Cysteinyl-leukotrienes induce vascular endothelial growth factor production in human monocytes and bronchial smooth muscle cells.

BACKGROUND: Cysteinyl leukotrienes (cysLTs) are suggested to be implicated in the process of airway remodelling in asthma. OBJECTIVE: We investigated the potential for cysLTs to modulate vascular endothelial growth factor (VEGF) expression, a growth factor involved in the angiogenesis of airway remodelling. METHODS: VEGF mRNA and protein were quantified by real-time PCR and ELISA, respectively. VEGF promoter activation was assessed using luciferase gene-tagged promoter constructs. RESULTS: We found that LTD(4) induction of VEGF in human monocytes and bronchial smooth muscle cells is cysLT1 dependent. Stimulation of HEK293 cells stably expressing cysLT1 or cysLT2 with cysLTs showed a concentration-dependent activation of the VEGF promoter and a time-dependent increase in VEGF mRNA and protein. For the cysLT1-mediated response, mutations of hypoxia-induced factor-1 (HIF-1) sites failed to reduce cysLT-induced VEGF promoter activation and 5' deletions showed that the proximal region containing one AP-1 and four specificity protein 1 (Sp1) sites was necessary. Pretreatment with inhibitors of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not p38, and an overexpression of dominant negative forms of c-Jun, c-Fos or Ras suggested the implication of mitogen-activated protein kinases and AP-1. Mutation of the AP-1-binding element failed to prevent VEGF transactivation suggesting that AP-1 might not act directly on the promoter. Moreover, inhibition of Sp1-dependent transcription by mithramycin completely inhibited VEGF promoter transactivation and VEGF mRNA expression by LTD(4) . Finally, mutations of Sp1 binding elements prevented VEGF promoter transactivation. CONCLUSION AND CLINICAL RELEVANCE: Our data indicate for the first time that cysLTs can transcriptionally activate VEGF production via cysLT1 receptors, with the involvement of JNK, ERK, the AP-1 complex and Sp1. These findings suggest that cysLTs may be important in the angiogenic process of airway remodelling and potentially provide a previously unknown benefit of using cysLT1 receptor antagonists in the prevention or treatment of airway remodelling in asthma.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

The presence of two cytochrome P450 aldosterone synthase mRNAs in the hamster adrenal.

We isolated a cDNA from a hamster adrenal cDNA library which was similar in sequence to those of the mouse and rat P450c18 cDNAs. The hamster P450c18 cDNA, however, was shorter than the rat and mouse P450c18 cDNAs at its 5'-end and the peptide leader sequence was absent. From a hamster genomic library we isolated and sequenced the first seven exons and a 5'-flanking region of the first P450c18 gene exon. With this information we were able to generate a P450c18 cDNA containing the peptide leader sequence using the polymerase chain reaction. Northern analyses were performed on adrenals from hamsters maintained on a low sodium diet for 0, 4, 7 and 10 days using a 32P-labeled sequence specific to P450c18; two mRNA bands were found at 2 and 3.4 kb. The intensity of both bands was increased about 3- to 5-fold under sodium restriction compared to controls. A distinct mRNA band of 2.3 kb hybridized with an oligonucleotide specific to P450(11) beta and its intensity did not change following low sodium intake. Immunoblotting analyses were performed using an antibovine adrenal P450(11) beta antibody that does not discriminate between P450(11) beta and P450c18 proteins. Three bands were detected at 52, 48 and 45 kDa in homogenate preparations of entire glands. Furthermore, the 45 kDa protein band was present in homogenates of the zona glomerulosa and absent in homogenates of the zone fasciculata-reticularis. In conclusion, these results show that the hamster adrenals express P450c18 as do mouse, rat and human adrenal glands. Furthermore, two P450c18 mRNAs, which are inducible by a low sodium intake, are present in the hamster adrenal vs one for the rat. The physiological role of these two hamster adrenal mRNA species remains to be elucidated.

The hamster adrenal cytochrome P450C11 has equipotent 11beta-hydroxylase and 19-hydroxylase activities, but no aldosterone synthase activity.

We have isolated a hamster adrenal P45OC11 cDNA which shared 90 and 84% homology, respectively, with the nucleotide sequence and the amino acid sequence of the hamster adrenal P450aldo. Both P450C11 and P450aldo cDNA coding sequences were inserted in the plasmid pBluescript SK, transcribed and then translated using a rabbit reticulocyte system in the presence of [35S]methionine. The reaction products were immunoprecipitated with an anti-bovine P450C11 antibody for P450C11 and with an anti-hamster P450aldo for P450aldo. Immunoprecipitated proteins were analyzed by polyacrylamide gel electrophoresis. A single 35S-labeled protein band was detected for P450C11 and for P450aldo, respectively. P450C11 and P450aldo cDNAs were then both inserted into the expression vector pCMV5 containing a viral sequence specific for the attachment of ribosomes to mRNA. These constructions were transfected in COS-1 cells. 24 h after transfection, the presence of P450C11 and P450aldo mRNAs was determined by Northern blot analysis. In a time study experiment we found that P450C11 transformed the labeled-steroid into [14C]corticosterone, [14C]19-OH-deoxycorticosterone and [14C]18-OH-deoxycorticosterone in ratios of 1:1.11:0.07, after 2 h of incubation; no [14C]aldosterone could be detected. Cells transfected with plasmids harboring the P450aldo cDNA transformed [14C]deoxycorticosterone to [14C]corticosterone, [14C]aldosterone, [14C]18-OH-corticosterone, [14C]18-OH-deoxycorticosterone, [14C]19-OH-deoxycorticosterone and [14C]11-dehydrocorticosterone in ratios of 1:0.25:0.45:0.04:0.04:0.04 after 12 h of incubation. These results indicate that one P450 catalyzes the ultimate step of glucocorticoid formation and a separate P450 is involved in the final steps of aldosterone formation in hamster adrenals. The capacity of the hamster adrenal P450C11 to hydroxylate at positions 11beta and 19 in nearly equal ratio makes this animal an excellent model to study the mechanism of synthesis and inhibition of 19-OH-deoxycorticosterone, the precursor of 19-nor-deoxycorticosterone, a very potent mineralocorticoid involved in the development of essential hypertension.

Ontogeny of epidermal growth factor (EGF), EGF receptor (EGFR) and basic fibroblast growth factor (bFGF) mRNA levels in pancreas, liver, kidney, and skeletal muscle of pig.

Epidermal growth factor (EGF), EGF receptor (EGFR), and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) levels were examined by Northern blot analysis in four tissues (pancreas, liver, kidney, and skeletal muscle) of pig from fetal 90 d to postnatal 180 d of age. The present study shows for the first time that EGF mRNA increased with advancing age in the kidney and skeletal muscle of pig. A high level of EGF mRNA was observed in the kidney compared with the liver and skeletal muscle. In the pancreas, high levels of EGF mRNA were found in fetuses and newborns and were low in older pigs. Pancreatic EGFR mRNA level parallelled its EGF mRNA, whereas in the kidney and skeletal muscle, patterns of EGFR mRNA were reversed to their EGF mRNA levels. In the liver, EGFR mRNA was abundant but EGF mRNA was undetected. In the pancreas and skeletal muscle, the highest levels of bFGF mRNA were found in fetuses of 90 d of age and then decreased with advancing age. In the liver and kidney, there were no major changes in bFGF mRNA levels during the examined developmental periods. These results show that EGF, EGFR, and bFGF mRNA levels are developmentally and tissue specifically regulated in pig. In the pancreas, mRNA levels of EGF, EGFR and bFGF were high in fetal and neonatal life and low thereafter. In the kidney and skeletal muscle, EGF mRNA increased with advancing age. EGF may play a role in muscle growth and maintenance in growing pigs during the later stage of development.

Herbal preparations: a primer for the aeromedical physician.

PURPOSE: This study was an effort to identify the botanical preparations of potential risk to the aviator and aviation safety, and to ascertain whether aviators are using dietary supplements despite extensive educational efforts discouraging over-the-counter medication use. Herbal preparations may be used by nearly 20% of the adult population. Although the aviator population may be presumed to use them as well, the actual degree of use among aviators is unknown. Use of such substances as health promotion or therapeutic agents may provide health benefits, but may also carry risk. Military and civilian aviators are not currently required to disclose such use, nor are examiners obligated to inquire or counsel aviators about them. This paper examines the trends in post-mortem toxicological samples suggesting botanical preparation use, and develops a rational method for determining suitability for use by the aviator. METHOD: The toxicological test results from 3177 mishap pilots performed at the Civil Aeromedical Institute from 1989-1997 were examined for the presence of substances suggesting botanical preparation use. The prevalence of positive test results for ephedrine among mishap pilots was compared with the prevalence of tests positive for chemically and biologically similar non-botanical substances among mishap pilots. A review of existing literature was also performed to identify substances posing possible risk to the aviator health or aviation safety. RESULTS: Ephedrine was found to be the only substance routinely screened on toxicological specimens that was suitable for association with botanical substance utilization. The percent of specimens positive for ephedrine increased three- to four-fold while the percent of specimens positive for similar non-botanical substances decreased overall. The literature revealed sufficient evidence that a number of open market botanical agents are capable of causing incapacitation by cardiovascular or neuropsychiatric mechanisms, yet are legally permitted for use by aviators. CONCLUSION: Aviators are using botanical products with increasing frequency, and many of those substances may pose significant risk of incapacitation, altered sensorium, or adverse health effects. The flight surgeon must be diligent in eliciting a history of use and assisting aviators to minimize personal risk and risks to flight safety. A rational approach to assessing risk is presented.

Survival at high altitudes: wheel-well passengers.

There have been 10 specific "wheel-well" passenger stowaway flights (the wheel-well area was entered just before take-off) documented in the N.Y. Times between 1947-1993. Five stowaways survived flights encompassing altitudes as high as 39,000 feet, with six dying in the process (one flight had two stowaways, one fatal, one surviving). Three Douglas DC-8 and four Boeing 707 aircraft, plus a Caravelle, an unknown jet, and a piston airliner were involved. Several of the wheel-well flight stowaways were reportedly politically motivated to attempt these international flights. This paper describes the unpressurized flight environment and the physiology that enabled human survival under conditions of extreme hypoxia and cold, inducing a virtual "hibernative" state. It is likely that similar attempts will continue, and alert airport security preventive measures are indicated.

Abnormal glucose levels found in transportation accidents.

BACKGROUND: The Federal Aviation Administration's Office of Aerospace Medicine is responsible for the certification of pilots with diabetic conditions. The present study evaluated the use of postmortem vitreous humor and urine glucose levels in transportation accident fatalities as indicators of potentially incapacitating medical conditions or performance impairment. METHODS: Vitreous humor and/or urine from 192 accident fatalities were analyzed for glucose using a hexokinase method. Cases with values below the lower limit of detection (10 mg x dl(-1)) and above 3 standard deviations (SD) from the mean were not included in the final statistics. All cases more than 5 SD above the mean were deemed abnormal and a full case history was evaluated based on the available medical history. RESULTS: The mean vitreous humor glucose concentration was 30+/-21 mg x dl(-1) (N=98), while it was 27+/-16 mg x dl(-1) in urine (N=127). Of the 192 cases, 9 were identified as having abnormal glucose levels. Abnormal glucose levels were found in 5 of the 8 cases with a known diabetic condition. Glycosuria or low renal threshold was reported in 2 fatal pilots; 1 of these pilots had an abnormal glucose level. CONCLUSIONS: Hyperglycemia can be established from the vitreous humor and urine glucose levels. All of the abnormal glucose cases detected were previously identified during the medical certification process or had a medical reason for the abnormal level. Elevated vitreous humor and urine glucose levels have proven useful in identifying individuals with a pre-existing diabetic condition that might have been a factor in the accident.

Monovision contact lens use in the aviation environment: a report of a contact lens-related aircraft accident.

BACKGROUND: The use of contact lenses to satisfy the distance visual acuity requirements for obtaining a civil airman medical certificate has been permitted since 1976. According to the Federal Aviation Administrations Guide for Aviation Medical Examiners, the use of monovision contact lenses is not considered acceptable for aviation duties. METHODS: A report is presented using information from a National Transportation Safety Board (NTSB) aircraft report (NTSB/AAR-97/03) of a nonfatal accident on a scheduled airline flight. Past studies on the use of contact lenses in the aviation environment are reviewed. RESULTS: On October 19, 1996, a McDonnell Douglas MD-88 aircraft, Delta Airlines Flight 554, was substantially damaged in an undershoot approach while landing at LaGuardia Airport, Flushing, New York. The approach, with less than favorable weather conditions, was over water to Runway 13 and the flight crew transitioned to visual references just above the decision height. During continued descent, the plane struck an approach light structure and the end of the runway deck, shearing off the main landing gear and sliding 2,700 feet down the runway. CONCLUSIONS: The NTSB determined that the probable cause of this accident was the inability of the pilot to overcome his misperception of the airplane's position relative to the runway, due to the use of monovision contact lenses. The adverse effects of wearing contact lenses in the aviation environment are discussed.

Ontogeny of IGFs and IGFBPs mRNA levels and tissue concentrations in liver, kidney and skeletal muscle of pig.

As far as we know, there is no available information about ontogenic changes of tissue concentrations of IGF-I and II and IGFBPs in large mammals. Serum, liver and kidney levels of IGFs and IGFBPs were examined in fetuses at 90 and 110 days of gestation and in pigs at 1d, 3 wk, 3 mo and 6 mo of age. Ontogeny of mRNA levels of IGFs, IGF type I and type II receptors (IGFI-R and IGFII-R), IGFBP-1 and -3 (IGFBPs) and growth hormone receptor (GHR) were also examined by Northern blot analysis in liver, kidney and skeletal muscle of pig. Serum IGF-I, IGF-II and IGFBP-3 concentrations were low during the fetal life and increased after birth. The highest level of IGF-II mRNA was found in fetuses for all studied tissues. In the liver, IGF-I mRNA level and its protein content peaked at 3 wk of age. The highest IGF-II concentration was found at 1d and 3 wk of age. The IGFII-R mRNA remained at a constant level during the whole development period. The most abundant IGFBP-1 mRNA and its protein content were found at birth. The level of IGFBP-2 was high during fetal and early postnatal life. The IGFBP-3 content was relatively low in fetuses and reached the highest level after 3 wk of age. In the kidney, IGFs, IGFBP-3, IGFI-R and IGFII-R as well as GHR mRNA levels were relatively high during the fetal and early postnatal life. The IGFs concentrations were the highest in newborns. In the skeletal muscle, IGFs, IGFBP-3 and IGFI-R mRNA levels decreased with advancing age. During the postnatal life, the high IGFs concentrations in the liver and the kidney correspond to fast growth periods of these organs.

Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of alpha-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides.

The genome of Saccharomyces cerevisiae contains five genes that encode type II transmembrane proteins with significant amino acid similarity to the alpha-1,3-mannosyltransferase Mnn1p. The roles of the three genes most closely related to MNN1 were examined in mutants carrying single and multiple combinations of the disrupted genes. Paper chromatographic analysis of [2-3H]mannose-labeled O-linked oligosaccharides released by beta-elimination showed that the MNT2 (YGL257c) and MNT3 (YIL014w) genes in combination with MNN1 have overlapping roles in the addition of the fourth and fifth alpha-1,3-linked mannose residues to form Man4 and Man5 oligosaccharides whereas MNT4 (YNR059w) does not appear to be required for O-glycan synthesis.

Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae.

The yeast actin cytoskeleton is polarized during most of the cell cycle. Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton. Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization. In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome. To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton. Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization. Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton. We conclude that depolarization of the actin cytoskeleton is a specific phenotype.