Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling technique.

Incorporation of thymidine into Novikoff rat hepatoma cells was analyzed with a rapid sampling technique which allowed collection of 12 time points in 20 sec. Transport was a rapid, saturable, nonconcentrative process with a Km of about 85 micrometer. The intracellular thymidine pool was also rapidly labeled in cells which phosphorylated thymidine, that a group translocation process involving thymidine kinase can be ruled out. Under all conditions examined, phosphorylation, not the transport, of thymidine was the rate-determining step in its incorporation into the acid-soluble pool. Estimation of transport rates from total incorporation into cells which phosphorylate the substrate is invalid in this cell system and must be questioned in all instances.     

Biosynthesis of membrane glycoproteins in rat hepatoma tissue culture cells

The early steps in the biosynthesis of glycoproteins associated with the plasma membranes of rat hepatoma tissue culture cells has been analyzed. By measuring the effect of tunicamycin on the incorporation of [3H] mannose and [3H] fucose into cell glycoproteins, it was determined that an interval of about 1 h was required to transfer the glycoprotein from site of mannosylation to the site of fucosylation. This result was corroborated by an analysis of the time required for the appearance of either mannose or fucose-labeled glycoproteins at the cell surface. The separation of membrane glycoproteins by a two-dimensional gel system allowed the visualization of the modifications leading to both size and charge heterogeneity of these proteins. By following the changes in electrophoretic mobility introduced into membrane glycoproteins during a chase period after a pulse labeling, the time course of these molecular alterations could be estimated. Several glycoproteins have apparently higher rates of synthesis than the bulk of membrane-associated glycoproteins. Most of these glycoproteins were released within 2 h after biosynthesis from the intracellular membrane fraction and appear after 3 h in the medium. In addition to the glycoproteins that contain both mannose and fucose and that show a high degree of charge heterogeneity, there are other membrane-bound species that are not noticeably modified by the incorporation of fucose or sialic acids. These glycoproteins could represent constituents limited to the internal membrane system of the HTC cell.     

Studies on responsiveness of hepatoma cells to catecholamines. V. Loss of adrenergic response of glycogen phosphorylase in rat ascites hepatoma AH130 cells

The beta-adrenoceptor-cyclic adenosine monophosphate (AMP) dependent glycogenolytic cascade was examined in normal rat hepatocytes and rat ascites hepatoma AH130 cells. The cyclic AMP content in AH130 cells was half of that in normal hepatocytes, and the cyclic AMP levels in both kinds of cells were clearly increased by isoproterenol (IPN). Cyclic AMP-dependent protein kinase activity was higher in AH130 cells than in normal hepatocytes. Phosphorylase kinase activities in 10000 x g supernatant of normal hepatocytes and AH130 cells were also increased in the presence of cyclic AMP. Phosphorylase a activities in the supernatant of both kinds of cells gradually decreased during incubation with 40 mM glucose at 37 degrees C, and the enzyme activity of normal hepatocytes was completely restored by the addition of Mg2(+)-adenosine triphosphate (ATP), but in the case of the hepatoma cells the recovery was small. The decreased phosphorylase a activity in the hepatoma cells was increased by additional glycogen but did not exceed the level before the incubation. In the case of normal hepatocytes it was not affected by glycogen. This indicates that glycogen contained in the cells influences the activation of phosphorylase; the glycogen content in AH130 cells was far less than in normal hepatocytes. On the other hand, when intact cells were incubated with a high concentration of glucose, phosphorylase a activity in the homogenate of normal hepatocytes was decreased and could be restored by IPN and dibutyryl cyclic AMP, but the enzyme activity in the homogenate of AH130 cells was very low and hardly changed after the incubation and treatment with these agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drug response assay system in a microchip using human hepatoma cells.

A microchip-based cell response assay system to an anticancer agent was developed. The hepatoma cell line HepG2 was used to assess the effects of an anticancer agent, doxorubicin. The required cell number was reduced by two orders, and the observation of the time course of cell response became possible. The system clearly showed that treatment with higher doses of the drug or longer exposure times gave more effects to cells. The possibilities of novel drug response studies or toxicity assay system were demonstrated.     

Metabolic routes along digestive system of licorice: multicomponent sequential metabolism method in rat

This study was conducted to establish the multicomponent sequential metabolism (MSM) method based on comparative analysis along the digestive system following oral administration of licorice (Glycyrrhiza uralensis Fisch., leguminosae), a traditional Chinese medicine widely used for harmonizing other ingredients in a formulae. The licorice water extract (LWE) dissolved in Krebs–Ringer buffer solution (1 g/mL) was used to carry out the experiments and the comparative analysis was performed using HPLC and LC‐MS/MS methods. In vitro incubation, in situ closed‐loop and in vivo blood sampling were used to measure the LWE metabolic profile along the digestive system. The incubation experiment showed that the LWE was basically stable in digestive juice. A comparative analysis presented the metabolic profile of each prototype and its corresponding metabolites then. Liver was the major metabolic organ for LWE, and the metabolism by the intestinal flora and gut wall was also an important part of the process. The MSM method was practical and could be a potential method to describe the metabolic routes of multiple components before absorption into the systemic blood stream. Copyright © 2015 John Wiley & Sons, Ltd.     

Glycosylated nucleoside lipid promotes the liposome internalization in stem cells

We report new glycosyl-nucleoside-lipid based liposomes decorated with sugar moieties. The GNL-liposomes feature a suitable glycosylated surface for their internalization into ADSC stem cells.     

Sensitivity to antitumor drugs and vinblastine binding to membrane in rat ascites hepatoma AH66 cells.

Rat ascites hepatoma AH66 cells have lower sensitivity to Vinca alkaloids and anthracycline antibiotics than AH66F cells, a subline of AH66 cells. AH66 cells expressed P-glycoprotein, while the protein was not detectable in AH66F cells. There are two affinity sites for [3H]vinblastine binding in the AH66 cell membrane, while AH66F cells have only one affinity site. The high affinity [3H]vinblastine binding in AH66 cells was inhibited by Adriamycin, verapamil, nicardipine, and reserpine. The high affinity site of the binding may be the multidrug transporter, P-glycoprotein. [3H]Vinblastine binding was not influenced by adenosine 3'-5'-monophosphate (AMP), adenosine triphosphate (ATP), or guanosine triphosphate (GTP). The multidrug resistance in AH66 cells may depend on P-glycoprotein which is not modulated by nucleotide.     

Enhancing the stability of kinesin motors for microscale transport applications

Biomolecular motors, such as kinesins, have great potential for micro-actuation and micro- or nanoscale active transport when integrated into microscale devices. However, the stability and limited shelf life of these motor proteins and their associated protein filaments is a barrier to their implementation. Here we demonstrate that freeze-drying or critical point-drying kinesins adsorbed to glass surfaces extends their lifetime from days to more than four months. Further, photoresist deposition and removal can be carried out on these motor-adsorbed surfaces without loss of motor function. The methods developed here are an important step towards realizing the integration of biological motors into practical devices, and these approaches can be extended to patterning and preserving other proteins immobilized on surfaces.     

Synthesis and structure of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic Acid

The first synthesis of (alphaS,betaS)-beta-hydroxy-alpha-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(alphaS,betaS)-11] has been achieved by OsO(4) oxidation of [S-(E)]-4-[4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7-yl]-2-[(methoxycarbonyl)amino]-3-butenoic acid methyl ester (13) followed by successive gamma-deoxygenation through the cyclocarbonates, separation from the (alphaS,betaR)-isomer by means of flash chromatography, and deprotection. On the other hand, the minor nucleoside of rat liver tRNA(Phe) was isolated on a scale of 100 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P(1), followed by reverse-phase column chromatography, complete digestion with nuclease P(1)/alkaline phosphatase, and reverse-phase HPLC. Comparison of this nucleoside with the synthetic one has unambiguously established its structure to be (alphaS,betaS)-11.     

Synthesis of a novel conformationally locked carbocyclic nucleoside ring system

[reaction: see text] A fast and efficient synthetic route to novel Northern locked carbocyclic nucleosides (as precursors of carbocyclic locked nucleic acids or cLNAs) is described. The target nucleoside with a oxabicyclo[2.2.1]heptane ring system was prepared from a simple starting material, diethyl malonate. Ring closure by intramolecular O-alkylation provided the target ring system as the major isomer over the [3.2.0] oxetane system. The adenine moiety was introduced through a reactive triflate after inversion of the stereochemistry of the corresponding alcohol by oxidation and reduction.     

Thermal stability of the system BaO-BaO2

The thermal stability of barium peroxide obtained via the oxidation of BaO was studied. The investigations were carried out with a Q1500D MOM derivatograph. The flow rate of air or oxygen-argon mixtures was 25–30 l/h. It was found that the thermal properties of the BaO-BaO₂ system and of BaO₂ depend on the partial pressures of oxygen and water vapour during the process of oxidation of BaO. The most stable BaO₂ can be produced from high-purity BaO with a special crystal structure.

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Zusammenfassung Es wurde die thermische Stabilität von Bariumperoxid, erhalten durch die Oxidation von BaO, untersucht. Die Untersuchungen wurden mittels eines MOM-Derivatographen Q-1500D ausgeführt. Der Durchsatzstrom der Luft bzw. der Sauerstoff/Argon-Gemische betrug 25–30 1/h. Es zeigte sich, daß die thermischen Eigenschaften des Systemes BaO-BaO₂ bzw. von BaO₂ vom partiellen Druck des Sauerstoffes bzw. vom Wasserdampfdruck abhängen, der bei der Oxida'tion von BaO vorherrscht. Das stabilste BaO₂ konnte aus hochreinem BaO mit einer speziellen Kristallstruktur gewonnen werden.

     

Thermal stability of the amorphous system As-Se-I

The effect of adding iodine to the binary systems As-Se on the thermal stability of the ternary glasses thus formed was investigated by DTA and DDTA. The investigated glasses corresponded to different regions of the phase diagram, i.e. they had different contents of As and I and, consequently, contained different types of structural units.In the investigated ternary systems with iodine, the crystallization effect appears at a lower temperature than in the binary glass As₄₀Se₆₀, and the same holds for the temperatures of softening and melting.For both binary and ternary systems, the activation energies of crystallization were determined. The glass As₄₀Se₆₀ exhibited a somewhat higher activation energy, i.e. a somewhat lower tendency to crystallize than the ternary system.The critical cooling rates of the melts for minimum degree of crystallinity were estimated. The obtained values are approximately equal for the binary (As₂Se₃) and ternary (AsSeI) systems.

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Zusammenfassung Mittels DTA und DDTA wurde untersucht, wie die Zugabe von Jod zu dem binären System As-Se die thermische Stabilität der so gebildeten ternären Gläser beeinflußt. Die untersuchten Gläser entsprechen verschiedenen Regionen des Phasendiagrammes, d.h. sie besitzen einen unterschiedlichen As- bzw. I-Gehalt und enthielten unterschiedlich geartete Struktureinheiten.Bei den untersuchten jodhaltigen ternären Systemen tritt der Kristallisationseffekt bei niedrigeren Temperaturen auf als bei den binären Gläsern As₄₀Se₆₀, das gleiche gilt für die Schmelz- als auch für die Erweichungstemperatur.Sowohl bei den binären als auch bei den ternären Systemen wurde die Aktivierungs energie für den Kristallisationsprozeß ermittelt. Das Glas As₄₀Se₆₀ zeigte eine etwas höhere Aktivierungsenergie, d.h. eine etwas geringere Neigung zur Kristallisation als das ternäre System.Für die kritische Abkühlgeschwindigkeit der Schmelzen zum Erreichen eines minimalen Kristallinitätsgrades wurden bei den binären (As₂Se₃) und ternären (AsSeI) Systemen annähernd gleiche Werte bestimmt.

     

Synthesis and stability of oligonucleotides containing acyclic achiral nucleoside analogues with two base moieties

[structure: see text] Nucleotide building blocks with two base moieties were synthesized and incorporated into oligonucleotides. One of the two bases is involved in base pairing within the double helix, while the other base is sticking out of the minor groove. This system may be used for presenting sequence information at the outside of the helix.     

Redifferentiation of human hepatoma cells induced by green tea polyphenols

A novel approach for the treatment of cancer is the differentiation therapy in which cancer cells are induced to attain a mature phenotype when exposed to differentiation inducers. To examine the effects of polyphenols extracted from green tea, i.e. ( – )-epicatechin (EC), ( – )-epigallocatechin (EGC), ( – )-epicatechin gallate (ECG) and ( – )-epigallocatechin gallate (EGCG), on the proliferation and redifferentiation of human hepatoma cell line SMMC-7721, we measured the changes of cell growth, cell surface charge and cell morphography after treament with green tea polyphenols. It was found that the growth curve of treated cells was decreased remarkably, cell surface charge of treated cells was decreased and the microvilli on the surface of treated cells were reduced obviously. It confirmed that green tea polyphenols could reverse malignant phenotypic characteristics and induced redifferentiation of SMMC-7721 cells. The ability of green tea polyphenols to inhibit reactive oxygen species (ROS)-mediated oxidative damage of DNA was also assessedin vitro by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular and linear forms. It was found that green tea polyphenols could significantly inhibit the oxidative damage of DNA induced by a water-soluble azo initiator 2,2-azobis(2-amidinopropane hydrochloride) (AAPH). However, they could promote the oxidative damage of DNA induced by H₂O₂ and Fe²⁺ at high concentrations. The relationship between the anti-cancer activity and antioxidation-prooxidation activity of green tea polyphenols is discussed.     

Identification of members of the annexin family in the detergent-insoluble fraction of rat Morris hepatoma plasma membranes

For proteomic analysis, plasma membranes of rat hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized according to the previously developed method [D. Josic, K. Zeilinger, Methods Enzymol. 271 (1996) 113-134]. If the Triton X100 insoluble pellet is subsequently extracted, several proteins can be solubilized. These proteins can be classified in two groups according to their molecular size. The proteins with apparent molecular weights in SDS-PAGE between 70 and 75 kDa belong to the first group. Smaller proteins, with apparent molecular weights between 30 and 45 kDa, are members of the second group. The main protein of higher molecular weight was also found in the Triton X100 insoluble extract from normal rat liver plasma membranes. This protein was identified as Annexin A6. The proteins from the second group are practically absent in the Triton X100 insoluble extract from rat liver. These proteins are present in relatively high concentrations in plasma membranes of Morris hepatoma 7777. Both groups of detergent-insoluble proteins from Morris hepatoma 7777 were further analyzed with SELDI-TOF and LC electrospray ionization mass spectrometry. From the first group, Annexin A6, together with two other integral plasma membrane proteins, was identified. In the second group of proteins with apparent molecular weights between 30 and 45kDa, further members of the annexin family, Annexins A1, A2, A4, A5 and A7 were identified. The possible role of these low molecular size annexins as potential cancer biomarkers is discussed.     

Thermal stability of rat uterus during development

Summary The age dependence of thermal denaturation was monitored in rats anaesthetized after they're born at 7^th, 14^th, 21^st, 28^th, 35^thand 42^nddays. The samples were stored in rigor or physiological saline solution. The DSC scans in the early age groups show a low temperature exotherm (connective tissues: from gel to liquid crystal transition) and one endotherm (it is very probably the myosin). During further development the endotherms became more and more complex (due to the development of contractile system). At 42 days the scans seem to be similar to the adult ones. In the two buffers the endotherms markedly differ showing that the ATPase activity is present. In adult uterus, treated with nucleotides or estrogen, this activity significantly differs from the skeletal muscle. On the basis of our results we suppose, that the age dependent changes are decisive processes in the development of rat uterus.