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Biomedical subjects

M Reed

Publications and source records attributed to M Reed.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1991

Obesity and polycystic ovary syndrome.

Our studies show that obese women with polycystic ovary syndrome are more likely to have hirsutism and menstrual disturbances than are lean women with PCOS. The most obvious biochemical differences between obese and lean women with PCOS is that SHBG concentrations are much lower in women with obesity. The SHBG levels are inversely related to insulin, and insulin has been shown to have a direct inhibitory action on SHBG secretion. Other factors, however, may contribute to the mechanism of the increased prevalence of hirsutism and anovulation in obese women with PCOS, such as a direct effect of insulin or increased activity of 5 alpha-reductase in peripheral tissues. Finally we have been able to show that weight reduction of more than 5% is associated with an improved biochemical profile and, importantly, with restoration of fertility.

Endocrine Glands↗
PubMed1991

Expression of rat hepatic lipase in heterologous systems: evidence for different sites for interface binding and catalysis.

Rat hepatic triglyceride lipase was expressed as a bacterial fusion protein and as a secreted protein in eukaryotic cells. The bacterial fusion construct coded for seven amino acids at the N-terminus which are not present in the hepatic lipase cDNA, but otherwise consisted of only the complete mature lipase sequence. Fusion protein was isolated as an insoluble product which did not have lipase or phospholipase activities; it was, however, active as an esterase when solubilized after preparative gel electrophoresis. The fusion protein was used to raise polyclonal antibodies that recognize native rat hepatic lipase and inhibit its activity. For eukaryotic expression, a full-length rat hepatic lipase cDNA clone was inserted into the metallothionein promoter expression vector pMTSV40polyA.Bam. Transfected CHO cells, induced with ZnSO4, secreted an immunoreactive protein of Mr approximately 57,000. A lipase-producing clonal cell line was isolated and used to characterize the enzyme. The protein was purified from serum-free medium by heparin-Sepharose and DEAE-Trisacryl M column chromatography. It was apparently identical to native rat hepatic lipase, with the exception of the conformation of the linkage of the sialic acids which form part of the N-linked carbohydrate complexes. The bacterial fusion protein, the CHO-produced lipase, and native rat hepatic lipase were all inhibited by phenylmethylsulfonyl fluoride, implying that they function catalytically as serine esterases. Substrate competition studies indicated that the esterase and lipase activities use the same active site; thus, the major defect in the fusion protein was probably in triglyceride substrate binding. These results suggest that interface binding and catalysis occur at different sites in the protein.

Animals↗
PubMed1990

The effect of hypoxia on angiotensin-stimulated release of PGI2 from the splanchnic bed.

The effect of hypoxia on splanchnic angiotensin II (AII)-stimulated prostaglandin (PG) release was examined in male rats. The superior mesenteric artery was cannulated and perfused in vitro with physiologic buffer at a pO2 of 460 or 60 torr. Splanchnic vessels were perfused isolated (SV) or in continuity with the small intestine (SV + SI). AII (10-300 ng) was infused as bolus injections. Quantitative analysis of SV + SI effluent by bioassay indicated the predominant PG released was PGI2. Quantitative analysis by radioimmunoassay confirmed PGI2 as the major PG released from the SV and SV + SI following AII stimulation. Relative hypoxia significantly decreased AII-stimulated release of PGI2 from the SV, and only modestly from the SV + SI. These data demonstrate two splanchnic sources of AII-stimulated vasodilator PG synthesis, the SV and SV + SI, with the SV more sensitive to relative hypoxia. Diminished release of splanchnic vascular (SV) vasodilator PGI2 may be of importance in AII-mediated splanchnic vasoconstriction seen in hypoxia and shock.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

A simple protocol for the screening and preliminary identification of antibodies to human IgA using unpurified proteins in the passive haemagglutination test.

Experiments have shown that suitably diluted, unpurified IgA paraprotein sera can be substituted for chemically purified IgA proteins in the passive haemagglutination test for the detection of anti-IgA antibodies. The substitution of unpurified IgA paraprotein was made possible by the enhanced serological activity obtained by pre-heating the unpurified immunoglobulin preparations at 56 degrees C for 60 min. Using a small screening panel of suitably coated tanned red cells, various population groups were investigated for the presence of anti-IgA antibodies. Results were similar to those obtained by other workers using purified IgA preparations in the passive haemagglutination test.

Antibodies, Anti-Idiotypic↗
PubMed1990

From food basket to food security. The food factor in nutritional surveillance.

One important indicator of nutritional surveillance is the one devoted to monitor food security. The experience toward the development of one of such indicators is presented. This includes the development of a food basket, defined as the group of foods that meet the characteristics such as is now consumed by important population segments of the community; it contributes a substantial portion of the calories and proteins purchased, and is responsible for an important proportion of the food budget. The concept implies a dynamic food basket, the quantities of which are calculated in a way that simulates the behavior of the consumer and the best nutrition knowledge. For this purpose we use linear program techniques. A measure of the risk of being unable to buy the foods needed for a family is presented, and is used as a proxy for food security risk. In the appendix, the mathematical expressions of the model used for a linear program is also presented.

Colombia↗
PubMed1989

Effect of cytoskeletal geometry on intracellular diffusion.

A method is presented for determining the retardation of diffusion of particles inside cells owing to cytoskeletal barriers. The cytoskeletal meshwork is treated as a repeating periodic two-dimensional or three-dimensional lattice composed of elements of given size, shape, and spacing. We derive an analytic expression for the diffusion coefficient relative to that of the cytosol. This expression is evaluated by solving numerically an appropriate boundary-value problem for the Laplace equation. For the two-dimensional case, e.g., diffusion in a membrane, the results are quantitatively similar to those obtained by Saxton (1987. Biophys. J. 52:989-997) using Monte Carlo methods. The three-dimensional results are quantitatively similar to experimental results reported by Luby-Phelps et al. (1987. Proc. Natl. Acad. Sci. USA. 84:4910-4913) for the diffusion of dextran and Ficoll particles in Swiss 3T3 cells. By accounting for geometrical factors, these results allow one to assess the relative contributions of geometrical hindrance and of binding to the cytoskeletal lattice from measurements of intracellular diffusion coefficients of proteins.

Cytoskeleton↗
PubMed1989

Intubation with low-dose atracurium in children.

The objective of this study was to compare intubating conditions and neuromuscular effects using smaller doses of atracurium (0.25 mg/kg and 0.3 mg/kg) with the recommended dose of 0.4 mg/kg for intubation in children anesthetized with halothane, N2O and oxygen undergoing strabismus repair. All patients (10 in each group) had good or excellent intubating conditions at 80% depression of twitch height [T1 of train-of-four (TOF) stimulation]. Mean times to intubation were 2.6 +/- 0.2 minutes following 0.25 mg/kg and 2.2 +/- 0.2 minutes following 0.3 mg/kg. These times were significantly longer (P less than 0.05) than the mean intubation time of 1.5 +/- 0.2 minutes following 0.4 mg/kg. Mean times to recovery, defined as times from injection of atracurium to return of T1 of TOF to 10%, 25%, and 95% of control measurements, were significantly shorter with the smaller doses. Atracurium at these low doses may provide an alternative to succinylcholine for intubating children during halothane anesthesia for surgical procedures lasting 20-30 min.

Anesthesia↗