Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes

Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al. (1983) Biochem. Pharmacol. 32, 3571-3575) suggesting specificity for the interaction with NADPH. The complex was seen to facilitate electron transfer from NAD(P)H to oxygen, generating oxygen-free radicals which were detected by the spin-trapping technique and to flavin nucleotides, giving rise to flavin semiquinone radicals which were demonstrated by direct ESR spectroscopy under anaerobic conditions. A twofold increase in oxygen uptake and hydroxyl radical generation by the NAD(P)H-PQ complex was observed in the presence of hemoglobin. This effect was independent of heme concentration (in the range 1 X 10(-5)-1 X 10(-4) M) and oxidation state of the iron. Under anaerobic conditions, the NAD(P)H-PQ complex reduces Fe-III to Fe-II hemoglobin, and under aerobic conditions about 65% of the heme chromophore is irreversibly destroyed. Superoxide dismutase inhibits hydroxyl radical generation by the NAD(P)H-PQ pair; this effect is not observed in the presence of hemoglobin. In the presence of microsomes there is a 10-fold increase in both oxygen consumption and hydroxyl radical generation by the NAD(P)H-PQ pair. The fact that both pyridine nucleotides are active, and the inability of SKF 525A in decreasing hydroxyl radical generation, suggests that microsomal reductases are involved in the catalysis.     

Information content of binding sites on nucleotide sequences

Repressors, polymerases, ribosomes and other macromolecules bind to specific nucleic acid sequences. They can find a binding site only if the sequence has a recognizable pattern. We define a measure of the information (R sequence) in the sequence patterns at binding sites. It allows one to investigate how information is distributed across the sites and to compare one site to another. One can also calculate the amount of information (R frequency) that would be required to locate the sites, given that they occur with some frequency in the genome. Several Escherichia coli binding sites were analyzed using these two independent empirical measurements. The two amounts of information are similar for most of the sites we analyzed. In contrast, bacteriophage T7 RNA polymerase binding sites contain about twice as much information as is necessary for recognition by the T7 polymerase, suggesting that a second protein may bind at T7 promoters. The extra information can be accounted for by a strong symmetry element found at the T7 promoters. This element may be an operator. If this model is correct, these promoters and operators do not share much information. The comparisons between R sequence and R frequency suggest that the information at binding sites is just sufficient for the sites to be distinguished from the rest of the genome.     

Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications

Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.     

The "suspicious" gynecologic smear

"Suspicious" gynecologic smears from 842 patients over a seven-year period were analyzed for their causes and outcomes. The frequency of the cytologic diagnosis of "suspicious" ranged between 0.5% in 1979 and 1.44% in 1975 of all smears examined. Review of the smears showed that this classification was used to report a variety of conditions, including equivocal possible precancerous changes as well as the presence of severe inflammation, degenerative or atrophic changes, abnormal glandular cells and metaplasia. The cytologic follow-up, following anti-inflammatory or hormonal therapy, showed a conversion to negative findings in 65.1% of all cases, usually within 12 months. In 294 cases, histologic analysis became necessary, revealing precancerous changes or cancer in 147 patients (17.5% of the study group). Smears of postmenopausal women with suspicious glandular or endometrial cells received special analysis. Significant numbers of such cases had histologic findings positive for malignancy (20% of smears with glandular cells and 21.3% with endometrial cells), as did also smears showing post-irradiative changes (34.6%) or atrophic and degenerative changes (17.1%). Therefore, "suspicious" smears in these groups were considered to indicate an increased risk of malignancy. A regimen for the proper management of cases with "suspicious" smears has been established.     

12 beta-dehydrogenation of bile acids by Clostridium paraputrificum, C. tertium, and C. difficile and epimerization at carbon-12 of deoxycholic acid by cocultivation with 12 alpha-dehydrogenating Eubacterium lentum.

12 beta-Hydroxysteroid dehydrogenating activities were detected in 13 strains of Clostridium paraputrificum, 1 strain of C. tertium, and 1 strain of C. difficile, together with a 3 alpha- and 3 beta-hydroxysteroid dehydrogenase system in many strains. Redox reactions a C-12 of disubstituted and trisubstituted bile acids were performed unspecifically by representative strains of C. paraputrificum. 3 alpha,12 beta-, 3 beta,12 beta-Dihydroxy-, 3 alpha, 7 alpha, 12 beta-trihydroxy-, and 3-keto,12 beta-hydroxy-5 beta-cholanoic acids, so far not known as bacterial bile acid metabolites, were identified. Epimerization of the 12 alpha-hydroxyl group of deoxycholate via the 12-keto intermediate was achieved by cocultivation of C. paraputrificum and Eubacterium lentum, elaborating a 12 alpha-hydroxysteroid dehydrogenase only. In addition, epimerization at C-12 was demonstrated with mixed human fecal cultures.     

12 beta-dehydrogenation of bile acids by Clostridium paraputrificum, C. tertium, and C. difficile and epimerization at carbon-12 of deoxycholic acid by cocultivation with 12 alpha-dehydrogenating Eubacterium lentum

12 beta-Hydroxysteroid dehydrogenating activities were detected in 13 strains of Clostridium paraputrificum, 1 strain of C. tertium, and 1 strain of C. difficile, together with a 3 alpha- and 3 beta-hydroxysteroid dehydrogenase system in many strains. Redox reactions a C-12 of disubstituted and trisubstituted bile acids were performed unspecifically by representative strains of C. paraputrificum. 3 alpha,12 beta-, 3 beta,12 beta-Dihydroxy-, 3 alpha, 7 alpha, 12 beta-trihydroxy-, and 3-keto,12 beta-hydroxy-5 beta-cholanoic acids, so far not known as bacterial bile acid metabolites, were identified. Epimerization of the 12 alpha-hydroxyl group of deoxycholate via the 12-keto intermediate was achieved by cocultivation of C. paraputrificum and Eubacterium lentum, elaborating a 12 alpha-hydroxysteroid dehydrogenase only. In addition, epimerization at C-12 was demonstrated with mixed human fecal cultures.     

A novel type of human T cell clone with highly potent natural killer-like cytotoxicity divorced from large granular lymphocyte morphology

Interleukin 2-dependent cloned lymphocytes derived from an allogeneic HLA class II-mismatched but class I-matched mixed lymphocyte culture were screened for cytotoxic activity on target cell lines known to be susceptible or resistant to lysis by natural killer (NK) cells. Of 24 clones, eight were found to display NK-like cytotoxicity. Two manifested extremely high cytotoxicity levels (50% lysis of K562 at an effector to target ratio of 1:1), whereas the remainder were only moderately active (about 20% lysis at 25:1). NK-like clones were studied with regard to cell surface markers defined by monoclonal antibodies, as well as for their morphologic and cytochemical characteristics, and were compared with clones displaying different functions. The moderately active NK-like clones exhibited characteristic large granular lymphocyte morphology (many azurophilic granules, indented nuclei, high cytoplasm to nucleus ratio, and a basophilic peripheral cytoplasmic zone). This was, however, also characteristic of the majority of lymphocyte clones displaying functions other than NK. Surprisingly, the two clones with high NK-like activity did not exhibit large granular lymphocyte morphology, with few granules, round nuclei, and low cytoplasm to nucleus ratio. The T3, T9, T10, and T11 markers, as well as HLA-DR determinants, were expressed on their surfaces, but in contrast to the other clones, they did not display OKT4-, OKT8-, or OKM1-defined antigens. No distinction between them was possible on the basis of a cytochemical profile in relation to their function, because all clones were positive for acid phosphatase, either focal or dispersed and negative for nonspecific esterase or chloracetate esterase. The highly active lytic clones were, however, distinguished by an exceptionally rapid growth rate in culture (cell doubling time: 9 hr as compared to 30 to 40 hr, as usually required). These results demonstrate two different types of human NK-active lymphocytes with remarkably disparate lytic capacity, cell surface markers, and morphology.     

Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.     

Mutants of Pachysolen tannophilus with Improved Production of Ethanol from d-Xylose

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of d-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanol-defective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. One also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.