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Biomedical subjects

J Brouwer

Publications and source records attributed to J Brouwer.

At least 91 records · Page 5Linked to original sources
PubMed1992

Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.

If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

The Escherichia coli UvrABC endonuclease is capable of initiating the repair of a wide variety of DNA damages. To study the binding of the UvrAB complex to the DNA at the site of a lesion we have constructed a synthetic DNA fragment with a defined cis-diamminedichloroplatinum(II) (cis-Pt).GG adduct. The cis-Pt.GG is the major adduct after treatment of DNA with the antitumor agent cisplatin. Binding to the DNA at the site of the defined lesion was studied with DNase I and MPE.Fe(II) hydroxyl radical footprinting. The results indicate that the UvrAB complex binds to the convex side of the kink in the DNA caused by the cis-Pt.GG adduct. Concerted incisions of the damaged strand by the UvrABC endonuclease were at the 8th phosphodiester bond 5' to and at the 4th bond 3' of the adjacent guanines. An additional incision was found at the 15th phosphodiester bond 5' to the damaged site. This extra incision was stimulated by a high concentration of UvrC.

Bacterial Proteins↗
PubMed1991

Mutational specificity of gamma-rays differs for the same target in plasmid DNA and double-stranded (RF) M13 bacteriophage DNA.

In the lacZ alpha gene of a pUC plasmid a 144 bp insert was cloned as target for mutagenesis. Irradiation of the plasmid in a diluted aqueous solution by 60Co gamma-rays under oxic conditions leads to a very specific mutation spectrum. The predominant type of mutation was a C/G to A/T transversion (29 out of 47 mutants) whereas C/G to G/C transversions were found 7 times and C/G to T/A transitions 10 times. Only one frameshift could be observed which was a deletion of an A/T base pair. The mutations were not randomly distributed along the mutation target but show a strong preference for a certain DNA sequence in which two thirds of the mutations were scored. In this DNA area a hotspot (24 of the 47 mutants) for mutagenesis was located and within 6 bp next to this hotspot another seven mutations were scored. The mutation spectrum in the same mutation target as part of double-stranded (RF) M13 phage was published before. In both systems the mutational hotspot is located at the same site, but the predominant type of mutation is different. In the M13 system the C/G to G/C transversion was the most important event.

Bacteriophages↗
PubMed1990

Determination of alpha 1-antitrypsin in fecal extracts by enzyme immunoassay.

An enzyme immunoassay (EIA) has been developed for the determination of alpha 1-antitrypsin (AT). The assay can measure AT in fecal extracts (0.2 g wet feces/ml) from all healthy individuals. Purified human AT was coupled to peroxidase by means of a heterobifunctional reagent and the conjugate was used as a labeled antigen in competitive immunoassays. Concentrations of AT in feces from healthy individuals were less than 0.55 mg/g wet weight or less than 2.2 mg/g dry weight. CV for the physiological day-to-day variance varied between 9 and 140%. After incubation for 48 h at 37 degrees C, the average recovery rates for AT were 78% in ileostomy fluids, 88% in fecal extracts and 92% in feces. Results obtained with EIA correlated well with those obtained with a commercial radial immunodiffusion assay (r = 0.95).

Adolescent↗
PubMed1990

Differential repair of UV damage in Saccharomyces cerevisiae is cell cycle dependent.

In the yeast Saccharomyces cerevisiae the transcriptionally active MAT alpha locus is repaired preferentially to the inactive HML alpha locus after UV irradiation. Here we analysed the repair of both loci after irradiating yeast cells at different stages of the mitotic cell cycle. In all stages repair of the active MAT alpha locus occurs at a rate of 30% removal of dimers per hour after a UV dose of 60 J/m2. The inactive HML alpha is repaired as efficiently as MAT alpha following irradiation in G2 whereas repair of HML alpha is less efficient in the other stages. Thus differential repair is observed in G1 and S but not in G2. Apparently, in G2 a chromatin structure exists in which repair does not discriminate between transcriptionally active and inactive DNA or, alternatively, an additional repair mechanism might exist which is only operational during G2.

Blotting, Southern↗
PubMed1990

The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG).

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.

Antineoplastic Agents↗
PubMed1990

Analysis of the morphology of the unipolar endocardial paced evoked response.

In a retrospective study we analyzed the unipolar endocardial evoked response signal (ERS) of 103 patients prior to pacemaker implantation. The objective of this study was to give a complete description of the ERS morphology and to evaluate influences on this morphology of both various electrode characteristics and pacing rate. In addition, spontaneous endocardial signals were studied. The results demonstrate that acute leads had both higher R wave and T wave amplitudes and a faster downslope of the T wave. In the acute leads those with porous titanium carbon coated tips showed a more pronounced T wave. Pacing rate influences the R wave amplitude and the stimulus to T wave interval. Both stimulus to maximum and stimulus to minimum T wave interval show an exponential correlation with the stimulus interval. The interval between maximum and minimum of the T wave and the absolute amplitude of T wave are not influenced by rate. Although there were significant correlations of the spontaneous endocardial signal with the ERS, the predictive value of the spontaneous signal for the ERS morphology is low. Prospective studies will be necessary to confirm the findings in this study.

Aged↗
PubMed1990

Differential repair of UV damage in rad mutants of Saccharomyces cerevisiae: a possible function of G2 arrest upon UV irradiation.

After UV irradiation, the transcriptionally active MAT alpha locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HML alpha locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, rad16, and rad24, were impaired in the removal of UV dimers from the inactive HML alpha locus, whereas they had generally normal repair of the active MAT alpha locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HML alpha locus.

Blotting, Southern↗
PubMed1989

Mutations induced by 60Co gamma-irradiation in double-stranded M13 bacteriophage DNA in nitrous-oxide saturated solutions are characterized by a high specificity.

Irradiation of double-stranded M13 mp10 DNA in a diluted aqueous solution under N2O leads to a very specific mutation spectrum. Fifteen of 28 mutations induced in a 144 base pair (bp) target are C/G to G/C transversions, the other five bp substitutions are C/G to A/T transversions. Six mutations were single bp deletions, one is a large deletion of 180 bp and one is a 10 bp duplication which is probably from spontaneous origin. The mutations are not randomly distributed throughout the 144 bp mutation target but concentrated around two sites. The differences and similarities with the radiation-induced mutation spectrum previously obtained under oxygen are discussed.

Base Sequence↗
PubMed1988

60Co gamma-rays induce predominantly C/G to G/C transversions in double-stranded M13 DNA.

Upon irradiation with gamma rays of an oxygenated aqueous solution of double-stranded M13 DNA, a very specific mutation spectrum was found with respect to both the type and the positions in the DNA sequence. Of the 23 mutations, which were sequenced, 16 represent a C/G to G/C transversion. A C/G to T/A transition was found once and a G/C to T/A transversion twice. The remaining 4 mutations are frameshifts, 2 are identical and formed by the insertion of a G/C basepair; the other 2 mutations are due to a duplication of 10 basepairs situated at different positions but with a remarkable homology in base sequence. Fourteen mutations, including the 2 duplications are found in the neighbourhood of a TGCT/ACGA sequence.

Base Sequence↗
PubMed1988

The role of the excision-repair enzymes in mutation-induction by cis-Pt(NH3)2Cl2.

Mutation induction by cis-Pt(NH3)2Cl2 (cisplatin) has been shown to be absent in E.coli strains carrying a deletion of the uvrB gene (1). This suggested that excision-repair, which is normally thought to be error-free, is involved in mutation induction with cisplatin. Here, the role of the excision repair enzymes UvrA, UvrB and UvrC is investigated using E.coli strains with different repair capacities. It is shown that cisplatin induced mutagenesis is dependent both on UvrA and UvrB but not on UvrC. Of the UvrB enzyme the N-terminal 113 aminoacids are sufficient for mutation induction by cisplatin.

Cisplatin↗
PubMed1988

Non-identity of human plasma lysozyme and 4-methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside hydrolase.

1. Using 4-methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside (MU-TACT) as substrate, it is possible to measure the activity of purified lysozyme and to demonstrate lysozyme activity in the urine of patients with acute monocytic leukemia, characterized by massive lysozymuria. 2. Notwithstanding this observation, we present evidence that in normal human plasma another acid endoglucosaminidase is hydrolyzing the substrate. 3. The following data support the hypothesis of the existence of a separate hydrolase: (a) Thermoinactivation is different for MU-TACT hydrolase and lysozyme. (b) In plasma and many other biological samples, the concentration of lysozyme is too low to be measured with the artificial substrate and there is no correlation between MU-TACT hydrolase and lysozyme. (c) Serum of lysozyme deficient rabbits has normal MU-TACT hydrolase activity. (d) On Sephadex G-200 and DEAE cellulose chromatography, lysozyme and MU-TACT hydrolase are eluted separately. (e) Immunoremoval of lysozyme from human plasma does not affect the activity towards MU-TACT. (f) The effect of N-acetylglucosamine and N-acetylmuramic acid on the activity of lysozyme and MU-TACT hydrolase is different.

Animals↗
PubMed1988

Identification of the uvrA6 mutation of Escherichia coli.

The uvrA6 mutation has been cloned on a multicopy plasmid by using a chloramphenicol resistance marker introduced next to the uvrA gene in the Escherichia coli chromosome. The mutation was shown to reside in the N-terminal part of the uvrA gene. Sequencing part of this region of the mutant gene revealed a frameshift mutation at positions 207 to 209, which leads to a stop codon at position 262. A marker rescue experiment showed that this frameshift is the only mutation responsible for the UV-sensitive phenotype of the UvrA6 mutant. The method presented is suitable for the cloning of every chromosomal uvrA mutation and can be useful for the study of the functional domains of the UvrA protein.

Chromosome Mapping↗
PubMed1988

Detection of antibodies against Aspergillus fumigatus: comparison between double immunodiffusion, ELISA and immunoblot analysis.

The performance of ELISA to detect IgG and IgM antibodies to Aspergillus fumigatus has been evaluated in strongly precipitin-positive, weakly precipitin-positive and precipitin-negative patient sera, with immunoblot analysis as the confirmatory test. All strongly precipitin-positive sera contained increased IgG titers and showed clearly positive immunoblot patterns. Most of the weakly precipitin-positive sera contained ELISA titers within the normal range established with sera of healthy blood donors and showed normal immunoblot patterns. Increased titers of IgG and/or IgM were measured in one-sixth of the precipitin-negative patient sera. Immunoblot analysis confirmed the presence of antibodies to A. fumigatus in 55% of the precipitin-negative sera with increased antibody titers. ELISAs for A. fumigatus-specific IgG and IgM are sensitive tests for screening of patient sera. However, positive results with ELISA should be confirmed by means of immunoblot analysis.

Antibodies, Fungal↗