Analytical techniques for assaying nitric oxide bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood). NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO(2)(-)) and nitrate (NO(3)(-)) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.     

Pterins and the regulation of lymphocyte activation on the mode of xanthopterin action

Some intermediates of pterin anabolism amplify the lectin-induced lymphocyte stimulation while the catabolites xanthopterin and isoxanthopterin terminate their proliferation (Ziegler, I. et al., Cancer Res. 43, 5356 (1983). In the present investigation, we analysed the effect of xanthopterin on total RNA synthesis and on DNA synthesis in both concanavalin A-stimulated lymphocytes and in the lymphoblastoid cell line L 1210. The time courses at various inhibitor concentrations indicated that xanthopterin inhibits RNA synthesis prior to DNA synthesis. Further analysis of the RNA species was performed by double-labeling and subsequent polyacrylamide-gel electrophoresis. Pulse and pulse-chase experiments revealed that an inhibition of 45 S pre-RNA is closer to the target of xanthopterin inhibition than is DNA synthesis.     

Imaging techniques in microbiology

Recent advances in optical imaging have dramatically expanded the capabilities of the light microscope and its usefulness in microbiology research. Some of these advances include improved fluorescent probes, better cameras, new techniques such as confocal and deconvolution microscopy, and the use of computers in imaging and image analysis. These new technologies have now been applied to microbiological problems with resounding success.     

Mechanosensing in T lymphocyte activation

Mechanical forces play an increasingly recognized role in modulating cell function. This report demonstrates mechanosensing by T cells, using polyacrylamide gels presenting ligands to CD3 and CD28. Naive CD4 T cells exhibited stronger activation, as measured by attachment and secretion of IL-2, with increasing substrate elastic modulus over the range of 10–200 kPa. By presenting these ligands on different surfaces, this report further demonstrates that mechanosensing is more strongly associated with CD3 rather than CD28 signaling. Finally, phospho-specific staining for Zap70 and Src family kinase proteins suggests that sensing of substrate rigidity occurs at least in part by processes downstream of T-cell receptor activation. The ability of T cells to quantitatively respond to substrate rigidly provides an intriguing new model for mechanobiology.     

Lipid rafts in lymphocyte activation

The existence of sphingolipid- and cholesterol-rich membrane microdomains called "lipid rafts", as well as their role in lymphocyte biology, has been widely debated during the last few years. Plasma membrane microdomains seem to be primarily involved in initiation and propagation of the signal transduction cascade associated with lymphocyte activation. In this review, we discuss the recent literature suggesting that, during lymphocyte activation and chemotaxis, lipid rafts act as platforms to compartmentalise signalling and facilitate specific protein-protein interactions.     

Two roles for Ia in antigen-specific T lymphocyte activation

In this study we examined the mechanism by which a PPD-specific murine T cell hybridoma, 8B2, recognized PPD associated with antigen-presenting cells (APC) in a manner genetically restricted by I-Ad. It was found that PPD-pulsed APC that were glutaraldehyde-fixed and treated with anti-Ia monoclonal antibody (abbreviated as PGM) were unable to stimulate the 8B2 T cells, as expected, due to inhibition caused by antibody binding to the Ia. However, addition of non-antigen-treated, glutaraldehyde-fixed APC (abbreviated as G) to cultures containing 8B2 T cells and PGM restored T cell activation, as determined by IL 2 production. This second non-antigen-specific function provided by the additional APC, G, was attributed to Ia and could be substituted by APC plasma membranes and by soluble membrane extracts. Genetic restriction analysis in which a variety of Ia-positive and Ia-negative cell lines and B cell blasts from different mouse strains were used as PGM or as G showed that each APC provided different Ia determinants that were specifically recognized by the T cells. PGM cells had to express I-Ad in order to present the PPD determinant, whereas the non-antigen-specific function was specific for I-Ad or I-Ab. These results suggest that the anti-Ia antibody does not interfere with the PPD/I-Ad-specific determinant bound by the antigen-specific T cell receptor, but prevents a second non-antigen-specific interaction with another region of the Ia molecule, which is provided by G. These two roles for Ia (antigen-specific and non-antigen-specific) were also found for activation of normal polyclonal PPD-specific T cell responses; thus they are not unique to the 8B2 T cell, but are generally applicable. In addition, T cell interactions with PGM and with G each provide different intracellular activation signals. This was determined by substituting the PGM or the G with either the tumor promoter phorbol 12-myristate 13-acetate (PMA) or the Ca++ ionophore, ionomycin. It was found that 8B2 T cells cultured with PGM and ionomycin, but not with PGM and PMA, were activated for IL 2 production. Neither PMA nor ionomycin in conjunction with G resulted in T cell activation. Taken together, these results indicate that 8B2 T cell activation involves APC Ia antigens in two different ways: one is to contribute to the presentation of the foreign PPD antigen, and a second is a non-antigen-specific Ia-T cell interaction necessary to provide additional intracellular activation signals.(ABSTRACT TRUNCATED AT 400 WORDS)     

A sensitive, continuous spectrophotometric method for assaying alpha-glycerophosphate dehydrogenase: activation by menadione

By use of a more sensitive method than standard assays, it is demonstrated that alpha-glycerophosphate dehydrogenase activity can be monitored continuously in rat liver mitochondria; maximum activity was obtained by sonication in the presence of Triton X-100. Vitamin K3 (menadione) seems to enhance the activity of the enzyme. The assay in the presence of menadione is linear over a much greater mitochondrial concentration (up to 250 micrograms of protein in the reaction mixture).     

Locus of inhibitory action of cAMP-dependent protein kinase in the antigen receptor-triggered cytotoxic T lymphocyte activation pathway

The mechanism of the cAMP involvement in regulation of cellular functions was studied here using a novel functional assay (antigen receptor-triggered exocytosis of granules) of cloned cytotoxic T lymphocytes (CTL). We suggest that cAMP-dependent protein kinase, protein kinase A, counteracts the protein kinase C and Ca2+-mediated stimulatory T-cell antigen receptor (TcR)-triggered biochemical pathway. This suggestion is supported by experimental results which satisfy criteria for protein kinase A involvement in cellular functions. Pretreatment of CTL with cholera toxin induces cAMP accumulation in CTL, partially inhibits TcR-triggered "lethal hit" delivery to the target cell, and almost completely blocks TcR-triggered exocytosis of granules from CTL. Other agents that raise the intracellular level of cAMP, including forskolin and isobutylmethylxanthine (IBMX) also inhibit TcR-triggered CTL activation. Involvement of cAMP-dependent protein kinase in an inhibitory pathway is suggested by the synergistic effects of cyclic nucleotide analogs 8-bromo-cAMP and N6-benzoyl-cAMP in inhibition of TcR-triggered exocytosis. Forskolin and IBMX inhibited TcR-triggered phosphoinositide turnover in CTL, suggesting that cAMP affected very early events in signal transduction that follow TcR cross-linking by a ligand. The ability of IBMX to inhibit CTL activation when the TcR cross-linking step was by-passed by the combination of phorbol myristate acetate and ionophore A23187 suggests that the locus of inhibitory effect of cAMP is at both the early and late stages of the TcR-triggered transmembrane signaling pathway.     

Stress stimuli-induced lymphocyte activation.

Oxidants, heavy metals, and heat shock, collectively known as stress stimuli, induce the synthesis of a variety of proteins, termed stress proteins, and enhance glucose uptake. In this study, we have demonstrated that stress stimuli enhance protein tyrosine phosphorylation (PTyr-P), modulate protein tyrosine phosphatase (PTPase) activity, activate the src family protein tyrosine kinase (PTK), p56lck, and enhance glucose uptake in human peripheral blood mononuclear cells. The heavy metal Hg2+ and heat shock stimulated PTPase activity at an optimal dose, whereas the oxidant phenylarsine oxide (PAO) was only marginally stimulatory. Treatment of lymphocytes with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stress stimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and p56lck activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PAO were effective in activating PTPase than those effective in eliciting p56lck activation. We could not demonstrate a correlation between an effective dose inducing PTyr-P and glucose uptake. Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p56lck, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or preventing dephosphorylation of target proteins which are involved in the biological response. Our data may also provide the biochemical basis for the previously reported mitogenic effects of Hg2+ on lymphocytes.     

Calcium and T lymphocyte activation

A prolonged (at least 2-4 hr) elevation of [Ca2+]i accompanies early T cell activation by TCR/CD3-specific ligands. Ca2+ is generally thought to be an essential second messenger for early activation, but the precise molecular events contingent upon the Ca2+ signal remain to be determined. The Ca2+ signal can be separated into an early transient peak due to InsP3-released Ca2+ from intracellular stores, and a sustained plateau due to altered transmembrane Ca2+ flux. Patch clamp studies have identified an InsP3-activated, Ca2+ permeable channel in the plasma membrane of T lymphocytes that may be responsible for the sustained elevation of [Ca2+]i during continuous TCR/CD3 occupancy. The Ca2+ signal can be further resolved at the level of the single cell into a series of repetitive oscillations between peak and trough levels with a period of 16-20 s. The oscillations may be part of a frequency-encoded signaling system. Several nonlinear internal feedback controls may contribute to the periodic nature of the Ca2+ signal: PKC-mediated phosphorylation of the CD3 gamma subunit, which is a feedback inhibitor of TCR/CD3 function; amplification of Ca2+ release from endoplasmic reticulum by a highly cooperative step in the opening of Ca2+ channels by InsP3, and Ca2+-dependent feedback enhancement of PLC function; autoregulatory negative feedback on Ca2+ influx by Ca2+, both by a direct effect on the plasma membrane Ca2+ channel and by induction of membrane hyperpolarization secondary to Ca2+-activated K+ efflux. In addition, several other internal feedback controls on TCR/CD3 function, by CD4-induced tyrosine-specific phosphorylation of the CD3 zeta subunit, or on the Ca2+ signal, by extracellular Cl- or by GM1 gangliosides, are also postulated. The question of whether a G protein couples TCR/CD3 to PI hydrolysis and to Ca2+ mobilization is unresolved, although some indirect evidence for the involvement of GTP binding proteins in T cell activation has recently been obtained with cholera toxin. There is also preliminary evidence that TCR/CD3 may structurally conform to G protein coupled receptors, i.e., having a core structure of seven alpha helical transmembrane spanning segments, a ligand recognition site, loci for regulatory phosphorylation, and a putative nucleotide binding site.     

Calcium and lymphocyte activation.

The role of ionized calcium in the early phases of activation of human peripheral blood lymphocytes was evaluated by stimulating the cells with a calcium ionophore A23187 (Lilly) or with mitogenic lections over a broad range of extracellular calcium concentrations (< 1 to > 1000 μM). A number of biochemical parameters shown previously to be altered during stimulation of these cells by mitogenic lectins were studied including: 1) amino acid transport, 2) phosphatidylinositol turnover, 3) cyclic nucleotide accumulation, and 4) calcium uptake. The ionophore (0.1–0.5 μg/ml) was shown to produce stimulatory effects in all of these systems with the changes closely simulating those produced by the lectins themselves both in regard to time course and magnitude. A23187 also produced 5–10 fold increases in DNA synthesis as measured at 48–72 hr after exposure of the cells to this agent. The responses to A23187 were shown to be almost completely dependent on the presence of ionized calcium. Since mitogenic lectins are known to stimulate calcium uptake and DNA synthesis appears to require extracellular calcium, the early responses to A23187 suggested that calcium was important both during the early and later phases of lymphocyte activation. However, short time course studies of amino acid transport, cyclic AMP accumulation, and phosphatidylinositol turnover in calcium deficient media failed to provide convincing evidence of calcium dependency in lectin stimulation since the three responses were well preserved (<25% inhibition) in “calcium free” medium containing 1–3 mM ethylene bis (ethylene oxynitrilo) tetraacetic acid (EGTA) (an estimated final Ca²⁺ concentration of <1 μM). Greater than 50% inhibition of the lectin response was seen only when the cells were incubated in calcium free, EGTA-containing medium for 30 min prior to stimulation with lectin. Thus despite the striking ability of A23187 complexed with calcium to mimic the action of mitogenic lectins, its effects may involve more than simple transport of calcium into the cell. A23187 may also exert a direct membrane action as suggested by its ability to produce rapid increases in cAMP and the occurrence of cytotoxicity at 5–10 fold higher concentrations (2–4 μg/ml). However, these data do not entirely exclude a mechanism of ionophore action whereby: 1) mobilization of intracellular stores of calcium and 2) diminished intracellular transport of ionized calcium at extracellular concentrations less than or equal to 1 μM combine to provide an effective stimulus for cellular activation.     

Lipid rafts in lymphocyte activation and migration

Functional polarization of leukocytes is a requisite to accomplish immune function. Immune synapse formation or chemotaxis requires asymmetric redistribution of membrane receptors, signaling molecules and the actin cytoskeleton. There is increasing evidence that compartmentalization of the plasma membrane into distinct lipid microdomains is pivotal in establishing and maintaining leukocyte polarity. Specific rafts assemble into large-scale domains to create plasma membrane asymmetries at specific cell locations, thus coordinating temporally and spatially cell signaling in these processes. In this review we discuss the roles of lipid rafts as organizers of T lymphocyte polarity during cell activation and migration.     

Imaging techniques for elements and element species in plant science

Revealing the uptake, transport, localization and speciation of both essential and toxic elements in plants is important for understanding plant homeostasis and metabolism, subsequently, providing information for food and nutrient studies, agriculture activities, as well as environmental research. In the last decade, emerging techniques for elemental imaging and speciation analysis allowed us to obtain increasing knowledge of elemental distribution and availabilities in plants. Chemical imaging techniques include mass spectrometric methods such as secondary ionization mass spectrometry (SIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron-based techniques such as X-ray fluorescence spectroscopy (SRXRF), and so forth. On the other hand, X-ray absorption spectroscopy (XAS) based on synchrotron radiation is capable of in situ investigation of local atomic structure around the central element of interest. This technique can also be operated in tandem with SRXRF to image each element species of interest within plant tissue. In this review, the principles and state-of-the-art of these techniques regarding sample preparation, advantages and limitations, and improvement of sensitivity and spatial resolution are discussed. New results with respect to elemental distribution and speciation in plants revealed by these techniques are presented.     

Core histone acetylation during lymphocyte activation

Histone acetylation has been followed in cultures of human lymphocytes, in PHA-stimulated lymphocytes and in mixed lymphocytes obtained from identical twins and from unrelated donors. A computer assisted analysis of two-dimensional gels and autoradiograms revealed that in cultured lymphocytes only H3 and H4 core histones incorporate labeled acetate and that two H3 variants greatly differ in their rate of acetate uptake.     

Effects of cadmium on lymphocyte activation

The effects of cadmium (Cd) on phytohemoagglutinin or phorbol myristate acetate-induced lymphocyte activation were investigated and a dose-dependent inhibition of cell proliferation was found. Kinetic studies revealed that the Cd-sensitive step is an early event of T cell stimulation. Failure of IL2 secretion and reduction of IL2 receptor expression in the Cd-treated cells are also reported. Regardless of which mechanism is responsible for Cd effects, our studies show that the inhibition of lymphocyte activation is associated with reduced [3H]phorbol dibutyrate binding to Ca2+-phospholipid-dependent protein kinase and altered breakdown of phosphatidylinositols. Thus, Cd interferes with two biochemical events which play a critical role in lymphocyte signal transduction and activation.