A simplified and rapid limiting dilution assay of T lymphocytes

We report here the development of an alternative limiting dilution assay (LDA) of T lymphocytes (T cells). Blood mononuclear cells were first stimulated for 60 hr with PHA and then cultured in microwells in the presence of recombinant interleukin-2 without feeder cells. After 4 days of culture, wells were scored for proliferation. Clonal expansion of T cells followed the single-hit model of the Poisson distribution. The progenitor frequency (f) in the mononuclear cells and E-rosette-positive cells from normal donors were 0.082 +/- 0.025 (n = 12) and 0.236 +/- 0.029 (n = 5), respectively, but this was markedly decreased in patients who underwent marrow-ablative chemotherapy and autografts with blood hematopoietic stem cells. This LDA system should be of value in routine use for the evaluation of T cell proliferative activities.     

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells

Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin.     

ADAMTS9 activation by interleukin 1β via NFATc1 in OUMS-27 chondrosarcoma cells and in human chondrocytes

ADAMTS9 is a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes, with aggrecan-degrading activity. It has also been characterized to be reactive and highly activated ADAMTS by IL-1β in both chondrosarcoma cells and human chondrocytes (Demircan et al. Arthritis Rheum 52:1451–1460, 2005). In order to understand the regulation of ADAMTS9 gene expression a functional 3.0 kb human ADAMTS9 promoter has been cloned and characterized. A sequence analysis of the promoter revealed the presence of putative binding sites for Nuclear Factor of Activated T cells (NFAT), which is commonly found in the ADAMTS4 and ADAMTS5 promoters. NFATc1 was up-regulated in an activated form by IL-1β in human chondrocytes. The IL-1β inducible ADAMTS9 expression was inhibited by NFAT inhibitors, FK506 and 11Arg (11R)-VIVIT. Furthermore, direct binding of NFATc1 on distal and proximal promoters of ADAMTS9 was demonstrated by a chromatin immunoprecipitation assay. Promoter-reporter assays supported those results. These findings may provide a better understanding of the regulation of ADAMTS9 expression induced by inflammatory cytokines.     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

Response of the Rhythmic Leaf Movements of Soybean (Glycine max L. Merr) to the Light Intensity of Light/Dark Cycles

The rhythmic (circadian) leaf movements of soybean were entrainedto various light/dark cycles. The phase relation of the rhythmto light/dark cycles varied depending on the light/dark schedules.The light intensity in the light periods, however, had no effecton the phase relation although the light intensity in continuouslight schedules had a strong effect on the free-running period.Leaf movements also were controlled by a non-circadian factorwhich occasionally affect the lowest leaf position that is usuallytaken as a phase reference point of rhythmic leaf movement. (Received June 7, 1983; Accepted September 10, 1984)     

Phase Transfer Catalysis; Nucleophilic Substitution of β-Bromohydrins with Anions in Solid-Liquid System

Nucleophilic displacements of β-bromohydrins with azide, phthalimide, acetate and thiocyanate anions were effected by tetramethylethylenediamine (TMEDA), triethylenediamine (TEDA) and 18-crown-6 (CE) in solid-liquid media, to afford the products in various yields and distributions.