Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine

Haemophilus influenzae was labeled with thymidine-³H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the ³H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.     

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

Formation of mengovirus-like particles in cell-free extracts from virus-infected cells

³H-labeled particles with the density of intact mengovirus in CsCl were detected following the incubation of cell-free extracts from mengovirus infected cells with ³H-UTP in a RNA polymerase reaction mixture. The ³H-particles contained complete strands of ³H-labeled 35 S mengovirus RNA. The viral-like particles were found in the region of a sucrose gradient (150–250 S) where viral-specific RNA polymerase activity is detected.     

The specific binding of ricin and its polypeptide chains to rat liver ribosomes and ribosomal subunits

Ricin from Ricinus communis was isolated and the binding of ³H-reductively alkylated or ¹²⁵I-iodinated ricin was studied by incubating the toxic protein with ribosomes and isolating the ricin-ribosome complex by centrifugation. Neither of the labeled ricin derivatives nor ³H-labeled A chain bound Escherichia coli ribosomes, but both bound rat liver ribosomes in a reproducible manner. ³H-labeled ricin bound in a ratio of 1 mol/mol of ribosomes with a dissociation constant of 3 μm as calculated from a Scatchard plot. Similarly, ³H-labeled B chain isolated from ricin also bound in a one-to-one complex with a dissociation constant of 1 μm. The binding of ricin and ricin B chain was sensitive to lactose, while the binding of reduced ricin or ricin A chain was not prevented by lactose. Reduced ¹²⁵I-labeled ricin in the presence of lactose and ³H-labeled A chain bound with a ratio of 2 mol/mol of ribosomes. It was further demonstrated that ³H-labeled ricin A chain bound only to the 60S ribosomal subunit and not to the 40S ribosomal subunit. The dissociation constant for the binding was 2 μm both in the presence and absence of lactose and 2 mol of A chain were bound per mole of 60S ribosomal subunit.     

An assay for adenyl cyclase based on a combination of sodium borate electrophoresis and paper chromatography.

We describe a method for the assay of adenyl cyclase in whole tissue homogenates. Adenosine 3′:5′-cyclic monophosphate (cAMP) formed from α-³²P-, ¹⁴C- or ³H-labeled adenosine 5′-triphosphate (ATP) substrate is isolated from all known ATP metabolites and an unknown metabolite by electrophoresis in 1% sodium borate for 40 min, followed by overnight descending chromatography in 95% ethanol:1 m ammonium acetate (70:30). The purity of the cAMP isolated is established by chromatographic techniques as well as by utilizing a purified cyclic nucleotide phosphodiesterase. The method described here also makes possible the measurement of phosphodiesterase activity in homogenates. It is rapid enough to allow routine assay of 180 samples per day, although the number of samples processed depends on the number of electrophoretic and chromatographic units available.     

Testing Air-Filtering Systems: I. Procedure for Testing High-Efficiency Air Filters on Exhaust Systems

A procedure was developed for evaluating high-efficiency filters mounted in exhaust ducts at the National Animal Disease Laboratory. An aerosol of the test organism, Escherichia coli B T₃ bacteriophage, was generated in a chamber attached to a ceiling exhaust register in concentrations of at least 1000 viable organisms per ft³ of air. Samples were collected from both the pre- and postfilter areas, and the number of organisms per ft³ of air was determined. The efficiency of the filter was calculated from these figures. A total of 269 high-efficiency filters were tested. Of these, 249 had efficiencies of 98% or greater. The remaining 20, with efficiencies of less than 98%, were repaired and retested. No filter was accepted with an efficiency of less than 98%.     

The determination of the radiochemical purity of phosphorus-32 and tritium-labeled diisopropylphosphorofluoridate (DFP).

A method is described for the determination of the radiochemical purity of labeled diisopropylphosphorofluoridate (DFP), based on the irreversible inhibition reaction with the enzyme α-chymotrypsin. The nature of the impurities in commercially available ³²P- and ³H-labeled DFP is discussed.     

Radioimmunoassay of an antibody to phi X 174 DNA.RNA hybrid.

A fast and simple radioimmunoassay (RIA) technique was developed for an antibody to DNA·RNA hybrid using protein A-bearing Staphylococcus aureus cells as immuno-adsorbent and a glass microfiber filter for the deparation of free antigen and antibody-antigen complex. A simple method for preparing ³H-labeled DNA·RNA hybrid using single-stranded circular DNA of viruses and Escherichia coli RNA polymerase (nucleosidetri-phosphate: RNA nucleotidyltransferase, EC 2.7.7.6) is also described. In comparison to a hybrid made of natural sequences, a synthetic homopolymer hybrid poly (A)·poly(dT) was found to be a poor competitor for the antibody by this RIA technique.     

Sequential analysis in subnanomolar amounts of peptides. Determination of the structure of a habituation-induced brain peptide (ameletin)

Autoradiography of³H-labeled dansyl amino acids was applied to the sequential analysis of peptides available only in subnanomolar quantities. The method employs time-course analysis of the digestion of the peptide by aminopeptidase M, carboxypeptidase B, and dipeptidylaminopeptidase I. The cleaved amino acids or dipeptides are identified by tlc of the³H-labeled compounds. The procedure was used for the sequential analysis of a learning-induced peptide extracted from trained rat brain, called ameletin. The sequence, confirmed by synthesis, was:p-Glu-Ala-Gly-Tyr-Ser-Lys.     

Improved Method for Determining Bacterial Filtration Rates in Zooplankton

Filtration rates were determined for a natural population of zooplankton grazers (Bosmina longirostris [Müll.], Cyclops vicinus vicinus [Ulianine], Acanthodiaptomus denticornis [Wierz.], and Daphnia longispina [Müll.]) by using ³H-labeled bacteria as food for these organisms. There was a relationship between filtration rates of the major zooplankton grazers and the prevailing algal and bacterial composition in the lake water. Low filtration rates were obtained in the presence of colonial and filamentous cyanobacteria. The rapid process of bacterial adhesion to the external organs of grazers can result in an overestimation of filtration rates. By using the simple method presented here, filtration rates, with simultaneous correction for bacterial adhesion, can be quickly determined.     

Respiration of Tritiated Substrates in Heterotrophic Activity Assays

The respiration percentage of ³H-labeled glucose by heterotrophic microorganisms in water samples was determined by a method based on equimolar addition of [³H]- and [¹⁴C]glucose to samples. The respiration percentage of [³H]glucose exceeded that of [¹⁴C]glucose by 10% over the assayed range of respiration rates. Respiration percentages diminished with decreasing temperature, the relation between respiration of [³H]- and [¹⁴C]glucose remaining the same. The significance of tritiated water respiration with regard to experimental design is emphasized.     

Ribonucleic acid-permeable mutant of Escherichia coli

An RNA-permeable mutant was isolated from a tryptophan amber auxotrophic strain of Escherichia coli after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rationale of the isolation was based on the suppression of an amber mutation. A strain was selected, which could grow in minimal medium supplemented with transfer RNA prepared from an suI-carrying strain but not from an su⁻ strain. This mutant incorporated³H-labeled bulk RNA into the cells at a rate 40 times higher than did the parent strain. The level of tryptophan requirement, susceptibility to the lytic action of lysozyme and RNase activity in the culture medium of the mutant strain did not differ from those of the parent strain. The mutant strain incorporated ³H-labeled ribosomal RNA equally as well as it incorporated ³H-labeled transfer RNA and the incorporation was competitively inhibited by any species of cold RNA. However, the fate of ³H-labeled rRNA after incorporation resulted in degradation to yield acid-soluble fragments whereas tRNA after incorporation remained intact in the cell.     

Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes

Summary A technique of in situ hybridization on metaphase chromosomes with biotinylated DNA probes is described. This technique was used to localize unique DNA sequences on chromosomes and allowed a localization of two probes 1.8 and 1.3 kb long. The hybridization signal appears like two, twin, spots on the two sister chromatids, allowing a clear distinction from the background. Moreover a chromosomal localization is possible by counting a relatively small number of mitoses compared with the technique using ³H-labeled DNA probes.     

Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

Ehrlich ascites tumor cells, loaded with ³H-labeled arachidonic acid and ¹⁴C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of ³H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of ¹⁴C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A₂ is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in ¹⁴C-labeled stearic acid release nor in the synthesis of phosphatidyl ¹⁴C-butanol in the presence of ¹⁴C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of ¹⁴C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A₁, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of ³H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP_βS or with AACOCF₃, an inhibitor of the 85 kDa, cytosolic phospholipase A₂. Based on these results we propose that cell swelling activates a phospholipase A₂—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response. Received: 23 July 1996/Revised: 17 June 1997     

Headspace analyses of H labeling of acetone: Enabling studies of fatty acid oxidation in vivo

We demonstrate that one can measure low levels of ²H labeling (e.g., <0.025% excess ²H) by exchanging hydrogen (deuterium) in water with acetone and subjecting samples to gas chromatography–pyrolysis–isotope ratio mass spectrometry. This analytical method circumvents the need to use typical off-line reduction methods that convert water to hydrogen gas prior to isotope ratio mass spectrometry or the need to purchase extra peripheral devices that would permit the direct analysis of water labeling. This method enables routine measurements of fatty acid oxidation in rodents; that is, one administers a ²H-labeled fatty acid(s) and then quantifies the production of ²H-labeled water.     

Sensitive detection of tritium in Southern blot and plaque hybridizations

A sensitive method for detecting ³H-labeled probes in Southern blot and plaque hybridizations is described. The method is a combination of dipping nitrocellulose filters in melted Permablend III and preflashing X-ray films. About 10 cpm per band or plaque can be detected after 1 week. This method is used to detect cloned rDNA from Aspergillus nidulans and cloned variant sequences of calf satellite I DNA.     

Use of (1 or 3-3H, U-14C)glucose to estimate the synthesis of glycerolipids via acyl dihydroxyacetone phosphate.

Turnover rates of tRNAs in Friend virus-infected mouse leukemia cells are reported. Cells were labeled for one generation with [¹⁴C]- or [³H]uridine. ³H-labeled cells were transferred to nonradioactive medium and allowed to grow exponentially for 72 hours. Low molecular weight cytoplasmic RNAs isolated from ¹⁴C- and ³H-labeled cells were cochromatographed on reverse-phase columns. The results indicate considerable heterogeneity of turnover rates of 4S RNAs, with the most labile species turning over at least 1.75 times as fast as the most stable species.     

Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters

The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm³. Cell volume ranged from 0.072 to 0.096 μm³ at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm³ in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from ³H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

The preparation of specifically deuterium- or tritium-labeled N-acetylneuraminic acid and cytidine-5′-monophospho-β-N-acetylneuraminic acid as precursors for glycoconjugate synthesis

This report describes the specific exchange of H-3ax for a deuterium- or tritium-atom in free Neu5Ac in aquous solution under mild alkaline conditions (p²H 9.0). This principle was applied to the synthesis of ³H-labeled cytidine-5′-monophospho-β-N-acetylneuraminic acid. Subsequently, ³H-labeled CMP-Neu5Ac was employed for the enzymic transfer of Neu5Ac to asialofetuin. The latter reaction is an example of the enzymic synthesis of sialoglycoconjugates, specifically labeled at C₃ of Neu5Ac