Occurrence of the nuclear inclusion parasite in Oregon razor clams (Siliqua patula).
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Animals↗
Biomedical subjects
J Pierce
Publications and source records attributed to J Pierce.
Cooperation of V-oncogenes in human epithelial cell transformation.
The development of tissue culture systems for propagation of human epithelial cells has aided the investigation of events that lead epithelial cells to become neoplastic. In the present study, nontumorigenic human epidermal keratinocytes, immortalized by Ad12-SV40 virus or pSv3-neo, were transformed by a variety of retroviruses containing bas, H-ras, fes, fms, erbB and src oncogenes. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of Ad12-SV40 virus and retroviral oncogenes and support a multistep process for neoplastic conversion. This in vitro system may be useful in studying the interaction of a variety of retroviral oncogenes and human epithelial cells.
Adenoviridae↗
Purification of human epidermal growth factor by monoclonal antibody affinity chromatography.
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Antibodies, Monoclonal↗
Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulose-bisphosphate carboxylase activity.
The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P(2)) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P(2) carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P(2) carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P(2) carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P(2) carboxylase. The compound was analyzed by GC/MS, (13)C NMR, and (1)H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2-C-phosphohydroxymethyl)-D-ribonic acid]. Verification of structure was obtained by comparison of the inhibitory activity of the isolated compound with that of 2-carboxy-D-arabinitol 1-phosphate synthesized in vitro. This compound (but not 2-carboxy-D-arabinitol 5-phosphate) inhibited Rbu-1,5-P(2) carboxylase in a way that was kinetically identical to that of the isolated, naturally occurring compound. The structure of the isolated compound differs from the Rbu-1,5-P(2) carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate (K(d) = 32 nM) compared with the bisphosphate (K(d) < 10 pM). The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P(2) carboxylase activity in vivo.
Journal Article↗
Three partial reactions of ribulose-bisphosphate carboxylase require both large and small subunits.
Three partial reactions of ribulose-bisphosphate carboxylase/oxygenase were measured in the presence and absence of small subunits using the enzyme from the cyanobacterium, Synechococcus ACMM 323, whose small subunits may be reversibly dissociated from its octameric, large-subunit core. These partial reactions were: the exchange of the proton at C-3 of the substrate, ribulose 1,5-bisphosphate, with the medium which is indicative of C-2, C-3 enolization; the hydrolysis of the 6-carbon reaction intermediate, 3-keto-2-carboxy-D-arabinitol 1,5-bisphosphate, to two molecules of 3-phosphoglycerate; and the decarboxylation of the 6-carbon intermediate, which is catalyzed only by the deactivated, divalent metal-ion-free carboxylase. None of these partial reactions was catalyzed by the small-subunit-depleted, large-subunit octamer to an extent greater than that expected from the residual small subunit content (about 3%), implying that small subunits are required for all three reactions. Clearly, the small subunit's influence is not restricted to any single stage of the catalytic sequence. Under conditions where it was possible to demonstrate tight binding of the reaction-intermediate analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, to the large-subunit octamer, no binding of the 6-carbon intermediate could be detected. We suggest that either the tight-binding form of the 6-carbon intermediate is the hydrated gem-diol, not the ketone, or the large subunits by themselves intrinsically possess a trace of catalytic activity which discharges any bound intermediate before it can be measured.
Carbon Dioxide↗
Reaction intermediate partitioning by ribulose-bisphosphate carboxylases with differing substrate specificities.
The carboxylated, 6-carbon reaction intermediate (3-keto-2-carboxyarabinitol 1,5-bisphosphate) from the ribulose-1,5-bisphosphate carboxylase reaction was obtained by denaturing the enzyme with acid during steady-state turnover. Carbon-13 NMR analysis indicates that this beta-keto acid exists in solution predominantly as the C-3 ketone (as opposed to the hydrate) form. In neutral solution the intermediate slowly decomposes (t1/2 approximately 1 h) by decarboxylation. This decarboxylation reaction is catalyzed by nonactivated (metal free) ribulose-1,5-bisphosphate carboxylase. Alternately, the activated enzyme predominantly catalyzes the hydrolysis of the intermediate to two molecules of glycerate 3-phosphate. The partitioning of the intermediate (i.e. hydrolysis/(hydrolysis + decarboxylation] by activated ribulose-1,5-bisphosphate carboxylase was studied using enzymes from three different sources and with different activating metal atoms. This afforded a series of catalysts whose relative specificities for the alternate substrates, carbon dioxide and oxygen, varied over a 50-fold range. When Mg2+ was the activating metal, the partitioning of the reaction intermediate varied only from 0.93 to 1 for all three enzymes. Even the Co2+ activated enzyme from Rhodospirillum rubrum, which is completely devoid of carboxylase activity, partitioned approximately 30% of added intermediate to products. It is probable that the 6-carbon intermediate's strong commitment to product formation is paralleled by a similarly strong forward commitment of the analogous intermediate in the oxygenase reaction. In this event, the variations in relative specificity for the gaseous substrates of enzymes from different natural sources must arise by interactions that take place on the enzyme prior to the formation of the intermediates.
Carbon Dioxide↗
The sites for catalysis and activation of ribulosebisphosphate carboxylase share a common domain.
The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.
Binding Sites↗
Ventilation skills of emergency medical technicians: A teaching challenge for emergency medicine.
We assessed the ability of 64 emergency medical technicians (EMTs) to ventilate a resuscitation manikin with a bag valve mask and with a pocket face mask to determine if their skill levels met the American Heart Association standard of 12 ventilations per minute, each with a tidal volume of 800 mL or more. All ventilation attempts were made during ongoing chest compressions (60 per minute). A successful ventilation was defined as a tidal volume of 800 mL +/- 40 mL. In a preliminary skills assessment, EMTs averaged 4.8 attempts with the bag valve mask and 2.9 attempts with the pocket face mask before a successful ventilation (P less than .01). In a formal skills assessment that lasted two minutes, successful ventilations per minute averaged 8.3 with the bag value mask and 9.9 with the pocket face mask (P less than 0.1). EMTs passed if they averaged ten or more successful ventilations per minute; 67% passed with the bag valve mask and 77% with the pocket face mask (NS). During a ten-minute extended skill assessment the EMTs averaged 9.6 ventilations per minute with the bag valve mask and 9.5 with the pocket face mask (NS). EMTs achieved initial success and maintained continued success better with the pocket face mask, but a reasonably high percentage (67%) met an objective standard when using the bag valve mask. We propose that objective standards be used to test the skills of EMTs for any ventilatory adjunct that they are permitted to use.
Allied Health Personnel↗
Determinants of substrate specificity and the role of metal in the reactions of ribulosebisphosphate carboxylase/oxygenase.
Recent studies have provided a fairly detailed view of the various intermediates involved in the reactions of ribulosebisphosphate carboxylase and the manner in which the catalytically essential metal atom might catalyze their interconversions. A better understanding of how the enzyme distinguishes between its alternate substrates, CO(2) and O(2), has also emerged. The results of these studies should prove useful in anticipating possible ways in which the enzyme's substrate specificity might be manipulated. Together, the techniques that are described constitute a powerful methodology for more refined experimentation aimed at understanding the curious reactivities of ribulosebisphosphate carboxylase.
Journal Article↗
Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins.
Transfection of chicken embryo cells with pMC29, a plasmid vector containing the sequences for the acute transforming virus MC29, and a cloned transformation-defective helper virus, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-myc (the product of the gag-myc oncogene), and the production of infectious virus. MC29 mutants bearing site-directed deletions within the gag-specific sequences or within the middle portion of the myc sequences efficiently induced transformation of chicken embryo cells in culture. However, variants containing deletions of sequences in the amino-terminal half or carboxy-terminal portion of the myc gene were defective for transformation. The gag-myc proteins encoded by these variants efficiently localized to the cell nucleus. Premature termination mutants were isolated which encoded gag-myc proteins lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the cytoplasm. Deletion of as few as 11 residues within the middle of the myc-specific sequences (residues Ile-239 to Glu-249) significantly reduced the efficiency of chicken hematopoietic cell transformation.
Animals↗
Automatic external defibrillation. Lay person use in the home.
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Electric Countershock↗
Induction of growth alterations in factor-dependent hematopoietic progenitor cell lines by cocultivation with irradiated bone marrow stromal cell lines.
We studied the production of hemopoietins by x-irradiated plateau-phase cultures of cloned marrow stromal cell lines derived from C3H/HeJ marrow, termed D2XRII and clone 11. The production of CSF in agar overlay of control or 10,000 rad irradiated stromal cultures was quantitated by induction of colonies in: overlaid fresh marrow, IL-3-dependent cell line 32D cl 3, or GM-CSF/IL-3-dependent cell lines FDCP-1 or bg/bg cl 1. Conditioned media were tested for CSF by bioassay using fresh marrow cells, for M-CSF (CSF-1) by RIA, and for IL-3 and GM-CSF by microwell proliferation assay with 32D cl 3 and FDCP-1 cells, respectively. X-irradiation to doses that decreased CSF-1 to 40% of control levels (greater than 5,000 rad) resulted in a 30-fold increase in growth of FDCP-1 or bg/bg cl 1 cells in liquid co-culture or agar culture overlay with no detectable growth of 32D cl 3. The frequency of subculture of nonautocrine, factor independent (FI) variant clonal lines of FDCP-1 or bg/bg cl 1 cells was increased over 1000-fold by 15 weeks cocultivation with irradiated stromal cell cultures. FI subclonal lines formed tumors in syngeneic mice and contained no detectable poly A messenger RNA for GM-CSF or IL-3, and no elevation in c-myc, c-abl, c-src, or erb-B onc gene-specific messenger RNA compared to parent factor-dependent lines. These data indicate that x-irradiated plateau phase marrow stromal cells produce increased levels of cell contact-mediated biologically active hemopoietin(s) other than M-CSF, GM-CSF, or IL-3 and induce nonautocrine factor-independent malignant cell lines in vitro.
Animals↗
Long-term culture of human granulocytes and granulocyte progenitor cells.
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Cell Adhesion↗
A case for the military pediatrician.
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Military Medicine↗
Automatic external defibrillators: clinical, training, psychological, and public health issues.
Automatic external defibrillators (AEDs) will be used by spouses, family members, emergency first-responders, and the citizenry at large. Such use, however, raises a number of clinical, training, psychological, and public health issues. Clinical issues: Is cardiac arrest to be verified by the operator or the AED? Second verification systems, such as breath detectors, produce errors of omission, but greatly expand the pool of potential users. The relative merits of high sensitivity and low specificity in arrest verification must be defined by clinicians relative to the setting and the potential users. AEDs require cessation of basic CPR during their assessment periods; clinicians must determine the tradeoff between long interruption of basic life support and much earlier delivery of countershocks. Training issues: Criteria for those to be trained include consideration of who the patient will be and who the AED operator might be. AEDs pose a familiar adult education problem, that is, acquisition of a new psychomotor skill and retention of that skill for long periods before performance. What are the best teaching techniques? Currently available AEDs have different designs for device-operator interaction. Which design is most likely to assure proper performance during an actual arrest? Psychological issues: What are the psychological effects of learning about, living with, and eventually using an AED? The development of the automatic external defibrillator constitutes the most recent attempt to achieve early defibrillation of patients in cardiac arrest. The potential public health effect of such devices is enormous.
Electric Countershock↗
Large-scale preparation of ribulosebisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability.
An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability.
Ampicillin↗
Emergency CPR instruction via telephone.
We initiated a program of telephone CPR (cardiopulmonary resuscitation) instruction provided by emergency dispatchers to increase the percentage of bystander-initiated CPR for out-of-hospital cardiac arrest. Cardiac arrests in King County, Washington were studied for 20 months before and after the telephone CPR program began. Bystander-initiated CPR increased from 86 of 191 (45 per cent) cardiac arrests before the program to 143 of 255 (56 per cent) cardiac arrests after the program. During the after period, 58 patients received CPR as a result of telephone instruction, 12 of whom were discharged. We estimate that four lives may have been saved by the program. A review of hospital records revealed no excess morbidity in the group of patients receiving dispatcher-assisted CPR.
Adult↗
Treatment of ventricular fibrillation. Emergency medical technician defibrillation and paramedic services.
We compared the effect of rapid defibrillation by emergency medical technicians (EMTs) combined with paramedic care with that of standard EMT and paramedic care on survival from 540 witnessed episodes of out-of-hospital cardiac arrest caused by ventricular fibrillation. More than 400 EMTs were trained in the recognition of ventricular fibrillation and operation of a defibrillator. For a portion of the three-year study, emergency care for 179 cases was randomized between the two types of services. For randomized cases, when the time interval between EMT and paramedic arrival was greater than four minutes there was significantly improved survival with EMT defibrillation and paramedic care (42%) compared with basic EMT and paramedic care (19%). Similar findings occurred when all cases were considered (38% v 18%). Defibrillation by EMTs combined with paramedic services can enhance survival from ventricular fibrillation, compared with basic EMT and paramedic care.
Aged↗