Heterogeneity of 125I-labeled epidermal growth factor.

Highly purified epidermal growth factor (EGF) was iodinated, and the labeled product with the same isoelectric point as underivatized EGF was isolated by isoelectric focusing. When the 125I-labeled EGF was analyzed by reverse-phase chromatography, the resulting profile of 125I activity was much broader than the profile obtained with underivatized EGF. Rechromatography of 125I-EGF fractions indicated that our highly-purified labelled EGF was indeed heterogeneous. Analysis of each HPLC column fraction demonstrated that degradation of EGF had not occurred. The column fractions containing 125I-EGF were pooled into five groups for analysis of cell binding characteristics. Scatchard plot analysis of the five 125I-EGF pools revealed markedly different binding behaviors. In contrast, they had equal potency in stimulating DNA synthesis, within the sensitivity of our assay. Specific activity measurements indicated that the five HPLC pools of 125I-EGF had varying numbers of 125I atoms per EGF molecule. The heterogeneity of the highly purified 125I-EGF and the binding characteristics of the 125I-EGF subfractions pose serious implications for all workers who use iodinated ligands for receptor binding studies.

Okadaic acid induces transcription of junB through a CCAAT box and NF-Y.

The shellfish toxin, okadaic acid (OA), is a potent tumor promoter that induces expression of the proto-oncogene junB in mouse keratinocyte 308 cells. Here we show, through deletion analysis of the junB promoter, that sequences near the TATA box conferred transcriptional induction by OA. Transient transfections of luciferase constructs bearing the junB promoter with single mutations in various cis elements demonstrated that a promoter containing a mutated CCAAT box could not be induced by OA. When this CCAAT box was inserted into a heterologous promoter construct, OA induction was dependent on an intact CCAAT box. Flanking cis elements located near the CCAAT box, although not required for OA inducibility, did play a role in the basal level of transcription. NF-Y was shown by EMSA to bind to the CCAAT box. OA induction from the junB CCAAT box was blocked by dominant negative NF-YA as well as the CCAAT box-dependent anticancer drug, ET-473. Expression of a lexA/NF-YA chimeric protein demonstrated that OA induction was dependent on the binding of NF-Y family members. These studies demonstrate that OA can mediate transcriptional activation of junB through the classical CCAAT box and that transcription factor NF-Y plays a functional role in the induction.

PCR/RFLP assay for copy number of mutant and wild-type alleles.

A PCR method for quantitating the copy number of mutant vs. wild-type alleles in DNA from cell lines is described. The assay can be used to detect a point mutation in any gene that creates or destroys a restriction site. The alleles of interest are amplified by nested PCR and labeled in a second round of PCR. The product is digested with a restriction enzyme specific for that site, resolved on a non-denaturing gel and quantified by phosphor imaging techniques. Cell types with known numbers of wild-type and mutant alleles of c-Harvey-ras are used to validate the assay. The method is then applied to a cell line, homogygous for the mutation, to determine the gene copy number. The applicability of the method to other genes is shown using the matrix metalloproteinase gene, matrilysin. A cell line transfected with a plasmid carrying a mutant, auto-activated form of the gene is compared to its parent cell line. Advantages of this technique compared with Southern analysis are ease of screening a large number of clones or foci and accuracy of quantitation.

Expression of metalloproteinase genes in human prostate cancer.

Twenty-five surgical specimens of malignant human prostate, 3 lymph nodes with metastatic prostate carcinoma, 11 normal human prostates, as well as 3 human prostate cell lines (DU-145, PC3 and LNCaP) were examined for the expression of the human matrix metalloproteinase-7 gene (MMP-7) from the human collagenase family (originally called PUMP-1 for putative metalloproteinase-1) [Quantin et al. (1989) Biochemistry 28:5327-5334; Muller et al. (1988) Biochem J 253:187-192; Matrisian and Bowden (1990) Semin Cancer Biol 1:107-115]. Northern blots were prepared using total RNA extracted from 18 prostate adenocarcinomas, 2 lymph nodes with metastatic prostate carcinoma and 11 normal human prostates. When the northern blots were hybridized with a 32P-labeled MMP-7 cDNA probe, a 1.2-kb mRNA was detected in 14 out of 18 prostate adenocarcinomas, 1 out of 2 metastatic lymph nodes, and 3 out of 11 normal prostates. The 3 human prostate cell lines did not show any evidence of the MMP-7 transcript. In situ hybridization was conducted to localize the MMP-7 mRNA to individual cells using a 35S-labeled MMP-7 cRNA. In situ hybridization was carried out on snap-frozen tissue sections of 7 prostate adenocarcinomas and 3 lymph nodes containing metastatic prostate adenocarcinoma using the same tissues previously probed by northern analysis as well as new samples. In situ hybridization revealed that the MMP-7 gene was expressed in the epithelial cells of primary prostate adenocarcinoma as well as in invasive and metastatic cells. MMP-7 expression was also seen focally in some dysplastic glands but not in stroma. Additional northern blot analysis was performed using probes to human type-IV collagenase, type-I collagenase and stromelysin I in human prostate adenocarcinoma as well as normal prostate tissue. Our results indicated that 6 out of 10 adenocarcinoma samples and none of the 4 normal samples were positive for type-IV collagenase transcripts. Tissue samples were also examined for the expression of type-I collagenase (9 adenocarcinomas and 4 normal) and stromelysin I (13 adenocarcinomas) by northern analysis. None of the tissues was found to express the transcripts of interest at detectable levels. These data suggest that certain metalloproteinases are present in prostatic adenocarcinoma and may play a role in invasion and metastasis.

Analgesic comparison of propiram fumarate with pentazocine, codeine, and placebo in postsurgical pain.

The safety and effectiveness of a single oral dose of 50 mg propiram fumarate as an analgesic was compared in a double-blind clinical trial trial against single doses of standard reference analgesics (50 mg pentazocine hydrochloride or 60 mg codeine sulfate) or placebo. Subjects were adult patients experiencing severe postsurgical pain. Mean pain scores and SPID scores showed all three active drugs to be favored (P less than 0.05) over placebo in patients with severe initial pain. The most common side effects seen were drowsiness, nausea, and dizziness. These were not severe enough to require treatment. Propiram fumarate (50 mg) was shown to be an effective and safe analgesic in the treatment of severe postsurgical pain.

Clinical investigation of the analgesic potency and respiratory depressant activity of fentanyl, a new narcotic analgesic.

A controlled clinical comparison of fentanyl and morphine in 75 postsurgical patients is presented. In addition, the ability of the two drugs to produce respiratory depression was determined in six healthy volunteers. The results of these studies indicate that 0.2 mg. fentanyl intramuscularly is equianalgesic with 10 mg. morphine intramuscularly, and it would appear that there is no advantage in the use of fentanyl for the relief of postsurgical pain.

Intracellular processing of epidermal growth factor. II. Intracellular cleavage of the COOH-terminal region of 125I-epidermal growth factor.

Epidermal growth factor (EGF) undergoes a specific series of alterations during the course of its binding and internalization into cultured fibroblasts. The modified EGF species can be distinguished from each other and from native EGF by their isoelectric points. We employed peptide mapping techniques to determine the nature of these alterations. We found that 125I-EGF with a pI of 4.55 was converted to a pI 4.2 species by removal of 1 or 2 amino acid moieties from the COOH-terminal end of the protein. A pI 4.35 species was generated by a trypsin-like cut between amino acid residues 48 and 49, for a total of 5 amino acid moieties removed from the native EGF. The pI 4.0 species was formed by removal of at least the COOH-terminal arginine from the pI 4.35 species. Thus, upon binding and internalization, EGF was sequentially cleaved in the COOH-terminal region. Removal of the COOH-terminal polypeptide has been shown to dramatically reduce the affinity of EGF for its receptor, raising the possibility that intracellular dissociation of EGF from its receptor may be a direct result of the intracellular processing of EGF.

Use of PEEP in acute respiratory distress syndrome in dogs.

This study evaluates the effectiveness of combining mechanical ventilation and 5 cm H2O positive end-expiratory pressure (PEEP) at the onset of adult respiratory distress syndrome (ARDS) in dogs. Five cm H2O PEEP applied at the onset of ARDS in oleic acid injured dogs resulted in a decrease in cardiac output (CO). This decrease was accompanied by beneficial effects including a relatively stable SaO2 and PaO2. Control group dogs (receiving mechanical ventilation only) showed a less dramatic changing in CO, but demonstrated a dramatic drop in saturation, compromising oxygen transport of the tissues. Thus, despite decrease in CO experienced by the PEEP group, oxygen extraction at the tissue level remained high.

Evaluation of a hands-on workshop as a teaching tool for critical care medicine.

Many medical students have difficulty correlating the principles of cardiorespiratory physiology with the practice of critical care medicine. We developed a workshop to make this transition easier. After a computer-administered preworkshop test, groups of four students intubated a dog, and inserted arterial, peripheral, and pulmonary artery catheters. After baseline cardiorespiratory variables were measured, noncardiogenic pulmonary edema was created by administration of oleic acid via the right atrial port of the pulmonary artery catheter. Measurements were repeated after equilibration and again during the application of 5, 10, and 15 cm H2O of positive end-expiratory pressure. Monitoring techniques and calculation of physiologic variables and their significance were stressed; emphasis was also placed on the interrelationship between respiratory and cardiovascular manipulations. The educational validity of this workshop was confirmed by a significant improvement in the mean score of a post-workshop test over the mean preworkshop test score.

Compared effects of selected colloids on extravascular lung water in dogs after oleic acid-induced lung injury and severe hemorrhage.

While the hemodynamic effects of hydroxyethyl starch (HES) have been reported, the effect of this material upon extravascular lung water (EVLW) has not been investigated. Twenty mongrel dogs were subjected to both an oleic acid-induced lung injury and a 2-h period of hemorrhagic shock (MAP = 40 mm Hg). After reinfusion of shed blood, 5 dogs in each of 4 groups were given either 0.5 L of lactated Ringer's solution or 0.5 L of 5% albumin, 6% dextran 75, or 6% HES. Lactated Ringer's solution was then given in sufficient quantity to keep the wedge pressure (WP) at 12-15 mm Hg and PaO2, P(A-a)O2, cardiac index (CI) and oxygen delivery were determined. EVLW was measured by thermal-green dye double-indicator technique with an Edwards Lung Water Computer (American Edwards Laboratories, Santa Ana, CA). Mean baseline EVLW was 6.9 +/- 0.3 ml/kg. Mean EVLW rose to 11.5 +/- 1.9 ml/kg after oleic acid. One h after reinfusion, EVLW increased to 40.5 +/- 4 ml/kg in the dogs given only lactated Ringer's solution and to 39.5 +/- 1.5 ml/kg in the dextran group. EVLW was 25.5 +/- 3 ml/kg in the HES dogs, and 29.5 +/- 2 ml/kg in the group given albumin. Differences between albumin and lactated Ringer's solution and between the HES and lactated Ringer's groups were significant (p less than 0.02 and p less than 0.05). Measurements of oxygen, ventilation, CI, and oxygen delivery were not significantly different between the albumin and HES subjects.

Quantitation of early clonal expansion of two mutant 61st codon c-Ha-ras alleles in DMBA/TPA treated mouse skin by nested PCR/RFLP.

It has been hypothesized that tumor promotion in mouse skin involves clonal expansion of initiated cells with activated c-Harvey (Ha)-ras oncogene to give rise to benign tumors. We have used the two stage mouse skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiator and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as the tumor promoter to quantitate the number of mutated c-Ha-ras alleles in mouse epidermal DNA. Epidermal samples were harvested over a 12-week period before the appearance of papillomas. Three 61st codon (i.e. CAA) c-Ha-ras mutations, CTA (T2), CGA (G2) and CAT (T3) were quantitated by newly developed nested PCR/RFLP assays. During TPA promotion the number of T2 mutant copies showed a progressive increase starting at 4 weeks after initiation and the number of T3 mutant alleles showed an increase starting at 6 weeks. By 12 weeks after initiation, TPA-promoted mouse epidermis averaged approximately 8x10(5) T2 mutant alleles per epidermis while the number of T3 mutant alleles averaged 3x10(4) per epidermis. The best-fit lines for the quantitation of mutant alleles derived from DMBA/TPA-treated mice from 4 to 12 weeks after initiation were exponential. These results were consistent with clonal expansion of epidermal cells carrying these mutations during tumor promotion. The slopes of the best-fit lines for the mutant copies indicated a trend in which cells with the T2 mutations had a growth advantage during TPA promotion over cells with the T3 mutation.

Helium-oxygen mixtures in airway obstruction due to thyroid carcinoma.

The management of a patient with severe airway obstruction secondary to a thyroid mass is reported. When breathing room air the patient appeared in severe respiratory distress but when inspiring 22 per cent oxygen in helium she reported almost instantaneous relief and there was a marked decrease in respiratory rate, and increase in tidal volume and arterial oxygen tension. This improvement was to be expected because in situations where turbulent flow predominates a decrease in the density of inspired gases will result in an increase in flow rates. Contrary to established dogma a marked improvement was sustained when the patient was breathing 50 per cent oxygen in helium. The concentration of oxygen in helium was adjusted to obtain subjective relief for the patient in conjunction with adequate oxygenation.

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of JunD and FosB is required for okadaic acid-induced activator protein 1 activation.

Previously, we reported that in papilloma-producing 308 mouse keratinocytes, the tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, increased binding of activator protein 1 (AP-1) to a consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element (Rosenberger, S. F., and Bowden, G. T. (1996) Oncogene 12, 2301-2308). In this study, we investigated the correlation between AP-1 DNA binding and transactivation and examined molecular mechanisms involved in this process. Using a luciferase reporter driven by region -74 to +63 of the human collagenase gene, we demonstrated induction of AP-1-mediated transcription following okadaic acid treatment. By performing in vitro kinase assays, we found elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. The ERK-1/2-specific inhibitor PD 98059 completely abrogated okadaic acid-induced AP-1 transactivation without altering AP-1 expression, DNA binding, or complex composition. Phosphorylation analyses indicated that inhibition of ERK-1/2 decreased okadaic acid-elevated phosphorylation of JunD and FosB. To further examine the role of JunD and FosB in okadaic acid-induced AP-1 transactivation, we generated fusion proteins of the DNA-binding domain of the yeast transcription factor Gal4 and the transactivation domain of either JunD or FosB. Cotransfection experiments of these constructs with a Gal4-luciferase reporter demonstrated that both JunD and FosB are required for okadaic acid-induced transcription. Treatment with PD 98059 reduced JunD/FosB-dependent transactivation, suggesting that ERK-1/2-mediated phosphorylation is a critical component in this process.

Constitutive AP-1 DNA binding and transactivating ability of malignant but not benign mouse epidermal cells.

The mouse epidermal cell line 308 contains an activated Ha-ras gene and forms benign papillomas when transplanted to the skin of athymic nude mice. A radiation-associated malignant variant of this cell line, 308-10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in malignant conversion of 308-10Gy5, we assessed the activator protein-1 (AP-1) binding and transactivating ability of 308 and 308-10Gy5. In nuclear protein extracts of 308, AP-1 sequence-specific binding to an oligonucleotide containing a single high-affinity AP-1 binding site was induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, as determined by gel shift analysis. Nuclear extracts of 308-10Gy5 bound to the AP-1 oligonucleotide without treatment with tumor promoters. Not only was sequence-specific AP-1 DNA binding constitutively active in malignant versus benign tumor cells, but so was transactivation of a unique AP-1-responsive chloramphenicol acetyltransferase reporter construct, pTiCTaK. Constitutive transactivation of this AP-1-responsive reporter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady-state transcript levels of the tumor-associated AP-1-responsive genes stromelysin, urokinase-type plasminogen activator, c-jun, and c-fos were higher in malignant 308-10Gy5 cells than in benign 308 cells. These results suggest that acquisition of constitutive AP-1 DNA binding and transactivation can result in sustained deregulation of gene expression. While malignant progression in keratinocytes is probably not due solely to the acquisition of constitutive cellular AP-1 activity, the effect of deregulated expression of AP-1-regulated genes, especially basement membrane-degrading enzymes, may be functionally related to malignant conversion.

Acute acid aspiration lung injury in the rat: biphasic pathogenesis.

The purpose of this study was to develop a reproducible model of acute acid aspiration-induced lung injury in the rat to explore the pathophysiology of aspiration pneumonitis. A biphasic injury pattern was observed with injury peaks at 1 hr and 4 hr. Histologic studies at 4 hr revealed significant increases in neutrophils in the alveolar interstitial space. These studies suggest that acid aspiration results in a biphasic acute injury. We hypothesize that the first phase results from a direct physiochemical process or is mediated via afferent (capsaicin sensitive) nerves or both. The second phase, occurring 2-3 hr later, is mediated by neutrophils and is consistent with an acute inflammatory response.

Changes in arterial oxygen saturation in cigarette smokers following general anaesthesia.

The effect of cigarette smoking on postoperative arterial oxygen saturation was evaluated in 45 adult patients using pulse oximetry. Patients were divided into a smoking group (n = 20) and a non-smoking group (n = 25) based on current smoking habits up until the time of surgery. The two groups were similar with respect to sex, ASA physical status, surgical procedure, duration of anaesthesia, narcotic and anaesthetic use and recovery characteristics. The non-smoking group was, however, significantly (P less than 0.05) older than the smoking group. Postoperative oxygen saturation (SaO2) decreased (P less than 0.001) during transport of both groups of patients from the Operating Room to the Recovery Room; a decrease which was significantly greater in the smoking group. The severity of hypoxaemia was also significantly greater in the smoking group than in the non-smoking group. This study suggests that cigarette smoking contributes to postoperative arterial oxygen desaturation following general anaesthesia and that supplemental oxygen should be administered to these patients during postoperative transport.

Overexpression of three ubiquitin genes in mouse epidermal tumors is associated with enhanced cellular proliferation and stress.

A mouse ubiquitin clone that recognizes multiple transcripts overexpressed in murine tumors compared to normal epidermis was isolated by differential screening of complementary DNA libraries from mouse squamous cell carcinomas. Coding region probes detected five ubiquitin transcripts. Oligonucleotides were designed for unique parts of three mouse ubiquitin gene complementary DNA clones. The overexpressed transcripts at 2.4, 2.8, 6.4, and 7.8 kilobases (kb) were detected by an oligonucleotide specific for a mouse UbC polyubiquitin clone. A 1.2-kb UbB overexpressed transcript was detected by an oligonucleotide for a mouse four-unit polyubiquitin, and a 0.7-kb UbA overexpressed transcript was recognized by an oligonucleotide for the mouse ubiquitin carboxyl-extension protein of 52 amino acids. All three classes of transcripts were induced in mouse skin by the hyperproliferative agent ethylphenyl propionate and by the tumor promoting agent 12-O-tetradecanoylphorbol-13- acetate. Heat shock of cultured keratinocytes induced both the 6.4- and 7.8-kb transcripts recognized by the UbC-specific oligonucleotide. Consistent with the overexpression of the ubiquitin transcripts, the level of free ubiquitin protein, as determined by Western analysis, was elevated in the tumors and proliferating epidermis as compared to normal epidermis. Our results indicate that the overexpression of ubiquitin genes could be related to a sustained state of proliferation and stress in the tumors compared to the normal resting epidermis.