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.

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.

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.

Effect of soluble and insoluble fiber diets on serum prostate specific antigen in men.

PURPOSE: We assess whether high fiber diets influence serum prostate specific antigen (PSA) related to effects on serum sex hormone levels and fecal steroid excretion. MATERIALS AND METHODS: A randomized crossover controlled trial was performed on 14 healthy men with hyperlipidemia on 2 metabolic diets 4 months in duration with each containing foods high in soluble or insoluble fiber and approximately 25 to 30 gm. dietary fiber per 1,000 kilocalories. Serum PSA, free testosterone and estradiol, and fecal bile acid and neutral sterol excretion were evaluated. RESULTS: Mean serum PSA was lower with the soluble than the insoluble fiber diet (0.07+/-0.03 ng./ml., p = 0.035). No treatment difference was seen in free testosterone or estradiol, although the latter decreased significantly with the insoluble fiber diet (9+/-3 pmol./l., p = 0.004). After 16 weeks total fecal bile acid output was greater with the soluble (341+/-56 mg. daily) compared to the insoluble (203+/-35, p = 0.001) fiber diet but no differences were seen in fecal neutral sterol elimination. The treatment difference in fecal lithocholic acid output related to the difference in serum PSA (r = 0.57, p = 0.035). CONCLUSIONS: A small but statistically significantly lower serum PSA was seen in healthy men consuming soluble fiber, which was not related to changes in serum sex hormones but was related to the increased lithocholic acid output as a possible marker of increased fecal steroid elimination. The effect of soluble fiber on prostatic disease may warrant further investigation.

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.

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.

Quantitative analysis of p53 protein in non-small cell lung cancer and its prognostic value.

Accumulation of mutant p53 protein occurs frequently in human malignancies, including 40-60% of non-small cell lung carcinomas. The implications of such p53 over-expression, usually assessed by immunohistochemical techniques, for the prognosis of lung cancer patients remain undetermined. In this study, we used a time-resolved immunofluorometric assay to measure p53 protein concentrations in extracts prepared from 86 primary non-small cell lung tumours and examined the associations between p53 protein levels (corrected for total protein) and other clinico-pathologic variables, including post-surgical disease-free and overall survival. Contingency tables analysed by chi2 tests revealed no significant relationships between p53 status, defined by a median cut-off point, and patient gender, age, disease stage, histologic grade and type, lymph node extension, smoking history and administration of adjuvant chemotherapy or radiation. However, multivariate Cox proportional hazard regression analysis demonstrated a dose-response relationship between p53 concentration, expressed as a 4-level, quartile-divided variable, and increased risk of relapse (p = 0.010) and death (p = 0.016). Patients whose tumours contained p53 concentrations exceeding the median value had over 3-fold higher risk of relapse (p = 0.002) and death (p = 0.007) than those whose tumours had lower p53 concentrations. We also provide evidence suggesting that the impact of p53 on survival is greater in patients with squamous cell carcinoma than in those with adenocarcinoma. Although the latter finding needs confirmation, our results suggest that application of an immunoassay of p53 protein on non-small cell lung tumour extracts may identify patients at increased risk of unfavourable outcome.

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.

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.

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.

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.

p53 protein is absent from the serum of patients with lung cancer.

p53 protein, which accumulates intracellularly in over half of all human tumours, has also been reported to be present in the sera of patients with various malignancies, including lung cancer. Using a quantitative immunoassay, we measured p53 protein concentrations in 216 sera from 114 lung cancer patients of whom 75 provided matched lung tumour tissues, which were also assayed for p53 protein. p53 protein levels above the detection limit of 0.04 ng ml-1 were detected in only two sera from lung cancer patients (0.14 ng ml-1 and 0.27 ng ml-1), but not in any of 13 sera from non-malignant lung disease patients or in 100 sera from normal non-diseased individuals. The presence of these apparent traces of serum p53 protein concentrations could not be related either to the p53 protein expression status of the primary lung tumours or to the tumour stage, grade or histological type. By pretreating these two sera with anti-p53 antibody linked to solid phase, and by the addition of mouse serum to neutralise possible heterophilic antibodies, the signals arising from these sera were shown to be non-specific and possibly caused by heterophilic antibodies. We conclude that our data do not support previous reports of p53 protein in the sera of lung cancer patients. Since immunoassays are subject to numerous sources of interference in serum, including heterophilic antibodies, we suggest that the results of p53 protein analysis of serum specimens should be interpreted with caution.

Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis.

The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens.

Time-resolved immunofluorometric assay of p53 protein.

A common feature of human tumor tissue is mutant p53 protein accumulation. Here we evaluate a new "sandwich" immunoassay for p53 protein incorporating modifications to a previously reported method, including the use of microtiter plates coated directly with the anti-p53 monoclonal antibody DO-1, a detergent- and mouse serum-containing sample diluent, and a labeled secondary antibody diluent containing goat serum. The use of CM-1 antiserum to probe the immunocaptured p53 and the detection of bound complexes by a labeled secondary antibody allows coupling to a time-resolved fluorescence detection system. The new assay yielded p53 concentrations comparable with those by the previous assay for breast tumor cytosols (n = 198), nondiseased breast tissues (n = 70), and five transformed cell lines, but showed differences in p53 values measured in sera from patients without cancer (n = 78). These serum differences were found to reflect nonspecific interferences affecting the original method, which implies that the new immunoassay has improved specificity for serum p53 quantification.

Prostate-specific antigen expression by various tumors.

There is a growing body of evidence indicating that prostate-specific antigen (PSA) may be present in many steroid hormone-stimulated epithelial tissues other than that of the prostate. In particular, breast tumor cell lines treated with steroid hormone receptor agonists, breast tumors, and normal human breast have recently been found by our group to contain PSA. To investigate whether PSA may also be present in other human tumors, we employed a highly sensitive immunofluorometric assay technique to quantify PSA immunoreactivity in tumor extracts. Using a PSA-positivity cutoff value of 0.005 ng per mg of protein, 23 of 43 diverse tumors tested positive for PSA protein. Confirmatory analyses for PSA by a commercially available method (IMx) on six samples demonstrated a high degree of concordance between the two methods. To establish the molecular weight of the immunoreactive species, the most highly positive tumor extracts of each tumor type were fractionated by high performance liquid chromatography. Whereas the majority of tumors had immunoreactivity eluting at both 100 KDa and 33 KDa, corresponding to PSA bound to alpha 1-antichymotrypsin and free PSA, respectively, the colon and parotid tumors displayed immunoreactivity only at the 33 KDa fraction. We conclude that in addition to breast tumors and normal breast, colon, ovarian, liver, kidney, adrenal, and parotid tumors can also produce PSA. The physiological role of PSA in these tumors is currently under investigation.

Immunoreactive prostate-specific antigen in lung tumors.

Prostate-specific antigen (PSA) is a glycoprotein produced by the epithelial cells of the prostate. PSA is currently used clinically to diagnose and monitor prostate carcinoma. In previous work we have demonstrated that 30% of breast tumors and, more rarely other tumors, contain significant amounts of PSA. PSA appears to be a favorable prognostic indicator in breast cancer. Here, using a sensitive assay, we demonstrated for the first time that lung adenocarcinomas and squamous cell carcinomas also contain PSA. PSA in lung tumor extracts was present mainly in its 33 KDa form (free PSA), at levels measurable by commercial methods. The presence of PSA was associated more closely with male patients and adenocarcinomas. The physiological role of PSA in lung tissue and the prognostic significance of PSA in lung cancer remain to be determined. These and our previous data as well as reports by other groups support the view that PSA is a ubiquitous biochemical marker of steroid hormone action.

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.

Influence of fetal pulmonary epithelium on thrombin activity.

Epithelial injury and intra-alveolar fibrin are present in lung injury. To determine whether healthy fetal and neonatal lung epithelium could regulate thrombin activity (hence fibrin formation) we collected amniotic and postnatal endotracheal tube fluids from humans and directly sampled lung and amniotic fluids from fetal guinea piglets, rabbit pups, and lambs. The coagulant properties of the cell surface and media conditioned by rat fetal type II alveolar epithelium were assessed. All fluids contained glycosaminoglycans (GAGs), but mass and biological assays demonstrated only some (heparan sulfate and to a lesser extent dermatan sulfate) had antithrombin activity. The presence of proteoglycans (greater than 1,000 kDa) yielding active GAGs with less than 100 kDa after base elimination were demonstrated by Sepharose CL4B chromatography. Epithelial-derived fluids contained a factor VII-dependent procoagulant activity, but concentrated conditioned media overlying primary cultures of type II epithelium demonstrated a net antithrombin effect. These studies demonstrate that the lungs of human and nonprimate mammalian fetuses and fetal type II epithelium secrete GAGs, some of which possess antithrombin activity, which would oppose intra-alveolar fibrin formation.