Cyclic peptides

The interaction of WLIP (white line-inducing principle), a member of the viscosin group of Pseudomonas lipopeptides, with tolaasin, a lipopeptide mycotoxin secreted by Pseudomonas tolaasii, enables identification of the mushroom pathogen relying on formation of a lipopeptide coprecipitate between confronted colonies of an indicator strain (designated Pseudomonas 'reactans') and P. tolaasii. The WLIP non-ribosomal lipopeptide synthesis system of the mushroom isolate P. 'reactans' LMG 5329 (Wip) was identified and shown to be most similar to the Pseudomonas fluorescens SBW25 viscosin system (Visc), but remarkably different from the WLIP-generating Wlp system previously identified in the rice rhizosphere isolate Pseudomonas putida RW10S2. The Wlp machinery is composed of modules most similar to those recruited for biosynthesis of the nonviscosin-type lipopeptides putisolvin and entolysin by strains from the P. putida clade. In line with the pronounced synteny between the wip and visc flanking regions, strain LMG 5329 was identified as an authentic P. fluorescens closely related to strain SBW25. In both P. putida and P. fluorescens, WLIP production confers similar phenotypes of microbial antagonism and surface colonization. Genotypes other than wlp or wip were not identified among WLIP producers isolated from mushroom, maize rhizosphere or water.

Bacterial and fungal pathogens involved in lung infection in cystic fibrosis patients utilize a particular family of glycan-binding proteins, characterized by the presentation of six fucose-binding sites on a ring-shaped scaffold. These lectins are attractive targets for anti-infectious compounds that could interfere in the recognition of host tissues by pathogens. The design of a cyclopeptide-based hexavalent structure allowed for the presentation of six fucose residues. The synthetic hexavalent compound displays liable geometry resulting in high-avidity binding by lectins from Aspergillus fumigatus and Burkholderia ambifaria. Replacing the fucose residue with a conformationally constrained fucomimetic does not alter the affinity and provides fine specificity with no binding to other fucose-specific lectins.

Two new bicyclic hexapeptides, RA-XXIII and RA-XXIV, were isolated from the roots of Rubia cordifolia L. (Rubiaceae). Their structures were determined by the analysis of their 2D NMR spectra, chemical methods, and X-ray crystallography. The IC 50 values of RA-XXIII and RA-XXIV against P-388 leukemia cells were 0.16 and 0.48 m mg/ml, respectively.

Objective-Classification of rheumatoid arthritis (RA) is increasingly important as new therapies can halt the disease in its early stages. Antibodies to cyclic citrullinated peptides (anti-CCP) are widely used for RA diagnosis, but are not in the 1987 American College of Rheumatology (ACR) Criteria for RA Classification. We developed and tested the performance characteristics of new criteria for RA classification, incorporating anti-CCP. Methods-We identified all subjects seen in our Arthritis Center with rheumatoid factor (RF) and anti-CCP tested simultaneously between January 1 and June 30, 2004 and reviewed their medical records for the ACR criteria, rheumatologists' diagnoses, RF and anti-CCP. We revised the ACR criteria in two ways: (1) adding anti-CCP, (2) replacing rheumatoid nodules and erosions with anti-CCP (CCP 6 criteria). We compared sensitivity and specificity of all criteria, in all subjects and in subjects with arthritis symptoms ≤ 6 months. Results-Medical records of 292 subjects were analysed: mean age was 54 years, 82% were women, and mean symptom duration was 4.1 years. 17% were RF+ and 14% were anti-CCP+ at initial testing. 78 (27%) had definite RA per treating rheumatologist at latest follow-up. The CCP 6 criteria increased sensitivity for RA classification for all subjects regardless of symptom duration: 74% vs. 51% for ACR criteria with a loss in specificity (81% vs. 91%). Sensitivity was greatly improved in subjects with symptoms ≤ 6 months: 25% vs. 63% for ACR criteria with a decrease in specificity. The CCP 6 criteria improved upon the sensitivity of the ACR criteria, most remarkably for subjects with symptoms ≤ 6 months and could be used for classification of subjects for RA in clinical studies.

Life-threatening complications such as graft versus host disease and infection remain major barriers to the success of allogeneic hemopoietic stem cell transplantation (SCT). While pretransplantation conditioning and posttransplantation immunosuppression are important risk factors for infection, the reasons that similarly immunosuppressed transplant recipients show marked variation in frequency of infection after allogeneic SCT are unclear. Mannose-binding lectin (MBL) deficiency is a risk factor for infection in other situations where immunity is compromised. We investigated associations betweenMBL2 gene polymorphisms and risk of major infection following allogeneic SCT. Ninety-seven related allogeneic donor-recipient pairs were studied. Clinical data including survival, days of fever, graft versus host disease incidence and severity, and infection were collected by case note review. Five single-nucleotide polymorphisms in the MBL2 gene were genotyped using the polymerase chain rea...

The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-) 4 , a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of α V β 3 -positive or negative tumors. We chose the human embryonic kidney cells HEK293(β 3 ) (high levels of α V β 3 ) or HEK293(β 1 ) (α V β 3 -negative but expressing α V and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-) 4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β 3 ) than in the HEK293(β 1 ) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer. In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-) 4 was indeed binding to the α V β 3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-) 4 in this novel pair of HEK293(β 3 ) and HEK293(β 1 ) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β 1 ) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists.

The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-) 4 , a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of α V β 3 -positive or negative tumors. We chose the human embryonic kidney cells HEK293(β 3 ) (high levels of α V β 3 ) or HEK293(β 1 ) (α V β 3 -negative but expressing α V and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-) 4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β 3 ) than in the HEK293(β 1 ) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer. In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-) 4 was indeed binding to the α V β 3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-) 4 in this novel pair of HEK293(β 3 ) and HEK293(β 1 ) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β 1 ) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg 6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser 195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 -S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp 9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg 6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser 195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 -S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp 9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of

There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the peptides' affinity to their targets. Using chimeras of human and murine uPA as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket leads to changed exosite interactions, and vice versa. These findings are of general interest when considering the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention and could pave the way for potential drug candidates for the treatment of cancer.

Introducción: La enfermedad poliquística renal autosómica (ADPKD), es considerada huérfana hereditaria. Con una prevalencia mundial de 2.7/100,000 hab. Presenta graves complicaciones con desenlaces fatales. Hasta hace poco, el tratamiento se limitó al control de las manifestaciones clínicas; con la aparición del tolvaptan, se empezó a manejar la enfermedad. La introducción terapéutica del inhibidor de glucosilceramida sintetasa (venglustat) y el acetato de lanreotida hacen necesario revisar el estado de estas opciones terapéuticas para estos pacientes.
Métodos: Se realizó una revisión narrativa de la literatura en PubMed, Cochrane Library, OVID, EBSCO y Google Scholar.
Resultados: Se obtuvieron 92 artículos, de los cuales 5 fueron incluidos en el análisis cualitativo. El Ensayo CLINICO TEMPO 3:4, demostró que el tolvaptan redujo la tasa de crecimiento anual en el volumen total de riñón (TKV) del 5.5% al 2.8% y la tasa anual de filtración glomerular estimada (eGFR) disminuyó de −3.70 a −2.72 mL/min/1.73-m2. El estudio THE DIPAK-1 evidenció que el acetato de lanreotida reduce el volumen renal total (4.2% frente al control: 5.6%; diferencia: -1.4% por año; sin diferencias significativas para la incidencia de empeoramiento de la función renal. Los ensayos clínicos con venglustat evidenciaron mejoría preliminar en la TKV y la eGFR, acorde a los resultados de los estudios preclínicos, sin embargo, aún se encuentran en estudio.
Conclusiones: La ADPKD, es una patología con pocas opciones terapéuticas y con recientes tratamientos incorporados, actualmente en investigación. Este artículo establece las bases para un futuro diseño de un estudio
cuantitativo.

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a Class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 upregulated HDAC4 protein levels, while levels were decreased by RNA-interference (RNAi)-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4, and which may contribute to tissue or cell-line specific expression of HDAC4.

Background: BI 201335 is a highly potent and specific HCV NS3/4A protease inhibitor. A phase 1 trial in treatmentexperienced HCV GT-1 patients demonstrated a mean viral load (VL) reduction of 5.3 log 10 (IU/mL) for BI 201335 given once daily after 28 days in combination with peginterferon alfa (PegIFN) 2a and ribavirin (RBV). We now describe a phase 1b trial which has assessed safety, short-term efficacy, and pharmacokinetics of BI 201335 in GT-1 patients with compensated liver cirrhosis and non-response to previous PegIFN/RBV, a difficult-to-treat HCV population with a high unmet medical need. Methods: In this open-label, sequential group comparison, HCV GT-1 patients with compensated liver cirrhosis who have never achieved undetectable VL under previous PegIFN/RBV were treated with 240 mg once (QD; n=6) or twice daily (BID; n=7) in combination with PegIFNa2a (180 mcg/week) and RBV (1000/1200mg/d) for 28 days. All patients received a single loading dose of 480mg of BI 201335 as the...

Objective: Endothelin-1 (ET-1) production increases during acute myocardial infarction (MI) and may contribute to the genesis of ventricular tachycardia (VT) and ventricular fibrillation (VF). However, the antiarrhythmic effects of ET-1 receptor blockade, examined shortly after MI, have been debated. In the present study, we examined the effects of such treatment on VT/VF during the first 24 h post-MI. Methods: Thirty-five Wistar rats (223 T 22 g) were randomly allocated to either the ET-1 receptor-A (ET A ) antagonist BQ-123 (0.4 mg/kg, BQ-123 group, n = 17), or normal saline (control group, n = 18) and were subjected to coronary artery ligation. A single-lead electrocardiogram was continuously recorded for 24 h post-MI, using an implanted telemetry system, and episodes of VT/VF were analyzed. Monophasic action potential (MAP) recordings were obtained from the left (LV) and right (RV) ventricular epicardium at baseline, 5 min after treatment and 24 h post-MI. Results: There were 15.94 T 19.35 episodes/h/rat of VT/VF in the control group and 1.66 T 2.22 in the BQ-123 group ( p = 0.010), resulting in a lower ( p = 0.030) arrhythmic mortality in treated animals. The mean episode duration was 7.40 T 7.16 s for the control group and 2.30 T 1.37 s for the BQ-123 group ( p = 0.011). The maximum decrease in VT/VF was observed during the 1st, 5th and 6th hours post-MI. In the control group, LV MAP duration increased 24 h post-MI, displaying an increased beat-to-beat variation, but remained unchanged in the BQ-123 group. Conclusion: Acute ET A blockade reduces the incidence of VT/V F during the first 24-h post-MI in the rat, through a decrease in the dispersion of repolarization.

A model is presented of the melanocortin 1 receptor (MC1R), constructed by use of an unbiased, objective method. The model is created directly from data derived from multiple sequence analysis, a low-resolution EMprojection map of rhodopsin, and the approximate membrane thickness. The model agrees well with available data concerning natural mutations of MC1Rs occurring in different species. A model is also presented of the most rigid ligand for this receptor, the cyclic pentapeptide cHFRWG, shown docked in the receptor model. The receptor-ligand complex model agrees well with available experimental data. The ligand is located between transmembrane region 1 (TM1), TM2, TM3, TM6, and TM7 of the receptor. Multiple interactions occur between ligand and receptor, including interactions with Leu-48 (TM1), Ser-52 (TM1), Glu-55 (TM1), Asn-91 (TM2), Glu-94 (TM2), Thr-95 (TM2) Ile-98 (TM2), Asp-121 (TM3), Thr-124 (TM3), Phe-257 (TM6), Phe-283 (TM7), Asn-290 (TM7), and Asp-294 (TM7) of the receptor.

We describe the thermodynamic characterisation of the self‐sorting process experienced by two homodimers assembled by hydrogen‐bonding interactions through their cyclopeptide scaffolds and decorated with Zn–porphyrin and fullerene units into a heterodimeric assembly that contains one electron‐donor (Zn–porphyrin) and one electron‐acceptor group (fullerene). The fluorescence of the Zn–porphyrin unit is strongly quenched upon heterodimer formation. This phenomenon is demonstrated to be the result of an efficient photoinduced electron‐transfer (PET) process occurring between the Zn–porphyrin and the fullerene units of the heterodimeric system. The recombination lifetime of the charge‐separated state of the heterodimer complex is in the order of 180 ns. In solution, both homo‐ and heterodimers are present as a mixture of three regioisomers: two staggered and one eclipsed. At the concentration used for this study, the high stability constant determined for the heterodimer suggests that t...

We describe the first examples of a series of cyclic pseudopeptide libraries that have been prepared in a systematic approach in order to facilitate both synthesis and subsequent deconvolution attempts. Our synthetic strategy involved the attachment of a trifunctional amino acid (Asp, Asn or Glu) to a polystyrene resin via its side chain, and stepwise chain elongation using either protected amino acids or a pseudodipeptide building block. Head to tail cyclic peptides were formed by removal of the temporary N-and C-terminal protecting groups followed by ring closure by amide formation. Cyclization of the hexa, hepta, and octapseudopeptides on the resin avoided dimer formation, as monitored by mass spectrometry. We utilized a 'psi-scan' approach in which a second fixed position was serially addressed by stepping a dipeptide surrogate, Proψ[CH 2 S]Gly around the rings to generate a group of cyclic pseudopeptide sub-libraries. Oxidation of ψ[CH 2 S] to ψ[CH 2 SO] helped validate the synthesis and also provides a strategy for forming a new set of pseudopeptide libraries (previously described as 'libraries from libraries'). Our results suggest that libraries of cyclic pseudopeptides are an efficient method of preparing and assaying these synthetically more challenging entities as potential drug leads.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR 88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR 88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

A cyclic peptide derived from the active domain of α-fetoprotein (AFP) significantly inhibited the proliferation of MCF7 cells stimulated with the epidermal growth factor (EGF) or estradiol (E 2 ). The action of these three agents on cell growth was independent of the presence of calf serum in the culture medium. Our results demonstrated that the cyclic peptide interfered markedly with the regulation of MAPK by activated c-erbB2. The cyclic peptide showed no effect on the E 2 -stimulated release of matrix metalloproteinases 2 and 9 nor on the shedding of heparin-binding EGF into the culture medium. We propose that the AFP-derived cyclic peptide represents a valuable novel antiproliferative agent for treating breast cancer.

R heumatoid arthritis (RA) is a chronic, multisystemic, autoimmune disease with no well-established etiology, which manifests itself with synovial hyperplasia, articular cartilage and bone destruction. In RA synovium, pannus formation occurs as a result of permanent and abnormal synoviocytes and various inflammatory cell proliferations (1, 2). The cause of this chronic inflammation is not yet clear; however, one of the basic, major hypothesis is inadequate apoptosis (3, 4). Many various molecules and receptors have been defined in the regulation of cell apoptosis; however, Fas-Fas ligand death receptor pathway is significantly involved in the pathogenesis of RA (5). Fas(CD95) is a molecule that is expressed on the cell surface and initiates the signaling of death after binding its ligand (FasL). While Fas is expressed

Secondary metabolites contained in marine organisms disclose diverse pharmacological activities, due to their intrinsic ability to recognize bio-macromolecules, which alter their expression and modulate their function. Thus, the identification of the cellular pathways affected by marine natural products is crucial to provide important functional information concerning their mechanism of action at the molecular level. Perthamide C, a marine sponge metabolite isolated from the polar extracts of Theonella swinhoei and endowed with a broad and interesting anti-inflammatory profile, was found in a previous study to specifically interact with heat shock protein-90 and glucose regulated protein-94, also disclosing the ability to reduce cisplatin-mediated apoptosis. In this paper, we evaluated the effect of this compound on the whole proteome of murine macrophages cells by two-dimensional DIGE proteomics. Thirty-three spots were found to be altered in expression by at least 1.6-fold and 29 proteins were identified by LC ESI-Q/TOF-MS. These proteins are involved in different processes, such as metabolism, structural stability, protein folding assistance and gene expression. Among them, perthamide C modulates the expression of several chaperones implicated in the folding of proteins correlated to apoptosis, such as Hsp90 and T-complexes, and in this context our data shed more light on the cellular effects and pathways altered by this marine cyclo-peptide.

Rheumatoid arthritis (RA) is a polygenic disease associated with accelerated atherosclerosis and increased cardiovascular (CV) mortality. JAK/STAT signalling pathway is involved in autoimmune diseases and in the atherosclerotic process. JAK3 is a highly promising target for immunomodulatory drugs and polymorphisms inJAK3gene have been associated with CV events in incident dialysis patients. Therefore, the aim of this study was to assess the potential role ofJAK3polymorphisms in the development of CV disease in patients with RA. 2136 Spanish RA patients were genotyped for the rs3212780 and rs3212752JAK3gene polymorphisms by TaqMan assays. Subclinical atherosclerosis was evaluated in 539 of these patients by carotid ultrasonography (US). No statistically significant differences were found when each polymorphism was assessed according to carotid intima-media thickness values and presence/absence of carotid plaques in RA, after adjusting the results for potential confounders. Moreover, ...

A rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) based on the biotin-streptavidin amplification system was developed for the determination of anticyclic citrullinated peptide (anti-CCP). Europium-labeled streptavidin derivatives combined with europium and anhydride of diethylene triamine pentaacetic acid were used to label streptavidin, biotin was coupled with rabbit anti-human IgG to form a biotin-anti-human IgG bridge between streptavidin-europium and the anti-CCP antibody in the immunoassay. The anti-CCP assay was carried out by measuring the fluorescence of Eu(3+) -streptavidin at 615 nm. The presented method produced a wide linear range from 0.58 to 9,463 U/ml, while it was only 591.4-18.48 U/ml when using an ELISA kit, and featured a detection limit up to 0.5 U/ml for anti-CCP. The values determined by the biotin-streptavidin-TRFIA and ELISA correlated well (R(2) = 0.8927). The method was applied to determine anti-CCP in serum samples with satisfied recoveries of ...

BackgroundThe major reason for graft loss is chronic tissue damage, as interstitial fibrosis and tubular atrophy (IF/TA), where complement activation may serve as a mediator. The association of complement activation in a stable phase early after kidney transplantation with long-term outcomes is unexplored.MethodsWe examined plasma terminal C5b-9 complement complex (TCC) 10 weeks posttransplant in 900 patients receiving a kidney between 2007 and 2012. Clinical outcomes were assessed after a median observation time of 9.3 years [interquartile range (IQR) 7.5–10.6].ResultsElevated TCC plasma values (≥0.7 CAU/ml) were present in 138 patients (15.3%) and associated with a lower 10-year patient survival rate (65.7% vs. 75.5%, P < 0.003). Similarly, 10-year graft survival was lower with elevated TCC; 56.9% vs. 67.3% (P < 0.002). Graft survival was also lower when censored for death; 81.5% vs. 87.3% (P = 0.04). In multivariable Cox analyses, impaired patient survival was significantly...

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The marine-facultative Aspergillus sp. MEXU 27854, isolated from the Caleta Bay in Acapulco, Guerrero, Mexico, has provided an interesting diversity of secondary metabolites, including a series of rare dioxomorpholines, peptides, and butyrolactones. Here, we report on the genomic data, which consists of 11 contigs (N50~3.95 Mb) with a ~30.75 Mb total length of assembly. Genome annotation resulted in the prediction of 10,822 putative genes. Functional annotation was accomplished by BLAST searching protein sequences with different public databases. Of the predicted genes, 75% were assigned gene ontology terms. From the 67 BGCs identified, ~60% belong to the NRPS and NRPS-like classes. Putative BGCs for the dioxomorpholines and other metabolites were predicted by extensive genome mining. In addition, metabolomic molecular networking analysis allowed the annotation of all isolated compounds and revealed the biosynthetic potential of this fungus. This work represents the first report of ...

Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. A pharmacokinetic-pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dos...

Objectives The prognostic significance of rheumatoid factor (RF) and anticyclic citrullinated peptide antibody (anti-CCP) in rheumatoid arthritis (RA) remains contentious due to the conflicting lines of evidence. This study aims to determine the association between RF isotypes and anti-CCP with disease severity in RA patients from three ethnic groups. Methods A total of 147 RA patients from three different ethnic groups (Malays, Chinese, and Indians) who fulfilled the 1987 American College of Rheumatology (ACR) revised criteria for RA were recruited into this study. The seroprevalence of RF isotypes immunoglobulin (Ig)A, IgG, and IgM, as well as anti-CCP was determined using commercial enzyme-linked immunosorbent assay (ELISA) kits. Multinomial regression analysis was performed to assess the independent effects of autoantibody status on the development of deforming and erosive RA and the presence of extra-articular manifestations (EAM). In Chinese patients, we found a significant association (p \ 0.05) between IgG RF and anti-CCP and the presence of erosive disease, as well as IgM RF and IgG RF with the presence of joint deformities. In Indian patients, IgM RF was associated with deforming disease, whereas none of the antibodies were associated with disease severity in Malay patients. Multinomial regression analysis revealed that IgG RF was the most important predictor variable for erosive disease in Chinese patients, and IgM RF the only predictor variable associated with deforming disease in both Chinese and Indian RA patients. Conclusions There is variability in the phenotypic association of RF isotypes and anti-CCP in relation to disease severity of RA in the three ethnic groups. RF, in particular, IgG and IgM, may be better prognosticators of severe disease in Chinese and Indian patients.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, PMI Bcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein PMI Bcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM. PMI Bcr/Abl-R6 effectively induced apoptosis of HCT116 p53 +/+ cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our proteinbased delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy *

This study was designed to test the efficacy of eugenol, a compound obtained from the essential oil of cloves (Syzygium aromaticum) in collagen-induced arthritis (CIA), a well characterized murine model of rheumatoid arthritis. Macroscopic clinical evidence of CIA manifests first as periarticular erythema and edema in the hind paws. Treatment with eugenol starting at the onset of arthritis (day 25) ameliorated these clinical signs of CIA. Furthermore, eugenol inhibited mononuclear cell infiltration into the knee joints of arthritic mice and also lowered the levels of cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-γ and tumor growth factor (TGF)-β) within the ankle joints. Eugenol treatment did not affect the in vitro cell viability as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Therefore, eugenol ameliorates experimental arthritis and could be useful as a beneficial supplement in treating human arthritis.

Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and cerebrocortical neurons 8 to 9 days in vitro . However, the precise recognition site for ATX on the VGSC remains to be defined. Inasmuch as elevation of intracellular sodium ([Na ϩ ] i ) may increase N-methyl-Daspartate receptor (NMDAR)-mediated Ca 2ϩ influx, Na ϩ may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na ϩ ] i and augmenting NMDAR function. ATX (30 -100 nM) robustly stimulated neurite outgrowth, and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA receptors. Na ϩdependent up-regulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) . The Src kinase inhibitor PP2 abrogated ATXenhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and the calmodulin-dependent kinase kinase (CaMKK) inhibitor, 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca 2ϩ -dependent CaMKK pathway. These results with the structurally and mechanistically novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin site 5 ligand. These data suggest that VGSC activators may represent a novel pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.

Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and cerebrocortical neurons 8 to 9 days in vitro . However, the precise recognition site for ATX on the VGSC remains to be defined. Inasmuch as elevation of intracellular sodium ([Na ϩ ] i ) may increase N-methyl-Daspartate receptor (NMDAR)-mediated Ca 2ϩ influx, Na ϩ may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na ϩ ] i and augmenting NMDAR function. ATX (30 -100 nM) robustly stimulated neurite outgrowth, and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA receptors. Na ϩdependent up-regulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) . The Src kinase inhibitor PP2 abrogated ATXenhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and the calmodulin-dependent kinase kinase (CaMKK) inhibitor, 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca 2ϩ -dependent CaMKK pathway. These results with the structurally and mechanistically novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin site 5 ligand. These data suggest that VGSC activators may represent a novel pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

Porcme leukocytes contained three homologous peptldes. PG-1, 2 and 3. that manifested potent mlcrobicidal activity against Escherrchra coli, Lmeriu nmocytogenes and Candida olbrcuns in vitro. The peptides ('protegrms') &ere composed of 16 (PG-2) or 18 amino acid residues, and, like tachyplesms (broad-spectrum antibiotic peptides of horseshoe crab hemocytes), they contained two intramolecular cystme disulfide bonds. Considerably smaller than defensins. protegrms nevertheless showed substantial homology to them. especially to the 'corticostatic' rabbit defensin. NP-3a. The relatively simple structure of protegrins should provide useful prototypes for constructmg congeners with selectively enhanced host defense actlvltles.

The potencies and selectivity of peptide CRF antagonists is increased through structural constraints suggesting that the resulting ligands assume distinct conformations when interacting with CRF 1 and CRF 2 receptors. To develop selective CRF receptor agonists, we have scanned the sequence -Gln-Ala-His-Ser-Asn-Arg-(residues 30-35 of [DPhe 12 ,Nle 21,38 ]Ac-hCRF 4-41 ) with an i-(i + 3) bridge consisting of the Glu i -Xaa-Xbb-Lys i+3 scaffold where residues i = 30, 31 and 32. When i = 31 stressin 1 -A, a potent CRF 1 receptor-selective agonist was generated. In vitro, stressin 1 -A was equipotent to h/rCRF to release ACTH. Astressin 1 -A showed a low nanomolar affinity for CRF 1 receptor (K i = 1.7 nM) and greater than 100-fold selectivity versus CRF 2 receptor (K i = 222 nM). Stressin 1 -A released slightly less ACTH than oCRF in adult adrenal-intact male rats, with increased duration of action. Stressin 1 -A, injected intra-peritoneally, induced pellet output (a CRF 1 receptormediated response) and did not influence gastric emptying and blood pressure (CRF 2 receptormediated responses).

Antidepressant drugs are reported to cause alterations in blood glucose homeostasis in adults with diabetes mellitus. We report a patient with persistent congenital hyperinsulinism (CHI) who developed recurrent hypoglycaemia following Fluoxetine therapy. A 15-year-old girl with persistent CHI was initially managed with diazoxide therapy. She developed troublesome hypertrichosis, which affected her quality of life adversely. Diazoxide was then slowly weaned and stopped with the introduction of Octreotide, to which she responded well. Subcutaneous Lanreotide (long acting somatostatin analogue) was subsequently commenced (30mg, once monthly) as injecting Octreotide multiple times a day was proving to be difficult for the patient. The continuous blood glucose monitoring on monthly Lanreotide injections revealed good glycaemic control. Six months later, she developed depression due to psychosocial problems at school. She was commenced on Fluoxetine by the psychiatry team. She subsequentl...

Ph.D.-C7C library (New England Biolab Inc., USA) was used, which consisted of randomized 7mer peptides, each flanked by a pair of cysteine residues. The library containing 1.2x10 9 independent clones was fused to pIII coat protein via Gly-Gly-Gly-Ser spacer. Since M13 is a male-specific coliphage, ER2738 carrying the F episome was used for the infection with M13. This library was used to select platinum (Pt) binding peptides. Initially, to remove surface impurities, Pt powder (polycrystalline Pt particles) was cleaned by sonication in several solutions including a methanol/acetone mixture, isopropanol, and potassium phosphate/sodium carbonate (PC) buffer (55mM KH 2 PO 4 , 45 mM Na 2 CO 3 , and 200 mM NaCl) containing 0.5% (v/v) detergent (Tween 20 and Tween 80, Merck, USA). The target binding, elution, and amplification were then carried out with the modified version of manufacturer's instructions. The procedure of selection of binders is described, briefly. The peptide library was incubated with cleaned Pt particles in PC buffer containing 0.1% detergent (50:50 (v/v) Tween 20 and Tween 80, Merck, USA), to reduce nonspecific phage-substrate interactions, for 30 min at room temperature. The Pt particles

Semirijid ve şişirilebilir protezler erektil disfonksiyon tedavisinde kullanılmaktadır. Penil protezlere ait bazı komplikasyonlar bilinmektedir. Bu yazımızda, 64 yaşında bir erkek hastadaki semirijid penil protez fraktürü magnetik rezonans görüntüleme (MRG) bulguları ile sunulmaktadır. Hastanın MR görüntülerinde sol korpus kavernozum yerleşimli protez materyalinde açılanma ve düzensizlik seçilmektedir. MRG özellikle klinik şüphe varlığında penil protez fraktürlerinin değerlendirilmesinde değerli bir yöntemdir. Literatür taraması sonucu elde ettiğimiz bilgilere göre, bu olgu MRG bulguları ile tanımlanmış ilk semirijid penil protezi vakasıdır. Abstract

The marine fish pathogen Vibrio sp. 60 has been used as a host for heterologous expression of the Escherichiu coli heat-labile enterotoxin B-subunit and derivatives carrying a C-terminal extension. In this study, a chimeric enterotoxin B-subunit with an extension corresponding to the carboxy-terminal nine amino acids -Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp- from the small subunit of herpes simplex virus type l-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single protease that is secreted by the host strain. Such protease behaves as a typical metalloprotease, being inhibited by EDTA but not by a serine protease inhibitor. Purification and amino acid composition analysis of the two proteolysis products revealed a specific cleavage of the peptide bond between amino acids glycine and alanine of the nine amino acid extension with loss of activity. The above observation is relevant for the biotechnological exploitation of Vibrio sp. 60.

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose t...

The ongoing resistance to antibiotics constraints the research activity to design and develop new molecules, intended to overcome these necessities. In the current study, a series of organic compounds have been investigated for their properties, correlated to the potential of crossing biological membranes, consequently targeting a specific protein that would trigger an infectious disease in the body. On the matter, quantitative structureactivity relationship (QSAR) properties and molecular docking studies have been carried out for seven thiourea derivatives. The highly promising compounds have been nominated by software that ascertain if a specific molecule has an intended behaviour in the internal medium of the organism. Rezistența continuă la antibiotice constrânge activitatea de cercetare spre dezvoltarea și proiectarea de noi molecule, menite să depășească aceste necesități. În studiul actual au fost investigate proprietățile unei serii de compuși organici, aceste proprietăți fiind corelate cu potențialul de traversare prin membranele biologice și, în final, cu țintirea unei proteine responsabile de instalarea infecției. În acest sens, proprietățile QSAR și studiile de andocare moleculară au fost efectuate pentru șapte derivați ai tioureei. Cei mai promițători compuși au fost selectați prin intermediul unor programe software, capabile să determine dacă o anumită moleculă are un comportament specific în mediul intern al organismului.

WF11899A, B and C, novel antifungal lipopeptide antibiotics were isolated from the culture broth of Coleophoma empetri F-11899. These compounds belong to the echinocandin type of lipopeptides. Of these compounds, WF1 1 899A showed good solubility in water. These three antibiotics possess potent in vitro antifungal activities against Candida spp. Echinocandin B1} is an antifungal lipopeptide antibiotic which is structurally characterized by a cyclic hexapeptide acylated with a long side chain. Other related lipopeptides from fungal origin are known as* aculeacins2), mulundocandin3) and pneumocandins4'5), which are produced by Aspergillus aculeatus, A. sydowi and Zalerion arboricola, respectively. These antibiotics have excellent anti-Candida activity attributed to selective inhibition of l ,3-/?-glucan synthesis. Furthermore, pneumocandins were reported to be active against Pneumocystis carinii6\ pathogen causing pneumonia in AIDS patients. They are, however, barely soluble in water. This insolubility is one of the reasons why they cannot be developed as a clinical use. In the course of our screening for new antifungal antibiotics, we found that a strain of Coleophoma empetri F-1 1899 produces water-soluble echinocandin analogs, WF1 1899A, B and C7) (Fig. ). This paper describes taxonomic studies on the producing strain, fermentation, isolation and physico-chemical properties of these compounds.

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