Friendship quality and social information processing in clinically anxious children.

The association between perceived friendship quality (FQ) and social information processing (SIP) was examined in three groups of children and their close friends aged 7-12 years: 16 anxiety disordered children with social phobia (SP); 12 anxiety disordered children without SP (No-SP); and 32 nonclinical children. Positive and negative FQ positively associated with target children's positive and negative responding on a vignette measure of SIP. SP children reported lower positive SIP than No-SP but not nonclinical children; and this was the only group difference in SIP. Target children and their friends were similar in negative but not positive SIP. Following discussion about the vignette with a close friend, all target children increased in positive SIP; negative SIP did not change. Lower FQ and a more socially anxious friend predicted higher negative target child SIP postdiscussion. Close friendships play an important role in the SIP of both clinical and nonclinical children.

An automated algorithm for the generation of dynamically reconstructed trajectories.

The lack of long enough data sets is a major problem in the study of many real world systems. As it has been recently shown [C. Komalapriya, M. Thiel, M. C. Romano, N. Marwan, U. Schwarz, and J. Kurths, Phys. Rev. E 78, 066217 (2008)], this problem can be overcome in the case of ergodic systems if an ensemble of short trajectories is available, from which dynamically reconstructed trajectories can be generated. However, this method has some disadvantages which hinder its applicability, such as the need for estimation of optimal parameters. Here, we propose a substantially improved algorithm that overcomes the problems encountered by the former one, allowing its automatic application. Furthermore, we show that the new algorithm not only reproduces the short term but also the long term dynamics of the system under study, in contrast to the former algorithm. To exemplify the potential of the new algorithm, we apply it to experimental data from electrochemical oscillators and also to analyze the well-known problem of transient chaotic trajectories.

The Cool Teens CD-ROM for anxiety disorders in adolescents : a pilot case series.

Five adolescents received a multimedia CD-ROM containing a self-help treatment program for young people with an anxiety disorder. Participants used the 8-module Cool Teens CD-ROM over a 12-week period on a home computer. Every 2 weeks, they received a brief telephone call from a clinical psychologist to monitor symptoms and progress and to discuss any problems with understanding content or implementing techniques. Based on structured interviews, two participants (40%) no longer met diagnostic criteria (self-report ADIS) for at least one clinical anxiety disorder immediately following treatment and these same participants no longer met diagnostic criteria for any clinical anxiety disorder at 3-month follow-up. Two other participants failed to make gains based on diagnostic criteria, but showed improvement in anxiety symptoms for one main fear. Participants were generally satisfied with the multimedia content, the modules, and the delivery format of the program.

Immunoprophylaxis and cytotoxic effector cells against EL 4 leukemia induced in syngeneic C57BL/6J mice by use of irradiated EL 4 cells.

Tumor-specific immunoprophylaxis was achieved in C57BL/6J mice against EL 4 leukosis cell challenge by sensitization of the syngeneic host with multiple ip injections of irradiated EL 4 cells. A minimal radiation dose was used to replication-block EL 4 cells before inoculation, as defined by dose-response analysis of irradiated EL 4 cells. Multiple ip injections of irradiated EL 4 cells stimulated development of significant, yet relatively low, levels of cytotoxic lymphoid activity (CLA) in lymphoid cells of the peritoneal exudate as measured by in vitro 51Cr-release cytotoxicity assays. The specific temporal and frequency dependencies of the inoculation regimen for achieving immunoprophylaxis indicated that, in addition to CLA, other, short-lived, immune processes were important in the tumor rejection. These observations showed the capacity of the C57BL/6J host for tumor-specific immune recognition and rejection of the syngeneic EL 4 leukemia. The tumor rejection could be elicited solely by inoculations of irradiated EL 4 cells and did not require exogenous amplifiers, such as immunoadjuvants, chemical modifiers, and/or allogeneic immune information transfer.

High resolution proton magnetic resonance spectral characteristics of water, lipid, and protein signals from three mouse cell populations.

The high resolution proton magnetic resonance spectral properties of three C57BL/6J mouse cell populations (EL4 ascites tumor cells, normal spleen leukocytes, and normal erythrocytes) were investigated. Packed cell pellets were investigated at 100 MHz and 13.56 MHz. The 100-MHz spectra contained identifiable non-water proton resonances as well as the water resonance. The major non-water resonances from the EL 4 cells and normal leukocytes resembled resonances from lipid extracts, whereas the non-water resonances from erythrocytes resembled resonances from hemoglobin solutions. The reciprocals of the water proton spin-lattice relaxation times were roughly proportional to the total sample water content at both 100 and 13.56 MHz. The estimated slopes of these plots were frequency-dependent. The water proton relaxation rate generally increased with increasing total protein content of the packed cell pellets.

Effects of guanine ribonucleotide accumulation on the metabolism and cell cycle of human lymphoid cells.

A deficiency of purine nucleoside phosphorylase activity is associated with marked depletion of T-lymphocytes which is felt to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. Human T-lymphoblasts incubated in the presence of 2'-deoxyguanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphate whereas B-lymphoblasts and mature T4+-cell lines accumulate GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a complete block at the G1-S interface of the cell cycle. In contrast 3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine incorporation into DNA or RNA but do cause a pronounced slowing in the progression of cells through S phase. B-lymphoblasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-triphosphate from 2'-deoxyguanosine and progress normally through the cell cycle, demonstrating a requirement for guanine salvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide pools but not with inhibition of protein synthesis or alterations in basal levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic guanosine monophosphate. We conclude that the accumulation of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and its analogues.

DNA content of murine fibrosarcoma cell lines with varying metastatic potential.

The DNA content of murine fibrosarcoma cell lines of various metastatic potential was the subject of the current investigation. The cell lines were derived from methylcholanthrene-induced tumors as described previously (J. Varani et al., J. Natl. Cancer Inst., 71: 1281-1287, 1983). Cells were maintained in vitro and used for DNA studies no more than 48 h after passage. DNA staining was accomplished using propidium iodide and flow cytometry was used to quantitate relative amounts of DNA. Trout and chicken erythrocytes and mouse thymocytes were used as internal DNA standards for each cell line. DNA indices were calculated as the ratio of the G0-G1 peak channel number of the tumor cells to the G0-G1 peak channel number of the thymocytes. Manual chromosome counts were also obtained from each cell line using Giemsa-stained preparations. All cell lines demonstrated a single aneuploid population. The two tumor lines with the highest metastatic potential were slightly hyperdiploid whereas three low metastatic lines were near tetraploid. A sixth line of moderate metastatic potential was also found to be near tetraploid. Chromosome counts and flow cytometric analyses were in close agreement indicating that DNA content was largely due to chromosome replication. These data suggest that, in this model, metastatic potential and DNA content are inversely related once diploidy is exceeded.

Effects of the tricyclic nucleoside 6-amino-4-methyl-8-(beta-D-ribofuranosyl)- pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine on the viability and cell cycle distribution of L1210 cells in vitro.

TCN (1 microM) totally inhibited the growth of L1210 cells in culture and caused progressive loss of cellular viability, as indicated by a decreased clonogenicity and nigrosin dye exclusion. After 24 h or more of TCN treatment, a significant fraction of the cells had shrunk in size but did not fragment into dye-impermeable vesicles as reported previously for N1S1-67 hepatoma cells (P. G. W. Plagemann, J. Natl. Cancer Inst., 57: 1283-1295, 1976). TCN-induced growth inhibition was accompanied by a block of cell cycle progression in G1 or at the G1-S boundary. At all TCN concentrations studied, progression of cells out from behind this block was evident as a depletion of the early S-phase population in comparison to controls, while increasing the concentration of TCN (0.1 to 1 microM) led to a progressive retention of cells in S phase, suggesting a slowing of progression through S phase. The fraction of S-phase cells incorporating [methyl-3H]thymidine and the amount of [methyl-3H]thymidine incorporated per labeled cell were both decreased by TCN treatment. Increasing the concentration of TCN (0.1 to 1 microM) progressively decreased DNA synthesis and increased cell lethality. Thus it appeared that inhibition of DNA synthesis might cause the retention of cells in S phase which is associated with TCN lethality.

The modulating effect of isoproterenol on DNA replication and protein synthesis. Synthesis patterns of the HMG proteins from electrostatically sorted salivary gland nuclei during the in vivo cell cycle.

Within 96 h after initial isoproterenol administration, DNA replication and cell cycling were activated, as reflected in the bimodal distribution of nuclear fluorescence determined by flow-microfluorometric techniques. A group of proteins, the cetyltrimethylammonium bromide extractable nuclear proteins (CTAB-proteins), isolated form electrostatically sorted nuclei of rat salivary glands, was shown by staining and autoradiography after two-dimensional electrophoresis to undergo differential synthesis during various phases of the in vivo cell cycle after isoproterenol administration. Stained chromatographs revealed quantitative differences in protein synthesis. Gel autoradiography was a more sensitive technique than staining for detecting nuclear protein synthesis during cell cycling. As observed in the autoradiographs of the CTAB-proteins, isoproterenol initiated two distinct periods of protein synthesis in the salivary gland cell cycle: one during the 2C population G0/G1), and one during the 4C population (G2/M). Protein synthesis after isoproterenol administration was much more dramatic in the 2C (isoproterenol) population, where five new spots were seen. There was less radioactive incorporation in the 4C (isoproterenol) population. Two spots 'a' and 'b' that demonstrate differential protein synthesis in stained gel chromatographs and gel autoradiographs were shown to have electrophoretic mobilities, molecular weights and amino acid compositions highly similar to those of HMG1 and HMG2, respectively. A positive correlation could also be drawn between quantitative levels of 'a' and 'b' and their levels of incorporation during cellular activity with HMG (high mobility group) proteins. For example protein 'b' (HMG2) was consistently more abundant in proliferating cell populations than in the quiescent ones. Autoradiographic patterns of the CTAB-proteins indicated that proteins 'a' and 'b' were synthesized during the G0/G1 phase of the cell cycle, as were the majority of CTAB-proteins.

Measurement of intracellular fluorescence of human monocytes relative to oxidative metabolism.

Human monocytes (MN) produce O2- and H2O2 when stimulated by agonists. Dichlorofluorescin diacetate (DCFH-DA) has been used as a substrate for measuring intracellular oxidant production in neutrophils. DCFH-DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. DCFH-DA can not be appreciably oxidized to a fluorescent state without prior hydrolysis. We have examined the utility of DCFH-DA for the assessment of monocyte oxidative responses. The levels of intracellular fluorescence measured by flow cytometry were considerably less than expected from reported levels of O2--production or chemiluminescence assays. Compared with neutrophils, monocytes produced minimal increases in DCF fluorescence after stimulation with phorbol myristate acetate as measured by flow cytometry, but both cell types showed increases in fluorescence when bulk cell suspensions were measured by spectrofluorometry. To determine the intracellular location of the DCFH, bulk fluorescence measurements were made on both whole and sonicated cell preparations. When intact mononuclear cells were preloaded with DCFH-DA, then sonicated and oxidized with added excess H2O2, the increase in fluorescence was only 30% of the fluorescence of mononuclear cell sonicates to which DCFH-DA was added and oxidized in a similar manner. These results suggest that a portion of the DCFH-DA incorporated by intact cells, is not susceptible to oxidation by the added H2O2. Addition of NaOH to induce hydrolysis of any residual DCFH-DA in the sonicates of DCFH-DA-loaded intact mononuclear cells resulted in a further increase in fluorescence upon addition H2O2, suggesting that a significant portion of the DCFH-DA was not hydrolyzed despite ample uptake of this dye by these cells. In contrast, no further increase in fluorescence was observed in sonicates of DCFH-DA-loaded intact neutrophils, suggesting complete hydrolysis of all incorporated DCFH-DA to DCFH. When monocytes were allowed to phagocytose DCFH-DA-loaded Staphylococcus aureus, intracellular fluorescence was measurable by flow cytometry, indicating intracellular oxidation of the fluorochromes. We therefore propose that in monocytes the mechanism of intracellular processing of these fluorochromes differs from that in neutrophils owing to differences in intracellular localization of fluorochromes, site of oxidant production, and/or accessibility of the DCFH-DA to esterolysis.

Children with social phobia have lower quality friendships than children with other anxiety disorders.

BACKGROUND AND OBJECTIVE: Whilst shy, socially anxious or socially withdrawn children in nonclinical community samples report lower friendship quality (FQ) than nonanxious children, no study has examined the FQ of clinically anxious children. The aim of the study was to examine the FQ of children with anxiety disorders; and whether it differs for clinical children with or without a diagnosis of social phobia (SP). DESIGN: The study design was cross-sectional self-report. METHODS: Clinical children - 39 anxiety-disordered children with SP and 28 anxiety-disordered children without SP (No-SP) - presented for psychological treatment, and 29 nonclinical children were recruited from the community. Same-sex close friends were invited to participate using an unrestricted nomination procedure. All children were aged between 7 and 13 years. Both target child and friend completed the Friendship Quality Questionnaire and the Spence Children's Anxiety Scale. RESULTS: Using multilevel modeling within the framework of the Actor-Partner Interdependence Model, SP dyads were found to report lower overall FQ than No-SP dyads. SP dyads did not report lower overall FQ than nonclinical dyads. CONCLUSION: Children with SP in their diagnostic profile may be unique in their friendship experiences relative to children with other anxiety disorders.

Plant growth-promoting hormones activate mammalian guanylate cyclase activity.

In vivo injections of plant growth-promoting hormones increase the growth of animals as well as plants. Plant growth-promoting hormones and positive plant growth regulators are known to increase RNA and protein synthesis. Since cyclic GMP also increases RNA and protein synthesis, the object of the present investigation was to determine whether physiological levels of plant growth-promoting hormones and positive plant growth regulators have part of their mechanism(s) of action through stimulation of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system. Representatives of the three classes of growth-promoting hormones were investigated. Thus, auxins (indole-3-acetic acid, indole-3-butyric acid, beta-naphthoxyacetic acid, and 2,4,5-trichlorophenoxy acetic acid), gibberellins (gibberellic acid), and cytokinins [N6-benzyl adenine, kinetin (6-furfuryl aminopurine), and beta-(2-furyl) acrylic acid] all increased rat lung, small intestine, liver, and renal cortex guanylate cyclase activity 2- to 4-fold at the 1 microM concentration. Dose response curves revealed that maximal stimulation of guanylate cyclase by these plant growth regulators was at 1 microM; there was no augmented cyclase activity at 1 nM. The guanylate cyclase cationic cofactor manganese was not essential for augmentation of guanylate cyclase by these plant growth-promoting regulators. The antioxidant butylated hydroxytoluene did not block the enhancement of guanylate cyclase by these plant growth-promoting factors. These data suggest that guanylate cyclase may play a role in the mechanism of action of plant growth-promoting hormones and even of positive plant regulators at the cellular level.

Effect of methionine-loading on methyl group synthesis and activation in rat brain and liver.

Much greater increases in S-adenosylmethionine concentrations are observed in the liver in response to methionine-loading than in the brain due to differences in the methionine adenosyltransferase activities in these tissues. Liver methione adenosyltransferase exhibits a bimodal saturation curve with a nonlinear Line-weaver-Burk plot, indicating that high methionine concentrations are required for saturation. In the brain the methionine adenosyltransferase is saturated in vitro at a methionine concentration less than the normal physiological concentration. The increased S-adenosylmethionine concentrations in the livers of methionine-treated rats also account for the observed inhibition of N5,N10-methylenetetrahydrofolate reductase activity in this tissue. No inhibition of this enzyme is observed in the brain of methionine treated animals. Nor are S-adenosylmethionine concentrations increased significantly in brain. Serine hydroxymethyltransferase activity responds to methionine-loading by decreasing in brain and increasing in liver.

In vitro and in vivo cytotoxicity of T cells cloned from rejecting allografts.

Epa-1 is a tissue-restricted, non-major histocompatibility (MHC) antigen that may be responsible for the extreme sensitivity of skin to allograft rejection and graft-versus-host disease (GVHD), especially with MHC-compatible donors and recipients. To confirm that Epa-1 serves as a target in allograft rejection and GVHD, we isolated Epa-1-specific cytotoxic T lymphocyte (CTL) clones completely in vivo from sponge-matrix allografts and from lymph nodes draining rejecting skin allografts. These clones induced GVHD-like skin lesions in antigen-specific, MHC-restricted fashion following intradermal inoculation into appropriate hosts. The in vivo-derived clones are conventional CTL since they are IL-2-dependent and express the Thy-1.2+, Lyt-1-, Lyt-2+, L3T4- phenotype. The results of this study also are pertinent to the controversy over which T-cell subset actually mediates allograft immunity, since the intragraft isolation and subsequent cloning of conventional CTL that induce necrotizing skin lesions are direct evidence that CTL are the proximal mediators of allograft rejection.

Nonparametric flow cytometry analysis.

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.

Differences in flow cytometry and 3H-thymidine analyses of perturbed human lymphocytes.

A calf thymocyte crude aqueous extract was tested for DNA synthesis inhibitory activity using phytohemagglutinin-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis was assayed using tritiated thymidine and flow cytometry. Although the calf thymocyte crude extract inhibited tritiated thymidine incorporation by over 50%, only very slight changes in the flow cytometric analysis were observed. When dibutyryl-cyclic adenosine monophosphate was used as an inhibitor, a correlation in terms of the inhibition of tritiated thymidine to the inhibition by flow cytometry was observed.

Multiparameter kinetic analysis of killer cell initiation by using immune RNA.

By treating nonsensitized C57BL/6J spleen derived lymphocytes with EL-4 tumor cell directed xenogeneic extracted RNA we were able to monitor early changes in cellular DNA content by flow cytometric (FCM) analysis and 3H-thymidine uptake. These kinetic parameters were correlated with cell mediated cytotoxicity which appeared as early as 8 hr after activation as measured by release of chromium-51 from labeled EL-4 target cells. Flow cytometric analysis and 3H-thymidine uptake data shown peak S phase activity at 72 hr. Maximum cytotoxicity was observed at 48 hr. Cell cycle kinetic parameters were correlated with the appearance of cell mediated cytotoxicity.

Flow cytometric analysis of human erythrocytes: I. Probed with lectins and immunoglobulins.

A recent review (Aminoff, 1988) summarized the evidence for and against our hypothesis for the role of glycophorin in the senescence and clearance of mammalian red blood cells (RBC) from circulation. This hypothesis postulates the loss of sialic acid from RBC surface in two forms: (a) as vesicles containing the sialoglycoprotein glycophorin, and (b) as free sialic acid residues from glycophorin molecules remaining on cell surface. In this report we demonstrate the applicability of flow cytometric procedures to explore, at the cellular level, time-dependent changes on RBC surface with change in cell size, and with in vivo age. The RBC are probed with fluorescein isothiocyanate (FITC) labelled lectins and goat anti-human-IgG and -IgM. The relative intensity of fluorescence is correlated to the change in RBC size as measured by forward lightscatter. Reactivity of RBC with FITC-labelled wheat germ agglutinin can be inhibited with either 0.2M N-acetylglucosamine or by removal of sialic acid residues with neuraminidase. The properties of the smallest RBC correspond to those of the oldest RBC in their: (a) decreased reactivity with FITC-labelled lectins that recognize sialic acid residues, wheat germ and Limax flavus agglutinins, and (b) increased reactivity with FITC-labelled goat anti-human-IgG and -IgM. These results are compatible with our glycophorin hypothesis. Moreover, they suggest that the initial loss of sialic acid as glycophorin containing vesicles is gradual, while the subsequent step involving the loss of sialic acid residues is rapid and exposes multiple disaccharide galactose beta(1-3)N-acetylgalacosaminyl residues. These unmasked disaccharide sites are recognized by autoimmune IgG, IgM, and lectin-like receptors on macrophages resulting in the clearance of senescent RBC from circulation.

Activity of acyclic halogenated tubercidin analogs against human cytomegalovirus and in uninfected cells.

Novel acyclic halogenated tubercidins (4-amino-5-halo-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidines) were examined for their ability to inhibit human cytomegalovirus (HCMV) in yield reduction assays. 5-Bromo acyclic tubercidin (compound 102) was a more potent inhibitor of virus replication than the chloro- and iodo-substituted analogs (compounds 100 and 104). At a 100 microM concentration, the bromo and chloro compounds were more potent than acyclovir but not ganciclovir. Virus titers were reduced more than 99% by compounds 102 and 104 whereas compound 100 and the equally potent acyclovir reduced titers by only 90%. Quantitation of viral DNA by DNA hybridization demonstrated strong inhibition of HCMV DNA synthesis by these compounds. The most potent inhibitor, compound 102, had a 50% inhibitory (I50) concentration (1.6 microM) comparable to that of ganciclovir (1.8 microM). Cytotoxicity in uninfected human cells was evaluated and revealed the following: cell growth rates slowed markedly in the presence of 10 microM compound 102 whereas the same concentration of compounds 100 and 104 led to only a slight prolongation of population doubling time; these compounds inhibited cellular DNA synthesis but not RNA or protein synthesis, as measured by incorporation of radiolabeled precursors into acid-precipitable macromolecules; flow cytometry indicated that compound 102 was a mid-S phase blocker, and adenosine antagonized the inhibition of [3H]dThd incorporation by compound 102. Together, these results demonstrate that compound 102 is a potent and selective inhibitor of viral and cellular DNA synthesis and that acyclic halogenated pyrrolo-pyrimidine nucleosides may have therapeutic potential.