Biomedical subjects
M M Coates
Publications and source records attributed to M M Coates.
Engineering human DNA alkyltransferases for gene therapy using random sequence mutagenesis.
O6-Alkylguanine-DNA alkyltransferase (AGT) is a suicide enzyme that repairs alkylation damage at the O6 position of guanine in DNA. The essentiality of a limited number of amino acid residues at the active site has been determined by site-directed mutagenesis. We used random mutagenesis techniques to create a plasmid library of > 10(6) human AGT mutants with substitutions at residues 150-172 and selected for clones with mutations rendering Escherichia coli resistant to both the alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the AGT inhibitor, O6-benzylguanine (BG). On sequencing surviving clones resistant to MNNG in the presence and absence of BG, we found that a majority of the clones contained multiple substitutions at mostly nonconserved positions. We selected nine mutants resistant to a combination of MNNG and BG, and the survival of these mutants was as much as 341-fold higher than that of cells harboring wild-type AGT under the same conditions. Each of the mutants contained at least three amino acid substitutions and as many as eight, suggesting that maximum resistance to MNNG in the presence of BG requires even more substitutions than resistance to MNNG alone. BG is being tested clinically as a way to sensitize tumors to chemotherapeutic alkylating agents. Therefore, our BG-resistant mutants hold strong potential as gene therapy candidates for protecting normal tissue in patients receiving BG in combination with alkylating agents for the treatment of cancer.
Creation of human alkyltransferases resistant to O6-benzylguanine.
O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents. Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression. One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment. We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene. A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli. Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG. One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E. coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.
Writing for publication: case reports.
A publishable case report in the field of lactation consulting informs readers about some new or unexpected facet of lactation (the production of milk) or breastfeeding (the act of infant feeding). It increases our understanding of this physiological process and improves our ability to resolve breastfeeding problems. The Introduction should state the topic and why it is important. The Description should vividly describe the essential elements needed to understand the case; the Summary should discuss evidence from one's own observations and from pertinent literature that defends one's own observations and from pertinent literature that defends one's conclusions, and it should state how this information will be useful clinically. Having decided on the intended readership and a journal to which to submit the manuscript, and searched the literature for similar cases, the investigator has enough perspective to begin preparing a case report.
Nipple pain related to vasospasm in the nipple?
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Breastfeeding during maternal or infant illness.
The continuation of breastfeeding during a hospital stay is not only possible, it can be beneficial for both the mother and the infant. The ill mother avoids breast engorgement and possible mastitis; the infant of an ill mother continues to receive, in breast milk, antibodies to the mother's illness. Through breast milk, an ill infant receives antibodies to his illness, liquids, and an easily digested, nourishing food. In addition, the physical and emotional comfort the mother and infant give each other eases the job of the nurse who cares for them. Hospital policies that take a woman's lactational status into account and encourage breastfeeding should be the accepted standard.
Hospitalized infants: one resource.
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Learning at the conference.
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Bottle-feeding like a breastfeeder: an option to consider.
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Assisting the newborn to latch on to the very large breast: help!
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