The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: binding of toxin and subsequent inhibition of catecholamine release.

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin.     

The concentration dependence of the depolarization of yeast by monovalent cations.

Monovalent cations decrease the initial rate of uptake of the membrane potential probe 2-(dimethylaminostyryl)-1-ethyl-pyridinium (DMP) into metabolizing cells, showing that the cells are depolarized. A steep decrease in this rate was found even at low cation concentrations, reaching 62%, 42%, 58%, 40% and 40% at high concentrations of K+, Rb+, Cs+, Na+ and Li+, respectively. The corresponding concentrations at which half-maximum decrease was found were 0.22, 0.36, 1.2, 17 and 17 mM. These values are of the same order of magnitude as the half-saturation concentrations for monovalent cation uptake by the yeast.     

Dissimilation curves as a simple method for the characterization of growth of plant cell suspension cultures

A simple non-invasive method for the characterization of growth of a plant cell suspension in a single culture flask is given. The dissimilation of sugars by a cell-culture causes a loss of weight of the contents of the culture flask, and can therefore be used to follow the growth in that single culture flask. Because a correction for water evaporation is necessary, accurate results can only be obtained when a stable closure is used (e.g. Silicosen T-type plugs). The dissimilation curves obtained in this way were correlated to the concentration of sugars in the medium, the dry weight and the fresh weight. From these correlations the amount of intracellularly stored carbohydrates could be estimated. Rate constants for CO₂-diffusion were determined for different types of closure. These values allowed the estimation of CO₂ levels inside the culture flasks from the dissimilation curves (CO₂ release curves). The dissimilation curves obtained using this method can easily be related to other types of growth curves. Different growth-phases can be clearly distinguished, e.g. lag-phase, exponential growth-phase and stationary-phase.     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

Reviewers for 1988 and 1989

Reviewers for 1988 and 1989     

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (_p), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (q_I).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (_exe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q _N and the efficiency of excitation capture (_exe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.Abbreviations F₀ basic fluorescence - F₀ basic fluorescence, thylakoid in energized state - F_m maximal fluorescence - F_m maximum fluorescence in energized state - F_s steady state fluorescence - F_v maximal variable fluorescence - PFD photon flux density - PS II_rc Photosystem II reaction center - q_F0 quenching of basic fluorescence - q_E energy related quenching - q_N non-photochemical quenching:-q_f-total quenching - q_I inhibition-related quenching - q_p photochemical quenching - q_r quenching due to state transition - R_d dark respiration - _p PS II efficiency of excitation capture of open PS II_rc - _pe extrapolated minimal value of _p - _p0 extrapolated maximal value of _p - _si quantum efficiency of linear electron transport, calculated from gas exchange measurements based on incident light - _sf quantum efficiency of linear electron transport, calculated from fluorescence measurements, based on incident measuring light     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

The Athena semi-automated karyotyping system

In this article we describe Athena, a system that provides for semi-automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris-convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well-accepted Macintosh user interface.