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Biomedical subjects

A Kroon

Publications and source records attributed to A Kroon.

17 recordsLinked to original sources
PubMed2003

Monitoring enzymatic reactions in nanolitre wells.

We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory-on-a-chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.

Adenosine Triphosphate↗
PubMed2001

The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

Amino Acid Sequence↗
PubMed2000

Sensitivity of characteristic riverine insects, the caddisfly Cyrnus trimaculatus and the mayfly Ephoron virgo, to copper and diazinon.

This study reports the effects of two model toxicants, copper and diazinon, on two characteristic riverine insect species, the caddisfly Cyrnus trimaculatus and the mayfly Ephoron virgo. It was demonstrated that these species are very sensitive to both compounds in comparison with aquatic insects traditionally used in ecotoxicity tests. For diazinon, the 96-h LC(50) value of Cyrnus trimaculatus (1.1 microg/l) is lower than for any other insect species known from the literature and for copper it was demonstrated that Ephoron virgo is among the most sensitive aquatic insect species. The observed low LC(50) values stress the importance of using these indigenous species in assessing the risk of environmental contaminants in large European rivers and in defining conditions for ecological recovery.

Journal Article↗
PubMed2000

How predatory mites learn to cope with variability in volatile plant signals in the environment of their herbivorous prey.

When the chemical cues co-occurring with prey vary in time and space, foraging predators profit from an ability to repeatedly associate chemical cues with the presence of their prey. We demonstrate the ability of a predatory arthropod (the plant-inhabiting mite, Phytoseiulus persimilis) to learn the association of a positive stimulus (herbivorous prey, Tetranychus urticae) or a negative stimulus (hunger) with a chemical cue (herbivore-induced plant volatiles or green leaf volatiles). It has been suggested that the rate at which the integration of information becomes manifest as a change in behaviour, differs between categories of natural enemies (parasitoids versus insect predators: specialist versus generalist predators). We argue that these differences do not necessarily reflect differential learning ability, but rather relate to the ecologically relevant time scale at which the biotic environment changes.

Animals↗
PubMed1998

Response to photoperiod during diapause development in the spider mite Tetranychus urticae.

To study the question whether photoperiodic time measurement in the spider mite Tetranychus urticae is based on a qualitative or quantitative principle, the duration of diapause development was determined in individual females at various constant photoperiods at 19 degrees C. Diapause duration at all four long-night treatments fluctuated around 64.5 days, varying from 62.2 at LD 12:12h to 66.4 at LD 10:14h. The within-treatment variation in diapause duration of the long-night groups appeared to be significantly correlated to the nightlength of the photoperiods used; the longer the nightlength, the higher the within-treatment variation. Frequency distributions of females completing diapause under the two regimes with nightlengths near the critical nightlength were skewed to the right. Mean diapause durations at these regimes, LD 13:11h and LD 14:10h, were 25.4 and 11.9 days, respectively. Mites completed diapause rapidly and synchronously under the three short-night photoperiods tested; within two weeks after transfer from cold storage at 4 degrees C to the diapause terminating regimes at 19 degrees C all females started reproduction. Mean diapause durations were 8.1, 6.4 and 6.5 days for the short-night treatments LD 15:9h, LD 17:7h and LD 19:5h, respectively. The coefficients of variation of diapause duration (variability within groups relative to the mean) of the short-night and the long-night groups varied from 18 to 42%; the coefficients of the two intermediate groups were 69and 81%. There was a clear difference in diapause duration between long-night and short-night groups, but no significant difference was present in this characteristic between different long-night groups on the one hand and only a small difference between different short-night groups on the other. These results support the hypothesis that photoperiodic time measurement in the spider mite is based on a qualitative principle; photoperiods are classified as either 'long' or 'short' in relation to a 'critical' photoperiod. However, around the critical nightlength, intermediate responses were observed which might hint at the quantitative nature of the underlying mechanism. Therefore, although most results are in agreement with the hypothesis of a qualitative mechanism, it cannot be excluded that photoperiodic time measurement in the spider mite is based on a quantitative principle.

Journal Article↗
PubMed1993

The same photoperiodic clock may control induction and maintenance of diapause in the spider mite Tetranchus urticae.

In the spider mite Tetranychus urticae, both diapause induction (which takes place during the larval and nymphal stages) and diapause maintenance (in the adult female) are under photoperiodic control. The question of whether or not the same photoperiodic clock is involved in both photoperiodic reactions was investigated in eight strains of the spider mite, originating from different localities in Europe. The methods employed consisted of (1) determination of the relative importance of the photophase and scotophase in the two photoperiodic reactions; (2) comparison of photoperiodic response curves for diapause induction and diapause maintenance; and (3) determination of the effect of light breaks on the capacity of long nights to maintain diapause, and comparison with the effect of light breaks in diapause induction experiments. The scotophase appeared to be much more important than the photophase for both diapause induction and diapause maintenance. In all strains the critical daylength for diapause maintenance, measured at the moment of saturation of the response to long daylengths, was identical to the critical daylength for diapause induction. However, the critical daylength for diapause maintenance appeared to be labile; it shifted gradually to shorter values as the mites were kept in the cold for a longer period of time, or were kept at a higher temperature for a progressively longer period of time after their stay in the cold room. This seems to reflect a gradual loss of photoperiodic control of diapause maintenance as diapause development proceeds. Photoperiods close to the critical daylength appeared to be less strong with regard to diapause maintenance than shorter daylengths. Quantitative differences in the "strength" of different daylengths were found in all strains investigated. Interruption of the night by short pulses of light revealed either one or two peaks of sensitivity in the night, or one broad "trough" where the two peaks had merged. However, in each case maximal sensitivity to the light breaks occurred at the same position in the night for diapause induction and diapause maintenance. The many similarities found lead to the conclusion that most probably the same photoperiodic clock mechanism is involved in both diapause induction and diapause maintenance in T. urticae.

Animals↗
PubMed1988

Autoreactive B cells in normal humans. Autoantibody production upon lymphocyte stimulation with autoantigen-xenoantigen conjugates.

Mononuclear cells (MNC) from the blood of healthy individuals cannot be stimulated in vitro with the soluble autoantigen thyroglobulin (Tg). However, when Tg or pepsin fragments of Tg were coupled with a carrier protein, tetanus toxoid (TT), MNC from four healthy TT vaccinated individuals responded to the carrier-autoantigen conjugates by generating anti-Tg antibody forming cells (AFC), as shown in a spot enzyme-linked immunosorbent assay. Generation of anti-TT and anti-Tg AFC after stimulation with the conjugates required the donors to be boostered with TT. The autoantibodies were exclusively of the IgM class, in contrast to the carrier-specific anti-TT antibodies, which were predominantly of the IgG isotype. Activation of normal B cells to anti-Tg production was dependent on the presence of T cells in the cultures and required physical linkage of carrier and autoantigen: no anti-Tg AFC could be detected when MNC were stimulated with uncoupled combinations of Tg and TT. The autoreactive and the carrier-reactive B cells exhibited almost identical conjugate dose-response profiles, which suggest that they responded in a similar way to regulatory signals. These findings indicate that normal blood B cells are competent to respond to the autoantigen Tg in conjunction with signals originating from xeno-antigen-stimulated T cells.

Antibodies, Bacterial↗
PubMed1987

Analysis of the human tonsil B cell repertoire by somatic hybridization: occurrence of both "monospecific" and "multispecific" (auto)antibody-secreting cells.

The purpose of our study was to investigate the (auto)antibody repertoire of human tonsil B cells. To this end, unseparated tonsil B cells and "large" and "small" tonsil B cells, obtained after fractionation on a discontinuous Percoll gradient, were fused with the (human X mouse) heterohybridoma K6H6/B5. The IgM antibodies secreted by individual clones were analyzed in spot-enzyme-linked immunosorbent assay for reactivity against a panel of 5 autoantigens and 1 xenoantigen. The results show that both "mono-" and "multispecific" (auto)antibody-secreting clones can be obtained by somatic hybridization of unseparated, "small" and "large" tonsil B cells. The patterns of IgM reactivities in these three collections of hybridomas did not differ significantly. Inhibition experiments revealed that "multispecific" antibodies can bind with high affinity to more than one (auto)antigen. The results of these experiments are discussed in relation to the state of activation of tonsil B lymphocytes.

Antibody Specificity↗
PubMed1986

Production of anti-thyroglobulin antibody by blood lymphocytes from patients with autoimmune thyroiditis, induced by the insolubilized autoantigen.

Mononuclear cells (MNC) from the blood of patients with autoimmune thyroiditis cannot be stimulated in vitro with the autoantigen thyroglobulin (Tg). However, when Tg was coupled with a solid matrix, MNC from 13 of 19 patients tested responded to this insolubilized Tg (i-Tg) by generating cells which produced IgG anti-Tg antibodies, as demonstrated in a "spot ELISA". This response was specific not only in the assay phase but also in the induction phase, because 1) cultures with insolubilized ovalbumin, bovine Tg, or porcine Tg were negative, and 2) i-Tg did not induce polyclonal IgG production. Generation of anti-Tg-producing cells was completely abrogated when free Tg was added to i-Tg cultures. Stimulation with i-Tg after separation of T and non-T MNC revealed that: 1) the response was independent of T cells; 2) in three of the six patients whose MNC did not respond, removal of T cells did result in anti-Tg production; 3) re-addition of the T cells--but not of irradiated T cells--abrogated the response in two of these three patients; and 4) preincubation of monocytes with i-Tg resulted in suboptimal anti-Tg production. These findings indicate that B cells from patients with autoimmune thyroiditis can be specifically stimulated with Tg coupled with a solid matrix, resulting in generation of anti-Tg-producing cells. The essence of this mode of presenting the antigen would appear to be the cross-linkage of surface immunoglobulin. The response was T cell-independent; in some patients, however, the presence of a radiosensitive suppressor T cell population can be demonstrated.

Antibody Specificity↗
PubMed1986

Antigen-specific activation of autoreactive B cells in normal human individuals.

The purpose of our study was to determine if normal human B cells can be activated to autoantibody production using an antigen-specific system. For this purpose we investigated the in vitro antibody response to the autoantigen thyroglobulin (Tg) employing soluble or insolubilized Tg (i-Tg) and a B cell growth and differentiation factor (BDGF) to stimulate lymphocytes from healthy individuals. Similar experiments were carried out with the xenoantigen ovalbumin (OVA). The presence of Tg and OVA-reactive B cells was demonstrable by stimulating lymphocytes from tonsil, spleen and blood with a combination of pokeweed mitogen and formalinized Staphylococcus aureus: mitogen stimulation resulted in the generation of IgM anti-Tg and IgM anti-OVA antibody-forming cells (AFC) as detected in a spot enzyme-linked immunosorbent assay. Soluble antigen failed to induce autoantibody production. However, i-Tg or i-OVA did activate normal tonsil and spleen B cells. Differentiation of these activated B cells to IgM AFC required the presence of BGDF, derived from a human T hybrid clone. Preincubation experiments with the particulate autoantigen show that a specific activation signal is provided by the antigen which subsequently renders the B cells responsive to BGDF. i-Tg-dependent stimulation of B lymphocytes could be inhibited by adding free Tg to the cultures; the same applied to i-OVA stimulation. We conclude that the normal human B cell repertoire contains B cells that can be activated to autoantibody production by the autoantigen Tg if the necessary T cell signals are provided. Thus, these B cells are not in an inherently anergic state. Similar mechanisms seem to play a role in the activation of B cells responding to i-OVA and i-Tg.

Antibody-Producing Cells↗
PubMed1985

Enumeration of (auto)antibody producing cells in human using the "spot-ELISA".

An enzyme-linked immunosorbent assay (spot-ELISA) for individual immunoglobulin secreting cells, which became recently available, was applied to the enumeration of human B lymphocytes secreting specific antibodies of thyroglobulin. Polyclonally activated B cells from patients with auto-immune thyroid disease are incubated in thyroglobulin coated plates. After removal of the cells specific antibodies are visualized by means of an immunoenzyme technique employing agarose to localize converted substrate. Individual specific antibody secreting cells are counted as blue spots using an inverted microscope. Numbers and isotype of spots correlate well with the amount and isotype of secreted antibody as detected with a conventional ELISA. This easy to perform, complement-independent technique offers a useful alternative to conventional plaque forming cell assays.

Antibody-Producing Cells↗
PubMed1979

ELISA for the serology of FIP virus.

An enzyme linked immunosorbent assay (ELISA) for feline infectious peritonitis (FIP) virus serology is described. The assay is analogous to a previously developed indirect heterologous immunofluorescence test (IFT) in which transmissible gastroenteritis (TGE) viral antigen was used. Comparative testing of selected feline sera in both assays resulted in corresponding titers, which justifies the conclusion that the ELISA is a reliable test for the serology of FIP virus.

Animals↗
PubMed1979

Laboratory of Physiological Chemistry, State University Groningen, Netherlands.

To obtain more information about the arrangement of Hind III restriction fragments in the tRNA-rRNA region of the Neurospora crassa mitochondrial (mt) DNA we have cleaved the mtDNA with Hpa I and Hind II. We could construct additional cleavage maps for these enzymes. Hybridization of rRNAs to Hind II fragments confirmed the existence of an intervening region of about 2,300 basepairs in the 24S rRNA (Hahn et al., Cell, in press). About seven tRNA genes, among which the genes for tRNA1Ser and tRNAMetM, are located in a segment of about 5,000 bp separating the 24S and 17S rRNA genes. Another cluster of 14 tRNA genes is found adjacent to the other end of the 24S gene. The genes for tRNALeu1 and tRNAMetF are located in this cluster.

DNA Restriction Enzymes↗
PubMed1977

Heterogeneous base distribution in mitochondrial DNA of Neurospora crassa.

The mitochondrial DNA of Neurospora crassa has a heterogeneous intramolecular base distribution. A contiguous piece, representing at least 30% of the total genome, has a G+C content that is 6% lower than the overall G+C content of the DNA. The genes for both ribosomal RNAs are contained in the remaining, relatively G+C rich, part of the genome.

Base Sequence↗