The link between the electrogenic and redox functions of the plasmalemma and the energy metabolism of the cell

A dependence of the plasmalemma redox activity, determined by the reduction of external electron acceptors (ferricyanide, nitro-blue tetrazolium), on the energy state of the cell, which was modified by light conditions or introduction of glucose into the media, was shown on leaves of Elodea canadensis Rich. Glucose (10 m M ) and light (40 W m^-2) caused hyperpolarization of the membrane potential and stimulated the redox activity of the plasmalemma. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) completely inhibited the light activation of electrogenic and redox functions of the plasmalemma. The light saturation intensity for membrane potential and ferricyanide reductase activity was 10–30% of the light saturation of photosynthesis. Membrane potential, K⁺ transport and plasmalemma redox activity changed in parallel in response to light and darkness and when DCMU was added. Ferricyanide reductase activity is suggested to be a simple parameter for characterizing the energy state of the cell. The functional significance of the light-induced hyperpolarization of the membrane potential is discussed.     

Characterization of amine oxidases from pisum, lens, Lathyrus and Cicer

Amine oxidases have been purified to homogeneity from Pisum sativum, Lens esculenta, Lathyrus sativus and Cicer arietinum. The enzymes have a M_r. of 150 000 and are composed of two identical subunits of 72 000. The amine oxidases showed an isoelectrophoretic heterogeneity.     

Embryonic stem cells derived from morulae,inner cell mass,and blastocysts of mink: Comparisons of their pluripotencies

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley-Liss, Inc.     

Adaptive Evolution of p53 Thermodynamic Stability

The thermodynamic stability of a protein plays an important role during evolution and adaptation in order to maintain a folded and active conformation. p53 is a tumour suppressor involved in the regulation of numerous genes. Human p53 has an unusually low thermodynamic stability and is frequently inactivated by oncogenic missense mutations. Here, we examined the thermodynamic and kinetic stability of p53 DNA binding domains from selected invertebrate and vertebrate species by differential scanning calorimetry and equilibrium urea denaturation. There is a correlation in the apparent melting temperature of p53 with the body temperature of homeotherm vertebrates. We found that p53 from these organisms has a half-life for spontaneous unfolding at organismal body temperature of 10-20 min. We also found that p53 from invertebrates has higher stability, bearing more resemblance towards p63 and p73 from humans. Using structure-guided mutagenesis on the human p53 scaffold, we demonstrated that the amino acid changes on the protein surface and in the protein interior lead to the elevated stability of p53 orthologs. We propose a model in which the p53 DNA binding domain has been shaped by the complex interplay of different selective pressures and underwent adaptive evolution leading to pronounced effects on its stability. p53 from vertebrates has evolved to have a low thermodynamic stability and similarly short spontaneous half-life at organismal body temperature, which is related to function.     

Tools,techniques, and future opportunities for characterizing the mechanobiology of uterine myometrium

The myometrium is the smooth muscle layer of the uterus that generates the contractions that drive processes such as menstruation and childbirth. Aberrant contractions of the myometrium can result in preterm birth, insufficient progression of labor, or other difficulties that can lead to maternal or fetal complications or even death. To investigate the underlying mechanisms of these conditions, the most common model systems have conventionally been animal models and human tissue strips, which have limitations mostly related to relevance and scalability, respectively. Myometrial smooth muscle cells have also been isolated from patient biopsies and cultured in vitro as a more controlled experimental system. However, in vitro approaches have focused primarily on measuring the effects of biochemical stimuli and neglected biomechanical stimuli, despite the extensive evidence indicating that remodeling of tissue rigidity or excessive strain is associated with uterine disorders. In this review, we first describe the existing approaches for modeling human myometrium with animal models and human tissue strips and compare their advantages and disadvantages. Next, we introduce existing in vitro techniques and assays for assessing contractility and summarize their applications in elucidating the role of biochemical or biomechanical stimuli on human myometrium. Finally, we conclude by proposing the translation of “organ on chip” approaches to myometrial smooth muscle cells as new paradigms for establishing their fundamental mechanobiology and to serve as next-generation platforms for drug development.     

Developmental and Environmental Effects on Sesquiterpene Lactones in Cultivated Arnica montana L.

The amount of sesquiterpene lactones and the lactone profile of Arnica montana L. in flowering and seed formation stages in vitro and in vivo propagated from seeds of German, Ukrainian, and Austrian origin and grown in two experimental fields were studied. It was found that in vitro propagated 2‐year plants in full flowering stage accumulated higher amount of lactones in comparison to in vivo propagated 3‐year plants and to the seed formation stage, respectively. Helenalins predominated in in vivo propagated 2‐year or in vitro propagated 3‐year plants. 2‐Methylbutyrate (2MeBu) was the principal ester in the samples with prevalence of helenalins, while isobutyrate (iBu) was the major one in the samples with predominance of 11,13‐dihydrohelenalins. The results revealed that the environmental conditions on Vitosha Mt. are more suitable for cultivation of A. montana giving higher content of lactones.     

Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide

Successful cancer gene therapy depends on the development of non-toxic, efficient, tumor cell- specific systemic gene delivery systems. Our laboratory has developed a systemically administered, ligand–liposome complex that can effectively and preferentially deliver its therapeutic payload to both primary and metastatic tumors. To further improve the transfection efficiency of this targeting complex, a synthetic pH-sensitive histidylated oligolysine K[K(H)KKK]₅-K(H)KKC (HoKC), designed to aid in endosomal escape and condensation of DNA, was included in the complex. The presence of HoKC increased the in vitro transfection efficiency over that of the original complex. Moreover, no increase in cytotoxicity was observed due to the presence of the HoKC peptide. In a DU145 human prostate cancer xenograft tumor model in athymic nude mice, inclusion of the HoKC peptide did not interfere with the tumor targeting specificity of the i.v. administered ligand/liposome/DNA complex. Most importantly, the level of transgene expression was significantly elevated in the tumors, but not in the normal tissue in those animals receiving the complex incorporating HoKC. The in vivo enhancement of transfection efficiency by this modified gene delivery vehicle could lead to a reduction in the number of administrations required for antitumor efficacy.     

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.