A plasmid-encoded anion-translocating ATPase

An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance.     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: application to isolated perfused organs

Of the available techniques used to identify free radicals, spin-trapping offers the unique opportunity to simultaneously measure and distinguish among a variety of important biologically generated free radicals. For superoxide and hydroxyl radical, the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) is most frequently used. However, this nitrone has several drawbacks. For example, its reaction with superoxide is slow, having a second-order rate constant around 10 M-1 s-1. Because of this, high concentrations of DMPO are essential in order to observe the corresponding spin-trapped adduct, 5,5-dimethyl-2-hydroperoxy-1-pyrrolidinyloxy. This may, in some cases, lead to cellular toxicity. In an attempt to circumvent this serious limitation, it has been proposed that an indirect approach be employed to detect and identify free radicals generated as a consequence of ischemia/reperfusion injury. In the direct (most frequently used) approach, the spin trap is first added to an isolated perfused organ under the appropriate experimental conditions. Then, the infusion buffer containing the spin-trap adduct(s) is placed into an quartz flat cell to be inserted into an ESR spectrometer. In the indirect method, the spin trap is added to the perfusate, which had previously exited the organ. Therefore, with this method one can prevent any spin-trap-mediated toxicities to the isolated perfused organ. However, because of the very rapid rate of free radical reactions catalyzed by either superoxide or hydroxyl radical, it is questionable whether ESR spectra recorded using this indirect method result from the actual spin-trapping of free radicals. In this report, we evaluated the indirect spin-trapping technique in light of the kinetic considerations discussed above.     

Postrhinoplasty "red nose": differential diagnosis and treatment by laser

Prior to anticipated nasal surgery, the nasal and facial skin should be examined for any vascular lesions. The skin type should be ascertained. A history of any prior nasal surgery, particularly on the nasal dorsum, should be noted. If rosacea is a clinical possibility, a trial of 1.5 to 2.0 gm q.d. of tetracycline for 6 to 8 weeks is warranted. If, after rhinoplasty, a diffuse "redness" on the nasal dorsum results and one can exclude other diagnoses, then argon laser therapy should be considered. A 3-mm punch biopsy should be obtained to see whether superficial ectatic vessels are present, a finding that would be indicative of a good result from laser therapy.     

Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis

Summary The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acids; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA1 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA1 either encodes a gene product or structure information that is responsible for incompatibility.     

Properties and function of the proton-translocating adenosine triphosphatase of Clostridium perfringens.

Growth of Clostridium perfringens was inhibited by compounds which dissipate or prevent the formation of electrochemical proton gradients. Membrane vesicles prepared from this organism exhibited Mg2+-dependent adenosine triphosphatase (ATPase) activity sensitive to N,N'-dicyclohexylcarbodiimide. Mg2+-ATPase activity was optimal of 50 degrees C, but no discrete pH optimum was observed. Adenosine triphosphate-dependent quenching of the fluorescence of the weak base quinacrine by everted membrane vesicles suggested that the Mg2+-ATPase is a proton pump capable of generating an electrochemical proton gradient. Adenosine triphosphate-dependent transport of Ca2+ by everted vesicles was sensitive to uncouplers and inhibitors of the Mg2+-ATPase.     

Measurement of stagnancy in a pipe line using a radioactive tracer and labelled bacteria

Summary A stainless steel T joint was used to simulate stagnant volume in a pipe-line. Residence time distribution in the lines was measured using a radiotracer (Cr⁵¹) which emitted radiation. The radiation was measured externally to the pipe line. From the experimental data the dead volume of the system was calculated. The effect of the depth of T and flow velocity on dead volume was evaluated. Labelled bacteria were used in some experiments instead of chromium. No significant difference in behavior between ionic chromium and bacteria labeled with chromium was found.     

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.