Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with β-trefoil folding and their dependence on the nature of the solvent

The galactoside-specific Viscum album L. agglutinin (VAA) is a potent biohazard akin to ricin and a mitogen for immune and tumor cells. These activities depend on cell surface binding to glycans. It is an open question whether the process of ligand binding alters the lectin's shape. Small angle neutron scattering (SANS) experiments revealed that the carbohydrate ligand lactose induced a decrease of the radius of gyration of dimeric VAA from 54.5 +/- 1 to 49.5 +/- 1 A in water. Apparently, VAA in aqueous solution and at the concentrations tested at 3.6 mg/ml and above adopts a compacted structure as response to ligand binding. In contrast to the behavior in aqueous solution, lactose binding in DMSO resulted in an increase of the lectin's radius of gyration from 49 +/- 1 to 55.5 +/- 1 A. Because shape changes may be reflected in the thermostability of the protein, this parameter was examined by activity assays of protein exposed to 60 degrees C and 70 degrees C and by differential scanning calorimetry (DSC). In line with the lactose-induced conformational alterations revealed by the SANS experiments, lactose presence enhanced the thermostability of VAA in water. Thus, binding of the carbohydrate ligand in solution can entail changes in shape and thermostability in the case of the tested plant lectin.     

Periodicity in the fine structure of histograms during nuclear decay

A theoretical explanation is offered for the appearance of periodicity in the fine structure of histograms describing the processes of nuclear decay. This effect is observed in a sample containing nonpolarized radioactive nuclei, with largely isotropic spin distribution over directions. However, because of fluctuations occurring in any real sample, the number of nuclei having spin directed along some isolated axis would differ somewhat from the number of nuclei with spin directed along another axis. The same is immobile relating to the Earth but rotates in space together with it. At the same time, the nuclear spins, owing to quite weak interaction with the environment, keep their directions in space. Therefore, the spin ensemble rotates both relative to the sample itself and relative to the detector of α-particles directed onto the sample. Since fluctuations in the number of nuclei with spins directed at a certain angle relative to the detector direction affect the fine structure of histograms, the latter proves to depend, among other things, of the rotation of the Earth. Consequently, when the Earth makes a full revolution and the distribution of fluctuations in the number of nuclei with given spin direction returns to the initial one, the shape of the fine structure of histograms will be repeated, and repeated over the same definite time intervals.     

Analysis of flow cytometric data of irradiated cell populations

Summary Flow cytometry (FCM) and autoradiography have been applied to determine changes in the cell kinetics of irradiated cells. Synchronized L-929 cells were irradiated with 10 Gy of X-rays when progressing from G₁-to S-phase of the cell cycle. In this study three methods to analyse DNA histograms were tested for applicability on FCM data obtained from cell populations blocked or retarded in the cycle: A) the Gaussian integral method, B) the peak-half-reflection method, and C) the rectangle method. Since histograms from synchronized cells are heavily distorted as compared to those obtained from exponentially growing cells and are quite similar to histograms from irradiated cells, they were used to test the suitability of the evaluation methods. Comparing the evaluated FCM data with the autoradiographic results from the same experimental series, the Gaussian integral method proved to be superior to the two other relatively simple approximation methods. The FCM histograms of irradiated cells were therefore analyzed only by the Gaussian integral method. It was shown that a considerable fraction of cells is still in the S-phase 25 h post irradiation, the DNA synthesis of which has ceased, as shown by autoradiography. This indicated that parallel measurements using FCM and autoradiography yield additional information on cell kinetic changes that cannot be obtained from applying one of the two methods used.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Mitogenic response to epidermal growth factor: Relationship to number,affinity, and down-regulation of EGF receptors in three murine embyro cell lines

Swiss 3T3 and C3H-M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H-10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10-nM EGF all three cell lines “down-regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.     

Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages

The rate of movement of different receptors and ligands through the intracellular endocytic apparatus was studied in alveolar macrophages. Cells were exposed to iodinated alpha-macroglobulin-protease complexes, mannose terminal glycoproteins, diferric transferrin, and maleylated proteins. By use of the diaminobenzidine density shift procedure, we demonstrated that these ligands were internalized into the same endocytic vesicle. We then compared the rates of transfer to the lysosome or recycling to the cell surface of different ligands/receptors contained in the same endosome. We found that although the rate constant for degradation was ligand specific, the lag time prior to the initiation of degradation was the same for all three ligands. We also found that molecules taken up nonspecifically by fluid-phase pinocytosis had the same lag time prior to degradation as ligands internalized via receptor-mediated endocytosis. These data suggest that different molecules within the same endocytic compartment are transferred to the lysosome (or degradative compartment) at the same rate. We measured the rate of return of receptors to the cell surface by either inactivating surface receptors by protease treatment at 0 degrees C, or by incubating cells with saturating amounts of nonradioactive ligand at 37 degrees C. We then measured the rate of appearance of "new" receptors on the cell surface. Using these approaches, we found that three different receptors were transferred from internal pools to the cell surface at the same rate. The rate of transfer was independent of whether receptors were initially occupied or unoccupied. Our observations indicate that receptor/ligands, once inside alveolar macrophages, are transported by vesicles which transfer their contents as a cohort from one compartment to another. The rate of movement of these receptors is determined by the movement of vesicles and is independent of their content.     

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation ( CV ) from 2 to 16%; (ii) constant measurement CV or CV 's increasing from G₁ to G₂ phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.
The fractions of cells in the G₁, S, and (G₂+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G₁ fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.     

Beyond Bar and Line Graphs: Time for a New Data Presentation Paradigm

Figures in scientific publications are critically important because they often show the data supporting key findings. Our systematic review of research articles published in top physiology journals (n = 703) suggests that, as scientists, we urgently need to change our practices for presenting continuous data in small sample size studies. Papers rarely included scatterplots, box plots, and histograms that allow readers to critically evaluate continuous data. Most papers presented continuous data in bar and line graphs. This is problematic, as many different data distributions can lead to the same bar or line graph. The full data may suggest different conclusions from the summary statistics. We recommend training investigators in data presentation, encouraging a more complete presentation of data, and changing journal editorial policies. Investigators can quickly make univariate scatterplots for small sample size studies using our Excel templates.     

Evidence of chlorophyll synthesis pathway alteration in desiccated barley leaves

In etiolated leaves, saturating flash of 200 ms induces phototransformation of protochlorophyllide (Pchlide) F655 into chlorophyllide (Chlide), then into Chl through reactions which do not need light sensibilisation. The synthesis of Chl is known to be slowed down in etiolated leaves exposed to desiccation stress. In order to analyse the intensity and time-course of Chlide transformation into Chl, we used the fluorescence emission of etiolated leaves previously exposed to a 200 ms saturating flash. We used low-temperature fluorescence spectroscopy to reveal the inhibition site of Chl synthesis in etiolated barley leaves exposed to water stress. Shibata shift appears as the main target point of the water deficit. It was found that water deficit inhibits partially active Pchlide F655 regeneration. Also, esterification of Chlide into Chl is impaired. It appears that these inhibitory effects alter the appearance of PSII active reaction centres.     

Ligand competition curves as a diagnostic tool for delineating the nature of site-site interactions: theory

A few molecular models have been developed in recent years to explain the mechanism of cooperative ligand binding. The concerted model of Monod, Wyman and Changeux and the sequential model of Koshland, Némethy and Filmer were formulated to account for positively cooperative binding. The pre-existent asymmetry model and the sequential model can account for negatively cooperative ligand binding. In most cases, however, it is virtually impossible to deduce the molecular mechanism of ligand binding solely from the shape of the binding isotherm. In the present study we suggest a new strategy for delineating the molecular mechanism responsible for cooperative ligand binding from binding isotherms. In this approach one examines the effect of one ligand on the cooperativity observed in the binding of another ligand, where the two ligands compete for the same set of binding sites. It is demonstrated that the cooperativity of ligand binding can be modulated when a competitive ligand is present in the protein-ligand binding mixture. A general mathematical formulation of this modulation is presented in thermodynamic terms, using model-independent parameters. The relation between the Hill coefficient at 50% ligand saturation with respect to ligand X in the absence, h(x), and in the presence of a competing ligand Z, h(x,z), is expressed in terms of the thermodynamic parameters characterizing the binding of the two ligands. Then the relationship between h(x) and h(x,z), in terms of the molecular parameters of the different allosteric models, is explored. This analysis reveals that the different allosteric models predict different relationships between h(x,z) and h(x). These differences are especially focused when Z binds non-cooperatively. Thus, it becomes possible, on the basis of ligand binding experiments alone, to decide which of the allosteric models best fits a set of experimental data.     

Asparagine-Requiring Tumor Cell Lines and Their Non-Requiring Variants: Cytogenetics, Biochemistry and Population Dynamics

Asparagine-requiring Jensen and Walker rat tumor cells and their asparagine-independent variants have been analyzed. The following results were obtained: (1) Both cell lines have very low levels of asparagine synthetase, and non-requiring revertants isolated from these lines have elevated levels of the enzyme. (2) No differences in chromosome number were detected between the parent Jensen line and five Jensen non-requiring revertants isolated from it. (3) Both Jensen and Walker cells undergo asparagineless death when deprived of this amino acid, although the Jensen cells do so at a more rapid rate. (4) Jensen requiring lines are at a selective advantage when grown in competition with non-requiring variants in complete medium, and their growth rate is more rapid when grown separately. The selective coefficients for the variant with respect to the asparagine-requiring parent ASN(-) line were 0.94 for the competition experiments and 0.83 for growth rate estimates. (5) A somatic cell hybrid between Chinese hamster cells (which require asparagine at low densities, and posses measurable synthetase activity) and the Walker line was found to be asparagine-independent, and it possessed enzyme levels equivalent to the hamster parent. The results of these investigations suggest a parallel with microbial auxotrophic mutants and can be understood in terms of alterations within nuclear structural genes.     

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.     

DNA binding properties of novel cytotoxic gold(III) complexes of terpyridine ligands: the impact of steric and electrostatic effects

Four gold(III) complexes of terpyridine derivatives 1–4 have been synthesized and characterized by spectroscopic methods. In vitro data demonstrated that all of them showed higher cytotoxicity than cisplatin against the human non-small-cell lung cancer cell line (A-549), the human stomach carcinoma cell line (SGC-7901), the human cervix carcinoma cell line (HELA), the human colon carcinoma cell line (HCT-116), the human liver carcinoma cell line (BEL-7402), the murine leukemia cell line (P-388) and the human acute promyelocytic leukemia cell line (HL-60). Complex 3 exhibits the highest activity, with growth inhibition rates of over 80% at 10⁻⁸ mol L⁻¹ against the A-549, HCT-116 and HELA tumor cell lines. Interestingly, ligands L1–L4 are also very cytotoxic against the cell lines tested. Complexes 1–4 are stable in aqueous solution for 2 days in the presence of the biological reducing agent glutathione. The inductively coupled plasma mass spectrometry data showed that DNA isolated from cells treated with complexes 1 and 3 contained gold with gold-to-nucleotide ratios of approximately 1:6,400 and 1:4,900, respectively. Fluorescence titration, UV and circular dichroism analyses proved that the steric and electrostatic effects of the ligand remarkably influence the interactions of their gold(III) complexes with DNA. The DNA binding ability of the complexes has been correlated with their cytotoxicity, which could potentially provide a new rationale for the future design of terpyridine-based metal complexes with antitumor potential.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Comparison of the ability of basic and acidic fibroblast growth factor to stimulate the proliferation of an established keratinocyte cell line: modulation of their biological effects by heparin, transforming growth factor beta (TGF beta), and epidermal growth factor (EGF)

The bioactivity of both bFGF and aFGF in the BALB/MK-1 cell line has been compared to that of EGF. Our results indicate that, for that cell type, aFGF was far more potent than bFGF in inducing cell proliferation. In the presence of heparin, aFGF was as potent as EGF. In addition, excess bFGF has an inhibitory effect on the proliferation of MK cells exposed to a saturating concentration of aFGF, therefore acting as a partial agonist of aFGF. Surprisingly, bFGF, although it had low biological activity, was capable of synergizing the effect of EGF. In its presence, cultures exposed to saturating concentration of EGF have a final cell density 3- to 4-fold higher than that of counterpart cultures exposed to EGF alone. TGF beta, which in previous studies has been shown to inhibit the growth of keratinocytes, also inhibited the growth of BALB/MK-1 cells in response to either bFGF or aFGF. These studies suggest a role for FGF in regulating BALB/MK proliferation. aFGF provides positive growth signals which can be negatively modulated by excess bFGF or TGF beta, while bFGF, although a poor mitogen, could act by potentiating the effect of subsaturating concentrations of EGF.     

Flow cytometry of postmortem human testicular tissue in cases of atherosclerosis

From forty-seven autopsy cases of atherosclerosis flow cytometry (FCM) of DNA and histology of both testes are compared with the histological sections of their supplying vessels arteriae testiculares and arteriae ductus deferentis at different levels. By this method, changes of spermatogenesis are judged separately for each side and the results can be related to the local conditions of blood supply. Four young men, dead after traffic accidents, served as control. In the majority of cases, the computer-assisted evaluations of the meiotic DNA histograms show no differences between the right and left testis, even when differences of the arterial diameters are found by histology. On the other hand, cases with distinct differences in the histograms can show insignificant pathological alterations of the vessels. Though most excessive forms of macroscopic and microscopic atherosclerosis do not necessarily lead to a significant reduction in spermatogenesis, some cases with moderate forms show a strong reduction or even a total loss. This discrepancy can best be explained by superposition of other diseases.     

Analysis of DNA synthesis rate of cultured cells from flow cytometric data

The rate of DNA synthesis along S phase is estimated from flow cytometric histograms on the basis of a mathematical model of a cell population. In the absence of loss, the model expresses the population kinetics in terms of DNA synthesis rate, S-phase influx, and population size. A single histogram is sufficient to determine the DNA synthesis rate when the population is in balanced exponential growth. Two suitably chosen histograms are necessary if the S-phase influx is exponential in a time interval longer than the S-phase duration. The analysis procedure was tested on published autoradiographic data and applied to three cultured cell lines (CM-S, 3LL, and M14 cells) that show various patterns of DNA distribution. In each case the cell-cycle fractions, the DNA synthesis rate, and the S-phase duration were obtained.     

Proliferation and apoptosis in a neuroblastoma cell line exposed to 900 MHz modulated radiofrequency field

The aim of this study was to examine whether a modulated radiofrequency of the type used in cellular phone communications at a specific absorption rate (SAR) higher than International Commission on Non-ionizing Radiation Protection (ICNIRP) reference level for occupational exposure, could elicit alterations on proliferation, differentiation, and apoptosis processes in a neuroblastoma cell line. The cell line was exposed for 24, 48, and 72 h to 900 MHz radiofrequency and proliferation and differentiation were tested by WST-I assay and by a molecular analysis of specific markers, two oncogenes and a cytoskeleton protein, in exponential growth phase and in synchronized cell cultures. Apoptosis was evaluated by caspase activation analysis and by molecular detection of Poly (ADP-ribose) polimerase (PARP) cleavage. Combined exposures to radiofrequency and to the differentiative agent retinoic acid or to the apoptotic inducer camptothecin were carried out to test possible interference between electromagnetic field and chemical agents. Overall our data suggest that 900 MHz radiofrequency exposure up to 72 h does not induce significant alterations in the three principal cell activities in a neuroblastoma cell line.     

Linked-function origins of cooperativity in a symmetrical dimer

The thermodynamic origins of substrate binding cooperativity in a dimeric enzyme that can bind one substrate (A) and one allosteric ligand (X) to each of two identical subunits are discussed. It is assumed that maximal activity is not subject to allosteric modification and that the substrates and allosteric ligands achieve binding equilibrium in the steady state. Each uniquely ligated form is assumed to be capable of exhibiting unique binding properties, and only the principles of thermodynamic linkage are used to constrain the system further. The explicit relationship between the Hill coefficient, the concentration of X, and the magnitudes of the relevant coupling free energies and dissociation constants is derived. In the absence of X only the homotropic coupling between substrate sites contributes to a nonhyperbolic substrate saturation profile. An allosteric ligand, X, can alter the cooperativity in two distinct ways, one mechanism being manifested when X is saturating and the only only when X is present at saturating concentrations. By evaluating the concentration of substrate required to produce half-maximal velocity as a function of [X], as well as the Hill coefficients when X is absent and fully saturating, the dissociation and coupling constants most important for understanding the mechanisms of allosteric action in an enzyme of this type can be determined.     

Future needs and expected trends in peripheral blood cell analysis: erythrocyte histograms

Erythrocyte histograms should be analysed in accordance with the ICSH recommended protocol to determine: How many red cell populations are present and the proportion of the total red cells which are included in each population. The central tendency (mode, median & mean) and dispersion (size ratio, geometric standard deviation & coefficient of variation) for each population. The proportion of microcytic or macrocytic cells in each population. Examples are given to show how the patient's diagnosis is assisted by careful analysis of the histograms and the effect of treatment monitored by sequential testing. Histograms can be generated from blood films or from aperture-impedance and light-scatter volume measurements. Volume measurements on these systems are influenced by the red cell shape and internal refractive index. Mean cell hemoglobin concentration affects both shape in the aperture-impedance orifice and internal refractive index in light-scatter systems. There is poor agreement between volume histograms obtained on the two measuring systems and the histograms available on current automated instruments provide no more useful information than can be obtained from the blood film. Automated instruments need to produce histograms with fewer artefacts and the histograms should then be examined in accordance with the ICSH protocol. This approach should maximise the diagnostic value of the complete blood count.     

Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl-l-methionine and magnesium chloride in sodium—potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl-l-methionine using this method are similar to values reported previously (0.51 and 14 μM, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2–3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.