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     

Cultivated potato chloroplast DNA differs from the wild type by one deletion — evidence and implications

Summary The chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned     

A convenient approach to the synthesis of medium size oligodeoxyribonucleotides by improved new phosphite method.

Improvement of the new phosphite method for the synthesis of oligodeoxyribonucleotides using the deoxyribonucleoside 3'-bis(1,1,1,3,3,3- hexafluoro-2-propyl) phosphite unit has been carried out via the hydrolysis and capping steps, without any side reaction products. The new phosphite unit and capping agent, bis(1,1,1,3,3,3-hexafluoro-2-propyl)-2-propyl phosphite, is readily activated by N-methylimdazole under very mild condition on a solid support. This operation involves a one pot reaction, which is an advantage over both the phosphite and H- phosphonate approaches. The mechanism of internucleotidic bond formation of the new phosphite method is also discussed.     

Who is the mother of the potato? — restriction endonuclease analysis of chloroplast DNA of cultivated potatoes

Summary Chloroplast DNA from 44 lines of 16 wild and 7 cultivatedSolanum species were compared by restriction endonuclease analysis. Seven chloroplast genome types were identified among them by 5 restriction enzymes: Type A (S. tuberosum ssp.andigena andS. maglia); Type S (S. goniocalyx, S. phureja, S. stenotomum, S. ×chaucha and a line of ssp.andigena); Type C (S. acaule, S. bukasovii, S. canasense, S. multidissectum andS. ×juzepczukii); Type T (S. tuberosum ssp.tuberosum); Type W (other wild species); Type W (S. chacoense f.gibberulosum) and Type W (S. tarijense). From this cytoplasmic identification, it was concluded thatS. goniocalyx, S. phureja, S. ×chaucha and ssp.andigena were derived fromS. stenotomum or its primitive type, which may have originally evolved itself fromS. canasense. The chloroplast genome of the European potato, however, was introduced from the Chilean potato, which might have been primarily constructed with the nuclear genome from ssp.andigena and with cytoplasm from other species. The cytoplasmic donor of the Chilean potato could not be determined.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 479. This work was done at Kyoto University when the author was a graduate student at Kobe University     

Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth

Yeasts of wild-type strains, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were shown to have the ability to form aminopropylcadaverine and aminopropylhomospermidine from cadaverine and homospermidine, respectively. A polyamine autotroph S. cerevisiae 179-5, which lacks ornithine decarboxylase, produced both aminopropylcadaverine and aminopropylhomospermidine, while another mutant S. cerevisiae Y 260 A, which lacks spermine synthase, formed only aminopropylcadaverine. Naturally-occurring triamines and tetraamines except norspermidine and norspermine stimulated the growth of S. cerevisiae 179-5. All the six aliphatic diamines with carbon chain length ranging from one to six were effective in activating the growth of S. cerevisiae 179-5, though all of them were not converted to either triamines or tetraamines.     

Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A

Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.     

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.