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Biomedical subjects

M Reed

Publications and source records attributed to M Reed.

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

FHIT gene expression in human ovarian, endometrial, and cervical cancer cell lines.

The fragile histidine triad (FHIT) gene, located at 3p14.2, has been shown to be altered in numerous epithelial cancers. Because previous studies have shown a loss of heterozygosity and cytogenetic abnormalities at the 3p region in ovarian, endometrial, and cervical carcinomas, we examined the status of the FHIT gene in 14 ovarian, 8 cervical, and 4 endometrial human cancer cell lines. RNA was isolated and subjected to reverse transcription-PCR to amplify the FHIT gene transcript. Sixty-three % (5 of 8) of cervical cell lines, 14% (2 of 14) of ovarian cell lines, and none (0 of 4) of the endometrial cell lines displayed aberrantly migrating FHIT transcripts. DNA sequencing demonstrated that the aberrantly migrating bands primarily lacked exons 5, 6, and 7 (with other exon losses also observed), resulting in shorter mRNA transcripts. Southern blot analysis of DNA from five of the cervical carcinomas demonstrated alterations in four of them, three of which had exhibited no normally sized FHIT transcripts. The results suggest that the expression of the FHIT gene may be altered in cervical tumor tissue, potentially implicating this gene in cervical tumorigenesis, whereas the involvement of this gene appears to be less important in the development of ovarian and endometrial cancer.

Acid Anhydride Hydrolases↗
PubMed1997

Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di-tert-butyl-4-methylphenol (BHT).

Several alkylphenols (e.g., 2,6-di-tert-butyl-4-methylphenol, BHT) form reactive quinone methide intermediates (e.g., 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity, we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine (BHT-NAC) was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand white rabbits. Rabbit serum contained an antibody which was highly specific for BHT-NAC, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHT-dependent protein alkylation over a wide molecular weight range. Prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74, and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, deuterated BHT, and the omission of NADPH. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT-QM as in the enzyme-mediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h postdosing and was dose-dependent. Two alkylated proteins were isolated and identified by N-terminal sequencing: a mitochondrial beta-oxidation enzyme, enoyl-CoA hydratase, and a plasma membrane/cytoskeletal linker protein from the ezrin/moesin/radixin family.

Alkylation↗
PubMed1997

Transcontinental shipping of pancreatic islets for autotransplantation after total pancreatectomy.

Islet autotransplantation prevents diabetes in some patients after total pancreatectomy. Pancreatectomy is done at most hospitals but islets are prepared at only a few centers. We report a case in which the pancreas was sent to a laboratory half a continent distant from the operative site, and islets were prepared and returned to the original hospital for autotransplantation 16 h after resection. At 10 months posttransplantation, the patient is normoglycemic and insulin independent, with an appropriate insulin secretion in response to glucose. Endocrine function can be retained after pancreatectomy even if the islets are isolated at a remote laboratory, and autotransplantation could be offered to patients without the need to travel. This outcome implies that the typical handling and processing of a pancreas destined to yield an islet allograft should not prevent the recovery of a sufficient number of viable beta cells to establish insulin independence in type 1 diabetic recipients.

Adult↗
PubMed1997

Reciprocal interference between the sequence-specific core and nonspecific C-terminal DNA binding domains of p53: implications for regulation.

The tumor suppressor p53 has two DNA binding domains: a central sequence-specific domain and a C-terminal sequence-independent domain. Here, we show that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain. Four previously described mechanisms for activation of specific DNA binding operate by blocking negative regulation. Deletion of the C terminus of p53 activates specific DNA binding only in the presence of large DNA. Three activator molecules (a small nucleic acid, a monoclonal antibody against the p53 C terminus, and a C-terminal peptide of p53) stimulate sequence-specific DNA binding only in the presence of both large DNA and p53 with an intact C terminus. Our findings argue that interactions of the C terminus of p53 with genomic DNA in vivo would prevent p53 binding to specific promoters and that cellular mechanisms to block C-terminal DNA binding would be required.

Animals↗
PubMed1997

Nurse-led general surgical pre-operative assessment clinic.

A nurse-led Pre-operative Assessment Clinic was introduced into the General Surgical Directorate at the Royal Hallamshire Hospital in August 1993 to provide a general medical and anaesthetic pre-operative assessment, to give information, both written and verbal, to patients about their operations and to identify social problems which might delay discharge. Patients were seen either at the time of their original clinic appointment or subsequently on a recall basis, prior to admission to hospital. The clinic was run by two out-patient nurses working to agreed protocols. Patients were given written information leaflets in addition to a verbal explanation about their admission and operation. In order to assess the effectiveness of the clinic, a group of patients attending during the first year were compared with similar patients who did not undergo pre-operative assessment. A questionnaire of satisfaction with information received was completed and compliance with the clinic protocols was recorded. In the pre-operative assessment group, patients were more satisfied with the amount of information received and there were significantly fewer cancellations due to unforseen medical problems in this group. Eighteen per cent of investigations were repeated unnecessarily because the reports were unavailable or considered out of date. However, only 1% of patients pre-operatively assessed had their operations cancelled after their admission, compared with 6% of non pre-operatively assessed patients. Given the total number of patients admitted for surgical procedures per year, the introduction of comprehensive pre-operative assessment for all patients could result in a substantial reduction in cancellations following admission.

Appointments and Schedules↗
PubMed1995

Inhibition of NAD(H)/NADP(H)--requiring enzymes by aurintricarboxylic acid.

Aurintricarboxylic acid (ATA), an inhibitor of Ca(2+)-dependent endonuclease activity, is often used to implicate a role for increased intracellular calcium in mechanistic toxicology studies. We report here on the ability of ATA to inhibit the activity of several NAD(H)/NADP(H)-requiring enzymes (purified or cellular homogenates), including lactic dehydrogenase, alcohol dehydrogenase, cytochrome c reductase, ethoxycoumarin o-dealkylase, isocitric dehydrogenase, glutathione reductase and glucose-6-phosphate dehydrogenase. These results were compared with the ability of ATA to inhibit micrococcal nuclease and rat liver Ca(2+)-dependent endonuclease activity in similar incubations. With the exception of alcohol dehydrogenase, ATA was a potent inhibitor of each of the purified enzymes, with IC50s ranging from 0.5 to 82 microM. In cell homogenates, however, ATA was from 10 to 100-fold less potent at inhibiting these enzymes. When exogenous protein was added to purified enzyme incubations, the effect of ATA was similarly diminished. Our results demonstrate that ATA inhibits a wide range of NAD(H)/NADP(H)-requiring enzymes in in vitro incubations using purified enzymes, but that the inhibitory effects are markedly reduced in incubations which more closely resemble a cellular milieu.

Animals↗
PubMed1995

The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation.

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.

Animals↗
PubMed1995

Serine phosphorylation in the NH2 terminus of p53 facilitates transactivation.

Murine tumor suppressor p53 is phosphorylated in the NH2-terminal transactivating domain at serines 9, 18, and 37. Change of any one of these serines to either alanine or aspartic acid did not alter p53 suppression of transformation of rat embryo fibroblasts by activated ras and E1A. Change of any two of these serines to alanines, however, led to a significant decrease in suppressor function. Substitution of alanines for all three serines caused the most severe loss of suppression and also reduced transactivation functions. The triple substitution had no apparent effects on intracellular accumulation or localization of p53, oligomerization, DNA binding, or interaction with the TFIID TATA-binding protein. In contrast, triple substitution of aspartic acid for serines 9, 18, and 37 had minimal effects on suppression and transactivation by p53. These results argue strongly that phosphorylation of serines 9, 18, and 37 facilitates the suppression and transactivation functions of p53.

Alanine↗
PubMed1995

Competing HMOs collaborate to improve preventive services.

BACKGROUND: In July 1993, an unusual collaboration developed between competing managed care plans and with competing primary care clinics as part of a federally funded research grant (IMPROVE from the Agency for Health Care Policy and Research). The goal of this collaboration is to scientifically test the ability of an health maintenance organization (HMO) to improve the delivery of eight adult preventive services by training and facilitating the use of continuous quality improvement and prevention systems by contracted private primary clinics. METHODOLOGY: In order to conduct this effectiveness study, it was necessary for two HMOs to come to a structural and functional understanding of how to operate jointly. Investigators recruited 44 private clinics for a randomized controlled trial in which 22 are being assisted in improving the process used to deliver these preventive services and 22 are being left alone as comparison clinics. The intervention is a train-the-trainer and consultation approach focused on clinics as collaborating customers. The comparison will be based on repeated surveys of patients and clinic personnel as well as chart audits to measure changes in systems and prevention rates. SUMMARY: Although this project was made possible by a number of unusual favorable factors, it can serve as a model for support of the clinician leadership that is essential to true health care delivery reform.

Adult↗
PubMed1995

p53-dependent activation of the mouse MCK gene promoter: identification of a novel p53-responsive sequence and evidence for cooperation between distinct p53 binding sites.

Transcriptional activation by p53 is dependent on the presence of a specific p53 binding site within control sequences of the target gene. One such target gene is the mouse muscle-specific creatine kinase (MCK) gene, which contains a p53 binding site between promoter residues -3182 and -3133 relative to the transcription start site. This DNA sequence is reported to be sufficient to confer p53-dependent activation on the MCK promoter. In contrast to this finding, evidence from promoter deletion studies suggests that sequences in the MCK promoter other than this p53 binding site also permit p53-dependent activation. To investigate this possibility, we have further examined sequences in the MCK promoter required for transcriptional activation by mouse p53. We report here identification of a second p53-responsive sequence within the MCK promoter. This novel sequence is situated between residues -177 and -81, and can confer p53-dependent, position- and orientation-independent activation on a heterologous promoter. Moreover, this sequence can specifically bind mouse and human p53. By promoter deletion studies, we provide evidence that these two elements cooperate to provide high-level, p53-dependent activation of the MCK promoter.

Animals↗
PubMed1995

Genetic transformation of nematodes using arrays of micromechanical piercing structures.

Foreign genes were expressed in the nematode Heterorhabditis bacteriophora using a new micro-mechanical device to inject DNA-coated microprobe through the nematode cuticle. After introduction of a plasmid in which a Caenorhabditis elegans 16-kDa heat shock promoter was fused to Escherichia coli, a beta-galactosidase gene was expressed in the progeny of the injected worms. The tip of the microprobe penetrated through the nematode cuticle without causing injury to the nematode, and about 8% of the total progeny tested expressed the foreign gene. We describe and validate a new and efficient genetic transformation system for nematodes. The method is quick and easy to use.

Animals↗
PubMed1994

p53 oligomerization and DNA looping are linked with transcriptional activation.

We examined the role of p53 oligomerization in DNA binding and in transactivation. By conventional electron microscopy (EM) and scanning transmission EM, we find that wild-type tetramers contact 18-20 bp at single or tandem 19 bp consensus sequences and also stack in apparent register, tetramer on top of tetramer. Stacked tetramers link separated DNA binding sites with DNA loops. Interestingly, the p53(1-320) segment, which lacks the C-terminal tetramerization domain, binds DNA consensus sites as stacked oligomers. Although the truncated protein binds DNA with reduced efficiency, it nevertheless induces DNA looping by self-association. p53, therefore, has a C-terminal tetramerization domain that enhances DNA binding and a non-tetrameric oligomerization domain that stacks p53 at consensus sites and loops separated consensus sites via protein-protein interactions. Using model promoters, we demonstrate that wild-type and tetramerization-deficient p53s activate transcription well when tandem consensus sites are proximal to TATA sequences and poorly when tandem sites are distal. In the presence of proximal sites, however, stimulation by distal sites increases 25-fold. Tetramerization and stacking of tetramers, therefore, provide dual mechanisms to augment the number of p53 molecules available for activation through p53 response elements. DNA looping between separated response elements further increases the concentration of local p53 by translocating distally bound protein to the promoter.

Animals↗
PubMed1994

p53 domains: structure, oligomerization, and transformation.

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

Animals↗
PubMed1993

p53 domains: identification and characterization of two autonomous DNA-binding regions.

We have investigated the DNA-binding, oligomerization, and trans-activation functions of isolated segments of murine p53. We find that p53 has two autonomous DNA-binding regions. One domain, from amino acid 280 to 390, forms stable tetramers and binds DNA nonspecifically. The biological significance, if any, of this DNA-binding activity is not known. A second domain, from amino acid 80 to 290, does not form stable tetramers under stringent conditions but binds DNA both specifically and nonspecifically. The specific DNA-binding function of p53, therefore, resides in the highly conserved central region of the protein and does not require stable tetramerization. Amino acids 1-290, which include both the specific DNA-binding domain and the amino-terminal acidic region, activate a p53-specific promoter in vivo. This finding strongly argues that the DNA-binding activity of p53 segment 80-290 is physiologically significant. The role of tetramerization in p53 function remains to be determined.

Amino Acid Sequence↗
PubMed1993

Multiple hereditary exostoses. An epidemiologic study of an isolated community in Manitoba.

From 1986 through 1988, 266 persons (149 adults and 117 children) were screened for multiple hereditary exostosis (MHE) in an isolated northern Ojibway community. Physical examination and confirmation by roentgenogram skeletal survey disclosed 21 children (19.4%) and 14 adults (9.5%) affected with MHE. Forty-one percent of children had lesions detectable before ten years of age, some as early as two years of age. Seventy-four percent of the lesions were characteristically sessile. Although lesions about the knee were most common, sites previously thought to be uncommon such as the metatarsals, hand, and spine were involved in 40% of the children. No cases of malignant degeneration have occurred in the adult population. Severity and multiplicity of lesions in successive generations point to an oncogenic gene origin. This study shows striking variance from current literature and provides a unique and valuable baseline assessment of research on the cause and natural history of MHE.

Adolescent↗
PubMed1993

p53 domains: suppression, transformation, and transactivation.

We investigated the suppression, transformation, and transactivation functions of isolated segments of wild-type murine p53. Intact p53, but no segment of p53, inhibited cellular transformation by the activated ras and adenovirus E1A proteins. We conclude that most of p53 is needed for suppression of cellular proliferation. Nevertheless, the transactivating domain of herpesvirus protein VP16 was able to substitute for the N-terminal transactivating domain of p53 in cellular suppression. Thus, unless the interchanged p53 and VP16 acidic segments share additional functions, transactivation is required for suppression by p53. Interestingly, we found that all p53 segments containing amino acids 320-360 enhanced transformation by ras and E1A. This region has been associated with the oligomerization of p53 (Milner et al., 1991; Sturzbecher et al., 1992). Furthermore, no p53 segment lacking amino acids 320-360 transformed cells. Amino acids 320-360, therefore, may account for the major transforming activity of p53. Intact p53 and chimeric VP16-p53 transactivated the CAT gene under control of a p53-specific promoter, while transforming segments of p53 interfered with transactivation by wild-type p53. Our findings argue that transactivation by p53 is required for cellular suppression and that any nontransactivating p53 that retains the capacity to oligomerize with wild-type p53 would have transformation potential.

Amino Acid Sequence↗