Metabolic interlock between the acetolactate synthase isoenzymes and lysine biosynthesis in Escherichia coli K-12

Summary Some of the strains containing mutations in the genes for the acetolactate synthase isoenzymes are temperature sensitive (ts). Suppression of the acetolactate synthase defect due to one of these mutations suppresses also the ts phenotype; moreover, a genetic cross shows that the two phenotypes cannot be dissociated.The ts phenotype is accompanied by a decreased efficiency of transduction with Pl phage. Observations at the light microscope show formation of abnormal cells. Under specific conditions diaminopimelate stimulates growth and restores normal transduction efficiency. The rate of diaminopimelate formed and excreted by non-growing cells decreases when an acetolactate synthase mutation is present.We give evidence that the ts phenotype is due to an increased formation of lysine from diaminopimelate; this causes a starvation for the latter and therefore cell wall abnormalities. In fact, even at the permissive temperature, the lysine pool is 8x increased in a strain with an acetolactate synthase defect, while a slight decrease in the diaminopimelate pool is observed. Moreover, introduction into a ts strain of a mutation in lysA (the gene coding for diaminopimelate decarboxylase) cures the ts phenotype. Finally among the temperature resistant revertants we found some lysine auxotrophs.     

Adipose conversion of Ob17 preadipocytes : Relationships between cell division and fat cell cluster formation

Adipose conversion of Ob17 preadipocyte cells proceeds after confluence with the formation of fat cell clusters, due to the coexistence of cells susceptible or not to adipose conversion. In order to determine whether commitment to differentiation occurs after quiescence or during exponential growth, the spatial arrangement of Obl7 cells was destroyed at different times before and after confluence by trypsinization followed by cell reinoculation. The resulting distribution of lipid-filled cells, as compared to non-replated control cells, indicates that both insusceptible and susceptible cells are present during the growth phase in the absence of insulin. It is shown that the formation of fat cell clusters of large size is due to mitoses of susceptible cells during a limited period of time after confluence. Blockade of post-confluent mitoses by selective elimination of cells in the S phase abolishes the formation of clusters of large size, but single differentiated cells and clusters containing a few cells remain present. Therefore post-confluent mitoses are not necessary for the differentiation to occur, but rather they serve to amplify the proportion of adipose cells relative to non-adipose cells.     

Regulation of adipose cell differentiation. II. Kinetics of induction of the aP2 gene by fatty acids and modulation by dexamethasone.

Fatty acids behave as activators of the aP2 gene expression in committed, lipid-free, non-terminally differentiated Ob1771 cells. Like fatty acids, dexamethasone provokes a dose-dependent accumulation of aP2 mRNA. However, fatty acids and dexamethasone act through different mechanisms to activate the aP2 gene expression since i) fatty acids and dexamethasone act in a synergistic manner; ii) the effect of dexamethasone is rapid and transient (maximal effect after 8 h), whereas that of fatty acids is slower, and maintained as long as the inducer is present and is fully reversible upon fatty acid removal; iii) the induction of the aP2 gene expression by dexamethasone does not require ongoing protein synthesis, while the response to fatty acids is completely prevented by cycloheximide; and iv) the induction of the aP2 gene expression by fatty acids but not by dexamethasone is confined to preadipocyte cell lines. This suggests that the process of activation by fatty acids, rather than the expression of the aP2 gene, is unique to adipose cells. Besides their effects on the aP2 gene, fatty acids activate the expression of the acyl CoA synthetase gene which encodes another protein involved in fatty acid metabolism. Activation of both genes by fatty acids appears not to be mediated by the CCAAT enhancer binding protein, a nuclear factor reported as transactivator of the aP2 promoter activity, since the enhancer binding protein mRNA is not expressed under these conditions.     

Dihydropyridine Modulation of Voltage-Activated Calcium Channels in PC12 Cells: Effect of Pertussis Toxin Pretreatment

In this study, we report the effect of pertussis toxin pretreatment on dihydropyridine modulation of voltage-sensitive calcium channels in PC12 cells. The rise in intracellular calcium concentration caused by potassium depolarization is not affected significantly by pertussis toxin pretreatment. Nicardipine, a dihydropyridine derivative, added either before or after potassium-induced depolarization, reduces the resultant elevation in cytosolic calcium level both in control and in pertussis toxin-treated cells. The dihydropyridine agonist Bay K 8644, when added before potassium, is able to enhance the potassium-induced spike of cytosolic calcium levels, an effect significantly reduced by pertussis toxin pretreatment. Moreover, the addition of Bay K 8644 after potassium holds the intracellular calcium concentration at a cytosolic sustained level during the slow inactivating phase of depolarization. This effect of Bay K 8644 is inhibited by nicardipine. Pertussis toxin pretreatment slightly weakens the effect of Bay K 8644 when added after potassium-induced depolarization, whereas it significantly reduces the nicardipine inhibition of cytosolic calcium rise stimulated by potassium and Bay K 8644, but not by potassium alone. In conclusion, our findings suggest that a pertussis toxin-sensitive guanine nucleotide regulatory protein could be involved in the interaction between dihydropyridine derivatives and voltage-dependent calcium channels.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

On coenzyme Q orientation in membranes: A linear dichroism study of ubiquinones in a model bilayer

Summary A general approach is developed to interpret linear dichroism (LD) spectra of ubiquinones (Q _n) in host bilayers. Information is reported in terms of guest-host mutual orientation and localization. The overall orientational anisotropy of guest ubiquinone molecules is described by a basic set of limiting orientation/localization modes. Assignments of the UV transitions of the ubiquinone chromophore were obtained by the liquid crystal-linear dichroism technique and molecular orbital (CNDO/S) calculations. The LD spectra of Q _n in the bilayers provided by the lyotropic nematic mesophase exhibited by water solutions of potassium laurate and decanol were interpreted on the basis of the above assignments. The resulting experimental evidence showed a multisite distribution in the host bilayer for the aromatic heads of all the investigated Q _n derivatives except Q₀. The orientational distribution suggested by the LD spectra fits the solubilization model recently proposed by G. Lenaz [J. Membrane Biol. (1988) 104:193–209] for ubiquinone in lipid membranes. Within this model Q _n molecules are located in the midplane and their headgroups oscillate transversally across the membrane. Q ₀ instead has a single site location, close to the polar bilayer interface. Experimental evidence that the headgroup carbonyls tend to grasp the polar interface of the host bilayer was also obtained. Orientation and location distributions of Q _n guest molecules are therefore likely to result from the tendency of their aromatic heads to grasp the polar heads of the host bilayer and from the concurrent tendency of their chains to settle into the hydrocarbon host interior.abbreviations AA average absorption - OD, OD optical densities for plane polarized radiations parallel () and perpendicular () to the sample optical axis - OD OD — OD - EPR electron paramagnetic resonance - LC-LD liquid crystal-linear dichroism - LD linear dichroism - LD _r reduced linear dichroism. - MO molecular orbital - N nematic - NMR nuclear magnetic resonance - S _jj order parameters of the directions j of the transition moments of the guest chromophore - S _ii order parameters of the orientational axes i of the guest molecule with respect to the magnetic field - S _ii order parameters of the axes i of the guest molecules with respect to the bilayer axis a - S _a order parameters of the host bilayer axis a with respect to the orienting magnetic field - _j,i deflection angles between the directions j and the axes i - O _i optical factors of the i axis see Eq. (A4)] - Q_n ubiquinone whose isoprenoid chain contains n isoprenoid units Dr. A. Rossi is gratefully acknowledged for the t.e.m. reduction of the spectra. Ubiquinone homologs were kind gifts from Eisai Co., Tokyo, Japan. This work was supported by M.U.R.S.T., and C.N.R. Target Project on Biotechnology and Bioinstrumentation, Rome, Italy.     

Interleukin 6 modulation of second messenger systems in anterior pituitary cells.

We investigated the effect of interleukin-6 (IL-6) on second messenger systems in anterior pituitary (AP) cells. The acute exposition of membranes derived from the pituitary gland to IL-6 did not modify basal and forskolin-stimulated adenylate cyclase (AC) activity, as well as inositol phosphate (IP) production and free [Ca(++)]i. Preincubation of AP cells with IL-6 for 20 min did not affect basal second messengers levels, while completely abolished the stimulation by VIP of AC activity, partially inhibited forskolin-stimulated cAMP formation and reduced TRH-stimulated IP production. Finally, the pretreatment of AP cells for 20 min with IL-6 also reduced the TRH-induced rise in free [Ca(++)]i.     

Aeration: A simple method to control vitrification and improve in vitro culture of rare australian plants

Summary Aeration of tissue cultured rare Australian plantsConostylis wonganensis S.D. Hopper (Haemodoraceae);Diplolaena andrewsii Ostenf.;Drummondita ericoides Harvey (Rutaceae);Eremophila resinosa F. Muell. (Myoporaceae);Eucalyptus ‘graniticola’ (Myrtaceae);Lechenaultia pulvinaris C. Gardner (goodeniaceae); andSowerbaea multicaulis E. Pritzel (Liliaceae) has been found to reduce vitrification in sensitive species as well as significantly improving shoot quality and transfer to soil in most study species. A simple 7-mm hole with a double-layer insert of filter paper in the polypropylene screw lids of the culture vessel decreased shoot vitrification over a 4-wk culture period. The method has implications for facilitating the tissue culture of other rare Australian plants and reducing the occurrence of this developmental abnormality.