Inhibition of hormone-stimulated lipolysis by clofibrate. A possible mechanism for its hypolipidemic action.

The present study was undertaken to investigate the mechanism of the antilipolytic action of clofibrate (p-chlorophenoxyisobutyrate). Clofibrate, in the dose range of 10-80 mg/199 ml, inhibited the initial rate of norepinephrine-stimulated lipolysis 17-44 percent in isolated rat fat cells. At a dose corresponding to therapeutic levels in vivo (10 mg/100 ml) clofibrate also inhibited hormone-stimulated lipolysis by 20-30 percent in fragments of human subcutaneous fat. Inhibition of lipolysis by clofibrate occurred at all concentrations of norepinephrine and ACTH (0.02-0.1 mug/ml) but did not occur with equilipolytic concentrations of dibutyryl cyclic AMP, suggesting a proximal site of action on the lipolytic sequence. Clofibrate reduced by 60 percent (315plus or minus40 vs. 120plus or minus25 pmol/g lipid; meanplus or minusSEM) the norepinephrine-stimulated initial rise in cyclic AMP, measured 10 min after addition of hormone. Because the antilipolytic effect occurred in the presence of glucose and without altering cellular ATP levels, the reduction in intracellular cyclic AMP levels could not be attributed to uncoupling of oxidative metabolism or to secondary effects of free fatty acid accumulation. In the secondary effects of free fatty acid accumulation. In the presence of procaine-HC1, which blocks hormone-stimulated lipolysis without inhibiting cyclic AMP accumulation, addition of clofibrate prevented the hormone-stimulated rise in cyclic AMP. Clofibrate did not affect the activity of the low-Km 3',5'-cyclic AMP phosphodiesterase in norepinephrine-stimulated adipocytes. These data suggest that the antilipolytic effect of clofibrate is due to its suppression of cyclic AMP production by inhibition of adenylate cyclase. The drug's hypolipidemic action may in part be explained by its antilipolytic effect, which deprives the liver of free fatty acid substrate for lipoprotein synthesis.

Expression of the prostate specific antigen gene by lung tissue.

We describe a female patient with lung adenocarcinoma whose tumor extract was highly positive for prostate specific antigen (PSA) immunoreactivity. PSA was present in its Mr 33,000 free form. Using reverse transcription-PCR, we were able to amplify a 754-bp fragment that specifically hybridized to a PSA RNA probe on Southern blots. The PCR fragment was sequenced and found to represent PSA cDNA and not human glandular kallikrein cDNA. PSA immunoreactivity in the lung tissue was localized by immunohistochemistry to normal epithelial cells adjacent to the tumor which was completely negative for PSA. Tissue culture experiments suggested that beclomethasone, a glucocorticoid used to treat the patient, was able to up-regulate PSA gene expression. This is the first report that unequivocally demonstrates PSA expression in lung tissue. We speculate that PSA expression was mediated by the exogenously administered steroid beclomethasone.

p21WAF1 protein expression determined by quantitative immunoassay in relation to non-small-cell lung cancer aggressiveness.

PURPOSE: p21WAF1, a cyclin-dependent kinase inhibitor, is an important mediator of the cell-cycle arrest and tumor suppression induced by the protein p53. Although alterations of the p53 gene and its overexpression are frequent in most malignancies, including non-small-cell lung cancer (NSCLC), and may be associated with poor patient prognosis, the clinical utility of p21WAF1 expression in NSCLC has not been established. METHODS: We have used a commercial enzyme-linked immunosorbent assay (ELISA) kit for p21WAF1 to test soluble extracts of 54 NSCLC specimens with known clinicopathological properties. RESULTS: There was no correlation between p21WAF1 and p53 concentrations, the latter being determined by a time-resolved immunofluorometric assay developed in-house. Furthermore, p21WAF1 levels were not associated with patient age, tumor/node/metastasis (TNM) stage, lymph node metastasis, histological grade or type, or smoking history, in Mann-Whitney analysis. chi2-tests, based on cutoffs equal to the 25th, 50th, or 75th percentiles of the p21WAF1 distribution, similarly did not reveal any statistically significant associations between p21WAF1 and other clinicopathological variables. Because of the small number of patients and the median follow-up of only 18 months, a meaningful survival analysis could not be performed. CONCLUSION: In summary, this preliminary study suggests that ELISA-quantified p21WAF1 levels in NSCLC extracts are weaker than p53 in terms of prognostic value and do not contribute to the further subclassification of patients.

Cardiac troponin I for the diagnosis of acute myocardial infarction in the emergency department.

Cardiac troponin I (TnI) was tested in 316 consecutive patients with chest pain who were admitted to the emergency department, of whom 62 were discharged with a diagnosis of acute myocardial infarction (AMI). The TnI level was abnormal in 49 patients with AMI compared with 27 for creatine kinase (CK)-MB in the first specimen obtained at admission. All 62 patients with AMI were correctly diagnosed at admission with a combination of TnI and myoglobin testing. The overall peak performance of TnI testing in samples received within 24 hours of admission indicated high sensitivity (97%) and specificity (98%) for the diagnosis of AMI. The TnI was positive in elderly patients with myocardial injury and low CK and normal CK-MB values. These data suggest that testing for TnI could replace CK-MB and, in combination with myoglobin, could facilitate the rapid and effective triage of patients with chest pain in the emergency department.

Comparison of immunofluorometry and immunohistochemistry for the detection of p53 protein in lung cancer specimens.

Although immunohistochemical techniques are widely used to demonstrate the presence of mutant p53 protein in a wide variety of malignant tissues, quantitative enzyme-linked immunosorbent assay (ELISA)-type immunoassays offer some advantages. In this study we compared immunohistochemistry, performed on formalin-fixed, paraffin-embedded sections of 91 primary lung tumor tissues, with a highly sensitive quantitative two-site immunofluorometric assay, on extracts of fresh-frozen specimens from adjacent regions of the same tissues. Monoclonal DO-7 antibody, and the related monoclonal DO-1 with polyclonal CM-1 antibodies, were used for immunostaining and ELISA, respectively. Concentrations of p53 were expressed relative to total protein, while an immunostaining score reflected the proportion of stained malignant cells, intensity of staining, and tumor cellularity. Strong concordance was shown between the two methods by Spearman correlation (P < .001), Wilcoxon rank sum (P < .001), and contingency table (P < .001) analyses. The use of ELISA-type assays for p53 quantification in lung tumor tissues may be an alternative to the more labor-intensive histologic techniques.

Frequency of expression of prostate-specific antigen mRNA in lung tumors.

The presence of prostate-specific antigen (PSA) protein and messenger RNA (mRNA) was studied in 52 primary lung tumor tissues. The PSA protein was detected more frequently and at higher levels in lung tumor extracts from men. The levels of PSA protein in tumor extracts correlated with preoperative and postoperative serum PSA levels, suggesting a possible contamination of the tumor extracts with PSA from residual blood in the tumor vasculature. The PSA mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization in 24 (68%) of 35 tumors from men, in 9 (53%) of 17 tumors from women, and in 5 (71%) of 7 adjacent normal lung tissue specimens. The levels of PSA protein did not associate with patient age, the tumor stage, grade, or histologic type, or the nodal status. Similarly, PSA mRNA was not associated with any clinicopathologic variables, but squamous cell carcinomas, especially in men, were more frequently positive. A by-product of the RT-PCR procedure was cloned and sequenced and found to be a 450-base pair sequence not previously deposited in the data bank. We conclude that PSA mRNA and protein frequently can be detected in lung tumors and normal tissues from men and women but at levels much lower than those seen in breast carcinomas in women. The significance of the new 450-base pair sequence remains to be determined.

Aminoethylprolyl (aep) PNA: mixed purine/pyrimidine oligomers and binding orientation preferences for PNA:DNA duplex formation.

[structure in text] The synthesis of (2S,4S)- and (2R,4S)-aepPNA monomers of adenine, guanine, and cytosine (3-5) and their incorporation at appropriate positions into aegPNA sequence 7 leads to mixed aeg-aep backbone/mixed nucleobase PNAs 8-11. The thermal stabilities of the derived duplexes with DNA are found to be dependent on nucleobase and backbone stereochemistry.

Aminoethylprolyl peptide nucleic acids (aepPNA): chiral PNA analogues that form highly stable DNA:aepPNA2 triplexes.

[formula: see text] The replacement of the glycyl component in the peptide nucleic acid (PNA) backbone by a prolyl unit bearing a nucleobase leads to the aminoethylprolyl (aep) PNAs, which are chiral and cationic. The homooligomeric aepPNA binds to complementary DNA sequences with high affinity and sequence specificity, forming highly stable triplexes.

Effect of caffeine on ventilatory responses to hypercapnia, hypoxia, and exercise in humans.

The effect of oral caffeine on resting ventilation (VE), ventilatory responsiveness to progressive hyperoxic hypercapnia (HCVR), isocapnic hypoxia (HVR), and moderate exercise (EVR) below the anaerobic threshold (AT) was examined in seven healthy adults. Ventilatory responses were measured under three conditions: control (C) and after ingestion of either 650 mg caffeine (CF) or placebo (P) in a double-blind randomized manner. None of the physiological variables of interest differed significantly for C and P conditions (P greater than 0.05). Caffeine levels during HCVR, HVR, and EVR were 69.5 +/- 11.8, 67.8 +/- 10.8, and 67.8 +/- 10.9 (SD) mumol/l, respectively (P greater than 0.05). Metabolic rate at rest and during exercise was significantly elevated during CF compared with P. An increase in VE from 7.4 +/- 2.5 (P) to 10.5 +/- 2.1 l/min (CF) (P less than 0.05) was associated with a decrease in end-tidal PCO2 from 39.1 +/- 2.7 (P) to 35.1 +/- 1.3 Torr (CF) (P less than 0.05). Caffeine increased the HCVR, HVR, and EVR slopes (mean increase: 28 +/- 8, 135 +/- 28, 14 +/- 5%, respectively) compared with P; P less than 0.05 for each response. Increases in resting ventilation, HCVR, and HVR slopes were associated with increases in tidal volume (VT), whereas the increase in EVR slope was accompanied by increases in both VT and respiratory frequency. Our results indicate that caffeine increases VE and chemosensitivity to CO2 inhalation, hypoxia, and CO2 production during exercise below the AT.

Validation of a simple rapid high performance liquid chromatographic method for amniotic fluid lecithin/sphingomyelin ratios.

A simple, rapid and sensitive HPLC method for the determination of L/S ratios in amniotic fluids is described. The method is based on isocratic separation in the normal phase with UV detection. The procedure has good precision and was validated clinically and by comparison with a routine TLC method. Although L/S ratios differed from those obtained by TLC, the clinical correlation between these methods was good. In single and serial samples from 39 mothers (42 babies) the HPLC method predicted respiratory distress syndrome (RDS) in all 9 babies with L/S ratios less than 7. No babies with ratios above 7 developed RDS. This method has potential clinical usefulness for the assessment of fetal lung maturity.

A multicenter evaluation of the Boehringer Mannheim ES 300 immunoassay system.

The ES 300 system, a fully automated multichannel immunoassay analyzer, was evaluated simultaneously for 9 weeks in four major centers. Precision, accuracy, carryover, comparison to in-house methods, and interferences were assessed for the following 17 tests: T4, T3, FT4, TSH, TBK, TBG, LH, FSH, prolactin, HCG, digoxin, cortisol, ferritin, IgE, insulin, AFP, and CEA. All centers reported good intra-lab and inter-lab precision. Accuracy was judged to be good based on correlation with in-house methods and recovery of target values in commercial and proficiency control materials. Linearity was evaluated for 14 analytes. Method biases were observed for T3 and insulin that were attributed to differences in standardization. No significant interferences from bilirubin, lipemia, and hemolysis were observed for all methods except insulin and AFP. Featuring random access capability, low daily maintenance, and high throughput, the ES 300 system performed well and met the stated claims of the manufacturer.

Systemic phospholipase A2 and cachectin levels in adult respiratory distress syndrome and multiple-organ failure.

In this clinical study we have prospectively measured plasma phospholipase A2 (PLA2) activity and tumor necrosis factor (TNF) levels in ventilated intensive care unit (ICU) patients with (n = 9) and without (n = 12) evidence of respiratory distress syndrome (ARDS) and multiple-organ failure (MOF). The median peak TNF concentration in control patients was 40 ng/L (range less than 40-100 ng/L) and in ARDS patients 231 ng/L (range 100-2550 ng/L; p less than 0.001). All of the control patients were discharged alive from the ICU, whereas 6 of 9 ARDS patients died in the ICU. In 6 ARDS patients, it was possible to measure more than 4 consecutive plasma TNF levels. Of these 6 patients, the 3 with persistent elevations in systemic TNF above 230 ng/L succumbed (p less than 0.05, one-tailed). Patients with ARDS also had parallel elevations in plasma PLA2 activity above controls. These elevations were significant for arterial PLA2 activity but not for venous PLA2 activity. Our study suggests that serial measurement of plasma (arterial or venous) TNF levels may have (1) prognostic and (2) etiologic significance in ICU patients with ARDS and MOF.

Serum pituitary and steroid hormone levels in the adult male: one value is as good as the mean of three.

The result of the hormone concentration of one blood sample was used to determine the accuracy of predicting not only the hormone concentrations of a second and third sample drawn 15 minutes apart, but also the mean value of the three samples. Three blood specimens from 73 men involved in two previously reported studies (A and B) were assayed individually for luteinizing hormone, follicle-stimulating hormone, prolactin, testosterone, androstenedione, dehydroepiandrosterone sulfate, estradiol, and cortisol. The predictive correlation of one single hormone value, when compared with the mean of three values, was 0.90 or greater, except for prolactin in study A (0.86) and testosterone in study B (0.86). Since the hormone level obtained for one sample has such a high predictive value for the hormone levels of the other two samples, drawing more than one sample is redundant.

Extracranial drainage of cerebrospinal fluid: a study of beta-transferrins in nasal and lymphatic tissues.

OBJECTIVE: To determine whether an alternative route of cerebrospinal fluid (CSF) drainage exists through the nasal mucosa and the cervical lymphatic system. STUDY DESIGN: A prospective study was carried out on 18 patients at a university teaching hospital. METHODS: Ten patients undergoing routine endoscopic sinus surgeries and eight patients undergoing neck dissections were recruited for this study. Tissues were sampled from the middle turbinate, nasopharynx, and upper septum in the first group; jugulodigastric lymph nodes and nasopharyngeal tissues were obtained from the second group. Specimens were subjected to immunofixation electrophoresis in an attempt to identify the presence of beta-1 and beta-2 transferrins. Serum samples were obtained from each subject to serve as controls. RESULTS: All tissue specimens contained beta-1 transferrin; none showed evidence of beta-2 transferrin. CONCLUSION: Using this technique, an alternate route of CSF drainage through the nose and the cervical lymphatic system could not be confirmed. Nevertheless, a new technique of performing immunofixation in solid tissues for the purpose of beta-transferrin identification is described.

Engineering preferences of hairpin PNA binding to complementary DNA: effect of N7G in aeg/aep PNA backbone.

AegPNA and aepPNA monomeric units bearing the N7-guanine nucleobase as a substitute for C+ have been demonstrated to bind to a GC base-pair of a duplex in a pH-independent manner when placed in the third strand. The aepPNA backbone exerts a preference for binding in the antiparallel Hoogsteen mode over the parallel Hoogsteen mode.

Selective determination of urinary free cortisol by liquid chromatography after solid-state extraction.

We have developed a selective and precise high-performance liquid chromatographic method for urinary free cortisol with an improved and efficient sample clean-up using C18 Sep-Pak cartridges. The urine sample (2 ml), with 11-deoxycortisol as internal standard, is applied to the Sep-Pak, which is then sequentially washed with acetone-water (1:4, v/v), water and hexane. Cortisol is eluted with diethyl ether, evaporated to dryness and redissolved in 2 ml of water. The wash cycle is repeated once using the same Sep-Pak cartridge. This double extraction greatly improves sample clean-up and allows modification of the mobile phase (tetrahydrofuran-methanol-water) so that cortisol is rapidly eluted as a single well resolved peak at 13 min. Chromatography is performed isocratically on a reversed-phase column with detection at 254 nm. Detection limits for urinary free cortisol by this procedure were two or three times lower than those obtained with two commercial radioimmunoassay kits. The chromatographic method was used successfully in the diagnosis of patients with hypercortisolism and Cushing's syndrome.

Uncoupling of lipolysis from cyclic AMP by procaine: a tool for studying the mechanism of action of antilipolytic agents.

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40-78%) by procaine-HCl (1-5mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine-HCl (0.1-1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine-HCl nor clofibrate had any effect on the low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.