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Objective: This study was designed to compare the incidence of anatomical variations displayed on computerized tomography scans of individuals that identify as Polynesian or New Zealand Europeans., Study Design: A retrospective analysis of computerized tomography of the paranasal sinuses. All scans were examined for a number of anatomical variations and the prevalence directly compared according to ethnic group., Setting: All scans were performed at a tertiary referral center (Dunedin Public Hospital)., Subjects: Participants underwent computerized tomography of the paranasal sinuses for various indications. Ethnicity was determined from the electronic hospital record., Results: There was no significant difference in the prevalence of anatomical variations between Polynesians and New Zealand Europeans. Our study results are comparable with other studies of Caucasian paranasal sinus anatomy., Conclusions: When performing endoscopic sinus surgery on those with Polynesian ethnicity, there are no specific anatomical variations to take into account.

Evidence-based practice must become a common, acceptable process for the bedside nurse. This paper demonstrates a process for engaging nurses in the use of evidence for direct practice. An example using the revision of a float policy is provided as a model for staff nurses to incorporate evidence in a mechanism to elicit change.

We isolated both the intact molecule (p185) and the ectodomain (p120) of c-erbB-2 oncoprotein from SK-BR-3 breast tumor cells. The p120 was extracted from the cells by 0.05 M phosphate buffer, pH 7.2, whereas the extraction of the p185 required the presence of a detergent, such as 1% Triton X-100 in 0.05 M Tris buffer. Protease inhibitors were also included in the extraction buffer during the isolation of p185 in order to prevent cleavage of p185 to p120 by an unknown protease apparently also present in the extract. In case there was any p120 in the p185 preparation, the p120 could be separated from p185 by chromatography on a Superose 12 column. Using the p120 and p185 as calibrators, we have established two microplate sandwich immunoassays: one measures both p185 and p120 (total assay) and the other is specific for the p185. Since capturing and detecting antibodies used in the total assay react against the extracellular domain of the c-erbB-2 oncoprotein, they can therefore be used to measure the p120 in serum and p185 in breast tumor tissue cytosol. On the other hand, the p185 specific assay uses the capturing antibody against the cytosolic domain of the oncoprotein and consequently can only measure p185 in breast tumor tissue cytosol.

We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate. By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained. We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation. More than 90% of the PSA from LNCaP cell cultures are fPSA. Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography. Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms. This was confirmed by chromatofocusing: the isoelectric point (pl) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid. We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer.

Three types of autoantibodies against the acetylcholine receptors (AChR) of skeletal muscle are detectable in patients with myasthenia gravis including binding, blocking, and modulating anti-AChR antibodies. Modulating autoantibodies correlate best with the severity of the disease, but are also technically most difficult to measure because the assay generally requires fresh human muscle cells. We have developed an assay for the modulation of anti-AChR antibodies using a rhabdomyosarcoma (RD) cell line expressing AChR on the cell surface. By decreasing the FetalClone III serum from 10% to 0.5% in Eagles Minimal Essential Medium (EMEM) we were able to increase the number of AChR on RD cells to meet the need of sensitivity of the assay. The extent of modulation was determined as the percent of AChR internalized in the presence or absence of modulating autoantibodies. Less than 6% modulation was found with the normal serum (n = 42). The CVs of both the intra- and day-to-day precision were less than 20%. When clinical samples (n = 105) were assayed in our laboratory and also at Nichols Institute, a correlation coefficient of 0.816 was obtained. The selection of RD cell line, the success of increasing the expression of the AChR on RD cells and the use of 125I alpha-bungarotoxin of high specific activity allowed the establishment of an assay which can be used in routine clinical laboratory for the measurement of modulating anti-AChR autoantibodies for the management of patients with myasthenia gravis.

We made an effort to identify a reliable source for obtaining large quantities of both free (PSA) and PSA-ACT complex for the preparation of the calibrator for the PSA assay. Using size exclusion chromatography, we found both free PSA and PSA-ACT complex in the conditioned cell medium of the LNCaP cell line, which was derived from a human metastatic adenocarcinoma of the prostate. An assay specific for PSA-ACT reacted only with the PSA-ACT complex from cells grown in serum-free medium, and not with the complex from the cell medium grown in 10% calf serum. We also found both free PSA and PSA-ACT complex in 15% of cytosols prepared from breast tumor tissues; the cytosol PSA concentrations ranged from 0.1 to 110 ng/ml. No correlation was found between cytosol PSA and concentrations of estrogen receptor, progestin receptor, epidermal growth factor receptor, cathepsin D, or the ectodomain of c-erbB-2 protein. Based on chromatographic characterizations and the slope of their dose-response curves, it appears that both free PSA and PSA-ACT complex found in the cytosols are similar to PSA complex from the cell medium and the serum of prostate cancer patients. Ectopic PSA was also detected in pooled sera from patients with breast, ovarian, pancreatic, and colon carcinoma. The PSA concentrations in these serum pools increased with the level of their dominant tumor marker. In any event, the LNCaP cell medium appears to be a reliable source for obtaining both free and ACT-complexed PSA of human tumor origin for the preparation of PSA assay calibrators.

We have identified and characterized c-erbB-2 protein molecules in sera from patients with carcinomas, in both cytosol and cell membrane extract from breast tumor tissue and in both the culture medium and cell extract of the SK-BR-3 cell line. These proteins were characterized by various chromatographic techniques and identified by the use of two immunoassays; one measures both the c-erbB-2 oncoprotein (p185) and its ectodomain (p120), and the other in-house assay reacts specifically for p185. We found that the majority of the immunoreactivity detected in the serum, tumor tissue cytosol, and conditioned cell medium was derived from the ectodomain molecule (p120) of the c-erbB-2 oncoprotein (p185), whereas only p185 was detected in the extracts from cell membrane of both tumor tissue and the SK-BR-3 cell line. The ectodomain molecules (p120) found in the serum, cytosol, and cell medium were very similar in terms of molecular size and charge property. The molecular weight was determined to be 120 kDa by the size exclusion HPLC method. Both p120 and p185 are glycoproteins and were retained by the ConA Sepharose column. Both molecules are also heterogeneous in charge and multiple peaks could be identified in the elution profiles of anion exchange HPLC and chromatofocusing. This information should not only facilitate the isolation of these molecules, but also improve preparation of specific antibodies, preparation of calibrators, and development of improved assays for these proteins.

A general approach is described for measuring homo- and heteronuclear spin coupling constants in polypeptides and small proteins. This method uses selective magnetization transfer to generate cross peaks similar to exclusive correlated spectroscopy (E.COSY) and a large direct spin coupling (1J) in one dimension to "pull apart" cross-peak components by frequencies much larger than the resonance line width. A general description of this method is presented, along with a brief discussion of spin topology and relaxation effects that must be considered in designing multidimensional nmr pulse sequences for measuring vicinal coupling constants. The principles are demonstrated in designs of several two-dimensional nmr experiments for determining coupling constants in polypeptides and proteins. These include experiments for measuring 3J(HN-H alpha), 3J(H alpha i-1-15Ni), 3J(15N-H beta), and 3J(H alpha-H beta) coupling constants, which depend on the polypeptide dihedral angles phi, psi, and chi 1. Multidimensional nmr experiments developed with this approach will allow measurements of many vicinal coupling constants in peptides, proteins, and other molecules. Coupling constants measured in these spectra can be used to determine backbone and side-chain conformations, to obtain stereospecific resonance assignments of prochiral atoms, and to characterize conformational distributions of dihedral angles. Combined with information obtained from nuclear Overhauser effect measurements, these data will provide more precise determinations of protein solution structures by nmr spectroscopy.