"It's time to make a good agency better": the Food and Drug Administration Modernization Act of 1997 and the First Amendment.
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Civil Rights↗
Biomedical subjects
A M Murphy
Publications and source records attributed to A M Murphy.
Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4.
Troponin I is a thin-filament contractile protein expressed in striated muscle. There are three known troponin I genes which are expressed in a muscle-fibre-type-specific manner in mature animals. Although the slow skeletal troponin I isoform is expressed in fetal and neonatal heart, the cardiac isoform is restricted in its expression to the myocardium at all developmental stages. To study the regulation of this cardiac-specific and developmentally regulated gene in vitro, the rat cardiac troponin I gene was cloned. Transient transfection assays were performed with troponin I-luciferase fusion plasmids to characterize the regulatory regions of the gene. Proximal regions of the upstream sequence were sufficient to support high levels of expression of the reporter gene in cardiocytes and relatively low levels in other cell types. The highest luciferase activity in the cardiocytes was noted with a plasmid that included the region spanning -896 to +45 of the troponin I genomic sequence. Co-transfection of GATA-4, a recently identified cardiac transcription factor, with troponin I-luciferase constructs permitted high levels of luciferase expression in non-cardiac cells. Electrophoretic mobility-shift assays demonstrated specific binding of GATA-4 to oligonucleotides representative of multiple sites of the troponin I sequence. Mutation of a proximal GATA-4 DNA-binding site decreased transcriptional activation in transfected cardiocytes. These results indicate that the proximal cardiac troponin I sequence is sufficient to support high levels of cardiac-specific gene expression and that the GATA-4 transcription factor regulates troponin I-luciferase expression in vitro.
3T3 Cells↗
Role of troponin I proteolysis in the pathogenesis of stunned myocardium.
Myocardial stunning is characterized by decreased myofilament Ca2+ responsiveness. To investigate the molecular basis of stunned myocardium, we performed PAGE and Western immunoblot analysis of the contractile proteins. Isolated rat hearts were retrogradely perfused at 37 degrees C for either 50 minutes (control group) or for 10 minutes, followed by 20-minute global ischemia and 20-minute reperfusion (stunned group), or for 20-minute ischemia without reflow. Another group consisted of hearts subjected to 20-minute ischemia in which stunning was mitigated by 10-minute reperfusion with low Ca2+/low pH solution. Myocardial tissue samples subjected to PAGE revealed no obvious differences among groups. Western immunoblots for actin, tropomyosin, troponin C, troponin T, myosin light chain-1, and myosin light chain-2 showed highly selective recognition of the appropriate full-length molecular weight bands in all groups. Troponin I (TnI) Western blots revealed an additional band (approximately 26 kD, compared with 32 kD for the full-length protein) in stunned myocardial samples only. In parallel experiments, skinned trabeculae were treated with calpain I for 20 minutes; Western blots showed a TnI degradation pattern similar to that observed in stunned myocardium. Such TnI degradation was prevented by calpastatin, a naturally occurring calpain inhibitor. The results show that (1) TnI is partially and selectively degraded in stunned myocardium; (2) this degradation could be prevented by low Ca2+/low pH reperfusion, which also prevented the contractile dysfunction of stunning; and (3) calpain I could similarly degrade TnI, supporting the idea that Ca(2+)-dependent myofilament proteolysis underlies myocardial stunning.
Animals↗
Embolic stroke after ligation of the pulmonary artery in patients with functional single ventricle.
In the setting of functional single ventricle with pulmonary overcirculation, pulmonary artery banding is frequently used to alleviate symptoms and to prepare for staged repair. At subsequent cavopulmonary anastomosis or Fontan procedure, the pulmonary artery may be ligated at the site of the pulmonary band. This article describes the association of embolic stroke and thrombus in a ligated or divided pulmonary artery stump in three patients with functional single ventricle. These events occurred from 1990 through 1992 among the 1700 inpatient pediatric cardiology admissions at two institutions. The patients, ranging in age from 15 months to 9 years, had cerebral infarctions documented by computed axial tomography scan or magnetic resonance imaging associated with the echocardiographic finding of thrombus in the proximal pulmonary artery stump after the embolic strokes. The strokes occurred 5 days to 5 years after surgery. Two patients had a second infarction within 2 to 5 weeks of the initial stroke. It is concluded that the presence of the ligated pulmonary artery stump may place patients at risk for embolic stroke. Surgical approaches to reduce the risk of thrombus formation should be considered prospectively in this patient group.
Cerebral Infarction↗
Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.
The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis.
Actins↗
The effects of antecedent color on reading for students with learning disabilities and co-occurring attention-deficit/ hyperactivity disorder.
The effects of color on the reading recognition and comprehension of 3 students with learning disabilities and attention-deficit/hyperactivity disorder were assessed in a single-subject design. Color did not enhance sight-word learning; for longer reading comprehension tasks, color had an immediate effect across and within sessions.
Attention Deficit Disorder with Hyperactivity↗
Antibody response and reactions to completion of a four-dose series with a two- or three-component acellular pertussis vaccine compared to whole cell pertussis vaccine.
We compared the reactions and immunogenicity of DT acellular pertussis (DTaP) vaccines containing pertussis toxoid (PT) and filamentous haemagglutinin (FHA) (2-component DTaP) or PT, FHA and pertactin (PRN) (3-component DTaP vaccine) with a whole cell (DTwP) vaccine as a fourth-dose booster in 158 children (15-20 months old) who had received 3 primary vaccine doses with the same vaccines at 2, 4 and 6 months of age. Randomization was 3:1 for DTaP:DTwP and all children received concomitant oral polio vaccine (OPV). Fever (> 38 degrees C), irritability, local injection site erythema (> 10 mm), swelling (> 10 mm), and pain (moderate or more) were assessed for 72 h after booster vaccination. DTwP vaccinees had a higher incidence of fever (29.4%) and injection-site pain (45.7%) than 3-component DTaP vaccinees (fever, 9.6%, p < 0.02; injection-site pain, 3.8%, p < 0.01); 2-component DTaP vaccinees had less injection-site pain (8.3%, p < 0.01). Pre- and post-vaccination immunoglobulin G (IgG) antibody was measured by enzyme-linked immunosorbent assay (ELISA). Pre- and post anti-PT levels were similar for all 3 vaccine groups. Anti-FHA antibody was higher pre- and post-vaccination for both DTaP vaccine groups compared with the DTwP vaccinees (p < 0.01 for all comparisons). For 3-component DTaP vaccinees, anti-PRN antibody was higher pre- and post-vaccination compared to DTwP vaccinees (p < 0.01 for both comparisons). Tetanus antibody was higher pre- and post-vaccination for DTwP versus both DTaP vaccine groups, and diphtheria antibody was similar pre- and post-vaccination for all 3 groups. These 2- and 3-component DTaP vaccines produce less common reactions and comparable or higher antibody to the components they contain (except tetanus) than DTwP vaccine when given as a booster to 15- to 20-month-old children previously primed with the same vaccine.
Adhesins, Bacterial↗
Contractile protein phenotypic variation during development.
A diverse set of contractile protein genes and alternatively spliced isoforms is expressed during the process of cardiac maturation. A large variety of functional sarcomeric units may occur at various stages of development, in different regions of the heart and in pathologic states. In this review, the developmental program of individual contractile protein gene families is discussed with an emphasis on the functional effects of maturational changes, particularly in the human myocardium.
Alternative Splicing↗
Modifier of rudimentary p1, mod(r)p1, a trans-acting regulatory mutation of rudimentary.
Modifier of rudimentaryp1 (mod(r)p1) is a hybrid dysgenesis-induced mutation isolated as a suppressor of rhd1, a hypomorphic rudimentary(r) allele in Drosophila melanogaster.mod(r)p1 has opposite effects on two of the rudimentary mutant phenotypes. It suppresses the wing truncation associated with hypomorphic r alleles, which was the phenotype used to isolate it. On the other hand, it does not suppress the sterility of r females and in fact decreases the fertility of wild-type females. This infertility is associated with a drastic decrease in r expression in mod(r)p1 females. P elementagging was used to clone the mutant allele, mod(r)p1. Subsequently, 28 kb of genomic DNA encompassing the wild-type mod(r) gene in the chromosomal region 1B was cloned. mod(r) encodes a 1.3 kb transcript which is not detected in the mod(r)p1 mutant. The sequences of mod(r) cDNA clones reveal that the gene encodes a protein of 200 amino acids in length. When compared to sequences in GenBank, the amino acid sequence did not reveal any long sequences similarities. However, the structure of the protein reveals similarities to known transcription factors. The N-terminal half of the protein is very acidic, whereas the C-terminal half is basic. The basic domain suggests a possible DNA-binding domain, while the acidic domain suggests a transcriptional activation domain. Consistent with this possibility is the fact that mod(r) acts through the 5' control region of the rudimentary gene to control its expression.
Amino Acid Sequence↗
The breathless FGF receptor homolog, a downstream target of Drosophila C/EBP in the developmental control of cell migration.
To investigate the molecular mechanisms responsible for the temporal and spatial control of cell movements during development, we have been studying the migration of a small group of follicle cells, called the border cells, in the Drosophila ovary. Timely initiation of border cell migration requires the product of the slow border cells (slbo) locus, which encodes the Drosophila homolog of the transcription factor C/EBP. Here we report evidence that one target of C/EBP in the control of border cell migration is the FGF receptor homolog encoded by the breathless (btl) locus. btl expression in the ovary was border cell-specific, beginning just prior to the migration, and this expression was reduced in slbo mutants. btl mutations dominantly enhanced the border cell migration defects found in weak slbo alleles. Furthermore, C/EBP-independent btl expression was able to rescue the migration defects of hypomorphic slbo alleles. Purified Drosophila C/EBP bound eight sites in the btl 5' flanking region by DNAse I footprinting. Taken together these results suggest that btl is a key, direct target for C/EBP in the regulation of border cell migration.
Animals↗
Nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase (DT-diaphorase) as a target for bioreductive antitumor quinones: quinone cytotoxicity and selectivity in human lung and breast cancer cell lines.
Bioreductive antitumor quinones require reductive metabolism to produce their cytotoxic effects. A series of these compounds was screened for relative rates of reduction by the two-electron reductase, NAD(P)H:quinone oxidoreductase (DTD). The antitumor quinones streptonigrin (SN), 2,5-diaziridinyl-3-phenyl-1,4-benzoquinone (PDZQ), 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinine (MeDZQ), and [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-propen ol] (EO9) were all excellent substrates for recombinant rat and human DTD. All four compounds were reduced by DTD at least 100 times faster than the clinically important bioreductive alkylating agent, mitomycin C (MC). Reduction of the antitumor quinones was generally 4-5 times more efficient by rat DTD than by human DTD. The exception was EO9, which, surprisingly, was reduced 23 times faster by rat DTD than by human DTD. The rate of reduction of each individual quinone was similar under either aerobic or anaerobic conditions, suggesting that DTD may be an important activating enzyme in the hypoxic fraction of solid tumors. The cytotoxicity of MeDZQ and MC was examined in a panel of human breast and lung cancer cell lines. The data showed good correlations between DTD activity and toxicity for both MeDZQ (r = 0.57, p = 0.054) and MC (r = 0.69, p = 0.020), confirming biochemical data that both compounds are bioactivated by DTD. In addition, IC50 values were in general lower for MeDZQ than for MC in cell lines containing elevated DTD, a finding that was consistent with metabolic data that indicated that MeDZQ was a better substrate for DTD than MC. SR, defined as the ratio of the IC50 value for the H596 NSCLC cell line (undetectable DTD activity) to the IC50 value for the H460 NSCLC cell line (high DTD activity), were determined for all five antitumor quinones. SN was the most selective (SR = 86) followed by EO9 (SR = 62), MeDZQ (SR = 17), and MC (SR = 11). Surprisingly, PDZQ, an excellent substrate for DTD, was toxic to both cell lines (SR = 1.8). These data suggest that antitumor quionones that are substrates for DTD may be selectively toxic to tumors with high DTD activity and may be useful in the treatment of those tumors.
Animals↗
Genetic evidence for a fourth terminal oxidase in Bradyrhizobium japonicum.
Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium, has a complex respiratory electron-transport chain, capable of functioning throughout a wide range of oxygen tensions. It does so by synthesizing a number of terminal oxidases, each appropriate for different environmental conditions. Several genes encoding terminal oxidases from B. japonicum have been cloned, but it is unknown what roles these individual oxidases play. In this paper, we describe the cloning and sequencing of the coxX gene encoding the large catalytic subunit for a fourth terminal oxidase from B. japonicum. The coxX gene encodes a 666-amino-acid (aa) protein (M(r) 74,527) that exhibits a high degree of homology to terminal oxidase proteins from a number of prokaryotic and eukaryotic species. This new oxidase exhibits greater homology to the Escherichia coli cytochrome o subunit I than any of the previously reported B. japonicum terminal oxidase genes.
Adaptation, Biological↗
Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation.
Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.
Actin Cytoskeleton↗
Differential regulation of slow-skeletal and cardiac troponin I mRNA during development and by thyroid hormone in rat heart.
We have examined mRNA levels for the cardiac troponin I (cTnI) and slow-skeletal (ssTnI) in perinatal rat hearts. Northern blots showed that hypothyroidism was associated with a delay in the expected isoform switching. RNA slot blots showed a six-fold increase in cTnI mRNA from day 3 to day 21 in hearts from postnatal euthyroid rats compared to a three-fold increase in cTnI for the same period in the hypothyroid hearts. On the other hand, ssTnI mRNA levels were higher after 3 days in hearts from the hypothyroid animals and fell to undetectable levels after 21 days. In euthyroid hearts ssTnI was not detectable after 14 days and was not re-expressed in adult hearts. In the ventricles from 28-day-old animals the most significant differences in cTnI mRNA levels were between the euthyroid and T3-treated hypothyroid preparations and in the 87 to 125-day-old group between euthyroid and hypothyroid ventricles. T3 treatment of the 87 to 125-day-old hypothyroid animals did not increase the cTnI mRNA above euthyroid levels despite elevated serum T3. These results show that thyroid hormone influences expression of the cTnI isoform in postnatal and young adult rats, but not to the same extent in animals greater than 28 days of age.
Animals↗
Enhancer of rudimentaryp1, e(r)p1, a highly conserved enhancer of the rudimentary gene.
A hybrid dysgenesis-induced mutation, enhancer of rudimentaryp1 (e(r)p1), is a recessive enhancer of a weak rudimentary mutant phenotype in Drosophila melanogaster. The e(r) gene was cloned using P element tagging and localized to region 8B on the X chromosome. It encodes a 1.0-kb and a 1.2-kb transcript. The 1.0-kb transcript is present in both adult males and females, while the 1.2-kb transcript is predominantly found in females. The difference in the lengths of the two e(r) transcripts is caused by two different polyadenylation sites spaced 228 bp apart. The amounts of both of these transcripts are drastically reduced in the e(r)p1 mutant. The P element in e(r)p1 is inserted in the 5'-untranslated leader region near the start of transcription. It may be producing its effect by suppressing transcription and/or by providing transcription termination and polyadenylation signals. The putative e(r) protein is 104 amino acids in length and bears no striking resemblance to protein sequences in GenBank or PIR. While its biochemical function is unknown at this time, sequence analysis indicates that the e(r) protein is highly conserved and, presumably, functionally very important. The amino acid sequences of the D. melanogaster and the Drosophila virilis proteins are 95% identical.
Amino Acid Sequence↗
A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds.
To determine the molecular defect accounting for the deficiency of pulmonary surfactant protein B (SP-B) in full-term neonates who died from respiratory failure associated with alveolar proteinosis, the sequence of the SP-B transcript in affected infants was ascertained. A frameshift mutation consisting of a substitution of GAA for C in codon 121 of the SP-B cDNA was identified. The three affected infants in the index family were homozygous for this mutation, which segregated in a fashion consistent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, but was not observed in 50 control subjects. We conclude that this mutation is responsible for SP-B deficiency and neonatal alveolar proteinosis in multiple families and speculate that the disorder is more common than was recognized previously.
Amino Acid Sequence↗
Health care reform: the Clinton proposal and congressional counterproposals--Part I.
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Community Health Services↗
Health care reform: the Clinton proposal and congressional counterproposals--Part II.
Note: Part I of this Article appears in the January 1994 issue of the JOURNAL OF HEALTH AND HOSPITAL LAW (V. 27, No. 1). Part II continues discussion of the potential effects of reform for the structure and financial security of various types of health care providers; identifies some possible effects of reform on health care facility operations; and describes proposed amendments to existing law appearing in various reform proposals.
Antitrust Laws↗