Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

Studies on detection of Candida antigen in the sera of mice inoculated orally with Candida albicans

Summary The authors succeeded in establishing a murine model of systemic candidiasis being disseminated from the primary gastrointestinal lesions caused by oral inoculation of Candida albicans. Using this model, an attempt was made for detecting the Candida antigen by enzyme-linked immunosorbent assay using avidin-biotin (AB-ELISA) from the serum of infected mice.Gastrointestinal candidiasis was formed in all of the 20 mice treated with the drugs (antibiotics, antineoplastic agents, hydrocortisone, etc.) and inoculated orally with C. albicans. Fourteen of these mice suffered from submucosal candidiasis, and C. albicans was cultured from the visceral organs in 12 of them. The assay by AB-ELISA was able to detect 1.0 ng/ml Candida mannan in the mouse serum. The Candida antigen was detected in the sera of 11 of the 14 mice with submucosal candidiasis. However, the antigen could not be detected in the sera of the 6 mice with intramucosal candidiasis.The assay by AB-ELISA is more sensitive and specific for the diagnosis of systemic candidiasis than other serological assays.     

In vitro test of self-incompatibility in Malus domestica

Summary An in vitro assay in which self-incompatible pollen of Malus domestica is selectively inhibited is described. This assay involves heat-labile substances diffusing from the stylar tissues — in particular, glycoproteins found in the protein extract of styles. In the presence of the self-style extract, a dramatic decrease in total protein concentration in the culture medium was revealed at 30-min germination. Pretreatment of the self-pollen with 100 mM glucose prevented this drop in protein level; moreover, tube growth was entirely restored. A possible explanation in terms of protein-carbohydrate complementation is suggested.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SLIDT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37° C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SLIDT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC. Another assay (e.g., electrical coupling, microinjection, metabolic cooperation, radioactive metabolite transfer, or fluorescence redistribution after photobleaching) should be considered to quantify changes in GJIC and construct chemical concentration-response curves.Abbreviations FBS, fetal bovine serum - GJIC, gap junctional intercellular communication - HBSS, Hank's balanced saline solution - SL/DT, scrape-loading/dye transfer - TPA, 12-O-tetradecanoylphorbol-13-acetate.     

The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine.

A stereospecific high-performance liquid chromatographic (HPLC) method was developed for the quantitation of the enantiomers of venlafaxine, an antidepressant, in dog, rat, and human plasma. The procedure involves derivatization of venlafaxine with the chiral reagent, (+)-S-naproxen chloride, and a postderivatization procedure. The method was linear in the range of 50 to 5,000 ng of each enantiomer per ml of plasma. No interference by endogenous substances or known metabolites of venlafaxine occurred. Studies to characterize the disposition of the enantiomers of venlafaxine were conducted in dog, rat, and human, following oral administration of venlafaxine. The Cmax, area under the curve (AUC) and (S)/(R) concentration ratios of the (R)- and (S)-enantiomers were compared. In rats, the mean plasma ratio of (S)-venlafaxine to that of (R)-venlafaxine over 0.5 to 6.0 h varied from 2.97 to 8.50 with a mean value of 5.51 +/- 2.45. The Cmax, AUC0-infinity, and t 1/2 values of the (R)- and (S)-enantiomers in dogs were not significantly different from one another (P greater than 0.1). The mean ratios [(S)/(R)] of enantiomers of venlafaxine in human over a 2 to 6 h interval ranged from 1.33 to 1.35 with an overall ratio of 1.34 +/- 0.26 (n = 12). These ratios of the enantiomers [(S)/(R)] were not statistically different from unity (P greater than 0.1) indicating that the disposition of venlafaxine enantiomers in humans is not stereoselective and is more similar to that in dogs than that in rats.