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Biomedical subjects

R M Taylor

Publications and source records attributed to R M Taylor.

At least 37 records · Page 2Linked to original sources

Neural stem cells are uniquely suited for cell replacement and gene therapy in the CNS.

In recent years, it has become evident that the developing and even the adult mammalian CNS contain a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to correct a variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Adult↗

A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair.

XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.

Amino Acid Sequence↗

Nanometre-scale rolling and sliding of carbon nanotubes.

Understanding the relative motion of objects in contact is essential for controlling macroscopic lubrication and adhesion, for comprehending biological macromolecular interfaces, and for developing submicrometre-scale electromechanical devices. An object undergoing lateral motion while in contact with a second object can either roll or slide. The resulting energy loss and mechanical wear depend largely on which mode of motion occurs. At the macroscopic scale, rolling is preferred over sliding, and it is expected to have an equally important role in the microscopic domain. Although progress has been made in our understanding of the dynamics of sliding at the atomic level, we have no comparable insight into rolling owing to a lack of experimental data on microscopic length scales. Here we produce controlled rolling of carbon nanotubes on graphite surfaces using an atomic force microscope. We measure the accompanying energy loss and compare this with sliding. Moreover, by reproducibly rolling a nanotube to expose different faces to the substrate and to an external probe, we are able to study the object over its complete surface.

Aluminum Silicates↗

Xenotransplantation.

Xenotransplantation is a potentially promising but exceedingly complex issue. It is critical that this subject be discussed within and outside the transplant community. The need for an expanded organ supply is urgent, but the scientific and ethical positions are complex. This article reviews the current status of xenotransplantation, including the potential benefits and risks; discusses multiple ethical issues; and makes recommendations for the transplant coordinator.

Animals↗

Role of the DNA ligase III zinc finger in polynucleotide binding and ligation.

Mammalian DNA ligase III exists as two distinct isoforms denoted alpha and beta. Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase. Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms. Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-alpha activity during DNA base excision repair in a cell-free assay. In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers. Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced approximately 10-fold. These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer. The implications of these results to the cellular role of the putative zinc finger are discussed.

Amino Acid Sequence↗

Role of a BRCT domain in the interaction of DNA ligase III-alpha with the DNA repair protein XRCC1.

The BRCT domain (for BRCA1 carboxyl terminus) is a protein motif of unknown function, comprising approximately 100 amino acids in five conserved blocks denoted A-E. BRCT domains are present in the tumour suppressor protein BRCA1 [1-3], and the domain is found in over 40 other proteins, defining a superfamily that includes DNA ligase III-alpha and the essential human DNA repair protein XRCC1. DNA ligase III-alpha and XRCC1 interact via their carboxyl termini, close to or within regions that contain a BRCT domain [4]. To examine whether the primary role of the carboxy-terminal BRCT domain of XRCC1 (denoted BRCT II) is to mediate the interaction with DNA ligase III-alpha, we identified the regions of the domain that are required and sufficient for the interaction. An XRCC1 protein in which the conserved D-block tryptophan was disrupted by point mutation retained the ability to interact with DNA ligase III-alpha, so this tryptophan must mediate a different, although conserved, role. XRCC1 in which the weakly conserved C-block was mutated lost the ability to interact with DNA ligase III-alpha. Moreover, 20 amino acids spanning the C-block of BRCT II conferred full DNA ligase III-alpha binding activity upon an unrelated polypeptide. An XRCC1 protein in which this 20mer was deleted could not maintain normal levels of DNA ligase III-alpha in transfected rodent cells, a phenotype associated with defective repair [5]. In summary, these data demonstrate that a BRCT domain can mediate a biologically important protein-protein interaction, and support the existence of additional roles.

Amino Acid Sequence↗

Impotence and the use of the internal iliac artery in renal transplantation: a survey of surgeons' attitudes in the United Kingdom and Ireland.

BACKGROUND: In 1996, a court in the United Kingdom ruled against a plaintiff who claimed that: (1) division of both internal iliac arteries at separate renal transplant operations had made him impotent and (2) the risk of his becoming impotent was sufficiently high that he should have been warned. The court ruled that there was not enough evidence in the medical literature to allow his claim to succeed. METHODS: A survey of the views of transplant surgeons in the United Kingdom and Ireland on attitudes toward using the internal iliac artery for transplantation was conducted. RESULTS: A 100% response rate was received. In a potent male patient, 11% of surgeons would never use the first internal iliac artery and 52% would use it infrequently. If one internal iliac artery had already been used, 61% would never use a second and 34% would use it infrequently. Eighty-nine percent believed the risk of impotence when the second internal iliac artery was used was 25% or higher, and 91% thought the risk should be explained to the patient. CONCLUSION: A very strong opinion exists among transplant surgeons in the United Kingdom and Ireland that the second internal iliac artery should not be used to revascularize a kidney transplant when the first has already been divided. The risk of impotence, if the second internal iliac artery is used, is greater than 25% and should be explained to the patient. This represents a very clear statement of currently accepted practice.

Adult↗

Dose-dependent enhancing and inhibitory effects of A77 1726 (leflunomide) on cytotoxic T lymphocyte induction.

Leflunomide is an immunosuppressive prodrug which prevents allograft rejection in several animal model systems and may, therefore, have clinical application in organ transplant recipients. Although cytotoxic T lymphocytes (CTL) are an important component of the allograft rejection response, the effect of leflunomide on CTL development has not been thoroughly explored. In this study we have determined the effect of A77 1726, the active metabolite of leflunomide, on CTL induction in C57BL/6 mouse T cell cultures stimulated with anti-CD3 monoclonal antibody. Conjugate formation with P815 target cells, granzyme B enzymatic activity in CTL lysates, and P815 cytolysis in a 51Cr-release assay were used as determinants of in vitro CTL function. At high concentrations (10-20 microM), A77 1726 strongly inhibited CTL generation. In contrast, a low concentration (0.5 microM) of A77 1726 promoted CTL development. These dose-dependent opposing effects of A77 1726 on CTL induction could not be attributed to alterations in CD8+ lymphocyte percentages, interleukin-2 or CD25 expression, or the ability to conjugate with P815 target cells. However, both interferon-gamma and granzyme B expression were significantly decreased when CTL were induced in the presence of 10-20 microM A77 1726, and were slightly, but not always significantly, elevated in the presence of 0.5 microM A77 1726. We conclude that at high concentrations A77 1726 is a potent inhibitor of CTL induction, but a low concentration of A77 1726 enhances CTL development.

Animals↗

Remyelination: cellular and gene therapy.

Dysfunctional myelination or oligodendroglial abnormalities play a prominent role in a vast array of pediatric neurological diseases of genetic, inflammatory, immunological, traumatic, ischemic, developmental, metabolic, and infectious causes. Recent advances in glial cell biology have suggested that effective remyelination strategies may, indeed, be feasible. Evidence for myelin repair is accumulating in various experimental models of dysmyelinating and demyelinating disease. Attempts at remyelination have either been directed towards creating myelin de novo from exogenous sources of myelin-elaborating cells or promoting an intrinsic spontaneous remyelinating process. Ultimately, some disorders of myelin may require multiple repair strategies, not only the replacement of dysfunctional cells (oligodendroglia) but also the delivery or supplementation of gene products (i.e., growth factors, immune modulators, metabolic enzymes). Although primary oligodendrocytes or oligodendroglial precursors may be effective for glial cell replacement in certain discrete regions and circumstances and although various genetic vectors may be effective for the delivery of therapeutic molecules, multipotent neural stem cells may be most ideally suited for both gene transfer and cell replacement on transplantation into multiple regions of the central nervous system under a wide range of pathological conditions. We propose that, by virtue of their inherent biological properties, neural stem cells possess the multifaceted therapeutic capabilities that many diseases characterized by myelin dysfunction in the pediatric population may demand.

Cell- and Tissue-Based Therapy↗

Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue.

A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach. The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pi) of 8.20. Alignment of the A. thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae showed good sequence homology (42-45% identity, 61-66% similarity), particularly around the active site. Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species. Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S. cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase. The level of the DNA ligase transcript was not increased in A. thaliana seedlings in response to DNA damage induced by a period of enhanced UV-B irradiation. However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells.

Amino Acid Sequence↗

Bending and buckling of carbon nanotubes under large strain.

The curling of a graphitic sheet to form carbon nanotubes produces a class of materials that seem to have extraordinary electrical and mechanical properties. In particular, the high elastic modulus of the graphite sheets means that the nanotubes might be stiffer and stronger than any other known material, with beneficial consequences for their application in composite bulk materials and as individual elements of nanometre-scale devices and sensors. The mechanical properties are predicted to be sensitive to details of their structure and to the presence of defects, which means that measurements on individual nanotubes are essential to establish these properties. Here we show that multiwalled carbon nanotubes can be bent repeatedly through large angles using the tip of an atomic force microscope, without undergoing catastrophic failure. We observe a range of responses to this high-strain deformation, which together suggest that nanotubes are remarkably flexible and resilient.

Carbon↗

Manipulation of individual viruses: friction and mechanical properties.

We present our results on the manipulation of individual viruses using an advanced interface for atomic force microscopes (AFMs). We show that the viruses can be dissected, rotated, and translated with great facility. We interpret the behavior of tobacco mosaic virus with a mechanical model that makes explicit the competition between sample-substrate lateral friction and the flexural rigidity of the manipulated object. The manipulation behavior of tobacco mosaic virus on graphite is shown to be consistent with values of lateral friction observed on similar interfaces and the flexural rigidity expected for macromolecular assemblies. The ability to manipulate individual samples broadens the scope of possible studies by providing a means for positioning samples at specific binding sites or predefined measuring devices. The mechanical model provides a framework for interpreting quantitative measurements of virus binding and mechanical properties and for understanding the constraints on the successful, nondestructive AFM manipulation of delicate samples.

Aluminum Silicates↗

Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of beta-glucuronidase.

A deficiency of beta-glucuronidase (GUSB) causes the multisystem progressive degenerative syndrome, mucopolysaccharidosis (MPS) type VII (Sly disease), which includes mental retardation. Animal homologues of MPS VII (ref. 3, 4) are models for testing somatic gene transfer approaches to treat the central nervous system in this and other lysosomal storage disorders. Previous attempts to correct murine MPS VII by gene therapy have successfully treated lesions in some organs but not in the brain. Other experimental modalities have forestalled some disease progression in the brain, but only if done at birth, before the onset of severe lesions, when the animals are phenotypically normal. We tested whether therapeutic amounts of GUSB could be delivered to the diseased adult brain by transplanting cells engineered to super-secrete the normal enzyme for export to surrounding neural tissues. Lysosomal distention was cleared from neurons and glial cells in the vicinity of the grafts, showing that the secreted enzyme could reach the diseased cells and reverse lesions in the severely diseased brain. The ability to correct established lesions will be important for the treatment of many lysosomal storage diseases affecting the brain, because most patients are not diagnosed until lesions are advanced enough to affect phenotype or developmental milestones in early childhood, and some forms of the diseases do not become apparent until later in life.

Animals↗

Glycosaminoglycan storage in cultured neonatal murine mucopolysaccharidosis type VII neuroglial cells and correction by beta-glucuronidase gene transfer.

The inherited deficiency of beta-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. beta-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal beta-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of beta-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.

Animals↗

The persistent vegetative state and anencephaly: problematic paradigms for discussing futility and rationing.

Medical futility has been a controversial issue for the last several years. Although anencephalic infants and patients in the persistent vegetative state (PVS) have often been suggested as paradigmatic cases for discussing the issue of medical futility, there is no consensus among physicians or ethicists that treatment of such patients is futile and courts have not supported the futility argument. Focusing on the issue of medical futility will not resolve the management issues surrounding the care of patients in PVS or anencephalic infants. Instead, an analysis of the benefits and burdens of treatments offers a potential solution by demonstrating that the real problem is the dissociation of the benefits and burdens of treatment for such patients. We propose the idea that anencephalic infants and patients in PVS provide an appropriate paradigm for discussing the issue of rationing of health-care resources.

Anencephaly↗

Reexamining the definition and criteria of death.

The whole-brain criterion of death was first formally proposed by the "Ad Hoc Committee of the Harvard Medical School to Examine the Definition of Brain Death" in a "Special Communication" published in JAMA in 1968. Since then, all states in the United States and many western countries have endorsed this definition of death. The strongest defense of the concept of "brain death" was provided by Bernat, Culver, and Gert in a series of papers published in the early 1980s, emphasizing the important distinctions between the definition and the criteria of death and the tests for death. Careful analysis, however, demonstrates that brain-related criteria of death are inconsistent with traditional concepts of death. Thus, although death is properly understood as a biological phenomenon, "brain death" is a social construct created for utilitarian purposes, primarily to permit organ transplantation. The best definition of death is "the event that separates the process of dying from the process of disintegration" and the proper criterion of death in human beings is "the permanent cessation of the circulation of blood." Nevertheless, because brain-related criteria of death have been widely accepted, and because our society has demonstrated a strong commitment to organ transplantation, abandoning the concept of brain death would create serious political problems. Abandoning the "dead donor rule" would solve the problem of obtaining organs for transplantation, but would create different, equally serious, political problems. Preserving the concept of brain death as a social construct, as a "legal definition of death," but distinct from biological death, is also problematic, but may be our most acceptable alternative.

Brain Death↗

Physicians' attitudes about the care of patients in the persistent vegetative state: a national survey.

OBJECTIVE: To study the attitudes and beliefs of physicians who have experience caring for patients in the persistent vegetative state (PVS). DESIGN: Mailed questionnaire survey. PARTICIPANTS: 500 physicians, 250 from the American Academy of Neurology and 250 from the American Medical Directors Association. MEASUREMENTS: Physicians' beliefs about diagnosis of the PVS, patient awareness and suffering, treatment withdrawal, appropriate use of health maintenance and life-prolonging therapies, organ donation, lethal injection, and the treatment they would want if they were in the PVS. RESULTS: 68% of surveyed neurologists and 60% of medical directors responded. Thirteen percent of responders believe that patients in the PVS have awareness and experience hunger and thirst; 30% believe they experience pain. Fewer than 9% believe that respiratory failure, cardiogenic shock, acute renal failure, or cancer should be aggressively treated. Eighty-nine percent believe that it is ethical to withdraw artificial hydration and nutrition. Almost two thirds of responders believe that it would be ethical to use the vital organs of patients in the PVS for transplantation, and 20% believe that it would be ethical to hasten the patient's death by lethal injection. CONCLUSIONS: When evaluating the appropriateness of treatments for patients in the PVS, neurologists and medical directors largely concur. Most physicians in both groups believe that patients in the PVS would be better off dead; that it is not necessary to provide aggressive therapeutic interventions; and that all therapeutic interventions, including artificial nutrition and hydration, can be withheld in certain circumstances. The areas of consensus are remarkable and suggest that an ethical standard that physicians believe should be followed when caring for these patients may be emerging.

Acute Disease↗