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Biomedical subjects

J T Wu

Publications and source records attributed to J T Wu.

At least 19 recordsLinked to original sources
PubMed1995

Evaluation of free PSA isoforms, PSA complex formation, and specificity of anti-PSA antibodies by HPLC and PAGE-immunoblotting techniques.

Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis-immunoblotting (PAGE-immunoblotting) procedures have been established for the study of isoforms of free prostate-specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti-PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS-PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA-ACT complex, which was consistently detectable by both HPLC and PAGE-immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA-A2M and PSA-AT complexes. The PSA-A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA-AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA-ACT and PSA-A2M complexes. However, more PSA-ACT than PSA-A2M was formed; the result was also confirmed by using 125I-PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14-fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti-PSA antibodies examined in this study reacted with PSA isoforms, PSA-ACT and PSA-A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values.

Antibody Specificity↗
PubMed1995

Correlation of serum concentrations of PSA-ACT complex with total PSA in random and serial specimens from patients with BPH and prostate cancer.

The current assays for serum prostate specific antigen (PSA) have failed to produce the same PSA values on the same specimens because of problems with antibody specificity and calibrator preparation. To eliminate these problems, we proposed to replace the current serum PSA assay with an assay specific for the PSA-ACT (PSA-alpha 1-antichymotrypsin) complex in the serum. An assay specific for the PSA-ACT complex was established using the anti-PSA antibody to coat the microplate for capturing the PSA complex and anti-ACT polyclonal antibodies for quantification. There was an excellent correlation between serum concentrations of PSA-ACT and total PSA, using either the Hybritech calibrator (gamma = 0.996) or a serum calibrator prepared in house (gamma = 0.993), in random as well as in serial specimens from 14 individual patients. Even though we did not find a gradual increase in the percentage of PSA-ACT with the increase of total PSA in cancer patients, a slightly higher percentage of free PSA was measured in pooled normal sera (18%) and in pooled sera containing only 12 ng/ml of total PSA (12%), compared to serum pools containing elevated PSA (> 100 ng/ml) level, in which most PSA was in the complex form (95%). Therefore, using an assay that specifically measures the PSA-ACT complex in the serum not only simplifies the preparation of calibrator but eliminates the difficulty of antibody selection, it also allows various assay kits to produce identical PSA values and also improve the test specificity for prostate cancer.

Humans↗
PubMed1995

Purification of PSA-ACT complex: characterization of PSA-ACT complex by various chromatographic procedures.

We have explored various chromatographic procedures with the intention of establishing an isolation procedure that would allow us to isolate a large quantity of PSA-ACT (prostate specific antigen-alpha 1-antichymotrypsin) complex either from patients' sera or from incubation mixtures of free PSA and protease inhibitors. We found that at pH 7.2, both free PSA and PSA-ACT molecules are negatively charged and bind to the DEAE-Sepharose column. However, they could be separated from each other using a linear gradient of NaCl at pH 7.2. Both free PSA and PSA-ACT molecules were also found to be retained by the Con A Sepharose column because of the carbohydrate moiety of the PSA molecule. These two molecules were not separable by Con A chromatography. These two molecules apparently differ in their isoelectric points and were well separated by chromatofocusing using a pH gradient from pH 9 to 6. It appears that chromatofocusing can also be used to identify the isoforms of free PSA because of its high resolving power. The large difference in molecular size between free PSA and PSA-ACT complex allowed their separation by gel filtration chromatography on a column containing either S-100, S-200, or S-300 gel. S-200 gel appeared to be the best for the separation of free PSA from PSA-ACT and for the removal of other contaminating serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography↗
PubMed1995

Identification and characterization of c-erbB-2 proteins in serum, breast tumor tissue, and SK-BR-3 cell line.

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.

Breast Neoplasms↗
PubMed1995

Measurement of c-erbB-2 proteins in sera from patients with carcinomas and in breast tumor tissue cytosols: correlation with serum tumor markers and membrane-bound oncoprotein.

Using a commercial kit with antibodies against the ectodomain of c-erbB-2 protein, we detected c-erbB-2 immunoreactivity in human serum. We found that the percentages of patients with elevated serum c-erbB-2 immunoreactivities were 35, 21, and 9% in breast, prostate, and ovarian carcinoma, respectively. The majority of the elevated immunoreactivities were associated with sera containing highly elevated tumor markers with the highest in breast carcinoma (35%) and lowest in ovarian cancer (9%). Excellent correlations were also observed between the serum levels of c-erbB-2 immunoreactivity and the dominant tumor markers in serial specimens from individual cancer patients. We could also detect the c-erbB-2 immunoreactivity in the cytosols prepared from the breast tumor tissue for estrogen and progesterone receptor (ER & PgR) measurements using the same commercial kit for serum studies, and the intact c-erbB-2 oncoprotein (p185) in the extracts of the tissue membrane fractions with a different kit designed for tissue extract. The level of c-erbB-2 immunoreactivity in the cytosol from 124 human breast tumor specimens had an excellent correlation with the cell membrane concentrations of p185 (gamma = 0.89). Most of the elevated cytosol c-erbB-2 immunoreactivities were also found to associate with breast tumor specimens containing low concentrations of ER & PgR. It appears that measuring the c-erbB-2 immunoreactivity potentially could be used as a prognostic marker without performing tissue biopsies and also as a serum tumor marker for managing cancer patients.

Biomarkers↗
PubMed1995

PSA immunoreactivity detected in LNCaP cell medium, breast tumor cytosol, and female serum.

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.

Breast Neoplasms↗
PubMed1995

Development of a microplate ELISA for free PSA and PSA-ACT complex in serum.

An ELISA on microplate was established for the total serum PSA. We selected the monoclonal antibody for the assay from commercial sources making certain that it reacted with both free PSA and PSA-alpha 1-antichymotrypsin (PSA-ACT) complex from human serum with similar affinity (so-called "equimolar"). We also chose a test format with polyclonal anti-PSA antibodies coated on the well and monoclonal anti-PSA antibodies for quantification to gain higher test sensitivity. Two different sample volumes from each specimen, 5 and 50 microliters, were used for the assay in order not only to further increase test sensitivity and improve precision at both low and highly elevated PSA concentrations, but also to widen the assay concentration range (0-500 ng total PSA per ml). Using two sample volumes also reduces any hook effect and shortens the turn-around time because repeated determinations are usually required when specimens contain highly elevated PSA concentrations. The use of pooled sera containing approximately 95% PSA-ACT complex and 5% free PSA as a calibrator allows for a close matching of the calibrator with serum specimens in immunoreactivity and PSA composition. Moreover, our assay shows no hook effect up to 15,000 ng/ml. The within-day precision (% CV) in the critical concentration range of 4-12 ng/mL is approximately 5%. The PSA values obtained from this assay correlate well with that of the Hybritech kit (gamma = 0.998, slope equals to 1.033), indicating that this kit can replace the Hybritech Tandem E PSA kit for serum PSA determination in clinical laboratories.

Antibodies, Monoclonal↗
PubMed1994

Assay for prostate specific antigen (PSA): problems and possible solutions.

The absolute tissue specificity of prostate specific antigen (PSA) allows the use of PSA test not only for detecting recurrence or metastasis at an early stage after radical prostatectomy but also for screening prostate cancer if combined with digital rectal examination. There is also a need to improve the current PSA test to better differentiate between prostate cancer and benign prostate hyperplasia (BPH). Because of these clinical applications, a much greater demand was placed on PSA test for extra sensitivity, accuracy, and precision even within the normal PSA concentration range. However, the current commercial assay kits for PSA do not provide correct PSA values. Many factors contributing to the problem include the specificity of the anti-PSA antibodies, the composition of the calibrator, the PSA values assigned to the calibrator, the PSA isoform used for anti-PSA antibody preparation, the test design, and the composition of the diluent. Most problems were derived from the failure of realizing earlier that the majority of the PSA exists in serum not as free PSA but as complexes with protease inhibitors. Other problems, such as constantly changing composition of various forms of PSA in serum specimens, and different clearance rates for various forms of PSA make almost impossible to develop an ideal assay for PSA. Therefore, we suggest that test should be designed for measuring PSA-ACT (PSA-alpha 1-antichymotrypsin) complex only. Changing the focus from the measurement of total PSA of various forms to the PSA-ACT complex alone may improve the differentiation between prostate cancer and BPH but may also simplify the selection of anti-PSA antibodies and the preparation of calibrator for the assay.

Biomarkers↗
PubMed1993

Development of radioimmunoassays on microplate: application for CA-GI and CA-Br tumor markers.

By taking advantage of a newly available microplate counter for radioactivity and the organic solvent-resistant, pigmented microplates, we have successfully established radioimmunoassays (RIA) for both CA-GI and CA-BR on microplate for routine clinical use. In the process of assay development, we found that both pigmented PicoPlate, made of acrylonitrile, and polystyrene Microlite 2 can be coated with antialpha fetoprotein (AFP) and antinerve growth factor (NGF) and used for setting up immunoassays for AFP and nerve growth factors. There were no problems following a test format of either competitive binding or sandwich design. Microlites 2 is recommended over PicoPlate because Microlites 2 is made of polystyrene, which is less expensive and separable into 8-well strips or even single wells. Single-well separation allows for the use of regular gamma counters in case Topcount is unavailable. We also found that the sensitivity of these tests was not significantly affected even though Topcount counts the weaker beta emissions. Similar dose-response curves could also be generated between original Biomira tube assays and assays using PicoPlate or Microlite 2 coated with protein antigens CA-Br and CA-GI. Excellent correlations were also obtained between the microplate assays and the Biomira tube assays for CA-GI and CA-Br using groups of serum specimens from cancer patients. We recommend the development of various RIAs on the microplate: it requires less reagents and less sample handling by the technologists and it can be essentially automated.

Antibodies↗
PubMed1993

Comparison of neural network and k-NN classification methods in medical image and voice recognitions.

We make a comparison of classification ability between BPN (Back Propagation Neural Network) and k-NN (k-Nearest Neighbor) classification methods. Voice data and patellar subluxation images are used. The result was that the average recognition rate of BPN was 9.2 percent higher than that of the k-NN classification method. Although k-NN classification is simple in theory, classification time was fairly long. Therefore, it seems that real time recognition is difficult. On the other hand, the BPN method is long in learning time but is very short in recognition time. Especially if the number of dimensions of the samples is large, it can be said that BPN is better than k-NN in classification ability.

Algorithms↗
PubMed1992

Low frequency and low level of elevation of serum CA 72-4 in human carcinomas in comparison with established tumor markers.

We evaluated a new circulating tumor marker, CA 72-4, by comparing its frequency of appearance and level of elevation with other established tumor markers in serial serum specimens from patients with various carcinomas. We found that CA 72-4, though highly expressed and widely found in various tumor tissues, is present at low concentration and frequency in the serum. In breast, colon, ovarian, and pancreatic carcinomas, only 21%, 30.9%, 16%, and 26.5% of specimens, respectively, showed elevated CA 72-4. When elevated, the level of elevation was also low, much lower than that of the dominant markers. Poor response of CA 72-4 to therapy was especially noticeable in serial specimens. In most cases, the CA 72-4 remained normal for the entire series while other markers remained at elevated levels. However, changes of the level of CA 72-4 usually paralleled those of other markers but at a much lower concentration. Simultaneous measurement of CA 72-4 and CA 19-9 appears useful to differentiate colorectal from pancreatic carcinomas when they all contained elevated levels of CA 19-9. There was a much higher ratio of CA 72-4 to CA 19-9 with colon than with pancreatic and other carcinomas (247 +/- 524 vs. 4.7 +/- 6.8).

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1992

Tumor markers CA 19-9 and CA 195 are also useful as markers for cystic fibrosis.

When monoclonal kits are used we can no longer detect highly elevated serum concentrations of carcinoembryonic antigen in cystic fibrosis (CF) patients as we could earlier (Pediatr Res 10:223-236, 1975). Instead, we find increased concentrations of CA 19-9 or CA 195 in the CF sera. The serum levels of CA 19-9 not only reflect the pulmonary condition of CF patients but also respond well to antibiotic therapy. Several lines of evidence suggest that the elevated serum concentration of CA 19-9 is derived from sputum and corresponds with the amount of sputum in the lung. Correlations between CA 19-9 and CA 195 in random and serial specimens from both patients with CF and patients with pancreatic carcinoma suggest that all sera contain heterogeneous, Lewis blood group-related epitopes and the proportions of various epitopes are different among individual patients. When monitored on multiple tumor markers, the pattern of CF is different from that of pancreatic carcinoma although both usually show elevated CA 19-9. Our study indicates that both CA 19-9 and CA 195 can be used as sensitive markers for the early detection of exacerbation in CF patients.

Adolescent↗
PubMed1992

Chromatographic characterization of CA 19-9 molecules from cystic fibrosis and pancreatic carcinoma.

We have compared the size, the binding to Concanavalin A (Con A), and the affinity for monoclonal antibody 1116NS-199 (Mab 19-9) among CA 19-9 molecules from sera of cystic fibrosis (CF) and pancreatic carcinoma patients and from sputum extracts. CA 19-9 molecules of two different sizes were found in all types of specimens by Sepharose 4B chromatography. While the smaller CA 19-9 molecule was predominant in CF patient sera, the larger molecule was associated with most of the sera from patients with pancreatic carcinoma. The majority of the sputum extracts contained the larger CA 19-9 molecule. All CA 19-9 molecules studied by Con A chromatography did not appear to bind to Con A, and almost 100% were found in the nonreactive fraction. The CA 19-9 molecules from sera of either CF or pancreatic carcinoma patients exhibited variable affinities for Mab 19-9, some approaching that of the standard curve but many also having lower affinities. The lowest affinity was displayed by CA 19-9 molecules from the sputum extract. It appears that development of more specific assays for CF and for carcinoma is possible if the correct CA 19-9 molecule is selected for antibody preparation and for use as standards.

Antibodies, Monoclonal↗
PubMed1992

The long-term modality effect: in search of differences in processing logographs and alphabetic words.

The visual superiority effect (a reverse long-term modality effect) has been consistently found with Chinese logographs. For its explanation in terms of script differences, it has been believed that lexical access is more direct or quicker for Chinese logographs than for alphabetic words. It has also been believed that Chinese logographs are more unique in shape or more discriminable than alphabetic words. Finally, Chinese logographs have been considered to facilitate recall through their graphic features that classify Chinese words into categories. The results of Experiments 1-5 show that these three assumptions can be ruled out. The results of Experiments 6-10, on the other hand, support the long-term priming interpretation of the visual superiority effect, which explains (a) why the visual superiority effect can be consistently obtained for recall of Chinese words by Chinese subjects, (b) why the effect cannot be consistently obtained for recall of English words by Western subjects, (c) why the effect can be also obtained for recall of English words by Chinese subjects, (d) why the effect can be easily obtained for recall of a set of words, but not for recall of a different set of words by Chinese subjects, and (e) why the effect can be easily obtained from Chinese subjects speaking a dialect that is different from Mandarin.

Adult↗
PubMed1992

[Leishmaniasis in Karamay. XI. The development of cutaneous leishmaniasis in monkey and man experimentally infected with Leishmania from Karamay big gerbil].

A monkey (Macaca rhesus) was inoculated subcutaneously with 2 Leishmania isolates (MRHC/CN/88/KXG-2 and MRHO/CN/90/KXG-56) from Karamay big gerbils (Rhombomys opimus) into the forehead and cheek, 36 days later ulceration with a diameter of 0.2cm occurred at 2 sites. The lesions persisted for 60 and 95 days respectively and then healed spontaneously. However, Leishmania could be detected from the dermal tissue for more than one year. When MRHO/CN/88/KXG-2 isolate was inoculated into the forearm or ear of the monkey, no ulceration but swelling was revealed at the sites of inoculation. Leishmania was found to be latent in the subcutaneous tissue of the forearm for more than 103 days. The monkey was dissected one year after inoculation, no evidence of visceral involvement was noted. Another isolate (MRHO/CN/87/KXG-12) was inoculated subcutaneously into the forearm of 2 healthy volunteers. One of them developed cutaneous leishmaniasis. An ulcer with a diameter of 0.4cm and a nodule as large as 0.9 x 1.5cm around the ulcer occurred at the inoculation site 128 days later. Leishmania parasites were found harboring in macrophages of the dermis and in the epithelial cells of hair follicles. The results indicate that Leishmania parasite of the Karamay big gerbils is pathogenic to monkey and man, in addition to the fact that human cutaneous leishmaniasis and gerbil's Leishmania co-exist in Karamay, it seems that big gerbil is a potential reservoir of human cutaneous leishmaniasis in this area.

Animals↗
PubMed1991

Ca(2+)-insensitive modulation of a K+ conductance by inositol polyphosphates.

Macrophages derived from phorbol ester-induced human leukemic (HL-60) cells exhibit a voltage-activated inward rectifying potassium conductance which was modulated by macrophage colony-stimulating factor (Wieland, S. J., Chou, R. H., and Gong, Q. H. (1990) J. Cell. Physiol. 142, 643-651). Roles of intracellular messengers in this regulatory mechanism were investigated. Intracellular dialysis with inositol 1,3,4,5-tetrakisphosphate (IP4) or inositol 1,4,5-trisphosphate during tight-seal whole cell recording produced a rapid increase in the inward rectifying conductance. Changes in intracellular Ca2+ levels alone did not reproduce the stimulatory effect of these modulators. Intracellular dialysis with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) resulted in profound inhibition of this conductance. These data suggest a novel cellular function for inositol polyphosphates, particularly IP4, and show antagonistic modulation with GTP gamma S on a human macrophage inward rectifier.

Calcium↗
PubMed1991

Acetylcholine receptors from normal human muscle: concentration, purification, and use in radioreceptor assays for autoantibodies.

We measured the concentration of acetylcholine receptor (AChR) in various normal human muscles, obtained at autopsy, in order to find a more reliable and convenient source than the amputated leg muscle from diabetic patients for the isolation of AChR. We found that human calf muscle contains the highest concentration of AChR, approaching the concentration of receptor required for the radioreceptor assay for autoantibodies in myasthenia gravis (MG). Because the amount of contaminant proteins in the receptor preparations affect the sensitivity and precision of the assay, various chromatographic techniques were tested to improve the purity of the receptor preparation. We found that both G-100 Sephadex and DEAE Sephacel chromatographies were effective in removing contaminant proteins. DEAE Sephacel chromatography is particularly useful because the procedure provides higher recovery of receptor. Against the same pool of autoantibodies from patients with MG, AChRs from diabetic and normal leg muscle exhibit similar affinities. We conclude that AChR from normal human calf muscle can be used in radioreceptor assays for measuring autoantibodies in patients with myasthenia gravis.

Autoantibodies↗