Search PubMed⌕ Search

Biomedical subjects

C A Myers

Publications and source records attributed to C A Myers.

At least 19 recordsLinked to original sources
PubMed2001

Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron's catalytic core. Previous studies suggested a model in which the protein binds first to the intron's P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr).

Base Sequence↗
PubMed1998

Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation.

We have previously described a 160-bp enhancer (BCE-1) in the bovine beta-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-beta and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-beta but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.

Acetylation↗
PubMed1996

A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.

Bacteriophage T4↗
PubMed1995

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments.

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

Animals↗
PubMed1992

Gene transfer in primary cultures of human hepatocytes.

Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli beta-galactosidase gene (beta-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-1 antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.

Avian Sarcoma Viruses↗
PubMed1992

A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression.

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.

Animals↗
PubMed1991

Paradoxical effects of blood alcohol concentration charts.

BACKGROUND: This study was designed to determine the association between alcohol drinking and the possession of blood alcohol concentration charts. METHOD: A total of 30 college-age subjects participated in the study. Subjects were randomly assigned to either an experimental or a control group. All subjects answered a short entrance questionnaire to determine their height and weight. Those assigned to the experimental group were supplied a copy of a blood alcohol concentration chart and instructed in its use. The volume of alcoholic beverages consumed was surreptitiously counted for all subjects. Prior to leaving the premises the subjects completed an exit questionnaire which asked them to estimate the amount of alcohol they had consumed, whether they had driven to the pub, and whether they intended to drive away. The time spent in the pub was noted for each subject. RESULTS: Among those in the control group there was a tendency to overestimate the volume consumed, and for those in the experimental group, a tendency to more accurately estimate their consumption. Those given blood alcohol concentration charts consumed alcohol at a significantly higher rate than did those in the control group. CONCLUSION: A likely explanation for this outcome is that the chart served as a stimulus to prompt a drinker to more quickly achieve a blood alcohol level consistent with his/her drinking expectancies.

Adult↗
PubMed1990

Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

Animals↗
PubMed1989

Determination of peak trabecular bone density: interplay of dietary fiber, carbohydrate, and androgens.

To elucidate mechanisms linking nutrition and sex hormones to magnitude of peak trabecular bone density, we studied 11 normal women aged 19-21 y. Trabecular bone density was related inversely to dietary fiber (r = -0.69, p = 0.02) and carbohydrate (r = -0.70, p = 0.02) and directly to serum free-and-albumin-bound testosterone (fab T) (r = -0.70, p = 0.02) and total testosterone (total T) (r = 0.66, p = 0.03). Dietary fiber was correlated negatively with fab T (r = -0.74, p = 0.009), total T (r = -0.70, p = 0.02), and androstenedione (Adione) (r = -0.72, p = 0.01). Controlling for the effect of fab T or Adione weakened the relationship between dietary fiber and bone density and the relationship was no longer statistically significant. Conversely, controlling for sex hormones did not abolish the effect of carbohydrate on bone density. The contributions of fab T and carbohydrate to bone density were independent. These results suggest that dietary fiber may depress serum androgens which in turn decrease trabecular bone density. Carbohydrate may also depress bone density but independently of sex steroid hormones.

Adult↗
PubMed1988

Effect of declining renal function on bone density in aging women.

The factors that are responsible for trabecular bone loss in aging women are not completely understood. To evaluate declining renal function as a possible factor, we studied 19 Caucasian women (average age 67) who were from 6 to 41 years postmenopausal. Trabecular bone density was quantitated by computerized tomography of the spine. Serum calcium, phosphorus, and creatinine were normal in all subjects. Creatinine clearance averaged 74 ml/min (range 38-122), decreased with age (r = -0.60, P = 0.003), and was inversely related to serum creatinine (r = -0.51, P = 0.01). Bivariate regression demonstrated that bone density decreased with age (r = -0.59, P = 0.004); controlling for the effect of creatinine clearance weakened this correlation to r = -0.45 (P = 0.03); controlling additionally for 1,25-dihydroxyvitamin D [1,25(OH)2D] and parathyroid hormone (PTH) reduced the correlation coefficient to r = -0.34 (P = 0.11). Bone density also decreased in direct proportion to the decrement in creatinine clearance (r = 0.44, P = 0.03); controlling for the effects of 1,25(OH)2D and PTH reduced this correlation coefficient to r = 0.34 (P = 0.11). These results suggest that occult renal insufficiency may contribute to bone loss in aging women, and that this effect may be mediated in part by 1,25(OH)2D and PTH. In this age group renal function should be assessed by measuring creatinine clearance rather than the serum creatinine concentration since renal insufficiency can be masked by apparently normal circulating creatinine levels.

Aged↗
PubMed1988

Determinants of atraumatic vertebral fracture rates in menopausal women: biologic v mechanical factors.

In menopausal women, the susceptibility to atraumatic vertebral fractures is thought to be governed by both mechanical factors (as represented by bone density) and by biologic factors such as age, body size, and dietary calcium. Whether these biologic factors independently influence fracture rates beyond the effect of bone density is a matter of controversy. To compare the relative importance of mechanical and biologic factors on vertebral fractures, we elucidated the determinants of atraumatic compression fractures in 63 menopausal women who had no chronic diseases other than osteopenia. Trabecular bone density was determined by quantitative computerized tomography of the spine. Fracture frequency was expressed as the number of compressed vertebrae per person between T5 and L4. The analysis showed that fracture frequency, bone density, and the biologic factors were closely interrelated. Fracture frequency was inversely correlated to bone density [R2 (spline model) = .40, P less than .0001], body size (r = -.26, P = .05), and dietary calcium (r = -.28, P = .04), and directly correlated with age (r = .46, P = .0002). Bone density decreased with age (r = -.65, P less than .0001), increased with body size (r = .37, P = .004), and tended to increase with dietary calcium (r = .24, P = .08). After controlling for bone density, there was no perceptible residual relationship between fracture frequency and any of these biologic factors. We conclude that mechanical factors overshadow age, body size, and dietary calcium as determinants of vertebral fracture frequency. The apparent influence of these biologic factors on fracture frequency is explained by their surrogate effects on bone density.

Aged↗
PubMed1988

In vitro evaluation of the cytotoxic potential of a novel man-made fiber, calcium sodium metaphosphate fiber (Phosphate Fiber).

As part of a comprehensive effort to evaluate the toxicological potential of calcium sodium metaphosphate fiber (Phosphate Fiber), the in vitro cytotoxicity of the fiber in cultured cells was studied. Two pulmonary-derived cell systems (rat alveolar macrophages, RAM; rat lung epithelial cells, LEC) and an established cell line (Chinese hamster ovary, CHO) were used. Release of lactate dehydrogenase (LDH) was used as an endpoint for cytotoxicity for all three cell types. In addition, inhibition of colony formation was used for CHO cells. The cytotoxicity of Phosphate Fiber was compared to a variety of mineral dusts and fibers including chrysotile asbestos, crocidolite asbestos, two glass fibers, calcium sulfate fiber, titanium dioxide, as well as the nonfibrous raw material, calcium sodium metaphosphate glass. Results with all three cell culture systems demonstrated that the Phosphate Fiber was less cytotoxic than the two asbestos fibers, similar in cytotoxicity to the glass fibers, and more cytotoxic than the calcium sulfate fiber and titanium dioxide. To further investigate the cytotoxicity of the Phosphate Fiber, it was fractionated by sedimentation into small and large fibers. The small Phosphate Fiber was found to be more cytotoxic and the large Phosphate Fiber to be less cytotoxic than the unfractionated Phosphate Fiber. The in vitro data suggest that Phosphate Fiber is less cytotoxic than asbestos, but further determination of safety can only be made after the in vivo data have been obtained.

Animals↗
PubMed1988

A comparison of the risk of vertebral fracture in menopausal osteopenia and other metabolic disturbances.

The risk of atraumatic compression fracture in postmenopausal women increases as vertebral trabecular bone density decreases. To determine whether the risk is similar for patients who have other metabolic disorders, we compared eight-nine patients who had various disturbances affecting bone and sixty-three postmenopausal women who had no evidence of underlying disease. Trabecular bone density was measured by quantitative computed tomography of the lumbar spine. The relationship between frequency of fracture and bone density was modeled mathematically with spline threshold, quadratic polynomial, and decaying exponential functions. Analysis of covariance showed that the diagnostic category did not influence the relationship between frequency of fracture and bone density in any of the three models. We concluded that the risk of atraumatic compression fracture, as assessed by measurement of vertebral trabecular bone density using quantitative computerized tomography, is independent of the underlying metabolic disturbance.

Adult↗
PubMed1983

Subcellular location of a soluble factor that stimulates DNA replication in permeable animal cells.

The subcellular location of a soluble DNA replication-stimulating factor released from lysolecithin-permeabilized cultured mammalian cells was investigated by cytochalasin B (CB) enucleation. In both Chinese hamster ovary cells and baby hamster kidney (BHK) cells, approx. 70% of the total activity of the replication-stimulating factor was associated with the karyoplast fraction. In neither cell line did the replication-stimulating factor show any significant change in activity or in subcellular location between G1 and S period synchronized cells. The general nature and mechanism of action of this stimulatory factor were characterized in order to show that it was similar to "cytoplasmic" factors reported to stimulate DNA replication in other subcellular systems. The predominantly nuclear location of the replication-stimulating factor supports a physiological role in DNA metabolism in animal cells.

Animals↗
PubMed1983

Identification of mammalian DNA repair factors using a reconstituted subcellular system. Partial characterization and subcellular location of a DNA repair-stimulating protein in hamster cells.

By reconstituting lysolecithin-permeabilized hamster cells with endogenous proteins, a protein(s) which stimulated bleomycin-induced DNA repair synthesis was identified. The repair protein was inactivated by proteinase K and had an apparent molecular weight of 12 000-15 000 D. The following enzymatic activities were not detected in the partially purified DNA repair protein: general endonuclease, apurinic endonuclease, exonuclease, DNA polymerase or DNA polymerase beta-stimulating activity. The subcellular location of the DNA repair-stimulating activity was investigated by cytochalasin B enucleation; approx. 80% of the activity was associated with karyoplasts, suggesting a nuclear location. Neither the activity nor subcellular location of the repair protein fluctuated appreciably during the cell cycle, consistent with a physiological role in DNA repair. Although the function of the DNA repair protein is not yet known, this approach should be useful in identifying and characterizing mammalian DNA repair proteins.

Animals↗