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Biomedical subjects

M T Muller

Publications and source records attributed to M T Muller.

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

The involvement of topoisomerase I in the induction of DNA-protein crosslinks and DNA single-strand breaks in cells of ultraviolet-irradiated human and frog cell lines.

Exposure of GM 4390 human skin fibroblasts and ICR 2A frog cells to 10 kJ m(-2) of ultraviolet B (UVB) radiation resulted in the formation of DNA-protein crosslinks (DPCs) and DNA single-strand breaks (SSBs). However, upon incubation, there were rapid increases in the yields of both DPCs and SSBs. An enhancement in these DNA alterations was detected within 12 min after irradiation and their levels continued to rise by 5-8-fold within 15 h after exposure to UV radiation. Using an antibody-based assay that measures covalent complex formation between topoisomerase (topo) I and genomic DNA, it was found that topo I is one of the proteins involved in these DPCs induced by UV radiation. The levels and rate of increase of topo I-DNA covalent complexes were similar to the UV-radiation-dependent formation of DPCs and SSBs. A UV-radiation-sensitive mutant frog cell line, DRP 153, was also examined and was found to be deficient in this induction of DPCs and SSBs by UV radiation. When these cells were transfected with the human SUVCC3 gene, the resulting transformant displayed kinetics for the induction of DPCs and SSBs similar to the human and parental frog cells. However, human topo I was not defected in the transformed frog cells, indicating that SUVCC3 does not encode topo I. It is likely that SUVCC3 encodes an associated enzymatic activity which permits normal stimulation of topo I-DNA covalent complexes in UV-irradiated cells.

Animals↗
PubMed1996

Characterization of topoisomerase II-DNA interaction and identification of a DNA-binding domain by ultraviolet laser crosslinking.

We have used ultraviolet laser crosslinking to characterize the DNA-binding properties of highly purified yeast topoisomerase II in the absence of ATP. A single 5 ns, 20 mJ pulse of 266 nm light produced optimal crosslinking to a short DNA duplex, with an efficiency of 0.25%. An equilibrium binding constant (Keq) of 1.2 +/- 0.5 x 10(8) M(-1) was determined from kinetic analysis. Topoisomerase II showed highest affinity for supercoiled DNA. Limited proteolysis of crosslinked topoisomerase II-DNA complexes showed a site of crosslinking to be within a 29-kDa fragment with Leu-681 at its amino-terminal end. This region contains the active Tyr-783 and is homologous to the amino-terminal region of the DNA-binding bacterial gyrase GyrA subunit, suggesting a conserved DNA-binding mechanism.

Base Sequence↗
PubMed1996

The role of the social network in active euthanasia and physician-assisted suicide.

OBJECTIVE: To obtain insight into the involvement of a patient's social network in the process leading to the administration of euthanasia and assisted suicide (EAS). DESIGN: Descriptive, retrospective. METHOD: Data were collected by means of anonymous questionnaires sent to a random sample of 521 general practitioners (GPs) in the Province of North Holland and 521 GPs from the rest of the Netherlands and all 713 Dutch nursing home physicians (NHPs). The data were collected over the period 1986 to June 1990 inclusive. RESULTS: GPs and NHPs had discussions most often with the partner of the patient about the patient's request for EAS, the doctor's intention to administer EAS and the actual administering of EAS. According to both the GPs and the NHPs the social network practically always agreed with the doctor's decision to administer EAS. The persons who were most often present at the patient's bedside when EAS was administered were the patient's partner and children (29%). Often, especially in the cases of GPs, the decision not to report EAS was determined by the desire of relatives and/or the physician not to involve the relatives in a judicial inquiry. CONCLUSION: To a large extent the social network was involved in the patient's request for EAS, the doctor's intention to administer EAS and the actual administering of EAS. This seems to be important both for good decision-making with regard to EAS and for helping relatives to come to terms with the loss of a dear one.

Communication↗
PubMed1996

Topoisomerase II alpha is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure.

A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.

Animals↗
PubMed1996

Voluntary active euthanasia and doctor-assisted suicide: knowledge and attitudes of Dutch medical students.

The objective of the study was to gain insight into the knowledge of and attitudes towards voluntary active euthanasia and doctor-assisted suicide (EEDAS) of Dutch medical students, and to determine whether knowledge and attitudes change after a 1-day informative conference about EDAS. Data were collected by means of two self-administered questionnaires. Questionnaire 1 had to be completed before the start of the conference and questionnaire 2 after the conference. In both questionnaires, students were asked by means of two open-ended questions to define euthanasia and doctor-assisted suicide. They were also asked to indicate which of eight statements met with the requirements for prudent practice. Finally, the students were asked to what extent they agreed or disagreed with each of seven statements about attitudes towards EDAS. To determine if a selection occurred among students who returned both questionnaires, their background characteristics, and knowledge and attitudes towards EDAS were compared with those who returned only the first questionnaire. Forty-seven students returned only the first questionnaire, while both questionnaires were returned by 137 students. No differences were found between students who returned both questionnaires and those who returned only the first questionnaire with regard to age, religion, knowledge of and attitudes towards EDAS. Students' knowledge of the definitions of EDAS and the requirements for prudent practice improved significantly. Students' reactions to the statements on attitudes towards EDAS showed that a large majority had a fairly positive attitude towards EDAS. There was no significant difference before and after the conference. Male students and students with a religion were more opposed to EDAS than female students and students without a religion. The fact that the students' knowledge of EDAS improved after a 1-day conference does not imply sufficient understanding of the issue. Because EDAS is allowed only under strict conditions in the Netherlands, medical students require special training. Only then will they be equipped to deal with requests for EDAS during their future careers.

Adolescent↗
PubMed1995

Eukaryotic topoisomerase II cleavage is independent of duplex DNA conformation.

Alternating purine-pyrimidine (RY) repeats have been identified in naturally occurring DNA and have many intriguing properties. Eukaryotic topoisomerase II displays significant cleavage activity at RY repeats (Spitzner et al. (1990) Nucleic Acids Res. 18, 1-11) due to the homology between RY repeat and the topoisomerase II consensus sequence. Cleavages are remarkably strong on duplex B form DNA. Certain RY elements are known to adopt altered DNA forms, such as Z-DNA, under the influence of superhelical stress. To investigate the dependence of topoisomerase II activity on DNA conformation, a plasmid containing a 40 bp of deoxyguanine-thymine repeat was constructed and the dependence of topoisomerase II cleavage patterns were compared. Although the degree of negative supercoiling strongly affected the overall efficiency of topoisomerase II cleavage, the sequence specificity was identical over a wide range of superhelical densities. These results suggest that topoisomerase II site specific action on duplex DNA is largely independent of DNA conformation. Moreover, since the GT target sequence is known to adopt a Z-DNA structure under conditions of superhelical density used in these experiments, the results reveal that topoisomerase II is a DNA binding protein capable of recognizing Z-DNA structure in eukaryotic cell.

Base Sequence↗
PubMed1995

Analysis of topoisomerase I/DNA complexes in patients administered topotecan.

As part of a Phase II clinical trial of topotecan, DNA breakage in vivo was measured by detecting covalent topoisomerase/DNA intermediates in peripheral blood. The ICE (in vivo complex of enzyme) bioassay was used to assess topotecan activity in the peripheral blood of patients before, during, and after infusion therapy. The results can be summarized as: (a) ICE bioassay is a specific, antibody-based assay for topoisomerase I-mediated DNA damage. Topoisomerase I/DNA complex formation can be monitored unambiguously in the absence of topotecan to establish a basal level of endogenous enzyme action on DNA; (b) infusion of topotecan significantly stimulated formation of covalent enzyme/DNA complexes. Complexes were detected within 5 min postinfusion and increased over the course of a 30-min treatment; (c) after termination of infusion, complex formation decreased by 3-4-fold within 30 min, showing that cleavage complexes quickly reseal after drug withdrawal; and (d) formation of complexes varied widely between patients. The ICE bioassay can evaluate the effects of topoisomerase I inhibitors on target tissues; thus, it may valuable in predicting response to these drugs.

Antineoplastic Agents↗
PubMed1995

Determination of 5' and 3' DNA triplex interference boundaries reveals the core DNA binding sequence for topoisomerase II.

Previous studies have shown that formation of intermolecular DNA triplexes at sequences that overlap protein binding sites inhibits DNA binding by these proteins. We show that DNA cleavage by eukaryotic topoisomerase II is blocked by triplex formation at sites overlapping and adjacent to the triple binding site. To map precisely the boundaries of triplex interference, we constructed a vector containing enzyme binding sites of different lengths and flanked both 5' and 3' by DNA triplexes. We call this method Triplex Interference Mapping by Binding Element Replacement (TIMBER). Triplex regions within 3 bases 5' or 7 bases 3' of cleavage sites blocked DNA cleavage; triplex formation outside of this region had no effect upon cleavage activity. We conclude that topoisomerase II binding requires unhindered access to the major groove of a duplex DNA binding site in this 10-base region. In addition, the inclusion of topoisomerase II inhibitors yielded the same results for the triplex interference assays despite alterations in DNA cleavage site selection. The statistical analyses of over 500 topoisomerase II cleavage sites (in the presence or absence of inhibitors) suggest a model consistent with the region spanning -3 to +7 (relative to the cleavage site) containing most of the base-specific contacts for topoisomerase II. This triplex interference assay may prove valuable in the characterization of DNA binding sites for other proteins as well, particularly in conjunction with deletion analysis.

Animals↗
PubMed1995

Attitudes of Dutch general practitioners and nursing home physicians to active voluntary euthanasia and physician-assisted suicide.

OBJECTIVE: To gain insight into the attitudes of Dutch general practitioners and nursing home physicians to voluntary active euthanasia and physician-assisted suicide. DESIGN: Descriptive study. METHOD: Data were collected by means of anonymous postal questionnaires to be completed by a random sample of 521 general practitioners from the province of North Holland, 521 general practitioners from the rest of the Netherlands, and 713 Dutch nursing home physicians who were members of the Dutch Association of Nursing Home Physicians. RESULTS: The written responses of general practitioners and nursing home physicians to six statements about voluntary active euthanasia and physician-assisted suicide showed that a large majority had a fairly positive attitude to euthanasia and suicide. This finding also emerged from the scores obtained on a scale compiled on the basis of the statements. General practitioners and nursing home physicians were more opposed to euthanasia and physician-assisted suicide if they had never performed it, if they belonged to a religious group, or if they were older. CONCLUSION: Dutch general practitioners and nursing home physicians have a fairly positive attitude toward euthanasia and physician-assisted suicide. However, the majority of these physicians favor a policy of voluntary active euthanasia and physician-assisted suicide under strict conditions.

Age Factors↗
PubMed1995

Active euthanasia and physician-assisted suicide in Dutch nursing homes: patients' characteristics.

We wished to obtain information about the principal and subsidiary diagnoses, sex, age, marital status, religion and background characteristics of Dutch nursing home patients to whom euthanasia/assisted suicide (EAS) was administered. We performed an exploratory, descriptive, retrospective study involving all Dutch nursing home physicians (NHPs) who in September 1990 were members of the Dutch Association of Nursing Home Physicians (NVVA; n = 713). An anonymous printed questionnaire in two parts was used. Part 1 was intended for all respondents and was expected to give insight into the nature and extent of EAS in Dutch nursing homes. Part 2 was intended only for respondents who had indicated in part 1 that they had administered EAS. They were asked to describe their last case of EAS. The study covered the period from 1986 to mid-1990. There was an 86% response. The respondents described 86 cases of EAS. Sixty-nine of these took place in a nursing home. The majority of patients to whom EAS was administered were suffering from a malignant neoplasm (53%). EAS was administered more often to men than to women. The average age of the patients was 70.9 years. When EAS was administered, the patients on average had been in the nursing home for 13.1 months. Dutch nursing home patients who were given EAS differed in various respects from 'the average nursing home patient'. The principal diagnosis for patients who were given EAS was a malignancy, whereas relatively few physically ill nursing home patients die as a result of a malignancy. The patients to whom EAS was administered were younger and more often male. EAS patients had been in the nursing home for a shorter time than the other somatic (physically disabled) patients who died during the study period.

Adult↗
PubMed1995

Clustering of Sp1 sites near the promoter region of ICP34.5 in herpes simplex virus type 1.

We report that a host nuclear protein of approximately 100 kDa binds to the tandemly reiterated DR2 sequence of herpes simplex virus type 1 (HSV-1). The DR2 sequence is a repeated component in the "a" sequence, which defines the signals for cleavage and encapsidation of viral DNA; the "a" sequence also contains the promoter regulatory signals for the gene encoding the viral neurovirulence factor, ICP34.5. Characterization of the host binding protein by means of gel shifts and DNase I footprinting revealed this protein is the eukaryotic transcription factor, Sp1. Furthermore, as judged from the sequence homology, the DR2 region contains clustered matches to the consensus binding site for Sp1. Comparison of the host factor and purified Sp1 (by means of gel shifts and footprinting) confirmed these findings. Since clustered DNA recognition elements represent unusually high affinity binding sites, these repeated Sp1 motifs proximal to the ICP34.5 gene suggest that this region may be a major Sp1 binding site in the viral genome.

Binding Sites↗
PubMed1995

Analysis of eukaryotic topoisomerase II cleavage sites in the presence of the quinolone CP-115,953 reveals drug-dependent and -independent recognition elements.

The quinolone derivative CP-115,953 [6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] has been shown to induce eukaryotic topoisomerase II-mediated breaks in DNA, producing cleavage patterns that are distinct from those induced by the anticancer drugs amsacrine, etoposide, and teniposide. High levels of the quinolone have been found to inhibit topoisomerase II activity via an interaction with the enzyme and not by DNA unwinding. Topoisomerase II cleavage sites were analyzed on nine DNA fragments, and 85 quinolone-induced sites were sequenced, as well as 86 amsacrine and 134 teniposide sites. A consensus sequence was derived for the quinolone sites that is different from those reported for other drugs; however, because topoisomerase II cleavage sites are double-stranded but not palindromic, different consensus sequences are not easily compared. For this reason, a new, double-stranded, consensus sequence method, the "unique-base analysis," was developed; this was applied to the quinolone sites as well as six other large sets of topoisomerase II sites determined in the absence or presence of drugs. For each of the seven sets of sites, conserved bases were found in the 16-base region spanning positions -6 to +10, relative to the enzyme cleavage site (DNA breakage between -1 and +1). The conserved bases were virtually identical in the regions flanking the cleavage site for all seven data sets. In contrast, the base preferences identified proximal to the cleavage sites were unique to the drug tested. These observations suggest that the selection of cleavage sites by topoisomerase II involves both enzyme-dependent and drug-dependent recognition elements. The single most preferred base in the quinolone sites was a cytosine at -1; the same preference was found with teniposide, and 60 of the 85 quinolone sites co-localized with teniposide sites.

Amsacrine↗
PubMed1995

Liposomal encapsulation increases the activity of the topoisomerase I inhibitor topotecan.

Topotecan, a topoisomerase I poison and water-soluble derivative of camptothecin, has shown promise in treating solid tumors; however, the drug is unstable under physiological conditions and converts to an inactive form within 30 minutes. Encapsulating topotecan in liposomes (LIP-TPT) minimizes inactivation. The efficacy of LIP-TPT was examined with a novel in vivo bioassay called ICE for In vivo Complexes of Enzyme. This bioassay uses antibodies to probe DNA for the presence of topoisomerase I covalent complexes and thereby allows direct quantification of topoisomerase I driven DNA adducts in living cells. We report that LIP-TPT was three- to fourfold more effective than free TPT in stabilizing covalent topoisomerase I-DNA intermediates inside tumor cells. These findings reveal that liposomal wrapping permitted effective delivery of camptothecin derivatives to active enzyme in the nucleus of the cell.

Antineoplastic Agents↗
PubMed1994

[Life-terminating actions by family practitioners and nursing home physicians without the patient's request].

OBJECTIVE: To describe deliberate and active termination of life without the patient's request by general practitioners and Dutch 'nursing home' physicians. DESIGN: Descriptive, retrospective investigation. METHOD: Data were collected through anonymous postal questionnaires sent to a random sample of 521 general practitioners in North Holland and 521 general practitioners in the rest of the Netherlands regarding 1986-1989 and to all 713 Dutch 'nursing home' physicians affiliated to the Dutch Association of Nursing Home Physicians regarding 1986-June 1990. RESULTS: 65 General practitioners (10%) and 28 nursing home physicians (5%) had at any time deliberately terminated the life of a patient without explicit request by the patient, 94 and 70 times, respectively. In almost all cases the general practitioners indicated that the patients were suffering intensely; the nursing home physicians indicated this in 65% of the cases; with 66% and 57% of the patients contact was not possible; in 50% and in 65% of the cases, respectively, the family was involved in the decision to terminate the patient's life. CONCLUSION: Termination of life without request by the patient occurs in the practices of Dutch general practitioners and Dutch 'nursing home' physicians, but is rare.

Advance Directives↗
PubMed1994

Voluntary active euthanasia and physician-assisted suicide in Dutch nursing homes: requests and administration.

OBJECTIVE: To learn how many requests for voluntary active euthanasia and/or physician-assisted suicide (EAS) are made to Dutch nursing home physicians (NHPs) and how often these requests are honored. DESIGN: Retrospective survey. SETTING: The Netherlands. PARTICIPANTS: All Dutch NHPs affiliated with the Dutch Association of Nursing Home Physicians (n = 713). MEASUREMENTS: An anonymous postal questionnaire was sent to all Dutch NHPs affiliated with the Dutch Association of Nursing Home Physicians (n = 713). Respondents were asked how often they had received an explicit request for EAS and whether they had complied with that request. Those who had complied were asked questions about the last occasion on which they had administered either voluntary active euthanasia or physician-assisted suicide. RESULTS: The response rate was 86% (n = 582). Of the respondents, 88% had never administered EAS in nursing homes. The remaining 12% (n = 69) had received 164 requests for voluntary active euthanasia and 53 requests for physician-assisted suicide in the period 1986 through mid-1990. Of these requests, 74 were granted (51 voluntary active euthanasia and 23 physician-assisted suicide). Dutch NHPs together receive an average of 300 requests for EAS a year. They comply with 25 of such requests annually. CONCLUSION: Not many requests for EAS are made in Dutch nursing homes. Of these requests, fewer than 1 in 10 result in the actual administration of EAS. The data presented are relatively constant for the 4.5-year period studied.

Adult↗
PubMed1994

Voluntary active euthanasia and physician-assisted suicide in Dutch nursing homes: are the requirements for prudent practice properly met?

OBJECTIVE: To acquire data about and an understanding of the way in which Dutch nursing home physicians (NHPs) who administer voluntary active euthanasia and/or physician-assisted suicide (EAS) cope with the requirements for prudent practice. These requirements include: the patient must experience his or her suffering as unbearable and hopeless; the wish to die must be well considered and persistent; the request must be voluntary; the NHP must consult at least one other physician; the physician is not allowed to issue a certificate testifying to natural death and is obliged to keep records. DESIGN, SETTING, PARTICIPANTS, MEASUREMENTS: See preceding paper. RESULTS: Sixty-nine NHPs (12%) indicated that they had administered EAS on at least one occasion. The state of the patient was described by the NHP as utterly hopeless in 88% of cases and as utterly unbearable in 64% of the cases. The period of time between the first discussion of the subject and the actual administration varied from less than a day to more than a year. The most frequently given reasons for the request were unbearable suffering (53%) and hopeless suffering (49%). The majority of the NHPs (85%) indicated that it was the patient who first broached the subject of EAS. Eighty-five percent of the NHPs also requested a consultation with another physician. In the majority of cases this second opinion was given by another NHP (63%); over 50% of these NHPs worked in the same nursing home. Ninety-one percent of the physicians consulted were convinced that the request was well considered, and 93% found that there was no alternative treatment available. The nurses involved were consulted informally: 94% were questioned about the request for EAS and 93% about the physician's intention to comply. Seventy-five percent of the respondents said they had made some sort of written notes regarding the last time they had administered EAS. The number of certificates testifying to death by natural causes fell after 1988. In 41% of the cases all requirements were met. CONCLUSION: The results of this study indicate that Dutch NHPs observe all the requirements for EAS in 41% of cases. In the remaining cases, shortcomings were found: NHPs allowed too little time between the first discussion and the actual administration; they did not always keep written records; or they signed a death certificate testifying that the patient had died a natural death.

Adult↗