activator. This is likely due to restricted access of hPg to the active site of the SakSTARg-hPm complex.

O-Mannosylation of Pichia pastoris cellular and recombinant proteins.

O-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan-protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (alpha1-2)-linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1-4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides. Only a very small quantity of N-linked oligosaccharides was present on the Asn289 consensus site. The major products were O-linked (alpha1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O-linked saccharides of mannose containing (alpha1-2) glycosidic linkages are present in P. pastoris cells and expressed proteins.

Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-kappaB and NF-IL6 binding sites in its promoter.

The sequence of a gene from Bombina orientalis was determined which codes for antibacterial peptides. The gene comprises two exons separated by a large intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for two identical bombinins and one bombinin H. The promoter region of the bombinin gene contains putative recognition sites for nuclear factors, such as NFkappaB and NF-IL6. In vivo experiments on B. orientalis have shown that a short contact with bacteria is sufficient to induce a marked increase in the amount of antibacterial peptides in the skin secretion of frogs. This increase was suppressed by pretreatment with glucocorticoids. In the latter case, a significant increase of I kappaB alpha in the secretion is also detectable.

The role of reflexive (algorithmic) testing in laboratory medicine: adapting to the new era.

Centralized Laboratory Services, Inc. (CLS) is a large, freestanding laboratory that is an affiliate of the Health Insurance Plan of New York, a managed care organization with more than 1 million members in New York and New Jersey. The laboratory work for this membership is consolidated at CLS, which thus serves an ambulatory patient population. The Medical Director at CLS is charged with optimizing laboratory utilization by clinicians who are part of the system.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin.

Gene-encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF-kappaB is an important transcription factor for many immunity-related mammalian proteins and also for insect immune genes. The activity of NF-kappaB is regulated by the interaction with an inhibitor, I kappaB. It was recently demonstrated that glucocorticoids induce the synthesis of I kappaB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF-kappaB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of I kappaB alpha. These results suggest that also in vertebrates peptide-mediated innate immunity is controlled by NF-kappaB-regulated transcription.

Characterization of the acidic oligosaccharides assembled on the Pichia pastoris-expressed recombinant kringle 2 domain of human tissue-type plasminogen activator.

The N-linked glycans assembled in Pichia pastoris on the recombinant kringle 2 domain of human tissue-type plasminogen activator (r-[K2tPA]) are composed of approx. 80% neutral and 20% charged species. After peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase-catalysed liberation of the oligosaccharides from the purified glycopeptide, the glycan mixture was resolved by HPLC on amino-silica-based resin. Oligosaccharide mapping of the resulting mixture by HPLC, gel filtration and time-of-flight matrix-assisted laser-desorption-ionization-with-delayed-extraction mass spectrometry (TOF-MALDI DE-MS) revealed that > 90% of the charged species consisted of a series of oligosaccharides possessing molecular masses that were consistent with a range of saccharides comprising phospho-Man10GlcNAc2-phospho-Man14GlcNAc2, with phospho-Man11GlcNAc2 representing the major species. The remaining material in the charged fraction contained identifiable phosphorylated glycans that were one or two mannose units shorter, and one to four mannose units longer, than those present in the above range of oligosaccharides. Treatment of the native charged glycan pool with alkaline phosphatase did not result in molecular-size alterations, showing that phosphomonoesters are not present. Mild acid hydrolysis of the glycans led to a decrease in the size of all charged glycans by one mannose residue, providing phospho-Man9GlcNAc2-phospho-Man13GlcNAc2. Following this procedure, treatment with alkaline phosphatase resulted in size decreases that were equivalent to the loss of one phosphate group from each glycan. This demonstrates that all charged glycans isolated contained phosphate in phosphodiester bonds to two mannose units. The present study shows that P. pastoris cells possess the capability of assembling phosphorylated glycans having the phosphate moiety present in phosphodiester linkages with two mannose units. These saccharides, like the neutral oligosaccharides, contain considerably smaller amounts of mannose than glycans present in other strains of yeast.

Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator.

The oligosaccharide structures present on Asn5 of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator [(r)-[K2tPA]] have been determined by a combination of techniques, including HPLC, FPLC, gel filtration, endoglycosidase digestions and mass spectrometry. The major oligosaccharides identified after their liberation by either hydrazinolysis or by the enzyme peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase, were in the oligomer range of (mannose)8(N-acetylglucosamine)2 (Man8GN2) to Man18GN2. The preponderance of these glycans spanned Man9GN2 to Man12GN2, and the major overall product was Man10GN2. An additional (less than 5%) amount of the polypeptide was hyperglycosylated. In contrast with glycoproteins produced in Saccharomyces cerevisiae, our results with specific mannosidase digestions were consistent with previous studies showing that (alpha 1,3)-linked mannose residues were not present in extensions of the core Man8GN2 unit. The results show that the N-linked glycosylation pathways in P. pastoris are substantially different from those found in S. cerevisiae, with shorter Man(alpha 1,6) extensions to the core Man8GN2 and the apparent lack of significant Man(alpha 1,3) additions representing the major processing modality of N-linked glycans in P. pastoris.

Temporins, antimicrobial peptides from the European red frog Rana temporaria.

A cDNA library from the skin of Rana temporaria has been screened using a cDNA fragment probe that encodes the signal peptide of the precursor of esculentin from the skin secretion of Rana esculenta. With this approach, the cDNAs encoding the precursors of three peptides were isolated. Subsequently, the peptides predicted from the sequence of the cloned cDNAs as well as several structurally related peptides could be isolated from the skin secretion of R. temporaria. These peptides, which were named temporins, have a length of 10-13 residues and show some sequence similarity to hemolytic peptides isolated from Vespa venom [Argiolas, A. & Pisano, J. J. (1984) J. Biol. Chem. 259, 10106-10111]. Natural and synthetic temporins have antibacterial activity against gram-positive bacteria, but they are not hemolytic. Temporins are the smallest antibacterial peptides hitherto found in nature.