Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.     

Variation in 3β-hydroxysteroid dehydrogenase activity and in pregnenolone supply rate can paradoxically alter androstenedione synthesis

The 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) enzymes are important in determining the balance of the synthesis of different steroids such as progesterone (P4), glucocorticoids, androgens, and estrogens. How this is achieved is not a simple matter because each of the two enzymes utilizes more than one substrate and some substrates are shared in common between the two enzymes. The two synthetic pathways, Δ(4) and Δ(5), are interlinked such that it is difficult to predict how the synthesis of each steroid changes when any of the enzyme activities is varied. In addition, the P450c17 enzyme exhibits different substrate specificities among species, particularly with respect to the 17,20-lyase activity. The mathematical model developed in this study simulates the network of reactions catalyzed by 3β-HSD and P450c17 that characterizes steroid synthesis in human, non-human primate, ovine, and bovine species. In these species, P450c17 has negligible 17,20-lyase activity with the Δ(4)-steroid 17α-hydroxy-progesterone (17OH-P4); therefore androstenedione (A4) is synthesized efficiently only from dehydroepiandrosterone (DHEA) through the Δ(5) pathway. The model helps to understand the interplay between fluxes through the Δ(4) and Δ(5) pathways in this network, and how this determines the response of steroid synthesis to the variation in 3β-HSD activity or in the supply of the precursor substrate, pregnenolone (P5). The model simulations show that A4 synthesis can change paradoxically when 3β-HSD activity is varied. A decrease in 3β-HSD activity to a certain point can increase A4 synthesis by favouring metabolism through the Δ(5) pathway, though further decrease in 3β-HSD activity beyond that point eventually limits A4 synthesis. The model also showed that due to the competitive inhibition of the enzymes' activities by substrates and products, increasing the rate of P5 supply above a certain point can suppress the synthesis of A4, DHEA, and 17OH-P4, and consequently drive more P5 towards P4 synthesis.     

Products of spontaneous conjugation of aflatoxins with bovine serum albumin: immunochemical properties

Products of spontaneous conjugation of aflatoxins B1, G1, and G2 with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by anti-aflatoxin antibodies. Antisera against BSA-B1, BSA-G1, and BSA-G2 were obtained and their specificity, determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.     

The Effect of Modification of Nucleotide-37 on the Interaction of Aminoacyl-tRNA with the A Site of the 70S Ribosome

To estimate the effect of modified nucleotide 37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNA^Phe _+Y and Phe-tRNA^Phe _–Y) with the A site of complex [70S · poly(U) · deacylated tRNA^Phe in the P site] was assayed at 0–20°C. As comparisons with native Phe-tRNA^Phe _+Y showed, removal of the Y base decreased the association constant of Phe-tRNA^Phe _–Y and the complex by an order of magnitude at every temperature tested, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA^Phe bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNA^Phe _–Y but not for Phe-tRNA^Phe _+Y. Thus, the modified nucleotide 3" of the Phe-tRNA^Phe anticodon stabilized the codon–anticodon interaction both in the A and P sites of the 70S ribosome.     

Conversion of L-cystine and L-cysteine to taurin by the enzyme systems of liver cells

The kinetics of conversion of sulfur-containing amino acids L-cystine and L-cysteine to taurin by the enzyme system of cattle liver cells was studied, and a mathematical model was developed. It was shown that L-cystine and L-cysteine conversion obeyed the Michaelis-Menten equations of serial-sequential conversions with regard to inhibition by the final product and inactivation. The yield of taurin under the optimized conditions of L-cystine and L-cysteine conversion (temperature, 40 degrees C; pH 1.5 and 3.0, respectively; and addition of enzyme preparations in five equal portions at 2-h intervals) was in the range 80-85% of the substrate weight.     

An efficient system for high-level expression and easy purification of authentic recombinant proteins

Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.     

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

Background  

The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown.     

Antibiotic properties of the strains of the basidiomycete Lentinus edodes (Berk.) sing]

Antibiotic properties of the extracts from the fermentation broth and mycelium of 15 strains of the edible and medicinal basidiomycete L. edodes were studied and it was shown that the extracts were active against grampositive and gramnegative bacteria, yeasts and mycelial fungi, including dermatophytes and phytopathogens. The strains differed by the set of the organisms susceptible to the action of the extracts. Strains of L. edodes combining marked antibiotic properties and high yields of water soluble polysaccharides were screened. The active compounds were detected by preparative TLC. Two of them were identified with UV- and mass spectrometry as lentinamycin B and erytadenine (lentinacin). Lentinamycin B was found to be the main component responsible for the antibiotic activity of the L. edodes strains.