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Biomedical subjects

A Houben

Publications and source records attributed to A Houben.

At least 37 records · Page 2Linked to original sources
PubMed1997

Histone H4 acetylation in plant heterochromatin is altered during the cell cycle.

Using polyclonal antibodies directed against acetylated isoforms of histone H4 (H4 acetylated at lysine positions 5, 8, 12, 16 and H4 tetraacetylated), indirect immunofluorescence revealed hyperacetylation for all H4 variants at the nucleolus organizer region (NOR) of metaphase chromosomes of the field bean Vicia faba. The transcriptionally inactive and late-replicating heterochromatin regions proved to be hypoacetylated at lysine positions 5, 8 and 12. The remaining chromatin showed average fluorescence. These patterns were altered when deacetylase was blocked by exposure of root tip meristems to trichostatin A for more than 2 h prior to fixation. Under these conditions, all lysine positions, except lysine 8, appeared to be hyperacetylated at the NOR and in addition at the prominent heterochromatin domains. This observation represents a hitherto unique switch of histone acetylation pattern during the cell cycle. This is apparently caused by deposition of acetylated H4. Ac5, 12 and 16 or by acetylation directly after replication, which later on becomes reduced (H4.Ac16) or even reversed (H4.Ac5 and 12) by deacetylase before cells enter mitosis.

Acetylation↗
PubMed1997

Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album.

A number of X chromosome DNA sequences have been isolated from a dioecious plant, Melandrium album (syn. Silene latifolia), using chromosome microdissection followed by degenerate oligonucleotideprimed polymerase chain reaction (DOP-PCR) amplification. Six DNA clones were selected and further characterized by DNA/DNA hybridization techniques to check their copy numbers, sex-specific methylation patterns, species specificity and positions on chromosomes. These clones were moderately to highly repetitive (approximately 10(3)-10(5) copies per haploid genome) and none of them gave a positive signal on Northern blots. One of the clones yielded a sex-specific methylation pattern: its abundant non-methylated CCGG island was found only in males. All the clones also hybridized to two closely related dioecious Melandrium species (M. rubrum and M. dicline). Nucleotide sequences of two X-derived clones showed a number of internal short direct repeats; one of them strikingly resembled a plant conservative telomere sequence (TTTAGGG). None of the clones hybridized to the X chromosome only, but all were localized at the telomeric heterochromatic regions (DAPI C-bands) of both arms of a vast majority of M. album chromosomes using the fluorescence in situ hybridization (FISH) technique. However, the non-homologous arm of the Y chromosome (contrary to the arm homologous to the X chromosome, possessing the pseudoautosomal region) showed neither a DAPI C-banding-stained heterochromatin nor a FISH signal with any of the DNA probes tested, thus indicating its evolutionary diversification.

Base Composition↗
PubMed1997

Differences of histone H4 acetylation and replication timing between A and B chromosomes of brachycome dichromosomatica.

Differences are demonstrated between A (transcriptionally active) and B (transcriptionally inactive) chromosomes that are characterized by a different level of histone H4 acetylation and a different timing of DNA replication. These differences between the chromatin of A and B chromosomes were found after immunolabelling of chromsomes of Brachycome dichromosomatica with antibodies specific for different acetylated forms (lysine 5, 8, 12 and 16) of histone H4. In contrast to the A chromosomes, which are labelled brightly in their entirety, the transcriptionally inactive B chromosomes are faintly labelled with antibodies against H4Ac5 and H4Ac8. No such difference between the chromosomes is found after immunostaining with the other antibodies H4Ac12 and H4Ac16. Analysis of DNA replication timing in root-tip meristems suggests that B chromosomes are labelled late in S-phase compared with A chromosomes. After C-banding the B chromosome appeared to have a similar amount of heterochromatin to the A chromosomes.

Acetylation↗
PubMed1997

Ribosomal RNA genes specific to the B chromosomes in Brachycome dichromosomatica are not transcribed in leaf tissue.

Ribosomal RNA genes are present near the end of the short arm and, to a lesser extent, near the centromere of the B chromosomes of some populations of Brachycome dichromosomatica. The internal transcribed spacer (ITS2) was amplified by PCR from total leaf DNA using primers within the conserved regions encoding the 5.8S and 25S stable rRNA species. Comparison of PCR amplified ITS2 sequences from several individual plants without B chromosomes with corresponding sequences derived from microdissected B chromosomes revealed two consistent differences between the rDNA of A and B chromosomes. One of these differences produced an SfcI restriction site that was present only in the ITS2 of the B-chromosome rDNA. Amplification by PCR of ITS2 from total genomic DNA from plants with and without B chromosomes showed an additive relationship between the amount of PCR product containing the SfcI site and the number of B chromosomes present. Quantitative analysis indicated that the proportion of total nuclear rDNA present on a single B chromosome varied between 2 and 4% in different A chromosome backgrounds. Similar experiments, with appropriate positive and negative controls, using reverse transcriptase PCR of the equivalent region within the 40S precursor rRNA, suggested that the B-chromosome rDNA was not transcribed. Similarly, PCR of reverse transcribed total RNA from plants containing B chromosomes using primers specific for the B chromosome ITS2 was unable to detect a transcript from the B chromosome.

Base Sequence↗
PubMed1996

Chromosome 'painting' in plants - a feasible technique?

It is shown that chromosome painting is as yet not possible for plants with very complex genomes, neither intra- nor interspecific. The reasons are inefficient blocking of dispersed repetitive sequences and insufficient signal intensity of short unique sequences. Future perspective are indicated.

Biotin↗
PubMed1996

Differential immunostaining of plant chromosomes by antibodies recognizing acetylated histone H4 variants.

Metaphase chromosomes of Vicia faba were exposed to antibodies recognizing defined acetylated isoforms of histone H4. After indirect immunostaining with antibodies directed against H4 acetylated on lysines 5, 8 and 12 respectively, the entire chromosome complement was labelled. The brightest signal appeared at the nucleolus organizing region (NOR). The large genetically inert heterochromatic regions, which are composed of late replicating tandemly repetitive DNA sequences, remained unlabelled. Thus, the chromosomal distribution of histones H4 acetylated at positions of lysine 5, 8 and 12 is broadly correlated with the intensity of transcription and the sequence of replication of the field bean chromatin during interphase. Antibodies against H4 acetylated at lysine 16 also caused a strong signal at the NOR but otherwise a uniform fluorescence along the chromosome.

Acetylation↗
PubMed1996

Molecular cytogenetic characterisation of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale).

The terminal heterochromatic segments of the long arms of 20 rye B-chromosomes were isolated by means of laser microdissection technology. Also the remaining portions of the long arms, along with the short arms of the same chromosomes were isolated. Each sample was used for degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplification reactions. The resulting products were used as probes for chromosome in situ hybridisation experiments, and in Southern hybridisation to digests of 0B and +B DNA. Competition hybridisation of these probes with 0B DNA allowed the detection of B-specific sequences. The terminal heterochromatin of the rye B-chromosome contains both B-specific sequences and sequences also present on the A-chromosomes of rye. The B-specific D1100 family is the major repeat species located in the terminal heterochromatin. Primers designed to the cloned sequence (E1100) were used to search for related low copy sequences in 0B DNA. The sequences of the PCR products revealed no similarities to that of the clone E1100 except for the primer sequences. The possible origin of this sequence is discussed in the context of models for the evolution of the rye B-chromosome.

Base Sequence↗
PubMed1996

The molecular organisation of a B chromosome tandem repeat sequence from Brachycome dichromosomatica.

A high copy, tandemly repeated, sequence (Bd49) specific to the B chromosome and located near the centromere in Brachycome dichromosomatica was used to identify lambda genomic clones from DNA of a 3B plant. Only one clone of those analysed was composed entirely of a tandem array of the B-specific repeat unit. In other clones, the Bd49 repeats were linked to, or interspersed with, sequences that are repetitious and distributed elsewhere on the A and B chromosomes. One such repetitious flanking sequence has similarity to retrotransposon sequences and a second is similar to chloroplast DNA sequences. Of the four separate junctions analysed of Bd49-like sequence with flanking sequence, three were associated with the same A/T-rich region in Bd49 and the fourth was close to a 25 bp imperfect dyadic sequence. No novel B-specific sequences were detected within the genomic clones.

Base Sequence↗
PubMed1995

Immunostaining and interphase arrangement of field bean kinetochores.

More than 100 sera from patients with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were tested in order to detect antigenic nuclear components of the field bean Vicia faba (2n = 12). Kinetochores of mitotic chromosomes and prekinetochores of interphase cells from root-tip meristems were specifically labelled via an indirect immunofluorescence procedure by antibodies of one of these sera. In 44% of interphase nuclei in which centromeres could be identified, only half (6) of the number of expected prekinetochores (12) was detected, circumstantially indicating at least transient association of homologous centromeres. Some nuclei showed clustering of centromeres at one pole (Rabl configuration). In metaphase chromosomes, each sister kinetochore contained a fluorescent spot. Western blotting of field bean nuclear proteins revealed four antigenic proteins of 28, 30, 64 and 68 kDa.

Blotting, Western↗
PubMed1994

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.

Base Sequence↗
PubMed1994

Polymerase chain reaction mediated localization of RFLP clones to microisolated translocation chromosomes of barley.

A new strategy has been devised and used for the physical localization of genetically mapped restriction fragment length polymorphism (RFLP) clones to barley chromosomes. Morphologically distinct translocation chromosomes from synchronized root-tip meristems were microisolated and their DNA was used as a template for polymerase chain reaction with sequence-specific primers. Four RFLP clones were assigned to cytologically defined segments of chromosome 5. This related approximately one-third of the map length of linkage group 5 to approximately one-fifth of the mitotic metaphase length of chromosome 5. The technique may substantially contribute to the connection of the RFLP-based genetic linkage maps with cytological markers of the barley chromosomes.

Base Sequence↗
PubMed1993

Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes.

A new technique is reported for the physical mapping of low copy DNA sequences on plant chromosomes. Individual chromosomes were microisolated and their DNA used as the target for the polymerase chain reaction in order to identify the chromosome carrying a specific gene sequence. The use of defined translocation chromosomes further refined the resolution of the method to a subchromosomal level. To demonstrate the applicability of the procedure genes have been localized coding for vicilin seed storage proteins on the field bean Vicia faba L. in a region which includes the centromere and the proximal parts of the short and the long arms of chromosome II.

Base Sequence↗
PubMed1990

High prevalence of antibiotic resistant Escherichia coli in faecal samples of students in the south-east of The Netherlands.

From December 1988 to March 1989 172 faecal samples from first and second year students at the University of Limburg, Maastricht, The Netherlands, were collected and analysed for the presence of Escherichia coli strains resistant to ampicillin, sulphamethoxazole, tetracycline, trimethoprim and nitrofurantoin. In addition, the antibiotic susceptibility (MIC) to these agents and six other compounds (i.e. aminoglycosides, nitrofurantoin, nalidixic acid and cephalothin) was determined. The prevalence of resistance of the samples analysed ranged from 86% (i.e. 148 out of 172) for sulphamethoxazole to 25% for trimethoprim. The prevalence figures for ampicillin, tetracycline and nitrofurantoin resistance were 76%, 47% and 29%, respectively. The percentages of the faecal samples with a high proportion of E. coli (greater than 50% of the total number of E. coli) resistant to ampicillin, tetracycline and sulphamethoxazole were 8%, 11% and 37%, respectively. For nitrofurantoin and trimethoprim the figures were 3% and 1%, respectively. Of the isolated E. coli (n = 797), 84% was resistant to ampicillin, 82% resistant to sulphamethoxazole. The lowest percentages (9%) were observed for both nalidixic acid and nitrofurantoin.

Ampicillin↗
PubMed1989

Inhibition of platelet-activating factor biosynthesis via the acetyltransferase by arachidonic and oleic acids in ionophore A23187-stimulated bovine neutrophils.

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation and allergy that is synthesized by several inflammatory cells including neutrophils. Addition of exogenous arachidonic acid to ionophore A23187-stimulated bovine neutrophils led to the inhibition of PAF biosynthesis assayed by incorporation of [3H]acetate into PAF and by bioassay; under the same conditions, leukotriene B4 (LTB4) formation was not decreased. The activities of the PAF metabolism enzymes indicated that the PAF synthesis inhibition by arachidonic acid is mediated via the acetyltransferase inhibition which is the last enzyme of the PAF formation. Another unsaturated fatty acid, oleic acid, exhibited the same inhibitory effect on [3H]acetate-PAF formation; however, the saturated stearic acid did not lead to any inhibition. These findings suggest that liberation of unsaturated fatty acids from membrane phospholipids, as a consequence of phospholipase A2 activation, would modulate PAF formation via inhibition of the acetyltransferase. In addition, the utilization of arachidonic acid oleic acids in activated neutrophils furnishes an easy means of blocking PAF synthesis in order to understand the role of this mediator in cellular processes.

Acetates↗
PubMed1987

[The effect of benzydamine on the generation and interaction of reactive oxygen species and cyclo- and lipoxygenase].

Pharmacological mechanisms of benzydamine (Tantum) are studied which are of relevance for the antiinflammatory properties of this non-steroidal antiinflammatory drug (NSAID). Benzydamine most effectively inhibits the generation of reactive oxygen species by murine neutrophils (IC50 1.7 X 10(-5) mol/l). Piroxicam, indomethacin and acetylsalicylic acid are ineffective. Benzydamine, however, does not interfere with xanthine oxidase-dependent superoxide anion radical formation or epinephrine oxidation. The other tested NSAID are as well inactive. The findings confirm the missing cyclooxygenase inhibition of benzydamine (IC50 greater than 10(-3) mol/l), contrary to the other NSAID which are strong (indomethacin IC50 6 X 10(-8) mol/l; piroxicam IC50 2 X 10(-7) mol/l) or moderate cyclooxygenase inhibitors (acetylsalicylic acid IC50 10(-5) mol/l). LTB4 generation via the lipoxygenase is only inhibited by indomethacin (EC50 3.6 X 10(-5) mol/l). Benzydamine appears unique among other NSAID by its relatively strong interference with the generation of reactive oxygen radicals and the lack of cyclooxygenase inhibition.

Animals↗