Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

Participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain

The participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain was examined. A reaction mixture of [1-14C]hexadecanoic acid with brain microsomes and NADPH formed two radioactive products having the same mobilities as pure hexadecanal (RF 0.61) and hexadecanol (RF 0.22), respectively, on TLC plates. The product of the RF 0.61 spot was further identified as hexadecanal using gas-liquid chromatography after methylation and TLC of its reduced product with LiAlH4 and semicarbazide. The ratio of hexadecanal to hexadecanol varied from 0.4 to 1.2 under the present experimental conditions. When solubilized rat brain microsomes were applied to a Sepharose 4B column coupled with the rabbit antibody raised against rat liver microsomal NADPH-cytochrome-c reductase, which reacts with aldehyde reductase from rat brain, the eluted fraction ceased to form [14C]hexadecanol but continued to form [14C]hexadecanal from [14C]hexadecanoic acid. These results strongly indicate that hexadecanal is the intermediate in the synthesis of hexadecanol from hexadecanoic acid in rat brain microsomes with the participation of microsomal aldehyde reductase.     

Isolation of chicken anemia agent with MDCC-MSB1 cells from chickens in the field

An attempt was made to isolate chicken anemia agent (CAA) from chickens suffering from anemia in the field by using MDCC - MSB1 , which was an established cell line derived from Marek's disease lymphoma. When 99 chickens of 15 flocks were examined, CAA was isolated from 58 chickens of 12 flocks. The rate of CAA isolation with MDCC - MSB1 cells was almost the same as that determined by an in vivo method by chick inoculation. It was shown that CAA was more closely concerned with anemic diseases of chickens in the field than fowl adenoviruses.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.     

Correlation between polyploidy and auxotrophic segregation in the imperfect yeast Candida albicans.

In order to clarify the relationship between polyploidization and the capability of phenotypic switching in the imperfect yeast Candida albicans, two types of variants were isolated as segregants from a fusant, which produced a proportion of the cell population with a higher ploidy than the rest, either in a temperature-dependent or -independent manner, when incubated at low (28 degrees C) and high (37 degrees C) temperatures. In the case of the temperature-dependent type of variants, high-ploidy cells appeared at 37 degrees C but rarely at 28 degrees C. This phenotype was named Pldts (temperature-sensitive polyploidization), and the temperature-independent phenotype was called Pld-. The appearance of high-ploidy cells in the culture of the Pldts strain at 37 degrees C was accompanied by a significant increase in the frequency of auxotrophic variants; these variants probably occur as a result of segregation of auxotrophic markers from the heterozygous to the homozygous state. Both Pldts and Pld- phenotypes were recessive in a fusion with a Pld+ parent. An adenine auxotrophic marker (ade1) was introduced into a Pldts strain in a heterozygous state, and the individual high-ploidy cells of this strain, grown at 37 degrees C, were micromanipulated to form colonies, which consisted of red and white sectors appearing at high frequency on a pink background. When the ade1 auxotrophy was introduced into Pld- strains, frequently sectored colonies were produced. These results suggested an increased level of chromosome missegregation in both types of Pld mutants. Analyses by pulsed-field gel electrophoresis of Ade-segregants, derived from a micromanipulated high-ploidy cell of a Pld(ts) strain, suggested the occurrence of nonreciprocal recombination, some of which includes chromosome loss.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.