Modification of the BrdU/H33258 technique for flow cytometry based on the pH-dependence of the fluorescence of H33258 stained DNA

Summary A simple analytical method is described for the evaluation of cell cycle progression data by modification of the BrdU/H33258 technique for flow cytometry. This procedure allows to obtain quenched and unquenched DNA-histograms of cells containing BrdU-substituted DNA by staining with one dye only, namely H33258. Quenched histograms are obtained at pH=7 and give information about how far cells have passed the cell cycle since the beginning of the incubation. The unquenched ones obtained at pH4 of the staining solution give information about the actual position of the cells in the cell cycle.     

对同一细胞内DNA、RNA和蛋白含量相关测定的显微荧光光度法

用荧光染料DAPI、PyroninY和FITC分别染同一细胞内DNA、RNA和蛋白.在紫外光、绿光和兰光顺序激发后,用MPVⅡ显微荧光光度计测量反映单个细胞内DNA、RNA和蛋白含量的荧光强度.根据荧光发射光谱分析,每种染料荧光之间的干扰是可以忽略的.测量结果与FCM获得的结果是一致的.显微荧光光度术对单个细胞多参数相关测量的优点是简单、便宜,并可用于对活细胞获得形态学和定量细胞化学的组合信息.     

Bacterial surface display of GFP(uv) on bacillus subtilis spores

To analyze a cotG-based Bacillus subtilis spore display system directly, GFP(uv) was expressed on the surface of Bacillus subtilis spores. When GFP(uv) was fused to the C-terminal of the cotG structural gene and expressed, the existence of a CotG-GFP(uv) fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This GFP(uv) displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.     

Base stacking in the dinucleoside phosphate rapa.

The pH-induced unstacking of rRpA has been investigated by batch calorimetry and uv spectroscopy. Equilibrium uv melting curves confirmed that the adenine bases in rApA are stacked at pH7 but unstacked at pH 1.5. The enthalpy change accompanying this pH-induced unstacking is +2.65 kcal (mole of A-A stack)-1 as measured by batch calorimetry. This represents the first direct determination of this important parameter for a dinucleoside phosphate. It is noted that the calorimetrically determined value reported here is considerably lower than published van't Hoff enthalpies but is consistent with values that can be derived from calorimetric data on polymers.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

Simultaneous determination of iodate and periodate by capillary zone electrophoresis: application to carbohydrate analysis

Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 microns i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates.     

Direct nanogram quantitation of nucleic acid and protein with a continuous-flow microcell

A simple method for rapid nanogram measurement of nucleic acids and proteins is described. It requires only 5 to 10 microliter of sample solution which is injected into the postcolumn flow stream of a high-performance liquid chromatograph. Samples are analyzed by uv detection at 260 nm for nucleic acids and 280 nm for proteins with a diode array detector. Analyzing speed is two samples per minute and the amount to be analyzed ranges from 3 ng to 80 micrograms for nucleic acids and 10 ng to 80 micrograms for bovine serum albumin, irrespective of the sample volume. The method is particularly useful for fast, accurate, and trace amount measurement of purified DNA, RNA, and protein samples in small volumes.     

Bile acids LIII: Application of reverse-phase high-pressure liquid chromatography to the analysis of conjugated bile acids in bile samples

The common conjugated bile acids of deproteinated bile from the human or the rat can be separated by high-pressure liquid chromatography and quantitated within 30 min with a 4-mm × 30-cm “fatty-acid analysis” column (Waters Associates) in 2-propanol/8.8 mm potassium phosphate buffer (pH 2.5) 160:340, coupled to a uv flow detector set at 193-nm wavelength. Detection limits were at least 0.1 nmol for the tauro-conjugates and 0.2 nmol for the glyco-derivatives.     

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.     

Photoreversible inhibition by ultraviolet light of germ line development in Smittia sp. (Chironomidae, Diptera)

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradition delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

Effects of ergosterol,palmitic acid and related simple lipids on the recovery of Candida albicans from ultraviolet irradiation

Summary Post-irradiation growth on sterols or long chain fatty acids promotes recovery of C. albicans from uv-induced lethal damage. The effect is observed only for cells which are not budding at the time of irradiation. Lipids have no effect on uv mutagenesis. A survey of a number of sporogenous and asporogenous yeasts indicates that the capacity for lipid-induced recovery from uv is a species specific characteristic of yeasts. The behaviors of cells damaged by uv and by amphotericin B, a membrane specific fungicidal antibiotic, suggest that lipids remediate an uv-induced derangement of the structure of the cell membrane critical for the initiation of cell division.     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

The effect of bromodeoxyuridine and ultraviolet light on 9L rat brain tumor cells

Incorporation of the thymidine analog bromodeoxyuridine (BrdUrd) into DNA increases the sensitivity of a cell to uv light. We have examined the effect of uv light on cell killing and alkaline elution profiles in 9L rat brain tumor cells pretreated with BrdUrd. Combination treatment with BrdUrd and uv irradiation produced a dose enhancement ratio of 3.8 at the 10% survival level compared with uv-radiated control cells; cell killing depended on both the time of treatment and the concentration of BrdUrd used for incubation. Sequential treatment caused single-strand breaks and DNA-protein crosslinks in the portion of DNA containing BrdUrd; uv irradiation alone caused very few strand breaks and no DNA-protein crosslinks. Because of the presence of both lesions in cells treated with BrdUrd and uv light, it was possible to calculate crosslinking factors without using a charging X-ray dose to induce strand breaks, the method commonly used with crosslinking drugs. Results of repair studies suggested that single-strand breaks are repaired more rapidly than are DNA-protein crosslinks.     

Molecular weights of glycosylated and nonglycosylated forms of recombinant human stem cell factor determined by low-angle laser light scattering.

The molecular weight of recombinant human stem cell factor (SCF) was determined using a low-angle laser light scattering combined with a differential refractometer and a uv detector. The protein samples were applied to these detectors through a gel filtration column by a high-performance liquid chromatographic pump. The Chinese hamster ovary (CHO) cell-derived SCF gave a molecular weight of 53,000 for the entire molecule and 35,000 for the protein moiety only at pH 7.0, indicating that the CHO cell-derived protein is glycosylated by 34%. Since the molecular weight of the polypeptide is 18,600, the results demonstrate that the CHO cell-derived SCF forms a dimer. The molecular weight of Escherichia coli-derived SCF was determined to be 39,000, similar to the above value (35,000). Essentially identical molecular weights were obtained at pH 3.0, indicating no dissociation of the dimer.     

Evidence for the generation of suppressor cells by ultraviolet radiation

The majority of murine skin tumor induced with ultraviolet (uv) light are unique in that they are of sufficient antigenicity to be consistently rejected when transplanted into normal syngeneic animals. However, the exposure of normal syngeneic mice to subcarcinogenic levels of uv prior to tumor transfer results in the progressive growth of transplanted uv-tumors. We report that normal mice can also be rendered tumor susceptible by the adoptive transfer of lymphoid cells from either tumor bearing or short term uv exposed donors. Further, the adoptive transfer of tumor susceptibility can be abolished by the pretreatment of cell suspensions from uv exposed donors with anti-theta and complement. These results suggest that uv irradiation may generate the development of T lymphocytes with suppressor activity.     

Globally asymptotic properties of proliferating cell populations

This paper presents a general model for the cell division cycle in a population of cells. Three hypotheses are used: (1) There is a substance (mitogen) produced by cells which is necessary for mitosis; (2) The probability of mitosis is a function of mitogen levels; and (3) At mitosis each daughter cell receives exactly one-half of the mitogen present in the mother cell. With these hypotheses we derive expressions for the and curves, the distributions of mitogen and cell cycle times, and the correlation coefficients between mother-daughter (_md) and sister-sister (_ss) cell cycle times.The distribution of mitogen levels is shown to be given by the solution to an integral equation, and under very mild assumptions we prove that this distribution is globally asymptotically stable. We further show that the limiting logarithmic slopes of (t) and (t) are equal and constant, and that _md0 while _ss0. These results are in accord with the experimental results in many different cell lines. Further, the transition probability model of the cell cycle is shown to be a simple special case of the model presented here.     

pH equilibration in human erythrocyte suspensions

Summary A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH^– or H⁺ was determined over a wide range of extracellular pH (4–11) in CO₂-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0>pH>11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH^– or H⁺ moves across the red cell membrane. Flux and velocity constants can be calculated from the initiald pH/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4–11), the greater the absolute value of total OH^– and/or H⁺ flux. Pretreatment with SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H⁺ and OH^– may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H⁺ and OH^– transport during CO₂-free pH equilibration.     

Determination of ADP-ribosyl arginine anomers by reverse-phase high-performance liquid chromatography

A simple and rapid method for the determination of ADP-ribosyl arginine anomers was devised. Analysis is performed by reverse-phase high-performance liquid chromatography on a 5-micron Cosmosil 5C18 column and uv detection. ADP-ribosylation of arginine by hen liver ADP-ribosyl-transferase and the effect of pH on anomerization are also presented.     

Immunologic effects of whole-body ultraviolet (uv) irradiation. III Defective splenic adherent cell function in alloantigen-stimulated T-cell proliferation

The daily exposure of a mouse to ultraviolet (uv) radiation causes a selective depletion of Ia-bearing adherent cells in that animal's spleen. This depletion manifests itself in functional deficiencies in the presentation of protein antigens and haptens to T cells. The present studies demonstrate a defect in splenic adherent cells (SAC) from uv-irradiated mice resulting in defective alloantigen presentation. We show that unfractionated splenocytes and SAC from uv-irradiated mice show decreased stimulatory activity in allogeneic MLR. We then utilize this phenomenon induced by uv radiation to characterize the stimulator cell in the M locus (Mls) determinant-driven MLR. We show that the stimulator cell in Mls determinant-driven MLR is an adherent cell and demonstrate that this stimulator cell bears Ia determinants by showing that whole spleen cells and SAC from mice treated with uv radiation are inefficient stimulators of the Mls determinant-driven MLR. The importance of the Ia determinant on the stimulator cells in Mls determinant-driven MLR is corroborated by the demonstration that a monoclonal antibody directed at this determinant fully blocks the Mls determinant-driven MLR. The significance of these studies to the problem of alloreactions in vivo is discussed.