Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases

The conjugates of an adenosine mimetic and oligo-l -arginine or oligo-d -arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l -methionine (SAM) and S-adenosyl-l -homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.)

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.     

Estimating seed dispersal distance: A comparison of methods using animal movement and plant genetic data on two primate‐dispersed Neotropical plant species

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Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Receptor interacting protein 140 regulates expression of uncoupling protein 1 in adipocytes through specific peroxisome proliferator activated receptor isoforms and estrogen-related receptor alpha

Expression of uncoupling protein 1 (Ucp1) mRNA is elevated in differentiated adipocytes derived from brown or white adipose tissue devoid of the nuclear receptor corepressor receptor interacting protein 140 (RIP140). Increased expression is mediated in part by the recruitment of peroxisome proliferator activated receptors alpha and gamma, together with estrogen-related receptor alpha, which functions through a novel binding site on the Ucp1 enhancer. This demonstrates that regulation of Ucp1 expression in the absence of RIP140 involves derepression of at least three different nuclear receptors. The ability to increase expression of Ucp1 by beta-adrenergic signaling is independent of RIP140, as shown by the action of the beta(3)-adrenergic agonist CL 316,243 to stimulate expression in both brown and white adipocytes in the presence and absence of the corepressor. Therefore, the expression of this metabolic uncoupling protein in adipose cells is regulated by inhibition as well as activation of distinct signaling pathways.     

Comparison of genovars and Chlamydia trachomatis infection loads in ocular samples from children in two distinct cohorts in Sudan and Morocco

Trachoma is a blinding disease caused by repeated conjunctival infection with different Chlamydia trachomatis (Ct) genovars. Ct B genovars have been associated with more severe trachoma symptoms. Here, we investigated associations between Ct genovars and bacterial loads in ocular samples from two distinct geographical locations in Africa, which are currently unclear. We tested ocular swabs from 77 Moroccan children (28 with trachomatous inflammation-follicular (TF) and 49 healthy controls), and 96 Sudanese children (54 with TF and 42 healthy controls) with a Ct-specific real-time polymerase chain reaction (PCR) assay. To estimate bacterial loads, Ct-positive samples were further processed by multiplex real-time qPCR to amplify the chromosomal outer membrane complex B and plasmid open reading frame 2 of Ct. Genotyping was performed by PCR-based amplification of the outer membrane protein A gene (~1120 base pairs) of Ct and Sanger sequencing. Ct-positivities among the Moroccan and Sudanese patient groups were 60·7% and 31·5%, respectively. Significantly more Sudanese patients than Moroccan patients were genovar A-positive. In contrast, B genovars were significantly more prevalent in Moroccan patients than in Sudanese patients. Significantly higher Ct loads were found in samples positive for B genovars (598596) than A genovar (51005). Geographical differences contributed to the distributions of different ocular Ct genovars. B genovars may induce a higher bacterial load than A genovars in trachoma patients. Our findings emphasize the importance of conducting broader studies to elucidate if the noted difference in multiplication abilities are genovar and/or endemicity level dependent.     

Molecular detection of Babesia canis in Dermacentor reticulatus ticks collected in Slovakia

Dermacentor reticulatus ticks are recognized as the most important vectors of Babesia canis, the aetiological agent of canine babesiosis occurring throughout Europe. Vector competence of D. reticulatus for B. canis is well described and experimentally determined; however, by using molecular analysis it was proven so by one recent study in Russia. Herein, the additional molecular evidence of B. canis infection in D. reticulatus ticks collected in Slovakia is provided. Using PCR followed by sequencing of distinctive amplicons we determined the presence of Babesia canis canis in one of 100 tested adult ticks. Two zoonotic pathogens, Francisella tularensis and Coxiella burnetii, were previously isolated from D. reticulatus ticks in Slovakia. In our samples, we detected only the presence of F. tularensis.