Antinociceptive Efficacy of the µ-Opioid/Nociceptin Peptide-Based Hybrid KGNOP1 in Inflammatory Pain without Rewarding Effects in Mice: An Experimental Assessment and Molecular Docking.

Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund's adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.

The C-terminal fibrinogen-like domain of angiopoietin-like 4 stimulates adipose tissue lipolysis and promotes energy expenditure.

Angptl4 (Angiopoietin-like 4) is a circulating protein secreted by white and brown adipose tissues and the liver. Structurally, Angptl4 contains an N-terminal coiled-coil domain (CCD) connected to a C-terminal fibrinogen-like domain (FLD) via a cleavable linker, and both full-length Angptl4 and its individual domains circulate in the bloodstream. Angptl4 inhibits extracellular lipoprotein lipase (LPL) activity and stimulates the lipolysis of triacylglycerol stored by adipocytes in the white adipose tissue (WAT). The former activity is furnished by the CCD, but the Angptl4 domain responsible for stimulating adipocyte lipolysis is unknown. We show here that the purified FLD of Angptl4 is sufficient to stimulate lipolysis in mouse primary adipocytes and that increasing circulating FLD levels in mice through adenovirus-mediated overexpression (Ad-FLD) not only induces WAT lipolysis in vivo but also reduces diet-induced obesity without affecting LPL activity. Intriguingly, reduced adiposity in Ad-FLD mice was associated with increased oxygen consumption, fat utilization, and the expression of thermogenic genes (Ucp1 and Ppargc1a) in subcutaneous WAT. Moreover, Ad-FLD mice exhibited increased glucose tolerance. Chronically enhancing WAT lipolysis could produce ectopic steatosis because of an overflow of lipids from the WAT to peripheral tissues; however, this did not occur when Ad-FLD mice were fed a high-fat diet. Rather, these mice had reductions in both circulating triacylglycerol levels and the mRNA levels of lipogenic genes in the liver and skeletal muscle. We conclude that separating the FLD from the CCD-mediated LPL-inhibitory activity of full-length Angptl4 reveals lipolytic and thermogenic properties with therapeutic relevance to obesity and diabetes.

Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry.

Posttranslational acetylation of lysine residues has been discovered as multifaceted regulatory modification for various nuclear, cytoplasmic, and mitochondrial proteins. The implementation of high-resolution and high-throughput mass spectrometry (MS) approaches has led to the identification of a hitherto underappreciated, large number of acetylation sites for a broad spectrum of cellular proteins. In this chapter, we describe a comprehensive protocol for the purification of an in vivo-acetylated, ectopically expressed, FLAG-epitope tagged nonhistone protein through immunoprecipitation (IP). The protocol also covers the sample preparation by SDS-PAGE, proteolytic digestion, and the analysis by LC-ESI MS. The success of this methodology, however, strongly depends on the physico-chemical properties of the respective protein(s) and the quality of selected peptide mass spectra.

Calcium feedback to cGMP synthesis strongly attenuates single-photon responses driven by long rhodopsin lifetimes.

Rod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R∗), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, when crossed into a line lacking calcium feedback regulation of cGMP synthesis, produced much larger alterations in SPR amplitudes. Analysis of SPRs from rods with and without feedback reveal that the consequences of trial-to-trial fluctuations in R∗ lifetime in normal rods are also dampened by feedback regulation of cGMP synthesis. Thus, calcium feedback trumps the mechanisms of R∗ deactivation in determining the SPR amplitude, attenuating responses arising from longer R∗ lifetimes to a greater extent than those arising from shorter ones. As a result, rod SPRs achieve a more stereotyped amplitude, a characteristic considered important for reliable transmission through the visual system.

Overexpression, purification and assessment of cyclosporin binding of a family of cyclophilins and cyclophilin-like proteins of the human malarial parasite Plasmodium falciparum.

Malaria represents a global health, economic and social burden of enormous magnitude. Chemotherapy is at the moment a largely effective weapon against the disease, but the appearance of drug-resistant parasites is reducing the effectiveness of most drugs. Finding new drug-target candidates is one approach to the development of new drugs. The family of cyclophilins may represent a group of potential targets. They are involved in protein folding and regulation due to their peptidyl-prolyl cis-trans isomerase and/or chaperone activities. They also mediate the action of the immunosuppressive drug cyclosporin A, which additionally has strong antimalarial activity. In the genome database of the most lethal human malarial parasite Plasmodium falciparum, 11 genes apparently encoding cyclophilin or cyclophilin-like proteins were found, but most of these have not yet been characterized. Previously a pET vector conferring a C-terminal His6 tag was used for recombinant expression and purification of one member of the P. falciparum cyclophilin family in Escherichia coli. The approach here was to use an identical method to produce all of the other members of this family and thereby allow the most consistent functional comparisons. We were successful in generating all but three of the family, plus a single amino-acid mutant, in the same recombinant form as either full-length proteins or isolated cyclophilin-like domains. The recombinant proteins were assessed by thermal melt assay for correct folding and cyclosporin A binding.

Identification of novel L-amino acid alpha-ligases through Hidden Markov Model-based profile analysis.

L-Amino acid alpha-ligase (Lal), catalyzing the formation of alpha-dipeptides from unprotected L-amino acids in an ATP-dependent manner, is used in cost-effective fermentative production of dipeptides. We searched for novel Lals by in silico screening using Hidden Markov Model-based profile analysis, and identified five novel Lals that showed low similarity and different substrate specificity from known Lals.

Molecular dynamics of some pentapeptides having a strong tendency to helical conformation.

Some pentapeptides with higher alpha-helical tendency possess typical sequence pattern, such as "+ - + + - - +", "+ - - + - - +", "+ - + - - - +", and " - + + - - - +" ( "+" = D,N,E,Q,K,R,T,C, or H; "-" = L,I,V, or A), especially symmetrical motifs (a pair of reverse sequences beside palindromic segments), such as ALALA, QQAQE/EQAQQ, and REALE/ELAER, hint that the nascent peptide can fold a certain conformation in a two-way folding fashion.

Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads.

Bacteria that survive under variable conditions possess an assortment of genetic regulators to meet these challenges. The group IV or extracytoplasmic function (ECF) sigma factors regulate gene expression in response to specific environmental signals by altering the promoter specificity of RNA polymerase. We have undertaken a study of PvdS, a group IV sigma factor encoded by Pseudomonas syringae pv. tomato DC3000 (DC3000), a plant pathogen that is likely to encounter variations in nutrient availability as well as plant host defences. The gene encoding PvdS was previously identified by sequence similarity to the Pseudomonas aeruginosa orthologue, which directs transcription of genes encoding the biosynthesis of pyoverdine, a siderophore involved in iron acquisition, and is responsible for the characteristic fluorescence of the pseudomonads. We identified 15 promoters regulated by PvdS in DC3000 and characterized the promoter motif using computational analysis. Mutagenesis of conserved nucleotides within the motif interfered with promoter function and the degree of the effect was different depending on which region of the motif was mutated. Hidden Markov models constructed from alignments of sequence motifs extracted from DC3000 and PAO1 were used to query genomes of DC3000 and other fluorescent pseudomonads for similar motifs. We conclude that the role of PvdS as a regulator of pyoverdine synthesis is conserved among the fluorescent pseudomonads, but the promoters recognized by PvdS orthologues may differ subtly from species to species.

Therapeutic and diagnostic applications of minor histocompatibility antigen HA-1 and HA-2 disparities in allogeneic hematopoietic stem cell transplantation: a survey of different populations.

Minor histocompatibility antigens (mHags) HA-1 and HA-2 are encoded by biallelic loci, with immunogenic variants, HA-1H and HA-2V, which induce strong HLA-A2-restricted alloreactive T-cell responses, and nonimmunogenic counterparts, HA-1R and HA-2M, which represent functional null alleles that are poorly presented by HLA class I molecules. HA-1 and HA-2 are potential targets of selective graft-versus-leukemia and graft-versus-tumor reactivity after allogeneic hematopoietic stem cell transplantation (HSCT); however, these applications are restricted to a limited number of patients. Here, we show that a far more frequent application of HA-1 and HA-2 disparity relies on their use as markers for the state of host chimerism after allogeneic HSCT. We have determined allelic frequencies of 29.3% and 70.7% for HA-1H and HA-1R, respectively, and of 83.7% and 16.3% for HA-2V and HA-2M, respectively, in >200 healthy individuals from northern Italy. Similar frequencies were observed in nearly 100 patients affected by hematologic malignancies or solid tumors, thus showing that HA-1 and HA-2 variability are not associated with the presence of cancer. On the basis of these data, we predict that HA-1 and HA-2 can be used in 32.8% and 23.5% of Italian transplant patients, respectively, as markers for the state of host chimerism, whereas exploitation of disparity for these mHags for targeted immunotherapy will be possible in 10.7% and 1.1% of Italian patients, respectively. Retrospective HA-2 typing of bone marrow aspirates obtained from a patient during complete remission or recurrence of acute myeloid leukemia after haploidentical HSCT showed the feasibility of using HA-2 as a surrogate marker for disease monitoring. Because of an apparent north-south gradient for HA-1 allelic frequencies, with higher frequencies for the HA-1H variant reported in white populations from Southern Europe as compared with Northern Europe and North America, the diagnostic applicability of HA-1 disparity will be slightly more frequent in transplant patients from the north. Taken together, our data show that determination of HA-1 and HA-2 variability can be an important parameter for the selection of allogeneic stem cell donors, in particular for patients affected by hematologic malignancies without a tumor-specific molecular marker.

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants.

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.

Visualization and tracking of single protein molecules in the cell nucleus.

A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protein molecules. Using the recombinant Escherichia coli beta-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of approximately 30 nm and a time resolution of 18 ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined to very small areas. In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion. However, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients. Importantly, both the diffusion coefficients and the fractions of these subpopulations were time-dependent. Our results suggest that proteins can move inside the nucleus over extended distances by diffusion. However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.

[Public health economic evaluation of screening for APC resistance (Leiden mutation) in new oral contraceptive users]. / Gesundheitsökonomische Evaluation des Screenings auf APC-Resistenz (Mutation Leiden) bei Neuanwenderinnen von Ovulationshemmern

BACKGROUND: Thromboembolic events in users of oral contraceptives rank among the most important complications with potential economic consequences. It is well known that a large part of thromboembolic complications correlates with hereditary thrombophilias. The relative high prevalence of the newly described resistance of activated protein C and an easy test for it rise up the question if general screening of new users of oral contraceptives is sensible of health economic view. METHOD: We conducted a cost-effectiveness analysis using decision analytic techniques, analysing the costs and outcomes in a hypothetical cohort of 10,000 women. RESULT AND CONCLUSION: From a third party payer perspective we estimate that screening for APC resistance by new users of oral contraceptives is more cost-effective than many other primary preventive methods. The cost per life-year gained of testing for APC resistance were in the order of DM 1544,--including direct medical cost only. From perspective of sickness insurance fund as cost unit we estimate that screening for APC resistance by new users of oral contraceptives is more cost-effective than many other primary preventive methods.

Functional analysis and modeling of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)

Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage.

An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors. This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd. A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed. The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1. Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described. Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames. Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.