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Biomedical subjects

A Meisel

Publications and source records attributed to A Meisel.

At least 37 records · Page 2Linked to original sources
PubMed1995

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.

Adenosine Triphosphatases↗
PubMed1995

Restriction endonucleases functionally interacting with two DNA sites.

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

Binding Sites↗
PubMed1995

Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites.

EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity. We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements. The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein. Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules. After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer. The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies. Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII. It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible. We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity.

Base Sequence↗
PubMed1995

The Consortium Ethics Program: an approach to establishing a permanent regional ethics network.

This paper describes the first three-year experience of the Consortium Ethics Program (CEP-1) of the University of Pittsburgh Center for Medical Ethics, and also outlines plans for the second three-year phase (CEP-2) of this experiment in continuing ethics education. In existence since 1990, the CEP has the primary goal of creating a cost-effective, permanent ethics resource network, by utilizing the educational resources of a university bioethics center and the practical expertise of a regional hospital council. The CEP's conception and specific components stem from recognition of the need to make each hospital a major focus of educational efforts, and to provide academic support for the in-house activities of the representatives from each institution.

Cost-Benefit Analysis↗
PubMed1995

The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes.

Studies on phage T3 and T7 have shown that these viruses avoid restriction not only by the phage-coded Ocr (and S-adenosylmethionine hydrolase) protein functions or by the complete loss of specific recognition sites for certain restriction endonucleases from their genomes, but also that there are two additional modes: resistance towards EcoP15 (which recognizes a non-symmetrical sequence) is achieved by an identical orientation of all the recognition sites in the virus genome (strand bias) and in the case of EcoRII by the extreme reduction in number and thereby greater distance between recognition sites in the genome. These observations led to the discovery that certain restriction endonucleases require the simultaneous cooperation with two DNA sites for their function, as well as to the ongoing elucidation of the molecular modes of action of these enzymes. Type II and type III enzymes display fundamentally different mechanisms of protein-DNA interaction. For EcoRII we favor a model of simultaneous binding of two DNA sites to a dimeric enzyme molecule (neighbouring sites of the same, looping, DNA molecule or sites located on different DNA molecules), while the action of EcoP15 seems to conform with a tracking-collision model of two enzyme molecules bound to inversely oriented recognition sites. In addition to podoviruses T3 and T7, strand bias of recognition sequences for different type III DNA modification-restriction enzymes is also observed in the inoviruses M13, IKE and PF3.

Bacteriophage T3↗
PubMed1995

Barriers to forgoing nutrition and hydration in nursing homes.

In the two decades since the Karen Quinlan case first brought the issues that now go under the heading of the "right to die" to the attention of the courts and the public, a well-accepted legal consensus has developed about the law governing the forgoing of life-sustaining medical treatment. Law and clinical medical practice do not always run in tandem, however, and what law prescribes does not always occur in practice. One aspect of the legal consensus-that artificial nutrition and hydration is a medical treatment and thus may be withheld or withdrawn according to the same procedures and standards as other life-sustaining medical treatments-is probably less well accepted than the remainder. For reasons that I will explain, this is understandable. But what is puzzling is that this element of the consensus seems to be even less well accepted in nursing homes than in acute-care hospitals.

Advance Directives↗
PubMed1992

Type III restriction enzymes need two inversely oriented recognition sites for DNA cleavage.

Type III restriction/modification enzyme recognize short, non-palindromic sequences that can be methylated on only one strand, with the paradoxical consequence that during replication of what is in effect hemimethylated DNA totally unmodified sites arise. Why the unmodified sites are not subject to suicidal restriction was not clear. Here we show that restriction requires two unmodified recognition sites that can be separated by different distances but which must be in inverse orientation. All of the unmodified sites in newly replicated DNA are of course in the same orientation, which explains why they are not restricted. This result may be of relevance to other manifestations of anisotropy in double-stranded DNA, such as genetic imprinting.

Base Sequence↗
PubMed1992

Informed consent.

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Disclosure↗
PubMed1992

The legal consensus about foregoing life-sustaining treatment: its status and its prospects.

The legal consensus that has evolved through adjudication and legislation since the Karen Quinlan case in 1976 is founded on the premise that there is a bright line between passive euthanasia and active euthanasia. Indeed, the term passive euthanasia is often eschewed in favor of less emotionally-laden terminology such as "forgoing life-sustaining treatment" or "terminating life support" so as to further sever any possible connection with active euthanasia. Legal approval has been bestowed upon passive euthanasia under certain circumstances while active euthanasia is routinely condemned. This consensus was put to a test in 1990 when the United States Supreme Court ruled on the Cruzan case. However, the Court's narrow decision did not upset the consensus, and in the most significant appellate decisions handed down by state courts since Cruzan, there has been a reaffirmation--and possibly even an extension--of the consensus. Two other threats to the legal consensus about forgoing life-sustaining treatment have begun to manifest themselves: the increasing pressure for mercy killing and "futility" cases. Both of these challenge the fundamental premises on which the consensus is grounded.

Advance Directives↗
PubMed1991

Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA.

The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.

Allosteric Site↗
PubMed1991

Legal myths about terminating life support.

There are a number of myths about what the law permits concerning the termination of life support, some of which spring from a fundamental misconception of what law is. A serious misunderstanding of the law can lead to tragic results for physicians, health care institutions, patients, and families. These misunderstandings are (1) anything that is not specifically permitted by law is prohibited; (2) termination of life support is murder or suicide; (3) a patient must be terminally ill for life support to be stopped; (4) it is permissible to terminate extraordinary treatments, but not ordinary ones; (5) it is permissible to withhold treatment, but once started, it must be continued; (6) stopping tube feeding is legally different from stopping other treatments; (7) termination of life support requires going to court; and (8) living wills are not legal.

Advance Directives↗
PubMed1990

Lessons from Cruzan.

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Government Regulation↗
PubMed1989

Evolution of clinical ethics teaching at the University of Pittsburgh.

The authors explain that several years of effort, by many faculty from a variety of disciplines, were required to expand medical ethics teaching at the University of Pittsburgh School of Medicine beyond the preclinical years. Since 1986, faculty associated with the school's Center for Medical Ethics have begun a comprehensive ethics teaching program for all four years and the residency period; they also are attempting to develop an ethics consultation service. The authors describe the program, its promise and plans, and the significant difficulties involved in establishing and maintaining it, not only problems of long-term funding but of the uninformed and negative attitudes of some students and faculty toward ethics teaching, especially in the clinical setting. Also discussed are the pros and cons of using cases in ethics teaching and the program's approaches to evaluation and to training clinical faculty in clinical ethics issues.

Education, Medical↗
PubMed1989

AIDS and dentistry.

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Acquired Immunodeficiency Syndrome↗