Adaptation of a load-inject valve for a flow injection chemiluminescence system enabling dual-reagent injection enhances understanding of environmental Fenton chemistry.

Environmental Fenton chemistry has been poorly constrained within the marine environment at a multi-component level. A simple, unique, reconfiguration of a flow-injection analytical system combined with luminol chemiluminescence allows quasi-simultaneously the measurement, using a single load-inject valve and a single photon multiplier tube, of reduced iron, Fe(II), and hydrogen peroxide. The system enables rapid, every 22s, measurements with good accuracy at environmentally relevant concentrations, less than 5% relative standard deviations on both a 5 nM Fe(II) standard and a 60 nM hydrogen peroxide standard. Limits of detection were as low as 40 pM Fe(II) and 100 pM hydrogen peroxide. The system showed excellent capability by measuring from within an organic rich seawater the photochemically induced production of Fe(II) and hydrogen peroxide and their subsequent cycling and Fenton like interactions.

Improved J-compensated sequences based on short composite pulses.

Efficient J-compensated sequences that are shorter in duration and use less RF pulses have been created from short but very efficient composite 90 degrees RF pulses. The improved J-compensation transforms in-phase into antiphase magnetization and can be incorporated in any pulse sequence that involves evolution of heteronuclear J-couplings. The compensated sequences were tested and incorporated into an HMBC sequence. J-compensated experiments referred to as HMBC-J45 + 90A and HMBC-J45 + 90B, were found to be effective over a wide range of J values.

Amelogenin-mediated regulation of osteoclastogenesis, and periodontal cell proliferation and migration.

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

31P MAS-NMR of human erythrocytes: independence of cell volume from angular velocity.

31P magic angle spinning NMR (MAS-NMR) spectra were obtained from suspensions of human red blood cells (RBCs) that contained the cell-volume-sensitive probe molecule, dimethyl methylphosphonate (DMMP). A mathematical representation of the spectral-peak shape, including the separation and width-at-half-height in the 31P NMR spectra, as a function of rotor speed, enabled us to explore the extent to which a change in cell volume would be reflected in the spectra if it occurred. We concluded that a fractional volume change in excess of 3% would have been detected by our experiments. Thus, the experiments indicated that the mean cell volume did not change by this amount even at the highest spinning rate of 7 kHz. The mean cell volume and intracellular 31P line-width were independent of the packing density of the cells and of the initial cell volume. The relationship of these conclusions to other non-NMR studies of pressure effects on cells is noted.

Thymosin beta4 promotes angiogenesis, wound healing, and hair follicle development.

New blood vessel formation is important in many physiological process, including development, wound repair, and tumor growth. In aged animals, angiogenesis is reduced resulting in poor wound healing. We have identified a novel small molecule, thymosin beta(4), that promotes angiogenesis and wound repair in both normal and aged rodents. It also promotes hair growth in normal and aged rodents. It acts by increasing angiogenesis and cell migration and is currently in clinical trials for wound repair.

Exploiting the Curtin-Hammett principle--recognition-mediated acceleration of an aldol reaction.

[figure: see text] Curtin-Hammett-Winstein-Holness kinetics are exploited in the acceleration of the aldol reaction between formylbenzo-15-crown-5 and acetylbenzo-15-crown-5. Potassium cations facilitate this acceleration through the fast, reversible formation of a 1:1:1 sandwich complex between the crown ethers and the metal cation.

Transfer of stereochemical information in a minimal self-replicating system.

[structure: see text]. The rational design, synthesis, and characterization of a minimal self-replicating system based on a 1,3-dipolar cycloaddition between a nitrone and a maleimide is presented. The importance of molecular recognition in this system is demonstrated using a competitive inhibitor. Doping experiments demonstrate that only one of the two diastereoisomeric products of the cycloaddition reaction is capable of acting as an efficient template for its own formation, accelerating the reaction between the nitrone and maleimide and controlling the stereochemical outcome of the reaction.

Understanding the structural properties of a homologous series of bis-diphenylphosphine oxides

A homologous series of bis-diphenylphosphine oxides (C6H5)2PO(CH2)(n)PO(C6H5)2 (with n = 2-8; denoted 2-8] have been investigated to explore the effects of a range of competing and cooperative intermolecular and intramolecular interactions on the structural properties in the solid state. The important factors influencing the structural properties include intramolecular aspects such as the conformation of the aliphatic chain and the intramolecular interaction between the two P=O dipoles in the molecule, and intermolecular aspects such as long-range electrostatic interactions (dominated by the arrangement of the P=O dipoles), C-H...O interactions, C-H...pi interactions and pi...pi interactions. Compounds 3 and 5 could be crystallized only as solvate co-crystals (3 water and 5 x (toluene)2], whereas the crystal structures of all the other compounds contain only the bis-diphenylphosphine oxide molecule. The crystal structures have been determined from single-crystal X-ray diffraction data, with the exception of 7 (which has been determined here from powder X-ray diffraction data) and 4 (which was known previously). The compounds with even n represent a systematic structural series, exhibiting characteristic, essentially linear P=O...P=O...P=O dipolar arrays, together with C-H...O and C-H...pi interactions. For the compounds with odd n, on the other hand, uniform structural behaviour is not observed across the series, although certain aspects of these crystal structures contribute in a general sense to our understanding of the structural properties of bis-diphenylphosphine oxides. Importantly, for the compounds with odd n, there is "frustration" with regard to the molecular conformation, as the preferred all-anti conformation of the aliphatic chain gives rise to an unfavourable parallel alignment of the two P=O dipoles within the molecule. Clearly the importance of avoiding a parallel alignment of the P=O dipoles becomes greater as n decreases. Local structural aspects (investigated by high-resolution solid-state 31P NMR spectroscopy) and thermal properties of the bis-diphenylphosphine oxide materials are also reported.

Using polarization effects to alter chemical reactivity: A simple host which enhances amine nucleophilicity

The rational design of a bis(phosphine oxide) host which is capable of binding a benzylic amine is presented. The ability of this host to increase the rate of addition of 4-fluorobenzylamine to N-phenylmaleimide is rationalized in terms of the enhancement of the nucleophilicity of the benzylic amine.

Lysosomal-mediated degradation of apoptotic thymocytes within thymic nurse cells.

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alphabetaTCR(lo)CD4(+)CD8(+) thymocytes in vitro was used in long-term coincubation experiments to determine the ultimate fate of thymocytes that remained within intracytoplasmic vacuoles of thymic nurse cells (TNCs). In an earlier report, a subset of the population released from the TNC interaction was shown to mature to the alphabetaTCR(hi)CD69(hi) stage of development, while thymocytes that bided within the TNC cytoplasm died through the process of apoptosis. Here, we show the presence of both apoptotic and nonapoptotic thymocytes within the cytoplasm of freshly isolated TNCs as well as in tsTNC-1 cells in culture. A microscopic analysis revealed total degradation of the cytoplasmic apoptotic thymocyte population that remained in tsTNC-1 cells after an 8- to 10-h incubation period. A quantitative analysis showed an increase of cytoplasmic thymocyte degradation over time to almost 80% after 9 h of incubation. However, in the presence of bafilomycin A1, which is used to inhibit acidification of lysosomal vesicles, degradation of apoptotic thymocytes never reached 10%. These data suggest that lysosomes within TNCs play a role in the degradation of apoptotic thymocytes. We examined tsTNC-1 cells before the addition of thymocytes to cultures and found lysosomes to be clustered around the nucleus in the cytoplasm of TNCs. Shortly after the internalization event, apoptotic thymocytes move to the area of the cytoplasm containing lysosomes. Using the confocal microscope, we obtained evidence that shows the degradation event to be facilitated through the fusion of lysosomes with the specialized vacuoles within TNCs containing apoptotic cells.

A thymic nurse cell-specific monoclonal antibody.

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alpha beta TCRloCD4+CD8+ thymocytes in vitro was used in the development of a monoclonal antibody that is specific to the cell surface of thymic nurse cells (TNCs) in the thymus. The rat monoclonal antibody ph91 showed specificity to cells of the subcapsular region of the thymic cortex. Upon mechanical dispersion of the thymus in vitro, ph91 recognized cells displaying the multicellular morphology unique to TNCs. Ph91 staining was not detected on fresh thymocytes, stromal cells of the inner thymic cortex, thymic medullary cells, B cells or fibroblasts. Ph91 recognized a 43-kDa protein on the surface of TNCs. Exposure of tsTNC-1 cells to ph91 in tissue culture significantly reduced the percentage of binding of the alpha beta TCRloCD4+CD8+ thymocyte subset previously shown to target TNCs. In organ culture, ph91 reduced the viability of developing thymocytes by 70%. The largest reduction was found in the alpha beta TCR+CD4+CD8+ thymocyte subset. These results represent the first report of a TNC-specific monoclonal antibody. Further, the antigen to which ph91 binds may play a role in the process of thymocyte binding and their subsequent internalization which is unique to TNCs and important to the T cell developmental process.

Positive selection by thymic nurse cells requires IL-1 beta and is associated with an increased Bcl-2 expression.

A temperature-sensitive line of thymic nurse cells (tsTNC-1) that maintains the ability to selectively internalize immature alpha beta TCRloCD4+CD8+ thymocytes in vitro was used in long-term coincubation experiments to determine nurse cell function during the process of MHC restriction. The thymocyte subset released from its association with TNCs contained both viable and apoptotic cells. The cells that remained within intracytoplasmic vacuoles died through the process of programmed cell death. Surviving or rescued thymocytes in the released population displayed an increase in Bcl-2 protein expression. The rescue activity of TNCs was drastically reduced with the addition of antibodies against either class I or class II MHC antigens to cocultures. A subset of the TNC-rescued population matured from the alpha beta TCRloCD69- phenotype to alpha beta TCRhiCD(69+)-expressing cells only when IL-1 beta was added to cocultures. These results suggest that TNC rescue of early double-positive thymocytes from apoptosis is associated with an interaction between the TCR and the MHC and the onset of Bcl-2 expression. Maturation of thymocytes within the TNC-rescued population requires the costimulatory effects of IL-1 beta.

Thymic nurse cell rescue of early CD4+CD8+ thymocytes from apoptosis.

We have now developed temperature sensitive lines of thymic nurse cells (TNCs), using the SV40 viral mutant tsA58, that maintain the ability to selectively internalize a subpopulation of alpha beta TCR+CD4+CD8+ thymocytes in vitro. One line, tsTNC-1, was shown to be able to rescue a subset of CD4+CD8+ thymocytes from programmed cell death at 32 degrees C, the temperature at which binding and internalization were detected. Rescue was significantly diminished at 38 degrees C, the temperature at which thymocyte binding was not observed. The rescued population of thymocytes showed a reduced level of apoptosis as measured by the DNA fragmentation assay. TNC rescue resulted in a shift of CD4+CD8+ thymocytes from immature TCRlow PNArhigh cells to the more mature TCRint PNArlow phenotype but no changes in cell surface levels of HSA nor CD69 were detected. The rescue activity of tsTNC-1 cells at 32 degrees C was significantly reduced with the addition of antibodies to either class I or class II MHC antigens. These results suggest that we have established TNC lines, using the SV40 viral mutant tsA58, that have the ability to rescue a subset of the TNC interactive thymocyte population from programmed cell death. The thymocyte population rescued by TNCs matures to a phenotype within the double positive stage of development that is indicative of positive selection.

The binding, internalization, and release of thymocytes by thymic nurse cells.

Recent studies in our laboratory have described the development of the SV40-transformed thymic nurse cell (TNC) line SVT-II2, that maintains the ability to internalize thymocytes in vitro. SVT-II2 cells were shown to bind and internalize a subset of the alpha beta TCR+, CD4+CD8+ thymocyte population exclusively. Also, SVT-II2 cells express cell surface class I and class II MHC antigens. These data are consistent with reports that suggest that TNCs may have a role in thymic education. In this report, we used scanning electron microscopy, transmission electron microscopy, and long-term video microscopy to study binding, internalization, and release of thymocytes by TNCs. The results of these experiments showed the internalization event to be selective and dynamic. The process appears to involve programmed cooperation between the two cell types that terminates with the release of selected thymocytes. Although no changes in thymocyte cell surface phenotype were detected as a result of their interaction with TNCs in vitro, over 90% remained viable after a 48-hr incubation period.

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.