Loss of CMS-specific mitochondrial DNA arrangement in fertile segregants of Petunia hybrids

The progeny of somatic hybrid Petunia plants derived from the fusion of a male-fertile line and a cytoplasmic male-sterile (cms) line were examined. Male-fertile progeny derived from three different male-sterile somatic hybrid plants did not exhibit the mitochondrial DNA (mtDNA) arrangement which has previously been correlated with cms in Petunia. The cms-associated mtDNA arrangement was present in the male-sterile predecessors of these fertile revertants. Thus, it is concluded that the loss of this mtDNA arrangement is associated with reversion to fertility in the progeny of the unstable somatic hybrid petunia plants.     

The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events : Studies with vascular smooth muscle cells and endothelial cells

We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFN alpha, recombinant human IFN alpha 2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent.     

Selective incorporation of 14C-arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to 14C-prostaglandins

A method is described for the efficient incorporation of radioactive arachidonic acid into the lipids of rabbit hearts and kidneys. Infusion of ¹⁴C-arachidonate through perfused tissues resulted in the quantitative removel of label from the media. Analysis of the lipids from tissues labeled by this procedure revealed that the majority of the ¹⁴C-arachidonate was incorporated into phospholipids. Essentially all of the radioactivity in phosphatidylcholine was found in the 2-position. Subsequent to the ¹⁴C-arachidonate infusion, stimulation of prostaglandin biosynthesis (e.g. by bradykinin) resulted in the release of radioactive prostaglandins. This suggests that the ¹⁴C-arachidonate is incorporated in a manner such that it is available for homone-stimulated prostaglandin biosynthesis. The method described allows both qualitative and quantitative analysis of arachidonate metabolism in intact tissues and offers significant advantages over other presently used methods.     

Bacillus subtilis DNA Polymerase III Is Required for the Replication of the Virulent Bacteriophage φe

The virulent phage phie of Bacillus subtilis which contains hydroxymethyluracil in its DNA requires host DNA polymerase III for its DNA replication. DNA polymerase III(ts) mutant cells infected with phie at restrictive temperatures do not support phage DNA synthesis. However, phie grows normally both at low and high temperatures in the mutant's parent strain and in spontaneous DNA polymerase III(+) revertants isolated from the mutant strain. Temperature-shift-down experiments with phie-infected cells having thermosensitive DNA polymerase III (pol III(ts)) indicate that at 48 C the thermolabile DNA polymerase III is irreversibly inactivated and has to be synthesized de novo after the shift to 37 C, before phage DNA synthesis can begin. Temperature-shift-up experiments with phie-infected mutant cells show that phage replication is arrested immediately after the temperature shift and indicate that phie requires DNA polymerase III throughout its replication stage.     

Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca²⁺-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca²⁺-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent K_m values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (V_max.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E₂ synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca²⁺ stimulated endogenous arachidonate deacylation and prostaglandin E₂ generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca²⁺-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: Pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to supress aggregation, through the action of their respective non-enzymatic breakdown products PGE₁, PGD₂, or PGD₃ all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cycloogygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A₂, and enhance formation of the anti-aggregatory metabolite, prostacyclin.Whereas EPA is not detectably metabolized by platelets, dihomo-γ-linolenic acid (8,11,14,-eicosatrienoic acid) is primariley converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH₁ and DLL which was partially compromised by the PGE₁ formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH₁. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A₂.     

Myo-Inositol Turnover in the Intact Rat Brain: Increased Production after d-Amphetamine

Abstract: Apparent turnover of myo -inositol in the brain of urethane-anesthetized rats was estimated in vivo from the rate of appearance of endogenous myo -inositol in the cerebroventricular compartment. Ventricular-cisternal perfusion technique combined with isotope dilution of [¹⁴C] myo -inositol was used to determine the rate of appearance of brain-produced myo -inositol and its modification by d -amphetamine. A mean value of 0.75 nmol/min was obtained for the rate of appearance in the cerebroventricular system. A dose dependent increase in this rate was seen after the administration of d -ampheta-mine. The endogenous removal of myo -inositol from the perfusate was also studied and found to be mediated in part by a saturable transport system that was not influenced by d-amphetamine. The rate of entry of myo -inositol from blood to the erebroventricular system was very low and accounted for only 2% of the total rate of appearance, indicating that the majority of myo -inositol in the rat cerebroventricular fluid originates in the brain.     

Properties of prostaglandin synthetase of rabbit kidney medulla.

The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.     

Indomethacin and aspirin inhibition of prostaglandin E2 synthesis by sheep seminal vesicles microsome powder

Sheep seminal vesicles microsome powder was used as a source of prostaglandin synthetase in studies on the nature of inhibition of prostaglandin synthesis by indomethacin and aspirin. Irdomethacin inhibition was found to be highly irreversible, although partial recovery of synthetase activity was obtained after extensive dialysis. A major difference was observed between the effects of aspirin and indomethacin on prostaglandin synthetase activity in seminal vesicles slices. Enzyme activity in microsomes prepared from slices incubated with aspirin was markedly inhibited while the activity in microsomes prepared after incubation with indomethacin was not affected. These results suggest that indomethacin may penetrate intracellularly very slowly, or not at all, and raise a question as to whether the inhibition by indomethacin $\text{in vivo}$ is mediated via direct inhibition of prostaglandin synthesis.