Facile construction of substituted pyrimido[4,5-d] pyrimidones by transformation of enaminouracil

The reaction of 6-amino-1,3-dimethylpyrimidine-2,4[1H,3H]-dione [1] as a binucleophile with primary aromatic or heterocyclic amines and formaldehyde or aromatic [heterocyclic] aldehydes in a molar ratio [1:1:2] gave the pyrimido[4,5-d]pyrimidin-2,4-dione ring systems 2-5. Treatment of 1 with diamines and formalin in molar ratio [2:1:4] gave the bis-pyrimido[4,5-d]pyrimidin- 2,4-diones 6-8. Furthermore, substituted pyrimido[4,5-d]pyrimidin-2,4-diones with uracil derivative 11 or spiro indole 16 were synthesized. Synthesis of pyrimido[4,5-d]pyrimidin-2,4-diones with different substitution at C-5 and C-7 was achieved to give 13 and 18, respectively

Rapid increase of cytosolic content of acetyl-CoA carboxylase isoforms in H9c2 cells by short-term treatment with insulin and okadaic acid

Mammalian acetyl-CoA carboxylase (ACC) is present in two isoforms, alpha and beta, both of which catalyze formation of malonyl-CoA by fixing CO2 into acetyl-CoA. ACC-alpha is highly expressed in lipogenic tissues whereas ACC-beta is a predominant form in heart and skeletal muscle tissues. Even though the tissue-specific expression pattern of two ACC isoforms suggests that each form may have a distinct function, existence of two isoforms catalyzing the identical reaction in a same cell has been a puzzling question. As a first step to answer this question and to identify the possible role of ACC isoforms in myogenic differentiation, we have investigated in the present study whether the expression and the subcellular distribution of ACC isoforms in H9c2 cardiac myocyte change so that malonyl-CoA produced by each form may modulate fatty acid oxidation. We have observed that the expression levels of both ACC forms were correlated to the extent of myogenic differentiation and that they were present not only in cytoplasm but also in other subcellular compartment. Among the various tested compounds, short-term treatment of H9c2 myotubes with insulin or okadaic acid rapidly increased the cytosolic content of both ACC isoforms up to 2 folds without affecting the total cellular ACC content. Taken together, these observations suggest that both ACC isoforms may play a pivotal role in muscle differentiation and that they may translocate between cytoplasm and other subcellular compartment to achieve its specific goal under the various physiological conditions.

Purification of Opioid Receptor in the Presence of Sodium Ion / 대한마취과학회지

BACKGROUND: Purification of opioid receptor is mandatory to improve opiate analgesic medication. Recently, it was reported that sodium ion increased the number of opioid binding sites for opioid antagonist. The importance of sodium ions lead us to design appropriate affinity chromatography and binding assay for the successful purification of mu-opioid receptor to homogeneity. METHODS: Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents, CHAPS and digitonin, in the presence of protease inhibitors and 1M NaCl. The solubilized material was passed through an opioid antagonist(10cd) affinity column and a wheat germ agglutinin(WGA) column, set up in series, to obtain a partially purified receptor preparation. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. RESULTS: Binding of opioid antagonist [H]diprenorphine to the partially purified or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed diffuse band with a medium molecular mass of 62KD upon electrophoresis. The average specific binding activity of the purified receptor was 18.8+/-2.3 pmol/mcg protein. CONCLUSIONS: Opioid agonists and antagonists either do not bind or bind with low affinity to G protein-dissociated free opioid receptors in the absence of sodium ions. However, the free opioid receptors have a high affinity for antagonists but not agonists in the presence of sodium ions.

ATP and Ca2+ homeostasis in Trypanosoma cruzi

Cell viability requires the perfect functioning of the processes controlling ATP and Ca2+ homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with disruption of ATP and Ca2+ homeostasis. Therefore, the study of the mechanisms by which different T. cruzi stages regulate the intracellular Ca2+ distribution and the ATP supply to maintain cell viability could provide new insights into the physiology of these parasites. One important objective of these studies is the identification of possible metabolic differences between host and parasite that could be exploited for the rational design of new and more effective trypanocidal drugs

Calcium homeostasis in Trypanosoma cruzi

By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin-permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium transport by Hela cell mitochondria

We studied mitochondria of digitonin-permeabilized Hela cells, since digitonin (60 ug/10 6 cells) increases plasma membrane Ca2+ permeability, in order to avoid problems such as low mitochondrial yield and the possibility of obtaining damaged or uncoupled mitochondria from tumor cells. Addition of Ca2+ to digitonin-permeabilized Hela cells gave rise to a cycle of respiratory stimulation. Ca2+ uptake was almost totally inhibited by antimycin A (6.0 ug/ml). Ca2+ release occurred upon addition of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) to digitonin-permeabilized Hela cells, under state conditions. An antimycin A-and FCCP-insensitive Ca2+ uptake was also detected in this preparation when ATP was added, which reflects Ca2+ capture by other organelles. The characteristics of the mitochondrial Ca2+ transport system in Hela cells are similar to those of other previously studied tumor cells. Mitochondria from Hela cells are resistant to the deleterious effects of massive Ca2+ loads

Efeito in vitro do esteviosídeo sobre a atividade lisossômica / In vitro effect of stevioside upon the lysossomal activity

O estudo da açäo do esteviosídeo sobre a fisiologia celular é amplamente justificado em funçäo da utilizaçäo deste glicosídeo na dieta e nas indústrias farmacêuticas e de alimentos. Neste trabalho, analisaram-se os efeitos que soluçöes de esteviosídeo, nas concentraçöes finais de 3,3 x 10*-9M, 3,3 x 10*-4M, provocaram sobre a estabilidade das membranas dos diversos componentes do compartimento lisossômicos, isolados de fígados de camundongos. Os resultados indicaram que as soluçöes nas concentraçöes 3,3 x 10*-6M, 3,3 x 10*-5M e 3,3 x 10*-4M têm efeito estabilizador sobre os complexos de membranas de lisossomos hepáticos, enquanto que as soluçöes nas concentraçöes 3,3 x 10*-9M, 3,3 x 10*-8M e 3,3 x 10*-7M näo determinaram nenhum efeito estatisticamente significante que pudesse ser avaliado através da metodologia aqui empregada

Detection of cholesterol by digitonin in coated membranes of the Golgi complex in the guinea pig spermatid

En la face de maduración o condensación del complejo de Golgi de la espermátide de cobayo, se presentan las membranas de las cisternas densas y de las vesículas de condensación con características especiales que las diferencian del resto de las membranas del Golgi. Las membranas de la fase de condensación son más gruesas, están en su mayor parte cubiertas por una trama poligonal del tipo característico de la clathrina y tratadas con la técnica de la digitonina forman los típicos rulos y agujas del complejo digitonina-colesterol. Esta observación revela que ambos componentes - clathrina y colesterol - pueden coincidir en la misma membrana, se concentran en la cara interna del Golgi y por fusión de membranas se transfieren a la membrana externa del acrosoma