Hydroxyurea treatment does not prevent initiation of DNA synthesis in Ehrlich ascites tumour cells and leads to the accumulation of short DNA fragments containing the replication origins

The ability of EAT cells to initiate DNA synthesis in the presence of high doses of hydroxyurea was examined using the recently developed method for crosslinking DNA in vivo. Since crosslinking blocks elongation but has little effect on initiation (Russev and Vassilev (1982) J. Mol. Biol. 161, 77-87), this approach permits a separate study of the two stages of the DNA replication. We found out that hydroxyurea did not greatly affect the initiation of DNA replication but strongly inhibited the elongation of the already initiated new DNA chains. This resulted in the formation of short fragments enriched in sequences synthesized at and around the sites where DNA initiation began. These fragments were not ligated to the high molecular weight chromosomal DNA and could be released under denaturing conditions in single-stranded form. The reassociation and electrophoretic analysis showed that they contained about 200 nucleotides long interspersed DNA sequences repeated approx. 10(4) times per haploid genome, that probably served as replication origins.     

Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

Life-cycle,delimitation and redescription of Catatropis verrucosa (Frölich, 1789) Odhner, 1905 (Trematoda: Notocotylidae)

The life-cycle of Catatropis verrucosa (Frölich, 1789) Odhner, 1905 has been completed experimentally starting from infected snails collected along the River Danube in Europe. Each stage of the life-cycle is redescribed. Taxonomic problems are discussed and the main features of the species are listed. Synonyms for C. verrucosa are Fasciola verrucosa Frölich, 1789, F. anseris Gmelin, 1790, Monostoma verrucosa (Frölich, 1789) Zeder, 1800, and Catatropis charadrii Skrjabin, 1915. Other names, such as Notocotylus triserialis Diesing, 1839, Notocotyle triseriale (Diesing, 1839) Diesing, 1850, Monostoma verrugueux Dujardin, 1845, “Monostoma sp. du canard” of Blanchard (1847), Notocotyle verrucosum (Frölich, 1789) Monticelli, 1892, N. verruqueux Railliet, 1895, and Distoma verrucosum (Frölich, 1789) Wolffhugel, 1900, were found to represent adults and/or larvae of C. verrucosa. Conversely, but less often, adults and larvae of other species were found described and illustrated as C. verrucosa. One of these, C. verrucosa of Joyeux (1922), was renamed Pseudocatatropis joyeuxi Kanev & Vassilev, 1986. Occasionally, authors actually working with C. verrucosa ascribed their results to different species. Based on experimental life-cycle studies, the following facts were demonstrated. (1) The first intermediate hosts are the prosobranch freshwater snails Bithynia tentaculata (Linnaeus, 1758) and B. leachi (Leach, 1818). (2) The same snails are also first intermediate hosts for Notocotylus imbricatus (Looss, 1893) Szidat, 1935, N. parviovatus Yamaguti, 1934, and N. ponticus Tschiaberaschvili, 1966. In all these species, the species characteristics are expressed by the adult morphology only, and the larvae cannot be identified by morphological criteria. It is proposed that tri-oculate monostome cercariae found in naturally infected B. tentaculata and B. leachi be referred to as “Cercaria imbricata group”. These cercariae include Cercaria imbricata Looss, 1893, C. helvetica I Dubois, 1928, C. triophthalmia Faust, 1930, C. fennica I Wikgren, 1956; C. ephemera of Lutta (1934); C. monostomi of Mathias (1925), Lutta (1934) and Zdun (1961), Cercaria Notocotylus attenuatus of Francalanci & Manfredini (1969), and Monostome cercaria I Emmel, 1943. (3) There is no second intermediate snail host in the life-cycle of C. verrucosa. (4) The final hosts are birds. (5) The adult worms possess, on the ventral body surface, a median ridge and two lateral rows of 12 (range 11–14) papillae per row.     

Production and properties of inulinase from Aspergillus niger

Summary A thermostable inulinase was identified in a strain of A. niger. The highest activity was observed at 50 °C (50 Lml^–1) and 77% and 34% of this was retained at 60° and 65°C, respectively. pH stability, the effect of thermal stabilizers such as Propylene glycol (10%) and Sorbitol (10%) and effects of different cations were investigated. It was found that the activity was completely inhibited by Ag⁺ and Hg²⁺, while Na⁺ had an activator effect.     

Contractile effects of prostaglandin E2 in rat rectum: sensitivity to the prostaglandin antagonists diphloretin phosphate and SC 19220.

Prostaglandin E2 (PGE2) applied cumulatively (1 nM-1 microM) induced concentration-dependent tonic contractions in the longitudinal muscle of isolated rat rectum. The PGE2 effects were not altered by guanethidine (50 microM), whereas atropine (3 microM), guanethidine plus atropine or tetrodotoxin (0.1 microM) reduced them to an almost equal extent and increased the EC50 values for PGE2. The after-contractions following electrical stimulation were enhanced by PGE2 (10 nM) and inhibited by atropine. Diphloretin phosphate (DPP, 100 microM) shifted the regression lines for PGE2 to the right in both untreated and tetrodotoxin-treated preparations, and thereby increased the EC50 values. Slopes of the concentration-effect lines for PGE2 before and after DPP differed in the presence of tetrodotoxin. The regression line for PGE2 with SC 19220 (100 microM) in tetrodotoxin-treated preparations was shifted to the right in a parallel fashion. It is concluded that PGE2 exerted both a neural (cholinergic) and a smooth muscle effect. There may be a competitive antagonism between SC 19220 and PGE2 but the block by DPP may be nonselective.     

ImmobilizedAspergillus terreus in itaconic acid production from glucose

Summary The production of itaconic acid by immobilizedAspergillus terreus TKK 200-5-1 was studied both in shake flask cultures, and in continuous column bioreactors. The effect of glucose and ammonium nitrate concentrations, and of pH were examined using a statistical experimental plan. The highest itaconic acid product concentration could be reached at the highest investigated glucose concentration of 150 g/l and the highest initial pH of 3.75, in the absence of ammonium nitrate. In a continuous packed bed column system operated fro 4.5 months itaconic acid was obtained at a productivity of 328 mg/d per gram of polyurethane foam carrier.     

Contractile effects of prostaglandin E2 in rat rectum: Sensitivity to the prostaglandin antagonists diphloretin phosphate and SC 19220

Prostaglandin E₂ (PGE₂) applied cumulatively (1 nM − 1 μM) induced concentration-dependent tonic contractions in the longitudinal muscle of isolated rat rectum. The PGE₂ effects were not altered by guanethidine (50 μM), whereas atropine (3 μM), guanethidine plus atropine or tetrodotoxin (0.1 μM) reduced them to an almost equal extent and increased the EC₅₀ values for PGE₂. The after-contractions following electrical stimulation were enhanced by PGE₂ (10 nM) and inhibited by atropine. Diphloretin phosphate (DPP, 100 μM) shifted the regression lines for PGE₂ to the right in both untreated and tetrodotoxin-treated preparations, and thereby increased the EC₅₀ values. Slopes of the concentration-effect lines for PGE₂ before and after DPP differed in the presence of tetrodotoxin. The regression line for PGE₂ with SC 19220 (100 μM) in tetrodotoxin-treated preparations was shifted to the right in a parallel fashion. It is concluded that PGE₂ exerted both a neural (cholinergic) and a smooth muscle effect. There may be a competitive antagonism between SC 19220 and PGE₂ but the block by DPP may be nonselective.     

An allied reprogramming,selection, expansion and differentiation platform for creating hiPSC on microcarriers

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs.     

Role of a JAK3-dependent biochemical signaling pathway in platelet activation and aggregation

Here we provide experimental evidence that identifies JAK3 as one of the regulators of platelet function. Treatment of platelets with thrombin induced tyrosine phosphorylation of the JAK3 target substrates STAT1 and STAT3. Platelets from JAK3-deficient mice displayed a decrease in tyrosine phosphorylation of STAT1 and STAT3. In accordance with these data, pretreatment of human platelets with the JAK3 inhibitor WHI-P131 markedly decreased the base-line enzymatic activity of constitutively active JAK3 and abolished the thrombin-induced tyrosine phosphorylation of STAT1 and STAT3. Following thrombin stimulation, WHI-P131-treated platelets did not undergo shape changes indicative of activation such as pseudopod formation. WHI-P131 inhibited thrombin-induced degranulation/serotonin release as well as platelet aggregation. Highly effective platelet inhibitory plasma concentrations of WHI-P131 were achieved in mice without toxicity. WHI-P131 prolonged the bleeding time of mice in a dose-dependent manner and improved event-free survival in a mouse model of thromboplastin-induced generalized and invariably fatal thromboembolism. To our knowledge, WHI-P131 is the first anti-thrombotic agent that prevents platelet aggregation by inhibiting JAK3.     

Excited-state relaxation paths of oxo/hydroxy and N9H/N7H tautomers of guanine: a CC2 theoretical study

We performed a theoretical investigation, at the CC2/aug-cc-pVDZ level, of the ring-deformation mechanisms of four guanine tautomers (oxo, hydroxy, N9H, and N7H). The study showed that the optimized conical intersections S₀/S₁ are accessible through the ¹ππ* excited states of tautomers. The optimized conical intersections S₀/S₁, which show deformation at the pyrimidine ring, have high energies. This means that the relaxations of the ¹ππ* excited states via internal conversion are disfavored. For two tautomers we found crossing points ¹ππ*/¹πσ* of the excited-state reaction paths, revealing the possibility of a population of the ¹πσ* excited state by the ¹ππ* excited state.