Method of electron-autoradiographic study of nucleic acid biosynthesis in bacteria during phagocytosis

Leukocytes from healthy men exposed to Staphylococcus epidermis were incubated with 3H-uridine and 3H-thymidine. The level of nucleic acid synthesis by phagocyted bacteria was examined by electron microscopic autoradiography. The method makes it possible to correlate bacterial and phagocyte functions with their ultrastructures.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in purified trachoma elementary bodies: effect of sodium chloride on ribonucleic acid transcription

Highly purified trachoma elementary bodies (T'ang strain), incubated in the presence of the four nucleoside triphosphates [Mg(2+), Mn(2+), 2-mercaptoethanol, tris(hydroxymethyl)aminomethane buffer (pH 7.5)] were found to incorporate (3)H-uridine triphosphate (UTP) into ribonucleic acid (RNA) molecules. Eighty-seven per cent of the labeled molecules were sensitive to ribonuclease treatment. In vitro RNA synthesis was almost completely inhibited by actinomycin D. Rifampin was also inhibitory, but allowed some initial RNA synthesis before complete inhibition occurred. When the reaction mixture lacked Mn(2+), trachoma elementary bodies synthesized, for a limited period, high-molecular-weight RNA species (23 to 24S, 16 to 17S, and 10 to 11S). Addition of 0.2 m NaCl to the same reaction mixture stimulated and prolonged (3)H-UTP incorporation into the same radioactive RNA species. Addition of 0.001 m Mn(2+) instead of NaCl also stimulated (3)H-UTP incorporation but prevented the synthesis of the high-molecular-weight RNA species.     

Electron microscopic observations of the phagocytosis and subsequent fate of Aeromonas salmonicida by Atlantic salmon neutrophils in vitro

Neutrophils isolated from the blood of Atlantic salmon (Salmo salar L.) were studied by electron microscopy at various time intervals after being incubated with opsonised and non-opsonised Aeromonas salmonicida strains, namely, the avirulent MT004 (A-layer negative) and the virulent MT423 (A-layer positive). After 15 min incubation with all four groups of bacteria (virulent, avirulent, opsonised or non-opsonised) a large number of neutrophils showed an elongated shape with the nucleus and all the organelles being located in one pole of the cell. Small vacuoles and clumping of glycogen granules were also observed. Neutrophils devoid of granules were noted after 30 min incubation, the majority containing engulfed bacteria. Degenerate neutrophils were also found in all the groups incubated with bacteria. Phagocytosis of bacteria was observed after 15 min incubation. The number of intracellular bacteria was very low, usually one or two per cell, although some neutrophils incubated with the opsonised avirulent strain MT004 contained a larger number of engulfed bacteria. Ingestion of bacteria was usually accompanied by the formation of phagocytic vacuoles containing an amorphous material of moderate electron-density as well as granule discharge into the vacuole. Both strains (MT004 and MT423), opsonised and non-opsonised, underwent morphological alterations after 3-7 h incubation suggesting that both A. salmonicida strains were killed by the neutrophils.     

Comparative radioautographic evaluation of RNA synthesis in vivo and in biopsy material]

The liver, kidney, skin biopsies are able to incorporate actively 3H-uridine, and the maximum rate of RNA synthesis over the hepatocyte nuclei in the peripheral zone of the biopsies corresponds to relevant rates in vivo. Only the skin biopsies were distinguished by total cell labelling. Liver, renal, skin capillary endotheliocytes can serve intertissue marker of the diffusion depth of 3H-uridine.     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Alteration of ringer pH and its effects on precursor incorporation into Drosophila salivary gland nuclei

Drosophila salivary glands were explanted and incubated with ³H-uridine (or ³H-thymidine) in Ringer's solution (Ephrussi-Beadle modified saline) adjusted to pH values in the integral range, 4 to 10. The results of autoradiographic investigations indicate a differential effect of altering Ringer pH on ³H-uridine as opposed to ³H-thymidine incorporation: a) Relatively uniform levels of chromosomal incorporation of ³H-thymidine occurred over the range of test pH. Some decrease of incorporation was noted at pH 5 and some increase at pH 9. b) Chromosomal incorporation of ³H-uridine was severely depressed at pH 4 and 7 relative to the high incorporation levels observed at other pH values. Controlling pH of Ephrussi-Beadle Ringer's solution in such experiments seems a necessity. This appears especially important for studies involving ³H-uridine incorporation.     

Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source.

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37 degrees C with 0.15 microM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 microM). In neutrophils from allergic patients, the leukotriene B4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 +/- 4.2 and 38.1 +/- 6.8 pmol/5 min per 2 X 10(6) cells in the absence and presence of 10 microM arachidonic acid, respectively; P less than 0.005; n = 8). The LTB4 level in neutrophils from healthy subjects was only 0.97 +/- 0.17 pmol/5 min per 2 x 10(6) cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-14C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB4 by the 5-lipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.     

The effect of age on the ability of oocytes to synthesize RNA and proteins during in vitro maturation

Summary The effect of age on the ability of the oocyte to resume meiosis in vitro and to incorporate ³H-uridine and ³H-leucine into RNA and protein, respectively, was examined in the rat. In comparison with the mature control oocyte, the nucleolus of the aged oocyte tends to be retained although the rate of germinal vesicle breakdown is not altered. The incorporation of ³H-uridine is reduced, while ³H-leucine incorporation is not impaired. It is concluded that the inability of the aged oocyte to synthesize RNA may be responsible for its inability to complete meiotic maturation in vitro.This work was supported in part by a grant from the Easter Seal Research Foundation of the National Easter Seal Society for Crippled Children and Adults     

Initiation of transcription and nucleologenesis in equine embryos

The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of ³H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with ³H-uridine (560 μCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 μCi/ml for 0.5–1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counter-stained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated ³H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of ³H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos. © 1995 wiley-Liss, Inc.     

Vitamin B6 deficiency and the lymphoid system: I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B₆-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate ³H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in ³H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for ³H-uridine incorporation.     

Incorporation of label from radioactive uridine into the stylar nucleic acids of Lilium longiflorum thunb. as affected by heat, 6-methylpurine and actinomycin D

Summary 5-³H-uridine injected into the stylar canal of detached lily stigma-styles was taken up initially into the rapidly-labeled-RNA of the nucleic acid profile of a methylated albumin kieselguhr (MAK) column but with increasing time was found in all portions of the RNA profile, but not in the DNA. Heat treatment of the style before injection of 5-³H-uridine greatly reduced the rate of incorporation of label into and the ultimate amount of label found in the RNA species of the lily style. Translocation of 5-³H-uridine through the ovary into heattreated pistils and the injection of 5-³H-uridine into styles which had been incubated for 1 or 2 days after heat treatment resulted in stylar nucleic acids more highly labeled than nucleic acids in control styles, with an incorporation pattern different than control styles. Heat treatment of lily pistils resulted in detectable changes in the proportion of stylar RNA species as separated on MAK columns and measured as absorbance units. Actinomycin D and 6-methylpurine treated styles incorporated label from a stylar injection of radioactive uridine in patterns different than each other, different than heat-treated styles and different than non-treated styles. 6-methylpurine and heat treatment of styles only slightly reduced the rate at which 5-³H-uridine was removed from the stylar canal into the stylar tissue.Paper number 8917 of the Scientific Journal Series, Minn. Agr. Exp. Sta., St. Paul, MN 55108.     

6-formylpterin intracellularly generates hydrogen peroxide and restores the impaired bactericidal activity of human neutrophils.

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H(2)O(2)) occurred. The H(2)O(2) generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H(2)O(2) independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.     

Biodegradation of hydrocarbon contamination by immobilized bacterial cells

This study examined the capacity of immobilized bacteria to degrade petroleum hydrocarbons. A mixture of hydrocarbon-degrading bacterial strains was immobilized in alginate and incubated in crude oil-contaminated artificial seawater (ASW). Analysis of hydrocarbon residues following a 30-day incubation period demonstrated that the biodegradation capacity of the microorganisms was not compromised by the immobilization. Removal of n-alkanes was similar in immobilized cells and control cells. To test reusability, the immobilized bacteria were incubated for sequential increments of 30 days. No decline in biodegradation capacity of the immobilized consortium of bacterial cells was noted over its repeated use. We conclude that immobilized hydrocarbon-degrading bacteria represent a promising application in the bioremediation of hydrocarbon-contaminated areas.     

Killing of Escherichia coli by mononuclear phagocytes and neutrophils stimulated in vitro with beta-1,3-D-polyglucose derivatives.

Human monocytes, human peritoneal macrophages, mouse peritoneal macrophages and human peripheral neutrophils pretreated with beta-1,3-D-polyglucose derivatives showed pronounced bactericidal capacity to Escherichia coli compared to control cells. The increased bactericidal capacity was detectable in mononuclear phagocytes over a wide range of concentrations of bacteria. Granulocytes, however, showed bactericidal capacity only at low concentrations of bacteria. The pretreated mononuclear phagocytes released significant amounts of IL-1 and PGE2. However, there was no significant release of tumor necrosis factor (TNF). By incubating unstimulated cells with purified IL-1 and TNF, the bactericidal activity of neutrophils and mononuclear phagocytes was enhanced. Our data indicate that the inability of neutrophils stimulated with beta-1,3-D-polyglucose derivatives to kill large numbers of bacteria could be overcome by a combined treatment with purified IL-1 or TNF in addition to beta-1,3-D-polyglucose derivatives. By incubating unstimulated cells with medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, the bactericidal activity of the cells was enhanced to the same extent as cells pretreated with purified TNF and IL-1. Cells incubated with IL-1-depleted medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, showed reduced bactericidal activity compared to cells incubated with undepleted medium. These studies demonstrate that beta-1,3-D-polyglucose-treated mononuclear phagocytes and neutrophils show enhanced bactericidal activity. The enhanced activity is partly caused by stimulation of the cells with IL-1 released from mononuclear phagocytes and partly by other unknown effects of beta-1,3-D-polyglucose derivatives on both mononuclear phagocytes and neutrophils.     

Incorporation of Radioactive Pyrimidine Nucleosides into DNA and RNA of Eimeria tenella (Coccidia) Cultured In Vitro*

Autoradiographic methods were used to study the incorporation of tritiated cytidine, thymidine, and uridine into asexual stages of Eimeria tenella cultured in embryonic chick kidney cells. Developing parasites did not incorporate ³H-thymidine either when host cells were labeled prior to infection or when the cultures were labeled for 30 min, 48–72 hr after infection. Continuous exposure of infected cultures to ³H-thymidine for up to 18 hr resulted in light labeling of cell cytoplasm and schizonts. ³H-cytidine and ³H-uridine were incorporated into parasites developing in cultures that were labeled before infection. When the cultures were labeled for 30 min, 48–72 hr postinfection and fixed immediately, schizonts were labeled lightly with ³H-cytidine but contained dense accumulations of ³H-uridine.     

Inefficient in vitro killing of virulent or nonvirulent Salmonella typhimurium by murine polymorphonuclear neutrophils

The bactericidal capability of murine peritoneal polymorphonuclear neutrophils against virulent and nonvirulent Salmonella typhimurium was examined in an in vitro system. Although preincubation of the bacteria in specific murine antiserum elicited greater chemiluminescence from phagocytizing neutrophils than did incubation in normal murine serum, antiserum did not enhance ingestion, as less than 5% of the challenge was taken up by neutrophils under any of the conditions studied. Nonvirulent salmonellae showed a transient decrease in viable numbers early during in vitro incubation with or without intact neutrophils. Virulent salmonellae, however, were able to multiply without a lag period except when these bacteria were pretreated with antiserum and incubated in association with intact murine neutrophils. Results of these in vitro studies suggest that the murine polymorphonuclear neutrophil and antisalmonella antibody must act together to effect neutrophil-associated bactericidal activity against virulent salmonellae, and thus, that the neutrophil alone does not play a major role in the protection of unvaccinated, sensitive mice from disease caused by S. typhimurium.     

3H-uridine incorporation in early porcine embryos.

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida-intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.