Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

Characterization of cardiac innervation in the nudibranch,Archidoris montereyensis

Summary The heart of the nudibranch mollusc Archidoris montereyensis is regulated by a small number of powerful effector neurons located in the right pleural and visceral ganglia. Two identifiable neurons in the pleural ganglion, a heart excitor (pl_HE) and a heart inhibitor (Pl_HI), are especially important regulators of cardiac function in that low levels of spontaneous activity in either cell significantly alters the amplitude and rate of heart contractions. These neurons have extensive dendritic arbors within the right pleural ganglion and branching axonal processes within the visceral ganglion. The visceral ganglion also contains a heart excitor neuron (V_HE) and at least two heart inhibitor neurons (V_HI cells), but their influence on cardiac activity is weaker than that of the pleural ganglion cells. All of these heart effector cells appear to be motor neurons with axons that terminate predominately in the atrio-ventricular valve region of the heart via the pericardial nerve. The simplicity and strength of these neuronal connections to the heart of Archidoris make this a favorable preparation for studies of cardiac regulation.Abbreviations Pl _HE pleural ganglion heart excitor neuron - Pl _HI pleural heart inhibitor neuron - V _HE visceral ganglion heart excitor neuron - V _HI cells, visceral heart inhibitor neurons - V _K visceral kidney excitor neuron - V _G visceral gill excitor neuron     

Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+ and Nadp+ in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant of Euglena Gracilis Klebs (Strain Z)

-We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP⁺ and NAD⁺, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CI), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2⁺-calmodulin) or for its substrate, NAD⁺. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.     

C1q,a collagen-like complement subcomponent,in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.     

Inhibited enzyme electrodes. Part 3: A sensor for low levels of H2S and HCN

It is shown that an inhibited enzyme electrode, using cytochrome oxidase, will respond to H₂S, HCN and azide ion. For all three inhibitors the kinetics of the inhibition and recovery processes have been analysed using the theoretical model presented previously (Albery et al., 1990a). Rearrangement of the differential equation describing inhibition and the development of the necessary software has enabled us to obtain values of the concentration of inhibitor in a matter of seconds after exposure of the sensor. The sensor will measure concentrations of H₂S down to 1 ppm in the gas phase and concentrations of HCN and azide ion down to 0·4 μmol dm⁻³ in the solution     

The role of phospholipase A2 in calcium-induced damage in cardiac and skeletal muscle

Summary This study compares the action of inhibitors of the eicosanoid cascade on calcium-induced myofilament damage in cardiac muscle of the perfused frog heart and incubated frog ventricle slices, and in skeletal muscle of incubated mammalian diaphragm and isolated and saponin-skinned amphibian pectoris cutaneous muscle. Mepacrine (10^-5M) and indomethacin (3×10^-6M) protected completely against myofilament damage induced by entry of calcium in the calcium-paradox in frog heart. However, inhibition of phospholipase A₂ (PLA₂) (with chlorpromazine, 2×10^-4M, or mepacrine, 10^-5M, 5x10^-5M), of cyclo-oxygenase enzymes (with indomethacin, 3x10^-6M to 10^-5M or BW755C, 3.8x10^-4M), or of lipoxygenase enzymes (with BW755C, 3.8x10^-4M or nordihydroguaiaretic acid, 2x10^-6M or 5x10^-6M) all failed in intact cardiac or skeletal muscle cells to prevent the myofilament damage that is rapidly triggered by 10^-2M caffeine, 6x10^-6M ruthenium red, 10^-4M DNP or 5 g ml^-1 A23187. These agents also failed completely to protect against myofilament damage in saponin-skinned amphibian skeletal muscle when [Ca]_i was raised to 8x10^-6M. Thus, inhibition of PLA₂ does not protect the myofilament apparatus against calcium released intracellularly, and it is suggested that mepacrine and indomethacin can block entry of calcium in the calcium-paradox in the amphibian heart. Chlorpromazine (2x10^-4M) and mepacrine (10^-3M) at zero [Ca] caused severe myofilament damage in skinned muscle, possibly due to an effect on membranes. Since inhibitors of PLA₂ and of lipoxygenases prevent efflux of creatine kinase and sarcolemma damage in mammalian skeletal muscle, it is evident that experimentally-induced rises in [Ca]_i (by caffeine or A23187) can trigger two separate pathways: (i) PLA₂ and the arachidonic acid cascade which culminate in membrane damage, and (ii) a different, Ca-activated system that causes rapid damage of myofilaments.     

In vitro and ex vivo effects of antidepressants on rat brain membrane-bound phosphatidylinositol synthetase activity

The in vitro and ex vivo effects of antidepressant drugs on membrane-bound phosphatidylinositol (PI) synthetase and PI: myo-inositol exchange enzyme activities were examined. In rat brain subcellular fractions, PI synthetase occurred exclusively in the microsomes. In comparison, the activity of CDP-diglyceride independent PI: myo-inositol exchange enzyme was low (3%). Of the various CDP-diglycerides tested for the activation of PI synthetase, CDP-dipalmitin was the most active. Addition of 1 mM of desipramine, amitriptyline, imipramine, iprindole, clomipramine and mianserin in vitro significantly inhibited (30–60%) PI synthetase activity, whereas the same concentration of zimelidine and fluoxetine had no effect. At low liponucleotide concentrations, PI synthetase activity was significantly enhanced by imipramine (1 mM), whereas the enzyme activity was inhibited at higher liponucleotide concentrations (>0.3 mM). In contrast, imipramine had no effect on the PI: myo-inositol exchange enzyme activity. No significant alteration in the PI synthetase activity was found following either acute (2 h) or chronic (21 d) treatment of rats with imipramine. The above results indicate that the de novo synthesis of PI is inhibited in vitro but not ex vivo by some antidepressant drugs. However, in view of the high concentration of the drugs required, the pharmacological significance of this inhibitory action with respect to their therapeutic effects is doubtful.