scholarly journals The Drosophila suppressor of sable protein binds to RNA and associates with a subset of polytene chromosome bands.

1997 ◽  
Vol 17 (4) ◽  
pp. 2291-2300 ◽  
Author(s):  
M V Murray ◽  
M A Turnage ◽  
K J Williamson ◽  
W R Steinhauer ◽  
L L Searles
Keyword(s):  
Salivary Gland ◽  
Polytene Chromosome ◽  
Consensus Sequence ◽  
Mrna Processing ◽  
Drosophila Embryo ◽  
High Affinity ◽  
Suppressor Of Sable ◽  
Exponential Enrichment ◽  
Chromosome Bands

Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5' splice site and a sequence that is found near the 5' end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands. Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.

scholarly journals Design of PSMA ligands with modifications at the inhibitor part: an approach to reduce the salivary gland uptake of radiolabeled PSMA inhibitors?

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Veronika Barbara Felber ◽  
Manuel Amando Valentin ◽  
Hans-Jürgen Wester
Keyword(s):  
Salivary Gland ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
In Vivo Studies ◽  
Tumor Xenograft ◽  
Tumor Uptake ◽  
Lncap Cells ◽  
High Affinity

Abstract Aim To investigate whether modifications of prostate-specific membrane antigen (PSMA)-targeted radiolabeled urea-based inhibitors could reduce salivary gland uptake and thus improve tumor-to-salivary gland ratios, several analogs of a high affinity PSMA ligand were synthesized and evaluated in in vitro and in vivo studies. Methods Binding motifs were synthesized ‘on-resin’ or, when not practicable, in solution. Peptide chain elongations were performed according to optimized standard protocols via solid-phase peptide synthesis. In vitro experiments were performed using PSMA+ LNCaP cells. In vivo studies as well as μSPECT/CT scans were conducted with male LNCaP tumor xenograft-bearing CB17-SCID mice. Results PSMA ligands with A) modifications within the central Zn2+-binding unit, B) proinhibitor motifs and C) substituents & bioisosteres of the P1′-γ-carboxylic acid were synthesized and evaluated. Modifications within the central Zn2+-binding unit of PSMA-10 (Glu-urea-Glu) provided three compounds. Thereof, only natLu-carbamate I (natLu-3) exhibited high affinity (IC50 = 7.1 ± 0.7 nM), but low tumor uptake (5.31 ± 0.94% ID/g, 1 h p.i. and 1.20 ± 0.55% ID/g, 24 h p.i.). All proinhibitor motif-based ligands (three in total) exhibited low binding affinities (> 1 μM), no notable internalization and very low tumor uptake (< 0.50% ID/g). In addition, four compounds with P1′-ɣ-carboxylate substituents were developed and evaluated. Thereof, only tetrazole derivative natLu-11 revealed high affinity (IC50 = 16.4 ± 3.8 nM), but also this inhibitor showed low tumor uptake (3.40 ± 0.63% ID/g, 1 h p.i. and 0.68 ± 0.16% ID/g, 24 h p.i.). Salivary gland uptake in mice remained at an equally low level for all compounds (between 0.02 ± 0.00% ID/g and 0.09 ± 0.03% ID/g), wherefore apparent tumor-to-submandibular gland and tumor-to-parotid gland ratios for the modified peptides were distinctly lower (factor 8–45) than for [177Lu]Lu-PSMA-10 at 24 h p.i. Conclusions The investigated compounds could not compete with the in vivo characteristics of the EuE-based PSMA inhibitor [177Lu]Lu-PSMA-10. Although two derivatives (3 and 11) were found to exhibit high affinities towards LNCaP cells, tumor uptake at 24 h p.i. was considerably low, while uptake in salivary glands remained unaffected. Optimization of the established animal model should be envisaged to enable a clear identification of PSMA-targeting radioligands with improved tumor-to-salivary gland ratios in future studies.

scholarly journals Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by the Drosophila melanogaster suppressor of sable Gene

2000 ◽  
Vol 20 (21) ◽  
pp. 8198-8208 ◽  
Author(s):  
Michael A. Turnage ◽  
Paul Brewer-Jensen ◽  
Wen-Li Bai ◽  
Lillie L. Searles
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Polytene Chromosomes ◽  
Ectopic Expression ◽  
Binding Activity ◽  
Suppressor Of Sable ◽  
Rna Accumulation

ABSTRACT The Drosophila melanogaster suppressor of sable gene,su(s), encodes a novel, 150-kDa nuclear RNA binding protein, SU(S), that negatively regulates RNA accumulation from mutant alleles of other genes that have transposon insertions in the 5′ transcribed region. In this study, we delineated the RNA binding domain of SU(S) and evaluated its relevance to SU(S) function in vivo. As a result, we have defined two arginine-rich motifs (ARM1 and ARM2) that mediate the RNA binding activity of SU(S). ARM1 is required for in vitro high-affinity binding of SU(S) to small RNAs that were previously isolated by SELEX (binding site selection assay) and that contain a common consensus sequence. ARM1 is also required for the association of SU(S) with larval polytene chromosomes in vivo. ARM2 promotes binding of SU(S) to SELEX RNAs that lack the consensus sequence and apparently is neither necessary nor sufficient for the stable polytene chromosome association of SU(S). Use of the GAL4/UAS system to drive ectopic expression of su(s) cDNA transgenes revealed two previously unknown properties of SU(S). First, overexpression of SU(S) is lethal. Second, SU(S) negatively regulates expression of su(s)intronless cDNA transgenes, and the ARMs are required for this effect. Considering these and previous results, we propose that SU(S) binds to the 5′ region of nascent transcripts and inhibits RNA production in a manner that can be overcome by splicing complex assembly.

scholarly journals Cell-Specific Aptamers as Emerging Therapeutics

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Cindy Meyer ◽  
Ulrich Hahn ◽  
Andrea Rentmeister
Keyword(s):  
Drug Delivery ◽  
Nucleic Acids ◽  
Small Molecules ◽  
Live Cells ◽  
Whole Cells ◽  
High Affinity ◽  
Recent Developments ◽  
Systematic Evolution ◽  
Exponential Enrichment

Aptamers are short nucleic acids that bind to defined targets with high affinity and specificity. The first aptamers have been selected about two decades ago by anin vitroprocess named SELEX (systematic evolution of ligands by exponential enrichment). Since then, numerous aptamers with specificities for a variety of targets from small molecules to proteins or even whole cells have been selected. Their applications range from biosensing and diagnostics to therapy and target-oriented drug delivery. More recently, selections using complex targets such as live cells have become feasible. This paper summarizes progress in cell-SELEX techniques and highlights recent developments, particularly in the field of medically relevant aptamers with a focus on therapeutic and drug-delivery applications.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

2021 ◽  
Vol 22 (15) ◽  
pp. 7848
Author(s):  
Annamaria Zannoni ◽  
Simone Pelliciari ◽  
Francesco Musiani ◽  
Federica Chiappori ◽  
Davide Roncarati ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Binding Mode ◽  
In Vitro Transcription ◽  
Target Sequence ◽  
Computational Alanine Scanning ◽  
Definition Of ◽  
Target Promoter

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

scholarly journals Hybrid-Type SELEX for the Selection of Artificial Nucleic Acid Aptamers Exhibiting Cell Internalization Activity

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 888
Author(s):  
Hiro Uemachi ◽  
Yuuya Kasahara ◽  
Keisuke Tanaka ◽  
Takumi Okuda ◽  
Yoshihiro Yoneda ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Surface Protein ◽  
Functional Screening ◽  
Nucleic Acid Aptamers ◽  
Hybrid Type ◽  
Cell Internalization ◽  
Promising Target ◽  
A Cell ◽  
Exponential Enrichment

Nucleic acid aptamers have attracted considerable attention as next-generation pharmaceutical agents and delivery vehicles for small molecule drugs and therapeutic oligonucleotides. Chemical modification is an effective approach for improving the functionality of aptamers. However, the process of selecting appropriately modified aptamers is laborious because of many possible modification patterns. Here, we describe a hybrid-type systematic evolution of ligands by exponential enrichment (SELEX) approach for the generation of the artificial nucleic acid aptamers effective against human TROP2, a cell surface protein identified by drug discovery as a promising target for cancer therapy. Capillary electrophoresis SELEX was used for the pre-screening of multiple modified nucleic acid libraries and enrichment of TROP2 binding aptamers in the first step, followed by functional screening using cell-SELEX in the second step for the generation of cell-internalizing aptamers. One representative aptamer, Tac-B1, had a nanomolar-level affinity to human TROP2 and exhibited elevated capacity for internalization by cells. Because of the growing interest in the application of aptamers for drug delivery, our hybrid selection approach has great potential for the generation of functional artificial nucleic acid aptamers with ideal modification patterns in vitro.

scholarly journals High-Affinity Nicotinic Receptors Modulate Spontaneous Cortical Up States In Vitro

2015 ◽  
Vol 35 (32) ◽  
pp. 11196-11208 ◽  
Author(s):  
C. Sigalas ◽  
P. Rigas ◽  
P. Tsakanikas ◽  
I. Skaliora
Keyword(s):  
Nicotinic Receptors ◽  
High Affinity ◽  
Up States
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