Foreign bodies in the pediatric patient.
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Child↗
Biomedical subjects
D G Johnson
Publications and source records attributed to D G Johnson.
Are scintiscans accurate in the selection of reflux patients for pyloroplasty?
BACKGROUND: Gastric emptying scintiscans are currently used to select reflux patients for added pyloroplasty at the time of fundoplication. The accuracy of this scan selection approach has been assumed. If preoperative scintiscans do not reliably predict postfundoplication gastric emptying, however, the decision to add pyloroplasty to the fundoplication operation may be inappropriate and even harmful. METHODS: The authors studied 27 children prospectively before and after gastric fundoplication. Gastric emptying at 60 minutes was measured by double isotopic labeling of liquid (111In) and solid (99mTc) phases of a test meal specifically designed for label fixation. The authors' question involved the accuracy of preoperative gastric scintiscans in predicting postfundoplication delay of gastric emptying (DGE). An evaluation of pyloroplasty as an effective treatment for DGE was not part of the study design. Pyloroplasty was performed as a secondary operation in three of the study children, however, because they persisted with unrelieved symptoms of retching, fullness, and abdominal discomfort. Scintiscan-documented postfundoplication delay in gastric emptying was present in all three patients at 18, 58, and 12 weeks, respectively. Additional scintiscans were performed in these patients after pyloroplasty. RESULTS: Gastric emptying of solids at 60 minutes did not show a significant change after a gastric fundoplication operation, although the trend was in the direction of a decrease (paired t test, P= .13). Liquid emptying at 60 minutes, however, was significantly increased (paired t test, P = .01). The variation in values between patients was wide, and the correlation between pre- and postoperative study results in the same patient was poor (r2 = 0.337 for solids and r2 = 0.116 for liquids). Most unexpectedly, scintiscans after postfundoplication pyloroplasty in the three patients with persistent symptoms showed no improvement in delayed gastric emptying on repeat scintiscan 42 to 117 weeks later. CONCLUSIONS: The data suggest that preoperative scintiscan evidence for postfundoplication DGE is probably accurate for solid emptying but not for liquids, at least as measured by the double isotope methodology of our study. Preoperative scintiscans that use a liquid phase label only may be highly misleading for the prediction of postfundoplication DGE. Furthermore, pyloroplasty may not be useful as treatment even when postfundoplication delay in gastric emptying can be accurately anticipated or confirmed. A fundamental motility disorder of the gastric body seems to be more important than muscular resistance at the gastric outlet as a cause for postfundoplication DGE, and the most effective treatment approach remains unclear.
Adolescent↗
Tracheobronchial injuries after blunt chest trauma in children--hidden pathology.
BACKGROUND: Blunt thoracic injuries in children are unique because the pliability of the chest wall allows transmission of massive external force directly into the mediastinum. Children presenting after blunt chest trauma may have complete disruption of the airway with little external sign of injury. Without prompt diagnosis and appropriate treatment, the risk for progressive respiratory failure is high. METHODS: Four children with tracheobronchial injuries were referred to a pediatric trauma center from 1994 to 1997. All children, age 18 months to 13 years, suffered unusual crush injuries. All diagnoses were based on unresolved pneumothorax or pneumomediastinum. RESULTS: Bronchoscopy identified the location of injury as posterior trachea (n = 1) and right mainstem bronchus (n = 2). A tertiary bronchial injury (n = 1) was missed by initial tracheogram and subsequent bronchoscopy but identified during surgical exploration. All children survived after thoracotomy and primary repair of the injury. CONCLUSIONS: Tracheobronchial disruption is a rare, life-threatening injury. Suspicion should be high when pneumomediastinum and pneumothorax are refractory to adequate pleural drainage. Flexible bronchoscopy with intubation distal to the injury may be necessary to prevent loss of the airway. Advance preparation should include setups for bronchoscopy, thoracotomy, and cardiopulmonary bypass. Patient survival depends on preparation and prompt surgical intervention.
Adolescent↗
Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus.
Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.
3T3 Cells↗
Synchronized proliferation induced by 12-O-tetradecanoylphorbol-13-acetate treatment of mouse skin: an in vivo model for cell cycle regulation.
Much of what is known about the mammalian cell cycle comes from studies using established cell lines in culture. In this study, cell cycle-regulatory events were analyzed in vivo after treatment of mouse epidermis with 12-O-tetradecanoylphorbol-13-acetate. A synchronized wave of basal keratinocyte proliferation occurred; over 80% of the cells were in S phase 15 h after treatment. c-myc protein expression was induced, and p57Kip2 protein levels dropped early after stimulation. Before S phase, cyclin D1 and cyclin-dependent kinase (CDK) 6 levels increased, and expression of cyclins E and A was induced. Rb was phosphorylated in late G1, and this correlates with the formation of cyclin D1/CDK4 and cyclin D1/CDK6 complexes. At the end of S phase, the p57Kip2 and p21Cip1 protein levels increased. These findings demonstrate that stimulation of basal epidermal cells by 12-O-tetradecanoylphorbol-13-acetate results in several classic cell cycle events and suggests that p57Kip2 plays a key role in regulating proliferation in the epidermis.
Animals↗
Design of sensitive fluorogenic substrates for human cathepsin D.
Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k(cat)/Km ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.
Cathepsin D↗
Pharmacological regulation of blood glucose levels in non-insulin-dependent diabetes mellitus.
Non-insulin-dependent diabetes mellitus is a metabolic disease that is common and is characterized by insulin insufficiency and resistance. Measures such as body weight reduction and exercise improve the metabolic defects, but pharmacological therapy is the most frequently used and successful therapy. The sulphonylureas stimulate insulin secretion. Metformin and troglitazone increase disposal and decrease hepatic glucose output without causing hypoglycemia. Acarbose is a dietary aid that spreads the dietary carbohydrate challenge to endogenous insulin over time. These pharmacological agents, either alone or in combination, can improve blood glucose regulation in patients with non-insulin-dependent diabetes mellitus.
Acarbose↗
High-affinity binding of the cell cycle-regulated transcription factors E2F1 and E2F4 to benzo[a]pyrene diol epoxide-DNA adducts.
Previous studies indicated that DNA adducts formed by a carcinogenic diol epoxide, 7r,8t-dihydroxy-9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. It was suggested that adduct-induced bends in the DNA were responsible for this behavior. The cell cycle-regulated transcription factor E2F is also known to bend DNA upon binding. When partially purified E2F was tested in a gel mobility-shift assay, binding to a target DNA containing two consensus E2F-binding sites was enhanced by prior modification of the DNA with BPDE. Recombinant human E2F1, E2F4, and DP1 fusion proteins were affinity purified from bacteria expressing these genes. A combination of either E2F1 or E2F4 with their dimerization partner, DP1, gave preparations that exhibited binding to the E2F site-containing DNA fragment. In both cases, the proteins exhibited much higher apparent affinity for BPDE-modified DNA than for unmodified DNA. In addition, BPDE-modified DNA was a better competitor for the binding than unmodified DNA. Heterologous DNA that contained no consensus E2F binding motifs also competed well for E2F binding when modified with BPDE. In contrast, transcription factor that does not bend DNA appreciably (GAL4) did not show enhanced affinity for BPDE-modified DNA. These findings suggest that numerous transcription factors that bend DNA may bind with anomalously high affinity to sequences that contain carcinogen-DNA adducts.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibit the expression and activity of Na+/K(+)-ATPase in FRTL-5 rat thyroid cells.
We recently reported that tumor necrosis factor-alpha (TNF-alpha) induction of the synthesis and secretion of transforming growth factor (TGF)-beta 1 by FRTL-5 cells is a thyroid-stimulating hormone (TSH)-dependent and age-dependent process. TNF-alpha is only cytotoxic to aged (> 40 passages) FRTL-5 cells grown in TSH-containing medium, whereas TGF-beta induces programmed cell death (apoptosis) in epithelial cells but not in FRTL-5 cells, which otherwise retain many properties of normal thyroid follicular cells. This cell line is, therefore, a convenient model for studies on the TSH-dependent and age-dependent inhibitory effects of these cytokines on epithelial cell growth, viability, and function. One prominent effect of TNF-alpha (and TGF-beta 1) on FRTL-5 cell function is suppression of iodide uptake, which is markedly stimulated by TSH. In aged FRTL-5 cells, iodide uptake is only about 10% that of young control cells. Na+/K(+)-ATPase activity, which drives iodide uptake by thyroid cells, is inhibited by TNF-alpha and TGF-beta. The following experiments quantitate the effects of TSH, aging, TNF-alpha, and TGF-beta 1 on the expression and activity of Na+/K(+)-ATPase activity in FRTL-5 cells. Young (< 20 passages) and aged (> 40 passages) FRTL-5 cells were treated with various doses (0-100 ng/ml) of recombinant human TNF-alpha or TGF-beta 1 for various times (0-3 days) with and without 2 U/liter TSH. These treatments reduced the rate-limiting Na+/K(+)-ATPase beta 1 mRNA level and Na+/K(+)-ATPase activity in parallel in a dose-dependent and time-dependent fashion. Aged FRTL-5 cells were more sensitive to the inhibitory effects of TNF-alpha, whereas young cells were more sensitive to the suppressive effects of TGF-beta 1 on the expression and activity of Na+/K(+)-ATPase. We conclude that inhibition of Na+/K(+)-ATPase activity by TNF-alpha and TGF-beta in FRTL-5 cells is differentially affected by aging and that this inhibitory effect can be dissociated from effects on cell viability.
Aging↗
Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences.
Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
On the resurgent population and food debate.
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Agriculture↗
Sulfonylurea binding to a low-affinity site inhibits the Na/K-ATPase and the KATP channel in insulin-secreting cells.
We have used hamster insulinoma tumor (HIT) cells, an insulin-secreting tumor cell line, to investigate modulation of the Na/K-ATPase and of the ATP-sensitive K channel (K(ATP)) by the sulfonylurea glyburide. Membrane proteins from cells cultured in RPMI with 11 mM glucose have at least two glyburide receptor populations, as evidenced by high and low binding affinity constants, (K(d) = 0.96 and 91 nM, respectively). In these cells K(ATP) channel activity was blocked by low glyburide concentrations, IC(50) = 5.4 nM. At 12.5 nM glyburide the inhibition developed slowly, tau = 380 s, and caused reduction of channel activity by 75 percent. At higher concentrations, however, inhibition occurred at a fast rate, tau = 42 s at 100 nM, and was almost complete. Na/K-ATPase activity measured enzymatically and electrophysiologically was also suppressed by glyburide, but higher concentrations were needed, IC(50) = 20-40 nM. Inhibition occurred rapidly, tau = 30 s at 50 nM, when maximum, activity was reduced by 40 percent. By contrast, cells cultured in RPMI supplemented with 25 mM glucose exhibit a single receptor population binding glyburide with low affinity, K(d)= 68 nM. In these cells inhibition of the Na/K-ATPase by the sulfonylurea was similar to that observed in cells cultured in 11 mM glucose, but K(ATP) channel inhibition was markedly altered. Inhibition occurred only at high concentrations of glyburide and at a fast rate; maximum inhibition was observed at 100 nM. Based on these data, we propose that glyburide binding to the high affinity site affects primarily K(ATP) channel activity, while interaction with the low affinity site inhibits both Na/K-ATPase and K(ATP) channel activities. The latter observation suggests possible functional interactions between the Na/K-ATPase and the K(ATP) channel.
Adenosine Triphosphate↗
Cytoskeletal proteins inside human immunodeficiency virus type 1 virions.
We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.
Actin Depolymerizing Factors↗
Cardiovascular and sympathoadrenal responses to heat stress following water deprivation in rats.
This study was designed to characterize the regional and systemic hemodynamic and sympathoadrenal responses to heating after 24 and 48 h of water deprivation in chloralose-anesthetized, male Sprague-Dawley rats (n = 7 per group). Water deprivation produced significant decreases in body weight of 8.1 and 13.7% in the 24- and 48-h groups (P < 0.05), respectively. After water deprivation, rats were exposed to an ambient temperature of 43 degrees C. After correction for body weight differences, heating rates were faster in the 48-h group compared with both euhydrated and 24-h groups. Mean arterial blood pressure (MAP), heart rate, and colonic (Tco) and tail (Ttail) temperatures increased above baseline in all groups during heating. Renal and mesenteric artery blood flow velocities decreased, and vascular resistances increased in response to heating. Compared with euhydrated controls, 48-h water-deprived rats exhibited attenuated pressor (delta MAP = 36 +/- 3 vs. 18 +/- 3 mmHg) and visceral vasoconstrictor (% delta in mesenteric resistance = 122.6 +/- 27.3 vs. 54.9 +/- 6.9%) responses during heating. Tail-skin blood flow estimated from Ttail was also lower at baseline and the onset of heating in water-deprived rats. However, peak Ttail and Tco values were similar across groups. Plasma catecholamines measured in separate groups of rats (n = 6 per group) were significantly higher at baseline and the end of heating in the 48-h group compared with euhydrated and 24-h groups. Despite this exaggerated sympathoadrenal response, the 48-h group exhibited attenuated hemodynamic responses to nonexertional heating compared with euhydrated and 24-h water-deprived rats. These data suggest that cardiovascular and thermoregulatory adjustments can compensate for small changes in hydration state (i.e., 24 h), but more severe levels of hypohydration significantly alter blood pressure and body temperature regulation during heat stress.
Adrenal Glands↗
Low level cyclic adenosine 3',5'-monophosphate accumulation analysis of [des-His1, des- Phe6, Glu9] glucagon-NH2 identifies glucagon antagonists from weak partial agonists/antagonists.
[des-His1, des-Phe6,Glu9]Glucagon-NH2 is a newly designed glucagon antagonist. This analog has a binding IC50 of 48 nM (compared to glucagon IC50 of 1.5 nM) and demonstrates pure antagonism in an adenylate cyclase assay. Although the number of glucagon antagonists has grown rapidly recently, closer examination suggested that many of these antagonists retained very low, almost imperceptible levels of cAMP accumulation that were sufficient to elicit an in vivo biological response. To investigate more carefully this secondary biological signal, we measured cAMP accumulation in a revised assay using isolated hepatocytes in the presence of the phosphodiesterase (PDE) inhibitor Rolipram. The PDE inhibitors Rolipram and isobutyl-1-methylxanthine (IBMX) increased the sensitivity of the cAMP accumulation assay from approximately 10-fold for the native hormone to 35-fold above basal levels. On the other hand, amrinone, another PDE inhibitor, did not affect the cAMP accumulation caused by glucagon. The use of PDE inhibitors indicated that three glucagon analogs that had previously been reported to have strong antagonist properties in classical adenylate cyclase assays were actually weak partial agonists in this new assay system. [N alpha-Trinitrophenyl-His1, homo-Arg12]glucagon, [des-amino-His1,D-Phe4,Tyr5, Arg12, Lys17,18,Glu21]glucagon, and [des-His1,Glu9]glucagon-NH2 demonstrated 233%, 21%, and 5.5% cAMP accumulation relative to the native hormone in the presence of 25 microM Rolipram. On the other hand, [des-His1,des-Phe6,Glu9]glucagon-NH2, a newly designed glucagon antagonist, did not activate adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 microM, indicating that it was a pure antagonist of glucagon-induced adenylate cyclase activity and also the first one in this class. This compound and others were tested in a glycogen phosphorylase assay. As [des-His1,des- Phe6,Glu9]glucagon-NH2 did not activate phosphorylase activity, it was chosen as our candidate for in vivo testing in streptozotocin-induced diabetic rats. An initial dose of 0.75 mg/kg was found to cause the greatest lowering of blood glucose levels (to 63% of the initial levels in 15 min) when the bolus was followed by continuous infusion of 25 micrograms/kgxmin for 1 h.
1-Methyl-3-isobutylxanthine↗
Oral antidiabetic drug use in the elderly.
Non-insulin-dependent diabetes mellitus (NIDDM) is a metabolic disease that is common in the elderly, and is characterised by insulin insufficiency and resistance. Measures such as bodyweight reduction and exercise improve the metabolic defects, but pharmacological therapy is the most frequently used and successful therapy. The sulphonylureas stimulate insulin secretion. Metformin and troglitazone increase glucose disposal and decrease hepatic glucose output without causing hypoglycaemia. Acarbose is a dietary aid that spreads the dietary carbohydrate challenge to endogenous insulin over time. These pharmacological agents, either alone or in combination, should improve blood glucose regulation in patients with NIDDM.
Administration, Oral↗
Regulation of E2F-1 gene expression by p130 (Rb2) and D-type cyclin kinase activity.
Previous experiments have demonstrated that the regulation of E2F-1 transcription factor activity is critical for the maintenance of normal cell proliferation control. Regulation of E2F-1 is accomplished through at least two mechanisms: posttranslational regulation by binding proteins such as Rb and transcriptional regulation of the E2F-1 gene. The E2F-1 gene promoter has recently been isolated to examine this latter aspect of E2F-1 regulation. Preliminary studies demonstrate that the E2F-1 promoter is under E2F-dependent negative control during the cell growth response, being transcriptionally repressed through E2F sites in G0 and early G1. We now demonstrate that the presence of an E2F DNA-binding complex containing the Rb-related p130 protein (Rb2) correlates with E2F-1 gene repression and that overexpression of p130 inhibits transcription from the E2F-1 promoter. Moreover, D-type cyclin-dependent kinase activity specifically activates the E2F-1 promoter by relieving E2F-mediated repression but is inhibited by coexpression of the cdk4 and cdk6 inhibitor p16 (CDKN2, MTS1, INK4). Taken together, these findings suggest that E2F-1 gene expression is controlled during cell cycle progression by a regulatory network involving at least one oncogene (cyclin D1) and several potential tumor suppressor genes.
Animals↗
Muscle sympathetic nerve activity during postural change in healthy young and older adults.
Recent evidence suggests that during orthostatic stress the reflex increase in muscle sympathetic nerve activity may be diminished in older adults. To test this hypothesis, we measured muscle sympathetic nerve activity, plasma noradrenaline concentrations, heart rate, and arterial blood pressure in twelve young (mean, 25 years; range, 19-29 years) adults and 14 older (mean 64 years; range, 60-74 years) healthy adults, while supine and during upright sitting. Supine control levels of muscle sympathetic nerve activity were higher in the older subjects (35 +/- 1 vs. 25 +/- 1 bursts/min, p < 0.05), but there were no differences in plasma noradrenaline concentrations, heart rate or arterial pressure. Despite higher supine control levels in the older group, the absolute unit increases in muscle sympathetic nerve activity in response to upright sitting (p < 0.05 vs. control) were not different in the two groups (7 +/- 1 vs. 7 +/- 1 bursts/min), nor were the increases in plasma noradrenaline concentrations. Heart rate did not increase above supine control in response to sitting in either group. Arterial pressure increased slightly (p < 0.05, supine vs. control), but there were no age-related differences. These results indicate that, contrary to recent findings, the reflex increases in muscle sympathetic nerve activity and plasma noradrenaline concentrations and regulation of arterial pressure during this natural orthostatic stress are well preserved in older healthy men and women.
Adult↗