Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells.

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.

Effects of thapsigargin, an intracellular calcium-mobilizing agent, on synthesis and secretion of cartilage collagen and proteoglycan.

The calcium-mobilizing agents thapsigargin and 2,5-di-(tert-butyl)-1,4- benzohydroquinone were shown to markedly elevate the intracellular calcium concentration of chick embryo chondrocytes in a dose-dependent manner. Under these conditions, the metabolism of macromolecules was variably affected. The synthesis and secretion of protein in general, and of collagen in particular, were significantly inhibited; in contrast, proteoglycan synthesis (but not glycosaminoglycan synthesis) was inhibited, whereas secretion was unaffected. Flunarizine, which prevented the thapsigargin-induced intracellular calcium elevation, and EGTA, which caused only a transient thapsigargin-induced intracellular calcium elevation, did not reverse these alterations. It was concluded, therefore, that the observed effects of thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone on chondrocyte macromolecule metabolism were not related to the ability of these drugs to increase the cytosolic free calcium concentration but may have been due to the specific depletion of the calcium sequestered in the endoplasmic reticulum. The differential effect of these drugs on protein and proteoglycan secretion suggests that the intracellular trafficking of these two classes of macromolecules may be controlled independently.

Reducing medication errors: potential benefits of bolus thrombolytic agents.

A recent Institute of Medicine report highlighted the high incidence of medical errors in clinical practice, and the important fact that errors are associated with increased mortality. The administration of thrombolytic therapy for acute myocardial infarction is a particularly high-risk situation for emergency physicians. The combination of extreme time pressure with a narrow "therapeutic window" increases the potential for adverse outcomes due to dosing errors. Numerous trials have found that the dose of thrombolytic therapy is closely related to outcomes, with too low a dose associated with lower rates of infarct-related artery patency and higher doses associated with increased bleeding and intracranial hemorrhage. In the GUSTO-I trial, 13.5% of patients treated with streptokinase and 11.5% of patients treated with tissue plasminogen activator (t-PA) had a medication error (i.e., incorrect dose or infusion length). Most importantly, 30-day mortality was significantly higher in patients with medication errors: for t-PA dosing errors mortality was 7.7% vs 5.5% for patients who received the correct t-PA dose (p < 0.001), with similar findings for streptokinase. More recent data from the InTIME2 trial and other studies showed that use of a bolus thrombolytic agent reduced the rate of medication errors. Thus, use of the simpler bolus thrombolytic agents may reduce emergency department medication errors, and thus improve overall clinical outcome.

Psychosis.

Psychosis is the term used to describe a mental state of dysfunction in behavior and thought processes. In patients with psychoses, mental capacity is grossly distorted and thought is disorganized. These factors cause an inability to recognize reality or relate to others in a meaningful way. A medical cause is found in approximately 20% of patients with acute psychosis. Emergency medicine physicians must differentiate psychotic symptoms caused by general medical conditions from psychosis caused by a primary psychiatric disorder. A careful evaluation must be performed to identify the cause of the psychosis. Correction of reversible causes, sedation, and appropriate disposition are the keys to the successful management of these patients.

Piriform sinus perforation during Esophageal-Tracheal Combitube placement.

The Esophageal-Tracheal Combitube is a new alternative airway device. Few complications of its use have been reported. This article reports a case of a 71-year-old female with angioedema of the tongue and airway obstruction who suffered piriform sinus rupture during Combitube placement by prehospital personnel, resulting in massive subcutaneous emphysema. Caution is required when using this device in all but the most controlled situations.

The effect of cocaine and amphetamines on vital signs in trauma patients.

The object of this study was to determine if the presence of sympathomimetics (cocaine, benzoylecgonine, or amphetamine), detected by routine urine toxicology screen, affect the initial presenting pulse, blood pressure, respiratory rate, or level of consciousness in trauma patients. A retrospective chart review was performed on 1,679 patients enrolled in an urban level 1 trauma registry between October 1987, and July 1992, for whom complete toxicology data were available. There were no clinically significant differences in the vital signs of patients with positive toxicology screens and those with negative screens. There was a statistically significant increase in prehospital respiratory rate among patients with benzoylecgonine or amphetamines on admission toxicology screens when potential confounding factors were controlled. However, these effects disappeared upon arrival at the hospital. The detection of sympathomimetics by toxicology screening had no effect on pulse and systolic blood pressure when age, sex, mechanism, type, and severity of injury, prehospital IV fluid volume, and alcohol intoxication were controlled.

Isolation and partial characterization of precursors to minor cartilage collagens.

Suspension cultures of cartilage cells were prepared from 17-day chick embryo sterna and radiolabeled with [14C]-proline under conditions which sought to minimize proteolytic conversion of procollagen to collagen. Collagenous proteins were isolated from the culture medium and cell fraction, were purified in their native state by (NH4)2SO4 precipitation and DEAE-cellulose chromatography, and were characterized by protease susceptibility, SDS-gel-filtration and SDS-polyacrylamide gel electrophoresis. Qualitatively, the precursor components present in the medium were similar to those in the cell extract; quantitatively, it appeared that the minor cartilage collagen precursor components derived from 1 alpha, 2 alpha, 3 alpha and type IX collagens were more prevalent in the cell extract. SDS-PAGE of unreduced samples showed that precursors to both of these collagens migrated as distinct high-molecular-weight aggregates. After chymotrypsin digestion, unreduced type IX collagen migrated as two disulfide-bonded aggregates--a large one (Mr approximately 210K) and a small one (Mr approximately 43K); whereas 1 alpha, 2 alpha, 3 alpha chains migrated identically whether reduced or unreduced. Reduction of undigested type IX aggregate yielded two components of Mr approximately 97K and 78K; whereas reduction of the chymotrypsin resistant 210K and 43 K aggregates gave a single component of Mr approximately 61K and a component which migrated at the dye front, respectively. The molecular origin of these components was confirmed by differential NaCl precipitation. It was concluded that this culture system synthesized precursors to 1 alpha, 2 alpha, 3 alpha and type IX collagens in addition to type II; type X collagen was not detected even though the 17-day sternum contained a population of cells morphologically similar to hypertrophic chondrocytes. The precursor chains to 1 alpha, 2 alpha, 3 alpha collagen had an apparent Mr greater than pro-alpha (II) and could be isolated as a disulfide-bonded aggregate(s); the precursor chains to type IX collagen had an apparent Mr less than pro alpha (II) and could also be isolated as a disulfide-bonded aggregate. All of the cartilage collagen precursors had protease-susceptible regions, but those in type IX appeared to be more sensitive to pepsin than to chymotrypsin.

Incorporation of hydroxyeicosatetraenoic acids into cellular lipids of adrenal glomerulosa cells: inhibition of aldosterone release by 5-HETE.

Metabolites of arachidonic acid appear to be involved in the regulation of aldosterone secretion. Adrenal cells metabolize arachidonic acid to several products including hydroxyeicosatetraenoic acids (HETEs). Since HETEs may be incorporated into the membrane lipids in some cells, we investigated whether HETEs were incorporated into lipids of adrenal glomerulosa cells and tested the influence of incorporation on aldosterone secretion. Cells were incubated with [3H] -arachidonic acid, -5-HETE, -12-HETE, -15-HETE or -LTB4. The cellular lipids were extracted and analyzed by TLC. Arachidonic acid was incorporated into all of the cell lipids with greatest accumulations in phospholipids (22%), cholesterol esters (50%), and triglycerides (21%). Uptake was maximal by 30 min. 5-HETE was incorporated into diglycerides and monoglycerides but not into phospholipids or other neutral lipids. The uptake followed a similar temporal pattern as arachidonic acid. 12-HETE was incorporated to a small extent into phospholipids, predominantly phosphatidylcholine. Neither 15-HETE or LTB4 were associated with cellular lipids. Angiotensin increased the uptake of 5-HETE and arachidonic acid into phosphatidylinositol/phosphatidylserine without altering uptake into the other lipids. When cells were pretreated with 5-HETE and washed to remove the unesterified HETE, basal aldosterone release as well as release stimulated by angiotensin, potassium and ACTH were significantly reduced. 15-HETE, which is not incorporated into cellular lipids, was without effect on aldosterone secretion. These studies indicate that 5-HETE may be incorporated into the cellular lipids of adrenal cells and may modulate steroidogenesis.

Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices.

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2'-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.

Post-translational alterations in newly synthesized cartilage proteoglycans induced by the glutamine analogue 6-diazo-5-oxo-L-norleucine. Time course of inhibition and recovery.

Incorporation of [35S]sulphate by cultures of matrix-free cells from chick embryo sterna in the presence of the glutamine analogue 6-diazo-5-oxo-L-norleucine (0.58 mM) was inhibited in a time-dependent manner to less than 15% of that in control cultures after 2 h. Characterization of the major cartilage proteoglycan synthesized under these conditions showed that it contained few, if any, normal-sized chondroitin sulphate chains and only about half of the normal complement of substituted serine residues. Subsequent addition of D-glucosamine hydrochloride (final concn. 2 mM) resulted in a time-dependent recovery of [35S]sulphate incorporation to 90% of control cultures after 2 h, but restored the chondroitin sulphate chains to normal size within 15 min. On the basis of these results, it is concluded that a 2 h preincubation is necessary to deplete the chondrocytes of the endogenous supply of UDP-N-acetyl-D-glucosamine required for optimal glycoconjugate synthesis, and that this situation results in the synthesis of a chondroitin sulphate proteoglycan with significantly altered properties, owing to the paucity of glycosaminoglycan chains; however, this condition is completely reversible if the D-glucosamine pool is repleted.

Emergency physicians and biological terrorism.

Recent developments, such as the bombings of the World Trade Center in 1993 and the Alfred P. Murrah Federal Building in Oklahoma City in 1995, the sarin attacks in Tokyo and Matsumoto, Japan, and US Embassy bombings in Kenya and Tanzania in 1998, have heightened fears of terrorist attacks. Future terrorist activities will continue to involve bombs and firearms, but may also include weapons of mass destruction, including biological agents. Recent US government initiatives have recognized the threats to our country from these weapons and have funded planning and response programs. These preparedness programs are being built on existing infrastructure of EMS and fire services' plans for hazardous materials response. Appropriate emergency department and hospital response, guided by public health principles, could significantly limit the morbidity and mortality of biological warfare agents. Inappropriate response by the medical community may worsen a chaotic and potentially devastating situation. This article discusses planning and response issues central to a potential bioterrorism event.

Mechanical stretch induces platelet-activating factor receptor gene expression through the NF-kappaB transcription factor.

In this study, we used the platelet-activating factor (PAF) receptor gene as a model of a mechano-sensitive gene to investigate how mechanical stimuli regulate gene expression and cell function. We utilized a culture system of pulmonary artery smooth muscle cells and a well-defined in vitro mechanical device that imparts an equibiaxial strain repeatedly to cells attached to an elastomeric membrane. Northern blot and immunohistochemical analyses revealed increased PAF receptor expression at both the mRNA and protein levels after 1 h exposure of the cells to a 5% strain at a frequency of 1 Hz. To investigate the mechanism of activation of this gene by stretch, we performed transfection experiments with a luciferase reporter gene linked to segments of the 5' flanking region of the receptor gene promoter. Expression of the transfected reporter gene bearing a 1.1-kb fragment of the promoter was enhanced in mechanically stretched cells indicating a direct effect on transcriptional activity. When truncated to leave the nucleotides between -610 to +27, the promoter-reporter construct lost stretch inducibility suggesting that the region between -1099 and -610 was required for stretch responsiveness. This region contains four copies of NF-kappaB binding sites. These elements are in close proximity to one another and can form a complex with nuclear proteins derived from stretched cells as demonstrated by gel mobility shift assay. Moreover, in experiments using cycloheximide, we found that de novo protein synthesis was not necessary for the induction of the PAF receptor gene expression by mechanical stretch. Conversely, preincubation of the cells with protein kinase C inhibitors suppressed mechanical stretch-induced PAF receptor gene expression at the mRNA levels and abrogated upstream events of NF-kappaB activation in the cytoplasm. These data strongly suggest that stretch-induced PAF receptor gene expression is mediated by NF-kappaB binding to the PAF receptor gene promoter and that protein kinase C activation is among the molecular features of NF-kappaB activation and translocation into the nucleus in mechanically stretched cells.

The effects of 6-diazo-5-oxo-L-norleucine, a glutamine analogue, on the structure of the major cartilage proteoglycan synthesized by cultured chondrocytes.

Incubation of embryonic chick chondrocytes with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, led to a dose-dependent inhibition of [35S]sulfate incorporation into proteoglycan. In the absence of exogenous L-glutamine, a maximal inhibition of 50-60% was achieved with DON concentrations greater than or equal to 1 microgram/ml (6 microM); the ED50 was approximately 0.2 microM. This inhibitory effect could be partially restored by the addition of 100-fold molar excess of either exogenous L-glutamine or M-glucosamine. The quantitative changes were due neither to inhibition of protein core synthesis nor to undersulfation of glycosaminoglycan chains. Rather, the proteoglycan synthesized in the presence of DON contained substantially fewer (approximately 50% of control) and smaller (10-15% of control, on the average) chondroitin sulfate chains as well as a paucity of keratan sulfate chains. The result of these structural changes was a proteoglycan with significantly lower molecular weight, buoyant density, and anionic charge. In spite of these modifications, the altered proteoglycan synthesized in the presence of DON was secreted normally and retained the ability to interact with exogenous hyaluronic acid and link proteins. The results of our experiments also indicate that DON substantially diminished the pool of hexosamine precursors required for glycosaminoglycan synthesis. We conclude that this decrease was responsible for the molecular alterations described above; and these, in turn, can account for the morphological changes previously seen in cartilage matrix synthesized in the presence of DON.