Isolation and characterization of cardiac progenitor cells from myocardial right atrial appendage tissue

Resident cardiac stem cells, known as “cardiogenic progenitor cells” (CPCs), are a heterogeneous population of immature cells residing in the myocardium and capable of self-renewal and differentiation into cardiomyocyte-like and vascular-like cells. CPCs are usually isolated by long enzymatic digestion of heart tissue and selection with stem cell markers. However, long exposure to enzymatic digestion and the small size of a myocardial sample significantly hinder acquiring a large number of viable cells. To avoid these problems, we developed a method based on CPC growth ex vivo and subsequent immunomagnetic selection.     

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ~ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ~ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.     

Cleavage of yeast tRNAPhe with complementary oligonucleotide conjugated to a small ribonuclease mimic

An oligonucleotide conjugate bearing a chemical construct mimicking the catalytic center of ribonuclease A has been designed and studied. The conjugate efficiently cleaves yeast tRNAPhe at a single site adjacent to the target complementary sequence.     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.     

Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs

Cell sheets (CS) from c-kit+ cardiac stem cell (CSC) hold a potential for application in regenerative medicine. However, manufacture of CS may require thermoresponsive dishes, which increases cost and puts one in dependence on specific materials. Alternative approaches were established recently and we conducted a short study to compare approaches for detachment of CS from c-kit+ CSC. Our in-house developed method using chelation by Versene solution was compared to UpCell? thermoresponsive plates in terms of CSC proliferation, viability, gap junction formation and engraftment in a model of myocardial infarction. Use of Versene solution instead of thermoresponsive dishes resulted in comparable CS thickness (approximately 100 mcm), cell proliferation rate and no signs of apoptosis detected in both types of constructs. However, we observed a minor reduction of gap junction count in Versene-treated CS. At day 30 after delivery to infarcted myocardium both types of CS retained at the site of transplantation and contained comparable amounts of proliferating cells indicating engraftment. Thus, we may conclude that detachment of CS from c-kit+ CSC using Versene solution followed by mechanical treatment is an alternative to thermoresponsive plates allowing use of routinely available materials to generate constructs for cardiac repair.     

Mechanism and Specificity of RNA Cleavage by Chemical Ribonucleases

Abstract

Cleaving of model RNA substrates by chemical ribonucleases constructed by conjugation of 1,4 diazabicyclo[2,2,2]octane with histamine and histidine was investigated. Similarly to RNase A, the chemical RNases produce fragments with 5′ hydroxy-group and 3′-cyclophosphate. The cleavage occurs as the catalytic reaction: more than 150 phosphodiester bonds in RNA can be cleaved by one molecule of RNase mimic.     

Interaction between kringle and growth-factor-like domains in the urokinase molecule: Possible role in stimulation of chemotaxis

The results presented in this paper suggest the presence of an interaction between the kringle- and the growth-factor-like urokinase domains. This interaction regulates chemotactic properties of urokinase. We also show that interaction of urokinase with its "classical" receptor (uPAR) has a "permissive" effect on the interactions between the kringle domain and other targets on the cell surface. On the basis of our data we can suggest that uPAR serves as an "adaptor" for urokinase, and the binding of urokinase kringle domain to its receptor causes immediate activation of intracellular signaling and induction of cell migration.