ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Vascular reactions to horseradish peroxidase in the guinea pig

Summary Albino guinea pigs were given intradermal injections of the protein tracer horseradish peroxidase. In a 0.1 mM concentration the tracer did not increase vascular permeability to Evans blue-labelled plasma proteins. In a 1 mM concentration, however, the peroxidase induced a local vascular leakage. This leakage was almost totally inhibited by pretreating the animals with acetylsalicylic acid, while antihistamine had only a weak inhibitory effect. We therefore believe that prostaglandins are important mediators in this HRP-induced vascular reaction.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a²⁺₃-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a²⁺₃-NO and recombination of NO with cytochrome a²⁺₃ (in the 30–70 K region) revealed no differences in structure between cytochrome a²⁺₃ in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a²⁺₃-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a²⁺₃-NO. Photodissociation of cytochrome a²⁺₃-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a²⁺₃ to cytochrome a³⁺. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20–40 K region) which differ completely from those observed in cytochrome a²⁺₃-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a²⁺₃-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu²⁺_B-NO compound. The triplet species with Δm_s = 2 EPR signals at g 4 and Δm_s = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

A substance released by metamorphosing larvae and young polyps ofHydractinia echinata induces metamorphosis in conspecific larvae

Summary A metamorphosis-inducing factor was isolated from medium conditioned by either metamorphosing larvae or 3-day postmetamorphic primary polyps. The factor has a molecular weight 8 kDa and is heatlabile. It does not induce metamorphosis of isolated posterior fragments and is therefore not identical to the internal signal described by Schwoerer-Böhning et al. (1990). The biological significance of the substance is currently unclear, therefore its inducing activity may be a side effect.     

Oxygenation of malignant tumors after localized microwave hyperthermia

Summary The oxyhemoglobin saturation (HbO₂) of single red blood cells within tumor microvessels (diameter: 3–12 µm) of DS-Carcinosarcoma was studied using a cryophotometric micromethod. In untreated control tumors (mean tissue temperature approx. 35° C) the measured values scattered over the whole saturation range from zero to 100 sat.%, the mean being 51 sat.%. Upon heating at 40° C for 30 min, the oxygenation of the tumor tissue significantly improved as compared with control conditions. After 40° C-hyperthermia a mean oxyhemoglobin saturation of 66 sat.% was obtained. In contradistinction to this, after 43° C-hyperthermia the tumor oxygenation was significantly lower and reached a mean HbO₂ saturation value of 47 sat.%. A further temperature rise to 45° C caused the oxygenation to drop drastically (mean oxyhemoglobin saturation value: 24 sat.%). This is due to a severe restriction of nutritive blood flow.The changes in tumor oxygenation after hyperthermia seem to be predominantly mediated through changes in tumor blood flow, including tumor microcirculation, which showed a similar temperature dependence. Metabolic effects probably play a minor role in the oxyhemoglobin saturation distribution within tumor microvessels.Supported by the Deutsche Forschungsgemeinschaft (Va 57/2-1). Presented in part at the International Symposium on Biomedical Thermology, June 30 to July 4, 1981, Strasbourg, France     

Storage of embryonically transcribed poly(A) RNA and its utilization during metamorphosis of the hydroidHydractinia echinata

Summary The formation of tentacles and stolons during metamorphosis is severely disturbed if inhibitors of mRNA metabolism are applied during certain phases of development. The periods of sensitivity to -amanitin are late gastrulation and the disk stage of metamorphosis. A cordycepin sensitive phase exists during the first hour of metamorphosis. In all drug sensitive phases an enhanced poly(A) synthesis is found indicating increased mRNA metabolism in these stages. Pulse-chase experiments show that planula larvae store a poly(A)-rich RNA population sedimenting between 28–18s. These long living molecules are of embryonic origin, are located in RNP particles and are degraded during metamorphosis. The particles in question appear to be stored mainly in interstitial cells. In early metamorphosis no uridine is incorporated but labelled poly(A) is added to preexisting molecules.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Metabolic activation and hepatotoxicity. Toxicity of bromobenzene in hepatocytes isolated from phenobarbital-and diethylmaleate-treated rats.

Hepatocytes freshly isolated from diethylmaleate-treated rats exhibited a markedly decreased concentration of reduced glutathione (GSH) which increased to the level present in hepatocytes from nontreated rats upon incubation in a complete medium. When bromobenzene was present in the medium, however, this increase in GSH concentration upon incubation was reversed and a further decrease occurred that resulted in GSH depletion and cell death. This was prevented by metyrapone, an inhibitor of the cytochrome P-450-linked metabolism of bromobenzene. Bromobenzene metabolism in hepatocytes was accompanied by a fraction of metabolites covalently binding to cellular proteins. The size of this fraction, relative to the amount of total metabolites, was increased in hepatocytes isolated from diethylmaleate-treated rats and in hepatocytes from phenobarbital-treated rats incubated with bromobenzene in the presence of 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase which, however, also acted as a GSH-depleting agent. In addition, the metabolism of bromobenzene by hepatocytes was associated with a marked decrease in various coenzyme levels, including coenzyme A, NAD(H), and NADP(H). Cysteine and cysteamine inhibited the formation of protein-bound metabolites of bromobenzene in microsomes, but did not prevent bromobenzene toxicity in hepatocytes when added at higher concentrations to the incubation medium (containing 0.4 mm cysteine). Methionine, on the other hand, did not cause a significant effect on bromobenzene metabolism in microsomes and prevented toxicity in hepatocytes, presumably by stimulating GSH synthesis and thereby decreasing the amount of reactive metabolites available for interaction with other cellular nucleophiles. It is concluded that, in contrast to hepatocytes with normal levels of GSH, hepatocytes from diethylmaleate-treated rats were sensitive to bromobenzene toxicity under our incubation conditions. In this system, bromobenzene metabolism led to GSH depletion and was associated with a progressive decrease in coenzyme A and nicotinamide nucleotide levels and a moderate increase in the formation of metabolites covalently bound to protein. Methionine was a potent protective agent which probably acted by enhanced GSH synthesis via the formation of cystathionine.