20-221 Sigma-AldrichNAD Cofactor

Nanostructured lipid carriers (NLC) are new drug systems composed of physiological lipid materials. The possibility of including different types of lipids into the NLC structure revealed the wide prospects for using biologically active natural oils for the development of the cutaneous preparations. In this study the formulation parameters of NLC on the basis of Siberian pine seed oil were evaluated including concentration of lipids, types of surfactants and storage conditions (4 degrees C, 20 degrees C, 40 degrees C). Size distribution and storage stability of formulations produced by hot high pressure homogenisation were investigated by laser diffractometry and photon correlation spectroscopy. The NLC were characterised by their melting behaviour using differential scanning calorimetry. The obtained data indicated the high physical stability of the developed NLC formulations.

Perfluorocarbons (PFCs) and their emulsions (PFCEs) were used in organ preservation before transplantation, but not in organ perfusion. Our purpose was to achieve organ perfusion with a PFCE at room temperature or at 37 degrees C, i. e. with oxygenation, to prevent damages related to reoxygenation after hypoxia. Therefore, we first investigated the effect of such emulsions on endothelial cells, the first cells to be in contact with the emulsion. A stem emulsion was prepared from perfluorooctyl bromide (90% w/v), emulsified with egg yolk phospholipids (2% w/v) and stabilized with a mixed fluorocarbon-hydrocarbon molecular dowel (1.4% w/v) (droplets of ca 0.2 micron in diameter). This emulsion was found to be stable when diluted with cell culture media or organ preservation fluids. Endothelial cells from human umbilical vein (HUVECs) were cultured in multiwell plates in M199 medium (with growth factors, 10% foetal calf serum and 5% human serum). Confluent cells were incubated overnight with 51Cr, washed and overlayed with M199 (control) or the above PFCE diluted 2x or 4x with M199 (test). After incubation, the cytotoxicity of the PFCEs was estimated by measuring 51Cr release and observing cell morphology by electron and light microscopy. The percentages of released 51Cr were identical to those of the control cells for the 2x, 3x or 4x diluted PFCEs at 4, 25 or 37 degrees C. After return to the M199 medium, the cells grew and multiplied normally. We conclude that the diluted PFCEs were devoid of cytotoxicity. The 2x diluted PFCE was however partially taken up by the cells: by microscopy, we observed intracellular PFC droplets and by density gradient analysis we found a slight increase in cellular density. The diluted PFCEs were compared to classical organ preservation solutions : HUVECs were incubated with UW (University of Wisconsin) or EC (EuroCollins) solutions at +4 and 37 degrees C (3, 17 or 24 h of incubation). The solutions were observed to be toxic to the cells under these conditions, with cell mortality after return to the M199 medium. This cytotoxicity may be attributed to the high K+ concentration of UW and EC, since similar assays performed on HUVECs with Hank's solution adjusted to 100 mM K+ showed a similar % of 51Cr release. UW and EC are therefore not acceptable as dilution media for PFCEs.

To assess whether plasma and urinary endothelin-1 (ET-1) values are related to the severity of diabetic nephropathy, we measured plasma and urinary ET-1-like immunoreactivity (ET-1-LI) in 14 healthy subjects, and in 50 normoalbuminuric (group 1), 13 albuminuric (group 2), and 10 renally insufficient (group 3) patients with Type 2 diabetes. Plasma ET-1-LI values were significantly increased in group 3, and correlated positively with serum creatinine levels (r = 0.579, p < 0.01). Urinary ET-1-LI excretion in group 3 (49.3 +/- 7.3 pmol day-1) was significantly higher than that in healthy controls (27.0 +/- 1.1 pmol day-1) and in group 1 (32.2 +/- 2.2 pmol day-1), while that of group 2 (38.8 +/- 5.9 pmol day-1) was also higher than in healthy controls. A significant positive correlation between urinary ET-1-LI and serum creatinine values was also found (r = 0.297, p < 0.05). Trend analysis showed significant linear and quadratic trends in the elevation of plasma ET-1-LI and a significant linear trend in urinary ET-1-LI levels from healthy controls to groups 1, 2 and 3. Our results demonstrate that an increase in plasma and urine ET-1-LI correlates with the severity of diabetic nephropathy.

The ability to identify and characterize low picomole quantities of gel-separated proteins has greatly benefited from recent advancements in mass spectrometric analysis methods, particularly peptide-mass search routines. We are investigating the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)) to gain as much mass information as possible from a single gel-separated protein species. This report details results obtained from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations, under nonreducing conditions, of known quantities of three proteins, followed by blotting to Immobilon-CD. Three methods were used to obtain MALDI-MS data from a single blotted protein band: (i) direct MALDI-MS of approximately 10% of the band, (ii) cyanogen bromide (CNBr) cleavage of another approximately 10% of the band, and (iii) enzymatic (endoproteinase Lys-C) digestion of the remaining approximately 70-80% of the band followed by MALDI-MS. At the level of 50 pmol of protein loaded onto the gel, data was obtained from all three approaches. At levels down to 5 pmol of protein loaded onto the gel, MALDI-MS data was obtained from the latter two methods, CNBr and Lys-C digestions, but not direct MALDI-MS. Sufficient peptide masses were obtained from the 5 pmol loads to identify two of the three test proteins using four mass search programs. Only limited peptide mass data was obtained from fetuin, a sialylated glycoprotein with six disulfides and no methionines, but it was identified.

Human prostate-specific antigen (PSA), a 33 kDa kallikrein-like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate-buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS-PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS-stable complexes with the two major extracellular protease inhibitors in blood, alpha 1-antichymotrypsin (alpha 1-ACH) and alpha 2-macroglobulin (alpha 2-M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors alpha 1-ACH and alpha 2-M.

Two rat IgG2a antibodies which define distinct epitopes on murine CD40 have been generated. These antibodies specifically bind recombinant murine CD40 expressed on L cells, and the soluble extracellular domain of murine CD40 coated onto microtiter plates. Both antibodies bind B220+ but not B220 murine spleen cells, and immunoprecipitate a 45-kDa protein from the surface of purified murine splenic B cells. These antibodies exhibit separate functional properties, consistent with the notion that they define two distinct CD40 epitopes. One of the monoclonal antibodies (designated 1C10) directly induces a specific proliferative response from mature murine B cells, up-regulates several B cell surface antigens, and rescues immature B lymphoma cells from anti-IgM-induced growth arrest. The other monoclonal antibody (designated 4F11) exhibits none of these properties, but is capable of synergizing with suboptimal amounts of either anti-IgM antibodies or the 1C10 agonistic anti-CD40 antibody to produce an optimal proliferative response of purified small dense B cells. Furthermore, 4F11 antibody synergizes with suboptimal amounts of 1C10 antibody to rescue B lymphoma cells from anti-IgM-induced growth arrest. The 1C10 and 4F11 antibodies were unable to cross-block each other's binding to recombinant CD40 expressed in L cells, providing strong support for the notion that the antibodies recognize distinct epitopes on CD40. The potential implications of two functionally distinct CD40 epitopes are discussed.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.

Two genes (SCR1 and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae. Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome, SCR1 and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes II and V, respectively. The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides. It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and pI 11.19. The deduced amino acid sequence shows a high degree of similarity to the L41 protein of the 60S ribosomal subunit from several eukaryotic organisms. The intron and the 5' non-coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes. A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80S ribosome since replacement of Gln56 in L41 from Schwanniomyces with Pro, by site-directed mutagenesis, confers cycloheximide sensitivity. SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid.

A monoclonal antibody (OKT3) directed against the T cell receptor (TcR)/CD3 molecular complex, as well as a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) were added to a culture of tumoral Jurkat T cells, in order to precise the sequence of intracellular signals leading to T cell activation. The experiments were performed in the presence or in absence of various stimulators of adenylate cyclase (AC) such as forskolin (FK), cholera toxin (CT) or prostaglandin E2 (PGE2). OKT3 increased inositol phosphate (IP) production; in parallel, it induced a slight accumulation of cAMP. The effect was markedly potentiated in presence of FK or CT, and to a lesser extent in the presence of PGE2. FK stimulated adenylate cyclase of Jurkat cell membranes, but the effect was not potentiated by OKT3, suggesting that potentiation of cAMP accumulation requires intact cells and is not mediated by direct receptor coupling. On the other hand, elevated cAMP accumulation induced a negative feedback on IP production. The effect of OKT3 on cAMP was mimicked by A23187, a Ca2+ ionophore, and abolished in the absence of extracellular Ca2+. PMA had the same effect as OKT3 on basal or FK- and CT-induced accumulation of cAMP. In contrast, it inhibited the PGE2 effect on the cyclic nucleotide. After desensitization of PKC by pretreatment with a high concentration of PMA, the phorbol ester was no longer effective. Under those conditions, facilitation by OKT3 of FK-induced accumulation of cAMP was preserved, whereas potentiation by the monoclonal antibody of the PGE2 stimulation of AC was even enhanced. The data indicate that cAMP accumulation indirectly elicited by phospholipase C activation is, at least partly, mediated by IP-dependent Ca2+ mobilization, while PKC is preferentially effective as an inhibitor of PGE2 stimulation.

In a previous paper, we described the kinetic characteristics of the inhibition exerted by the protease inhibitors tosylphenylalanyl and tosyllysyl chloromethanes on superoxide production by human polymorphonuclear leukocytes when stimulated by phorbol esters [E. C. Conseiller & F. Lederer (1989) Eur. J. Biochem. 183, 107-114]. The results suggested the existence of a specific target which was affinity labeled by the inhibitors. The target appeared to be neither a protease, nor intracellular enzymes which can be inhibited in vitro by the chloromethanes (protein kinase C, hexokinase and enzymes of the hexose monophosphate shunt). In the present work, using the cell-free reconstitution assay for superoxide production, we substantiate the hypothesis that the chloromethanes, target is on the plasma membrane. We have radiolabeled the membranes of cells inactivated before or after phorbol ester stimulation, using either [3H]KBH4 reduction after reaction with unlabeled inactivator, or tritiated tosylphenylalanyl chloromethane. In all cases, besides a certain background of non-specific labeling, a radioactive band of Mr 15,000 can be observed upon SDS/PAGE of radiolabeled membranes. We suggest that it is the chemical modification of this protein which is responsible for inactivation of superoxide production. Its identity and its role in the oxidative burst remain to be determined.