33 results on '"Roberts EF"'
Search Results
2. Early Life Exposure in Mexico to ENvironmental Toxicants (ELEMENT) Project.
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Perng W, Tamayo-Ortiz M, Tang L, Sánchez BN, Cantoral A, Meeker JD, Dolinoy DC, Roberts EF, Martinez-Mier EA, Lamadrid-Figueroa H, Song PXK, Ettinger AS, Wright R, Arora M, Schnaas L, Watkins DJ, Goodrich JM, Garcia RC, Solano-Gonzalez M, Bautista-Arredondo LF, Mercado-Garcia A, Hu H, Hernandez-Avila M, Tellez-Rojo MM, and Peterson KE
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- Adult, Age Factors, Female, Humans, Infant, Newborn, Male, Mexico, Pregnancy, Young Adult, Bone and Bones metabolism, Environmental Exposure adverse effects, Environmental Pollutants adverse effects, Lead adverse effects, Lead metabolism, Prenatal Exposure Delayed Effects etiology, Prenatal Exposure Delayed Effects metabolism
- Abstract
Purpose: The Early Life Exposure in Mexico to ENvironmental Toxicants (ELEMENT) Project is a mother-child pregnancy and birth cohort originally initiated in the mid-1990s to explore: (1) whether enhanced mobilisation of lead from maternal bone stores during pregnancy poses a risk to fetal and subsequent offspring neurodevelopment; and (2) whether maternal calcium supplementation during pregnancy and lactation can suppress bone lead mobilisation and mitigate the adverse effects of lead exposure on offspring health and development. Through utilisation of carefully archived biospecimens to measure other prenatal exposures, banking of DNA and rigorous measurement of a diverse array of outcomes, ELEMENT has since evolved into a major resource for research on early life exposures and developmental outcomes., Participants: n=1643 mother-child pairs sequentially recruited (between 1994 and 2003) during pregnancy or at delivery from maternity hospitals in Mexico City, Mexico., Findings to Date: Maternal bone (eg, patella, tibia) is an endogenous source for fetal lead exposure due to mobilisation of stored lead into circulation during pregnancy and lactation, leading to increased risk of miscarriage, low birth weight and smaller head circumference, and transfer of lead into breastmilk. Daily supplementation with 1200 mg of elemental calcium during pregnancy and lactation reduces lead resorption from maternal bone and thereby, levels of circulating lead. Beyond perinatal outcomes, early life exposure to lead is associated with neurocognitive deficits, behavioural disorders, higher blood pressure and lower weight in offspring during childhood. Some of these relationships were modified by dietary factors; genetic polymorphisms specific for iron, folate and lipid metabolism; and timing of exposure. Research has also expanded to include findings published on other toxicants such as those associated with personal care products and plastics (eg, phthalates, bisphenol A), other metals (eg, mercury, manganese, cadmium), pesticides (organophosphates) and fluoride; other biomarkers (eg, toxicant levels in plasma, hair and teeth); other outcomes (eg, sexual maturation, metabolic syndrome, dental caries); and identification of novel mechanisms via epigenetic and metabolomics profiling., Future Plans: As the ELEMENT mothers and children age, we plan to (1) continue studying the long-term consequences of toxicant exposure during the perinatal period on adolescent and young adult outcomes as well as outcomes related to the original ELEMENT mothers, such as their metabolic and bone health during perimenopause; and (2) follow the third generation of participants (children of the children) to study intergenerational effects of in utero exposures., Trial Registration Number: NCT00558623., Competing Interests: Competing interests: None., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2019
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3. Gods, Germs, and Petri Dishes: Toward a Nonsecular Medical Anthropology.
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Roberts EF
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- Humans, Anthropology, Medical, Medicine, Religion, Science
- Abstract
This commentary calls on medical anthropology to become programmatically non-secular. Despite recent anthropological critiques of secularity, within and outside of anthropology, most contemporary medical anthropologists continue to leave deities and religiosity out of their examinations of healing practices, especially in their accounts of biomedicine. Through a critical, relational constructionist lens, which traces how all entities are both constructed and real, a non-secular medical anthropology would insist that when deities are part of medical practice, they are integral to analysis. Importantly then, within the symmetrical nature of this same constructionist lens, biomedical entities like germs and petri dishes need to be accounted for just as much as deities.
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- 2016
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4. Nonsecular Medical Anthropology.
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Whitmarsh I and Roberts EF
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- Humans, Anthropology, Medical, Religion
- Abstract
A nonsecular medical anthropology insists on the ways medicine and science have constituted 'the secular' itself through the 'secular self'-how medical knowing has been used to craft the secular political subject. As James Boon noted, too often in social theory, "religion gets safely tucked away-restricted theoretically to 'meaning' rather than power" (1998:245). The authors of the six articles in this special issue 'untuck' religiosity from within the norms and numbers of medicine itself, and examine how 'secular' medicine has relied on religious traditions to produce political secularity. These articles demonstrate that 'secular' medicine relies on religious others whose exclusion bespeaks latent religious commitments of citizenship in the modern political realm of health.
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- 2016
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5. Discovery of (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide, a potent and orally efficacious mGlu5 receptor negative allosteric modulator.
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Hao J, Dehlinger V, Fivush AM, Rudyk HC, Britton TC, Hollinshead SP, Vokits BP, Clark BP, Henry SS, Massey SM, Peng L, Dressman BA, Heinz BA, Roberts EF, Bracey-Walker MR, Swanson S, Catlow JT, Love PL, Tepool AD, Peters SC, Simmons RM, Iyengar S, McKinzie DL, and Monn JA
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- Allosteric Regulation drug effects, Amides pharmacokinetics, Animals, Behavior, Animal drug effects, Cyclopropanes pharmacokinetics, Glutamic Acid chemistry, Glutamic Acid metabolism, Indazoles chemistry, Indazoles pharmacokinetics, Indazoles pharmacology, Male, Rats, Rats, Sprague-Dawley, Receptor, Metabotropic Glutamate 5 chemistry, Thiazoles chemistry, Thiazoles pharmacokinetics, Thiazoles pharmacology, Amides chemistry, Amides pharmacology, Cyclopropanes chemistry, Cyclopropanes pharmacology, Receptor, Metabotropic Glutamate 5 metabolism
- Abstract
A novel series of selective negative allosteric modulators (NAMs) for metabotropic glutamate receptor 5 (mGlu5) was discovered from an isothiazole scaffold. One compound of this series, (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide (24), demonstrated satisfactory pharmacokinetic properties and, following oral dosing in rats, produced dose-dependent and long-lasting mGlu5 receptor occupancy. Consistent with the hypothesis that blockade of mGlu5 receptors will produce analgesic effects in mammals, compound 24 produced a dose-dependent reduction in paw licking responses in the formalin model of persistent pain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
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- 2013
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6. Reproductive governance in Latin America.
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Morgan LM and Roberts EF
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- Abortion, Induced, Catholicism, Contraception, Humans, Latin America, Morals, Socioeconomic Factors, Government, Human Rights, Politics, Public Policy, Reproductive Behavior
- Abstract
This paper develops the concept of reproductive governance as an analytic tool for tracing the shifting political rationalities of population and reproduction. As advanced here, the concept of reproductive governance refers to the mechanisms through which different historical configurations of actors - such as state, religious, and international financial institutions, NGOs, and social movements - use legislative controls, economic inducements, moral injunctions, direct coercion, and ethical incitements to produce, monitor, and control reproductive behaviours and population practices. Examples are drawn from Latin America, where reproductive governance is undergoing a dramatic transformation as public policy conversations are coalescing around new moral regimes and rights-based actors through debates about abortion, emergency contraception, sterilisation, migration, and assisted reproductive technologies. Reproductive discourses are increasingly framed through morality and contestations over 'rights', where rights-bearing citizens are pitted against each other in claiming reproductive, sexual, indigenous, and natural rights, as well as the 'right to life' of the unborn. The concept of reproductive governance can be applied to other settings in order to understand shifting political rationalities within the domain of reproduction.
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- 2012
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7. Abandonment and accumulation: embryonic futures in the United States and Ecuador.
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Roberts EF
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- Anthropology, Cultural, Ecuador, Humans, United States, Embryo Research ethics, Fertilization in Vitro ethics
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When frozen embryos are publically debated in the United States, they are most often positioned as having two possible future trajectories: (1) as individual humans and (2) as contributors to stem cell research. Long-term embryo accumulation threatens both of these futures. An accumulated embryo is stuck in a clinic, held back from having an individual future or from contributing to science. There are other kinds of futures, though. For some patients in the United States and Ecuador, where I conducted ethnographic research, future reckoning involves a vision of responsibility toward embryos embedded within a specific family. For these patients, frozen embryo donation to another family or to science constitutes abandonment. The future at stake is not that of an individual embryo's life, but a group's future who would abandon one of its own. These patients would rather destroy embryos than freeze them for a future away from their relations. [Ecuador, United States, in
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- 2011
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8. God's laboratory: religious rationalities and modernity in Ecuadorian in vitro fertilization.
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Roberts EF
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- Ecuador, Female, Humans, Male, Attitude to Health ethnology, Catholicism, Fertilization in Vitro ethics, Religion and Medicine, Social Change
- Abstract
Catholicism is the only major world religion that unequivocally bans the use of in vitro fertilization (IVF). Nevertheless, in Ecuador, Catholic IVF practitioners declare God's dominion over their IVF laboratories and clinics in explaining pregnancy outcomes. My analysis of this routine combination of spiritual and material causal models in Ecuadorian IVF contributes to two ongoing discussions about (1) the tensions between "institutional" and popular forms of Catholic religiosity and (2) the proper boundaries of science in modernity. The Catholic Church's historical and contemporary struggle to determine control of the miraculous has usually been characterized as a conflict between educated clergy and humble peasants. In the case of Ecuadorian IVF, we find, instead, educated elites and middle classes participating in this same contestation with the Church, proclaiming their direct ability to harness the power of God to effect material change on earth. This spiritual power to affect clinical outcomes does not take place just anywhere, but in clinic and lab, disrupting another set of presumptions about modern scientific practice and subjectivity. Like other Ecuadorian elites and middle classes, IVF practitioners are heirs to Enlightenment thought, and experience themselves as modern in their participation in these high-tech endeavors. But their spiritual approach to laboratory rationality does not trouble these IVF practitioners' experience of themselves as moderns, prompting a reevaluation of the narratives of scientific modernity that limit their scope to Europe and North America.
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- 2006
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9. Derivation and characterization of a monoclonal antibody against human glycinamide ribonucleotide formyltransferase.
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Dotzlaf J, Carpenter J, Luo S, Roberts EF, Solenberg PJ, Qian YW, Lin A, He X, Sandusky GE, McClure DB, Chen VJ, and Zuckerman SH
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- Amino Acid Sequence, Animals, Epitopes genetics, Epitopes immunology, HeLa Cells, Humans, Hybridomas, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Phosphoribosylglycinamide Formyltransferase genetics, Antibodies, Monoclonal chemistry, Phosphoribosylglycinamide Formyltransferase immunology
- Abstract
Glycinamide ribonucleotide formyltransferase (GARFT) is a trifunctional enzyme involved in purine biosynthesis. Its central role in folate metabolism has made it an obvious target for the development of GARFT inhibitors, primarily for oncology. While the crystal structure, enzyme kinetics, and mechanism of action of GARFT inhibitors are reasonably well understood, GARFT regulation at the protein level remains unclear. The present study reports the development and characterization of a monoclonal antibody (MAb) specific for human GARFT. This MAb, an IgG1kappa, designated PHR1, recognizes human GARFT by both Western blot and by immunohistochemistry from non-small-cell lung carcinoma and colon adenocarcinoma tissue biopsies, has a KD of 1.14 x 10(10) M, and has been epitope mapped at residues 59-78 of the GARFT functional domain. The ability of PHR1 to recognize both sodium dodecyl sulfate (SDS)-denatured as well as native GARFT should make this MAb an important research tool in determining GARFT protein levels in both normal and neoplastic tissues.
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- 2006
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10. Nursing faculty's handling of academic dishonesty.
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Roberts EF
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- Attitude of Health Personnel, Decision Making, Education, Nursing, Baccalaureate standards, Health Knowledge, Attitudes, Practice, Humans, Motivation, Students, Nursing statistics & numerical data, Deception, Education, Nursing, Baccalaureate methods, Ethics, Nursing, Faculty, Nursing statistics & numerical data, Students, Nursing psychology
- Published
- 1999
11. Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs.
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Schultz RM, Chen VJ, Bewley JR, Roberts EF, Shih C, and Dempsey JA
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- Antimetabolites, Antineoplastic metabolism, Biological Transport, Drug Screening Assays, Antitumor, Enzyme Inhibitors metabolism, Folic Acid Antagonists metabolism, Glutamates metabolism, Guanine metabolism, Guanine pharmacology, Humans, Pemetrexed, Quinazolines pharmacology, Thiophenes pharmacology, Tumor Cells, Cultured, Antimetabolites, Antineoplastic pharmacology, Drug Resistance, Neoplasm, Enzyme Inhibitors pharmacology, Folic Acid Antagonists pharmacology, Glutamates pharmacology, Guanine analogs & derivatives, Thymidylate Synthase antagonists & inhibitors
- Abstract
Prior studies have indicated that MTA requires intracellular polyglutamation for optimal cytotoxic effect and that these polyglutamates potently inhibit several key enzymes of folate metabolism, including thymidylate synthase (TS), dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). In the present studies, we have investigated the mechanistic basis for resistance to MTA in several human tumor cell lines. The cell lines were developed for resistance by the gradual exposure to stepwise (fivefold) increases in the concentration of MTA over a 5-month period. The degree of resistance was 140-fold for GC3 colon carcinoma, 117-fold for HCT-8 ileocecal carcinoma, and 729-fold for CCRF-CEM leukemia cells adapted to 2 micromol/L MTA. The lines had strong cross-resistance (>3,200-fold) to raltitrexed. Only modest resistance was noted for methotrexate and the GARFT inhibitor, LY309887. The cytotoxicity of MTA in wild-type cells was only partially alleviated by thymidine addition (5 micromol/L) and complete protection required the addition of both hypoxanthine (100 micromol/L) and thymidine. In contrast, thymidine alone totally lacked protective activity in the MTA-resistant lines. The cells either demonstrated a GARFT-like reversal pattern (complete protection by hypoxanthine) for GC3MTA or a dihydrofolate reductase-like reversal pattern (complete protection by the combination of hypoxanthine and thymidine) for HCT-8MTA and CCRF-CEM(MTA) cells. Cellular resistance was multifactorial and stable on removal of selective pressure. Only GC3MTA cells showed increased TS activity (approximately 40-fold). Accumulations of 3H-MTA at 24 hours in CCRF-CEM(MTA), HCT-8MTA, and GC3MTA cells were 2%, 6%, and 46% of wild-type values, respectively. We also evaluated the cytotoxic activity of MTA in MCF-7 breast carcinoma and H630 colon carcinoma cells selected for resistance to raltitrexed and 5-fluorouracil, respectively, via TS amplification (provided by Dr P.G. Johnston, Belfast, Ireland). These cells demonstrated more than 200-fold less resistance to MTA compared with raltitrexed and MTA-induced cytotoxicity was prevented by hypoxanthine. These studies suggest that in addition to TS modulation, secondary targets emerge during the development of MTA resistance.
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- 1999
12. Academic misconduct in schools of nursing.
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Roberts EF
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- Humans, Moral Development, Ethics, Nursing, Professional Competence, Professional Misconduct, Schools, Nursing standards, Students, Nursing psychology
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- 1997
13. Activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) in human platelets.
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Kramer RM, Roberts EF, and Jakubowski JA
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- Arachidonic Acid blood, Calcium blood, Cytosol enzymology, Enzyme Activation, Gene Expression Regulation, Enzymologic, Group VI Phospholipases A2, Humans, Phospholipases A biosynthesis, Phospholipases A chemistry, Phospholipases A2, Blood Platelets metabolism, Phospholipases A blood
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- 1997
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14. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.
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Kramer RM, Roberts EF, Um SL, Börsch-Haubold AG, Watson SP, Fisher MJ, and Jakubowski JA
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- Arachidonic Acid blood, Blood Platelets drug effects, Calcium pharmacology, Calcium-Calmodulin-Dependent Protein Kinases antagonists & inhibitors, Cytosol enzymology, Enzyme Inhibitors pharmacology, Humans, In Vitro Techniques, Kinetics, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Peptide Fragments pharmacology, Phospholipases A2, Phosphorylation, Proline, Receptors, Thrombin, p38 Mitogen-Activated Protein Kinases, Blood Platelets metabolism, Calcium-Calmodulin-Dependent Protein Kinases blood, Mitogen-Activated Protein Kinases, Phospholipases A blood, Platelet Activation, Thrombin pharmacology
- Abstract
The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.
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- 1996
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15. Cytosolic phospholipase A2 (cPLA2) and lipid mediator release in the brain.
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Kramer RM, Stephenson DT, Roberts EF, and Clemens JA
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- Animals, Cytoplasm metabolism, Humans, Phospholipases A2, Brain metabolism, Lipid Metabolism, Phospholipases A metabolism
- Abstract
The Ca(2+)-sensitive 85 kDa cytosolic PLA2 (cPLA2) is a receptor-regulated enzyme that may initiate the cascade of events leading to the production of free fatty acids and lysophospholipids for subsequent conversion to eicosanoids and PAF. At least two early events are necessary for full activation of cPLA2: (1) increased concentration of cytosolic free Ca2+ promoting association of cPLA2 with its membrane phospholipid substrate and (2) phosphorylation by stimulated proline-directed kinases converting cPLA2 into an enzyme of enhanced catalytic efficiency. Moreover, pro-inflammatory cytokines, such as IL-1 and TNF may induce de novo synthesis of cPLA2 thus further potentiating the mobilization of arachidonic acid and subsequent production of eicosanoids and PAF. Increased levels of fatty acids and PLA2-derived products, including eicosanoids and PAF are amongst the hallmarks of cerebral ischemia and reperfusion, and thought to mediate pathophysiological alterations and cellular processes which may lead to cell injury and death. There is substantial evidence to indicate that cPLA2 is present in the brain and appears most abundant in astrocytes. Therefore, cPLA2 may be an important component in the cascade of events leading to acute and delayed destructive cellular processes in the brain and accordingly represents an attractive target for the development of novel therapies to prevent brain damage triggered by ischemic and inflammatory insults.
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- 1996
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16. Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat.
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Clemens JA, Stephenson DT, Smalstig EB, Roberts EF, Johnstone EM, Sharp JD, Little SP, and Kramer RM
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- Animals, Astrocytes enzymology, Blotting, Western, Brain Ischemia genetics, Cell Death, Cytosol enzymology, Doxorubicin analogs & derivatives, Gene Expression Regulation, Enzymologic, Hippocampus enzymology, Humans, Male, Microglia enzymology, Nerve Degeneration, Phospholipases A genetics, Phospholipases A2, Prosencephalon enzymology, Rats, Rats, Wistar, Brain Ischemia enzymology, Neuroglia enzymology, Phospholipases A analysis, Prosencephalon blood supply
- Abstract
Background and Purpose: Phospholipid breakdown has been reported to be an early event in the brain after global cerebral ischemia. Our earlier observations showing the localization of cytosolic phospholipase A2 (cPLA2) to astrocytes in aged human brains and the intense glial activation observed after global forebrain ischemia prompted us to investigate the cellular localization of cPLA2 in the rat brain subjected to global ischemia., Methods: Immunohistochemistry was performed in sections through the dorsal hippocampus in rats subjected to 30 minutes of four- vessel occlusion. PLA2 was localized with the use of a highly selective antiserum. Double immunofluorescent localization was performed to colocalize cPLA2 with various glial cell types. cPLA2 levels were also measured by enzymatic assay and Western blot analysis., Results: A marked induction of cPLA2 was observed in activated microglia and astrocytes in the CA1 hippocampal region at 72 hours after ischemia. Only a subset of astrocytes and microglia were immunoreactive for cPLA2. Twenty-four hours after ischemia, numerous cPLA2 immunoreactive astrocytes were observed. Western blot analysis of hippocampal homogenates at 72 hours after ischemia showed induction of a 100-kD band that comigrated with purified human cPLA2, and a threefold induction in cPLA2 activity was demonstrated by enzymatic assay., Conclusions: These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA2. Induction of cPLA2 was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.
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- 1996
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17. Thrombin induces activation of p38 MAP kinase in human platelets.
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Kramer RM, Roberts EF, Strifler BA, and Johnstone EM
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- Amino Acid Sequence, Calcium-Calmodulin-Dependent Protein Kinases drug effects, Calcium-Calmodulin-Dependent Protein Kinases isolation & purification, Chromatography, Ion Exchange, Humans, Molecular Sequence Data, Peptide Fragments pharmacology, Peptides chemistry, Peptides metabolism, Phosphotyrosine metabolism, Platelet Activation, Receptors, Thrombin physiology, Signal Transduction, Substrate Specificity, p38 Mitogen-Activated Protein Kinases, Blood Platelets enzymology, Calcium-Calmodulin-Dependent Protein Kinases blood, Mitogen-Activated Protein Kinases, Thrombin pharmacology
- Abstract
In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca(2+)-dependent cytosolic phospholipase A2 (Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., and Jakubowski, J.A. (1995) J. Biol. Chem. 270, 14816-14823). Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.
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- 1995
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18. Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2.
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Kramer RM, Roberts EF, Hyslop PA, Utterback BG, Hui KY, and Jakubowski JA
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- Amino Acid Sequence, Blood Platelets enzymology, Calcium metabolism, Cytosol drug effects, Cytosol enzymology, Enzyme Activation, Humans, In Vitro Techniques, Kinetics, Molecular Sequence Data, Phospholipases A2, Phosphorylation, Proline metabolism, Receptors, Thrombin agonists, Blood Platelets drug effects, Peptide Fragments pharmacology, Phospholipases A metabolism, Protein Kinases metabolism, Thrombin pharmacology
- Abstract
The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A2 by human platelets. We found that while SFLLRN evokes an initial transient increase in cystolic free calcium concentration ([Ca2+]i) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca2+]i declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca2+]i may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the "activating" phosphorylation of cytolic phospholipase A2 (cPLA2). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr669 derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA2 and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca2+]i and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA2 in the absence of ERK1/2 stimulation suggest that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA2.
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- 1995
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19. Phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE.
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Currie S, Roberts EF, Spaethe SM, Roehm NW, and Kramer RM
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- Animals, Arachidonic Acid metabolism, Cytosol enzymology, Eicosanoids biosynthesis, Enzyme Activation, Humans, Kinetics, Leukotriene C4 biosynthesis, Phospholipases A2, Phosphorylation, Sensitivity and Specificity, Calcium physiology, Mast Cells enzymology, Mast Cells ultrastructure, Phospholipases A metabolism, Receptors, IgE physiology
- Abstract
In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC4.
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- 1994
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20. Serine 228 is essential for catalytic activities of 85-kDa cytosolic phospholipase A2.
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Sharp JD, Pickard RT, Chiou XG, Manetta JV, Kovacevic S, Miller JR, Varshavsky AD, Roberts EF, Strifler BA, and Brems DN
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- Amino Acid Sequence, Catalysis, Consensus Sequence, Cytosol enzymology, Humans, Hydrolysis, Lysophospholipase metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Phospholipases A chemistry, Phospholipases A2, Sequence Homology, Amino Acid, Phospholipases A metabolism, Serine metabolism
- Abstract
The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).
- Published
- 1994
21. Characterization by electrospray mass spectrometry of human Ca(2+)-sensitive cytosolic phospholipase A2 produced in baculovirus-infected insect cells.
- Author
-
Becker GW, Miller JR, Kovacevic S, Ellis RM, Louis AI, Small JS, Stark DH, Roberts EF, Wyrick TK, and Hoskins J
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Cloning, Molecular, Gene Transfer Techniques, Humans, Kinetics, Molecular Sequence Data, Phospholipases A chemistry, Phospholipases A2, Phosphorylation, Protein Processing, Post-Translational, Recombinant Proteins, Baculoviridae genetics, Calcium pharmacology, Cytosol enzymology, Gene Expression, Mass Spectrometry, Moths, Phospholipases A genetics
- Abstract
The 85-kD cytosolic phospholipase A2 (cPLA2) is a novel receptor-regulated phospholipase that is thought to initiate the production of inflammatory lipid mediators. Since cPLA2 is present only in minute amounts (less than 0.01% of total cellular protein) in various cells and tissues, we have used the baculovirus expression system to produce sufficient quantities of cPLA2 for structural and functional analysis. The cDNA for cPLA2 was cloned into a baculovirus expression vector and, upon infection of Spodoptera frugiperda Sf-21 cells with the recombinant virus, cPLA2 was produced at high levels (9% of total cellular soluble protein). Gel electrophoresis and immunoblot analysis demonstrated that the recombinant protein has properties indistinguishable from cPLA2 present in human monocytic U937 cells. Structural analysis of recombinant cPLA2, using electrospray mass spectrometry in conjunction with automated sequence analysis, confirmed the expected sequence and revealed two post-translational modifications of the protein, phosphorylation on at least one site, and acetylation of the N-terminal serine residue after removal of the initiating methionine. In spite of the presence of six potential N-glycosylation sites, there is no evidence that any of them is glycosylated. The baculovirus expression system should prove useful for production of cPLA2, and electrospray mass spectrometry is a rapid and accurate method for the analysis of post-translational modifications.
- Published
- 1994
- Full Text
- View/download PDF
22. Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets.
- Author
-
Kramer RM, Roberts EF, Manetta JV, Hyslop PA, and Jakubowski JA
- Subjects
- Amino Acid Sequence, Cytosol enzymology, Enzyme Activation, Humans, Immunochemistry, In Vitro Techniques, Kinetics, Molecular Sequence Data, Phospholipases A2, Phosphorylation, Blood Platelets enzymology, Calcium metabolism, Phospholipases A metabolism, Thrombin pharmacology
- Abstract
Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phospholipase A2 to liberate arachidonic acid for further conversion to thromboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets contain significant amounts (60 ng/10(9) platelets) of the recently identified Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic labeling of human platelets with 33Pi revealed that the extent of phosphorylation of cPLA2 was greatly increased after thrombin treatment. Upon stimulation of platelets with thrombin, cPLA2 exhibits enhanced catalytic activity, as well as a change in its electrophoretic and chromatographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demonstrating that they are the consequence of thrombin-stimulated phosphorylation. Thrombin-induced phosphorylation and activation of cPLA2 is rapid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-dependent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thrombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization and further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets, we found that while activated cPLA2 exhibits the same Ca2+ requirement and apparent substrate affinity (Km), its catalytic activity (Vmax) is increased compared with control cPLA2. We conclude that 1) cPLA2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.
- Published
- 1993
23. Triploid abortus presenting as an ectopic pregnancy.
- Author
-
Montgomery EA, Roberts EF, Conran RM, and Hitchcock CL
- Subjects
- Adult, DNA genetics, Fallopian Tubes pathology, Female, Flow Cytometry, Humans, Hydatidiform Mole pathology, Pregnancy, Pregnancy, Tubal genetics, Pregnancy, Tubal pathology, Uterine Neoplasms pathology, Hydatidiform Mole complications, Ploidies, Pregnancy, Tubal complications, Uterine Neoplasms complications
- Abstract
In this article we present a case of an ectopic gestation having morphologic features of a partial hydatidiform mole and demonstrating triploidy by flow cytometry in a patient presenting at 9 weeks' gestation. We include brief comments on partial hydatidiform mole.
- Published
- 1993
24. Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) in human platelets.
- Author
-
Kramer RM, Roberts EF, Manetta JV, Sportsman JR, and Jakubowski JA
- Subjects
- Calcium pharmacology, Cloning, Molecular, Cytosol enzymology, GTP-Binding Proteins metabolism, Humans, Immunochemistry, Phospholipases A genetics, Phospholipases A immunology, Phospholipases A2, Receptors, Cell Surface metabolism, Thrombin pharmacology, Blood Platelets enzymology, Phospholipases A blood
- Published
- 1993
25. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells.
- Author
-
Kramer RM, Roberts EF, Manetta J, and Putnam JE
- Subjects
- Blotting, Western, Cell Line, Chromatography, Liquid, Cytosol enzymology, Electrophoresis, Polyacrylamide Gel, Humans, Immunohistochemistry, Kinetics, Molecular Weight, Phospholipases A isolation & purification, Phospholipases A2, Precipitin Tests, Calcium metabolism, Phospholipases A metabolism
- Abstract
Human monoblast U937 cells contain a soluble phospholipase A2 (PLA2) that is activated over the range of 150-600 nM Ca2+ and is stable only at neutral pH. We have purified this PLA2 over 34,000-fold to near homogeneity using sequential ion exchange, hydrophobic interaction, and gel filtration chromatography steps. The protein has a Mr of approximately 100,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 5.1. Four lines of evidence indicate that this 100-kDa polypeptide represents the PLA2. (i) The intensity of staining of the 100-kDa protein was proportional to the degree of purification of PLA2 activity, (ii) the relative staining intensity of the 100-kDa protein precisely paralleled the elution profile of PLA2 activity during chromatography steps, (iii) the PLA2 activity recovered from a nondenaturing gel (greater than 60% of the total activity applied) coincided exactly with the major high molecular weight protein detected by silver staining, and (iv) monoclonal antibodies against the 100-kDa protein immunoprecipitated the PLA2. We conclude that the cytosolic PLA2 isolated from U937 cells represents a novel, high molecular weight PLA2 responding to physiological (intracellular) changes in Ca2+ concentration and therefore may play a critical role in cellular signal transduction processes and the biosynthesis of lipid mediators.
- Published
- 1991
26. Effect of streptozocin-induced diabetes on insulin-receptor tyrosine kinase activity in obese Zucker rats.
- Author
-
Slieker LJ, Roberts EF, Shaw WN, and Johnson WT
- Subjects
- Animals, Diabetes Mellitus enzymology, Glucose Tolerance Test, Insulin Resistance, Intercellular Signaling Peptides and Proteins, Liver enzymology, Liver metabolism, Male, Muscles enzymology, Muscles metabolism, Peptides metabolism, Phosphorylation, Rats, Rats, Zucker, Diabetes Mellitus metabolism, Diabetes Mellitus, Experimental metabolism, Diabetes Mellitus, Type 2 metabolism, Obesity, Protein-Tyrosine Kinases metabolism, Receptor, Insulin metabolism
- Abstract
We examined insulin binding, insulin-stimulated autophosphorylation, and phosphorylation of poly(Glu.Na,Tyr)4:1 by liver and skeletal muscle insulin receptor from lean, obese, and obese streptozocin-induced diabetic Zucker rats. Induction of diabetes with streptozocin (30 mg/kg) lowered the lasting insulin level from 11.4 to 3.8 ng/ml, which was not significantly greater than the lean control level. Autophosphorylation and tyrosine kinase activity of liver insulin receptors were increased 70-100% in the obese control group (relative to lean rats), but diabetes reversed this hyperresponsiveness to insulin. In muscle, obesity was associated with a 40-50% decrease in autophosphorylation and tyrosine kinase activity, which was also reversed in the diabetic state. Autophosphorylation and tyrosine kinase activity were significantly correlated in liver and muscle and were also correlated with fasting insulin levels. These data suggest that insulin-receptor tyrosine kinase activity is regulated differently in liver and muscle and that the abnormalities in kinase activity associated with the obese Zucker rat are at least partly secondary to hyperinsulinemia.
- Published
- 1990
- Full Text
- View/download PDF
27. Structure of transfer RNAs: listing of 150 additional sequences.
- Author
-
Singhal RP, Roberts EF, and Vakharia VN
- Subjects
- Animals, Bacteria genetics, Drosophila genetics, Humans, Neoplasms genetics, Nucleic Acid Conformation, Species Specificity, Structure-Activity Relationship, RNA, Transfer genetics
- Published
- 1983
- Full Text
- View/download PDF
28. A new development in antihistaminic therapy.
- Author
-
ROBERTS EF
- Subjects
- Histamine Antagonists
- Published
- 1948
29. Prolonged blood concentrations of penicillin following intramuscular benzathine penicillin G.
- Author
-
PUTNAM LE and ROBERTS EF
- Subjects
- Humans, Penicillins analogs & derivatives, Blood, Injections, Intramuscular, Penicillin G Benzathine
- Published
- 1954
30. Antibiotics and newer advances with emphasis on the synthetic penicilins.
- Author
-
ROBERTS EF
- Subjects
- Penicillin V analogs & derivatives, Anti-Bacterial Agents, Chloramphenicol, Colistin, Demeclocycline, Dihydrostreptomycin Sulfate, Erythromycin, Kanamycin, Methicillin, Penicillins, Streptomycin, Troleandomycin, Vancomycin
- Published
- 1962
31. Body fluid concentrations of penicillin following intramuscular injection of single doses of benzathine penicillin G and/or procaine penicillin G.
- Author
-
WRIGHT WW, WELCH H, WILNER J, and ROBERTS EF
- Subjects
- Body Fluids, Injections, Intramuscular, Penicillin G Benzathine, Penicillin G Procaine, Penicillins administration & dosage
- Published
- 1959
32. Variations in Tuberculin Allergy.
- Author
-
Roberts EF
- Published
- 1930
33. Review of antibiotic therapy in pediatric infections.
- Author
-
ROBERTS EF
- Subjects
- Child, Humans, Infant, Anti-Bacterial Agents therapeutic use, Infections, Pediatrics
- Published
- 1959
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