Dansylated analogues of the opioid peptide [Dmt1]DALDA: in vitro activity profiles and fluorescence parameters.

Dansylated analogues of the potent and selective micro opioid peptide agonist [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) were prepared either by substitution of N(beta)-dansyl-alpha,beta-diaminopropionic acid or N(epsilon)-dansyllysine for Lys(4), or by attachment of a dansyl group to the C-terminal carboxamide function via a linker. All three analogues displayed high micro agonist potency in vitro and the C-terminally dansylated one retained significant micro receptor selectivity. The three analogues showed interesting differences in their fluorescence emission maxima and quantum yields, indicating that the dansyl group in two of them was engaged in intramolecular hydrophobic interactions. These dansylated [Dmt(1)]DALDA analogues represent valuable tools for binding studies, cellular uptake and intracellular distribution studies, and tissue distribution studies.

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A highly potent peptide analgesic that protects against ischemia-reperfusion-induced myocardial stunning

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00193.2002 ◽

2002 ◽

Vol 283 (2) ◽

pp. H783-H791 ◽

Cited By ~ 33

Author(s):

Dunli Wu ◽

Yi Soong ◽

Guo-Min Zhao ◽

Hazel H. Szeto

Keyword(s):

Myocardial Stunning ◽

No Reduction ◽

Specific Binding ◽

Ischemia Reperfusion ◽

Opioid Peptide ◽

Binding Studies ◽

Cardiac Membranes ◽

Effect Binding ◽

Perfused Hearts

We recently discovered an opioid peptide analgesic, 2′,6′-dimethyltyrosine (Dmt)-d-Arg-Phe-Lys-NH2([Dmt1]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt1]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt1]DALDA inhibited [3H]NE uptake into cardiac synaptosomes in vitro (IC50 = 3.9 μM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt1]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [3H][Dmt1]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt1]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt1]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.

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Imaging Endogenous Opioid Peptide Release with [11C]Carfentanil and [3H]Diprenorphine: Influence of Agonist-Induced Internalization

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.117 ◽

2014 ◽

Vol 34 (10) ◽

pp. 1604-1612 ◽

Cited By ~ 24

Author(s):

Darren R Quelch ◽

Loukia Katsouri ◽

David J Nutt ◽

Christine A Parker ◽

Robin J Tyacke

Keyword(s):

Confocal Microscopy ◽

Neurotransmitter Release ◽

Radioligand Binding ◽

Subcellular Fractionation ◽

Opioid Peptide ◽

Binding Studies ◽

Endogenous Opioid ◽

Double Labeling ◽

Endogenous Opioid Peptide

Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [11C]carfentanil, but not [3H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [11C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that μ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in μ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.

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The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656083 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0949-0954 ◽

Cited By ~ 8

Author(s):

J Prins ◽

F R Lues ◽

Y Y van der Hoek ◽

J J.P Kastelein ◽

B N Bouma ◽

...

Keyword(s):

Case Control Study ◽

Amino Acid Position ◽

Case Control ◽

Binding Studies ◽

Binding Characteristics ◽

Apolipoprotein A ◽

Significant Independent Risk Factor ◽

And Gender ◽

Control Study

SummaryElevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr12-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (DesafibTM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafibTM-X was observed for the Pro12-subtype Lp(a).

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Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

10.26434/chemrxiv.12710159.v1 ◽

2020 ◽

Author(s):

Sean A. Newmister ◽

Kinshuk Raj Srivastava ◽

Rosa V. Espinoza ◽

Kersti Caddell Haatveit ◽

Yogan Khatri ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Molecular Basis ◽

Hydrophobic Interactions ◽

Cytochromes P450 ◽

Targeted Mutagenesis ◽

P450 Monooxygenase ◽

Bond Functionalization ◽

Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br>

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Synthesis and Biological Evaluations of Organoruthenium Scaffolds: A Comprehensive Update

Current Organic Synthesis ◽

10.2174/1570179414666170703143049 ◽

2018 ◽

Vol 15 (2) ◽

pp. 179-207

Author(s):

Ashaparna Mondal ◽

Priyankar Paira

Keyword(s):

Singlet Oxygen ◽

Anticancer Agents ◽

Ruthenium Complexes ◽

Chemotherapeutic Agents ◽

Quantum Yields ◽

Ros Generation ◽

Standard Drug ◽

Cellular Environment ◽

Theranostic Agents

Background: Currently ruthenium complexes are immerging as effective anticancer agents due to their less toxicity, better antiproliferative and antimetastatic activity, better stability in cellular environment and most importantly variable oxidation and co-ordination states of ruthenium allows binding this molecule with a variety of ligands. So in past few years researchers have shifted their interest towards organoruthenium complexes having good fluorescent profile that may be applicable for cancer theranostics. Nowadays, photodynamic therapy has become more acceptable because of its easy and effective approach towards killing cancer cells. Objective: Objective of this review article is to shed light on synthesis, characterization, stability and fluorescence studies of various ruthenium [Ru(II) and Ru(III)] complexes and different bioactivity studies conducted with the synthesized compounds to test their candidacy as potent chemotherapeutic agents. Methods: Various heterocyclic ligands containing N,O and S as heteroatom mainly were prepared and subjected to complexation with ruthenium-p-cymene moiety. In most cases [Ru(η6-p-cymene)(µ-Cl)Cl]2 was used as ruthenium precursor and the reactions were conducted in various alcohol medium such as methanol, ethanol or propanol. The synthesized complexes were characterized by 1H NMR and 13C NMR spectroscopy, GC-MS, ESI-MS, elemental analysis and single crystal X-ray crystallography methods. Fluorescence study and stability study were conducted accordingly using water, PBS buffer or DMSO. Stable compounds were considered for cell viability studies. To study the efficacy of the compounds in ROS generation as photosensitizers, in few cases, singlet oxygen quantum yields in presence of light were calculated. Suitable compounds were selected for in vitro & in vivo antiproliferative, anti-invasive activity studies. Result: Many newly synthesized compounds were found to have less IC50 compared to a standard drug cysplatin. Those compounds were also stable preferably in physiological conditions. Good fluorescence profile and ROS generation ability were observed for few compounds. Conclusion: Numerous ruthenium complexes were developed which can be used as cancer theranostic agents. Few molecules were synthesized as photosensitizers which were supposed to generate reactive singlet oxygen species in targeted cellular environment in presence of a particular type of light and thereby ceasing cancer cell growth.

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In Vitro Cellular Uptake Studies of Self-Assembled Fluorinated Nanoparticles Labelled with Antibodies

Nanomaterials ◽

10.3390/nano11081906 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1906

Author(s):

Mona Atabakhshi-Kashi ◽

Mónica Carril ◽

Hossein Mahdavi ◽

Wolfgang J. Parak ◽

Carolina Carrillo-Carrion ◽

...

Keyword(s):

Flow Cytometry ◽

Cellular Uptake ◽

Hydrophobic Interactions ◽

Intrinsic Properties ◽

Wide Range ◽

Self Assembled ◽

Uptake Studies ◽

Targeting Ability ◽

Fluorinated Nanoparticles

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

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Structural and Genetic Aspects of Proline-rich Proteins

Journal of Dental Research ◽

10.1177/00220345870660021201 ◽

1987 ◽

Vol 66 (2) ◽

pp. 457-461 ◽

Cited By ~ 77

Author(s):

A. Bennick

Keyword(s):

General Structure ◽

Single Gene ◽

Conclusive Evidence ◽

In Vitro Translation ◽

Binding Studies ◽

Nmr Studies ◽

Post Translational Modifications ◽

Single Precursor ◽

Made In

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

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Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils

Nature Communications ◽

10.1038/s41467-021-24362-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Pijush Chakraborty ◽

Gwladys Rivière ◽

Shu Liu ◽

Alain Ibáñez de Opakua ◽

Rıza Dervişoğlu ◽

...

Keyword(s):

Amyloid Fibrils ◽

Corticobasal Degeneration ◽

Binding Studies ◽

Rigid Core ◽

Tau Aggregation ◽

The Brain ◽

Tau Aggregate ◽

Disease Specific ◽

Tau Fibrils

AbstractPathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

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Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

The Journal of Cell Biology ◽

10.1083/jcb.105.3.1463 ◽

1987 ◽

Vol 105 (3) ◽

pp. 1463-1469 ◽

Cited By ~ 60

Author(s):

C Fornieri ◽

M Baccarani-Contri ◽

D Quaglino ◽

I Pasquali-Ronchetti

Keyword(s):

Hydrophobic Interactions ◽

Isonicotinic Acid ◽

Lysyl Oxidase ◽

Acid Hydrazide ◽

Elastic Fibers ◽

Amino Groups ◽

Lysine Residues ◽

Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

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Intra-domain communication between the N-terminal and DNA-binding domains of the androgen receptor: modulation of androgen response element DNA binding

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01723 ◽

2005 ◽

Vol 34 (3) ◽

pp. 603-615 ◽

Cited By ~ 45

Author(s):

Jacqueline Brodie ◽

Iain J McEwan

Keyword(s):

Androgen Receptor ◽

Dna Binding ◽

Fluorescence Emission ◽

Progesterone Receptors ◽

Fluorescence Emission Spectrum ◽

Response Element ◽

Amino Terminal ◽

Binding Domains ◽

Receptor Modulation

The androgen receptor (AR) is a ligand-activated transcription factor that recognises and binds to specific DNA response elements upon activation by the steroids testosterone or dihydrotestosterone. In vitro, two types of response element have been characterised - non-selective elements that bind the androgen, glucocorticoid and progesterone receptors, and androgen receptor-selective sequences. In the present study, the allosteric effects of DNA binding on the receptor amino-terminal domain (NTD) were studied. Binding to both types of DNA response element resulted in changes in the intrinsic fluorescence emission spectrum for four tryptophan residues within the AR-NTD and resulted in a more protease-resistant conformation. In binding experiments, it was observed that the presence of the AR-NTD reduced the affinity of receptor polypeptides for binding to both selective and non-selective DNA elements derived from the probasin, PEM and prostatin C3 genes respectively, without significantly altering the protein–base pair contacts. Taken together, these results highlight the role of intra-domain communications between the AR-NTD and the DNA binding domain in receptor structure and function.

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