Two-dimensional electrophoretic analysis of membranous protein from human thyroid tissues and cancer cell lines

Thyroid neoplasm is the most commonly encountered neoplastic disorder in endocrine clinics. Thyroid scan, ultrasonography, and fine needle aspiration cytology (FNAC) are used as diagnostic tools to differentiate a malignant nodule from a benign lesion. There are certain limitations and pitfalls in FNAC, especially in the diagnosing of follicular tumors. The lack of characteristic findings or a specific tumor marker are the most common problems in the preoperative diagnosis of thyroid follicular carcinoma. Although serum thyroglobulin level has been used as a tumor marker for post-operative, well-differentiated thyroid cancer, the assay cannot be used for preoperative diagnosis of thyroid carcinoma. In this study, various thyroid tissues and cancer cell lines including CGTH W-1, CGTH W-3, RO 82 W-1, SW 579 cell lines were used for the investigation of tumor markers. Specific spots were identified in the area near the 60 kDa molecular mass protein and isoelectric point (pI) 5.9 of the CGTH W-1 cell line. These spots could not be found in the papillary or anaplastic thyroid cancer cell lines. Another spot with a molecular weight of about 9.8 kDa with a low pI of 4.8 was present in the CGTH W-1 and RO 82 W-1 cell lines. This spot appeared to be a tumor marker of follicular cancer cells. This spot could not be found in the papillary and anaplastic cancer cell lines and other benign thyroid tissues. Specific proteins that were identified in this study may be useful as tumor markers for follicular thyroid carcinoma.     

Charge heterogeneity of macrophage migration inhibitory factor (MIF) in human liver and breast tissue

Macrophage migration inhibitory factor (MIF) is an ubiquitous protein playing various immunological and hormonal roles. Theoretical electrophoretic coordinates calculated from protein sequence in the SWISS-PROT database (AC P14174) are 12 kDa and pI 8.24. Using two-dimensional (2-D) immunoblotting, we have detected isoelectric forms at ca. 11.9 kDa, with pI values of 7.8 and 6.98 in human liver tissue, breast tissue and a cell line and in preparations of human MIF expressed in E. coli. This evidence suggests that MIF charge heterogeneity originates from a post-translational modification not requiring eukaryote-specific enzymes. We have also detected in human liver a minor immunoreactive spot at pI 6.23, which coincides with the MIF spot in the liver map in SWISS-2DPAGE. The pI 6.23 isoform also conceivably derives from post-translational modification, as MIF is known to be encoded in the human genome by a single copy gene.     

In vitro viability of cryopreserved equine embryos following different freezing protocols

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4 degrees C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C

BACKGROUND: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. METHODS: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed. RESULTS: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pls) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was beta-GlcNAc. Lactoferrin showed glycation with NANA-alpha-2-6-Gal or NANA-alpha-2-6-GalNAc, whereas the sugar specificity of sIgA was NANA-alpha-2-3-Gal. CONCLUSIONS: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-alpha-2-3-Gal.     

Effects of renovascular hypertension on myocardial protein patterns: analysis by computer-assisted two-dimensional gel electrophoresis

Hypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer-assisted two-dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9-week-old rat siblings. Sham-operated animals served as controls. Systolic blood pressure (197 +/- 19 mm Hg) and heart/body weight ratios (0.36 +/- 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 +/- 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann-Whitney test) as well as a multivariate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern.     

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells

Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.     

Purification and characterization of a human sialoglycoprotein antigen expressed in immature thymocytes and fetal tissues

The monoclonal antibody UN1 was previously produced in our laboratory on the basis of selective reactivity with human thymocytes and has been classified as unclustered by the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. The antigen recognized by mAb UN1 was found to be expressed on the cell surface of immature human thymocytes, a subpopulation of peripheral T lymphocytes and on several fetal tissues including thymus. The UN1 antigen is purified from children's thymus by ion-exchange and affinity chromatography. Two-dimensional electrophoresis shows that the purified antigen displays microheterogeneity appearing as multiple spots over a pI range 4.4-5.0 at 100-120 kDa. Treatment with neuraminidase results in a retarded migration in SDS-PAGE, an increase in isoelectric point and a reduction in carbohydrate content, indicating a substantial content of sialic acid. Glycosidase digestion and lectin-binding analysis indicate that the carbohydrate residues are essentially O-linked. A preliminary analysis has detected the UN1 antigen in human breast carcinoma tissues but not in normal breast. The biochemical features and the pattern of expression of the UN1 antigen indicate that this molecule may have the characteristics typical of the family of cell-membrane-associated mucin-like glycoproteins; a number of these molecules are thought to have a role in cell-cell interaction, tumor progression and metastasis.     

Isolation and characterization of AFP-binding proteins from tumor and fetal human tissues

The AFP receptor (rAFP) was discovered in embryonal and tumor tissues with a high level of proliferation. The AFP-binding protein (AFPbp) possibly containing the AFP-receptor (rAFP) was isolated from human embryos and human breast cancer tissue using affinity chromatography on an AFP-Sepharose column. The similarity of molecular weight, subunit composition, and immunological characteristics was shown for embryonal and tumor AFPbp using immunoblotting, gel-filtration, and PAAG electrophoresis. Judging from Superose-12 gel-filtration data, the protein molecular weight made up to 320-380 kDa. The presence of an IgG-binding site was detected in embryonal and tumor AFPbp by Western blot analysis.     

Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5-200 kDa. For the first-dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second-dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.     

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.     

Electrical properties of the rod syncytium in the retina of the turtle

1. Intracellular responses were recorded from rods in isolated eye-cups of the snapping turtle. Chelydra serpentina. Responses to flashes of small (less than 100 mum diameter) and large (1000 mum diameter) spots of 500 nm light were studied. 2. Responses produced by small and large diameter spots which delivered less than 0-3 photons mum-2 had the same shape. The responses produced by large spots were, however, nearly ten times greater in amplitude. The difference in amplitude is termed enhancement. 3. Perfusing an eye-cup with a Co2+-containing medium blocked synaptic transmission from receptors to horizontal cells but did not affect the responses of rods. 4. The membrane conductance of a single rod, estimated by three independent methods, was approximately 1-2 X 10(-9) MHo. 5. Enhancement can be predicted by a mathematical model which treats rods as an electrical syncytium. The space coefficient describing the spread of current is approximately 65 mum indicating that the coupling conductance between rods was relatively high. 6. When the intensity of a small spot was increased from 0-3 photons mum-2 up to 6 photons mum-2, the shape of the response was unchanged. When the intensity of a large spot was increased to more than 0-3 photons mum-2, the voltage during the recovery phase was decreased. This decrease is termed disenhancement. 7. The voltages produced by bright, large and small diameter spots which delivered the same quantity of light to the impaled rod were compared. The voltage produced by a large diameter spot became for a short period during the recovery phase less than the voltage produced by a small diameter spot. This observation indicates that the response to a large spot included during recovery an active process which is not apparent in the response to a small spot.     

Cryopreservation of teeth. Organizational aspects of a tissue bank for tooth tissues

Squamous cell carcinoma (SCC) is one of the most common cancers in human. SCC, particularly, esophageal and lung SCC are relatively resistant to currently available regimens of chemotherapy or radiation therapy. Therefore, development of a specific immunotherapy using tumor specific cytotoxic T lymphocytes (CTL) would be important to offer other treatment modalities. However, generation of HLA class I-restricted CTL recognizing SCC has been rarely reported. We established the HLA A2601-restricted CTL cell line recognizing a peptide antigen expressed on SCC. This CD4- CD8+ cytotoxic T lymphocyte (KE-4 CTL) cell line was established in a patient with esophageal cancer. The KE-4 CTL recognized a peptide antigen on esohageal and lung SCC in an HLA A2601-restricted manner as evaluated by cytotoxity against a panel of tumor cells, transfection experiments with HLA A2601 cDNA, and reconstitution with eleted peptides. None of normal cells tested was lysed by this CTL. These results suggest the exstence of HLA A2601-restricted CTL precursors recognizing a peptide antigen on SCC in a patient with esophageal cancer.     

Subcellular localization of enterokinase (enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3% in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (Mr, 2 - 10(6)) and two larger peaks of free enzyme (Mr, 3 - 10(5) and 9 -10). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Testicular Sertoli cell tumor with a heterologous sarcomatous component: immunohistochemical assessment of Sertoli cell differentiation

OBJECTIVE: Immunohistochemical staining is reported to be useful in distinguishing ovarian Sertoli-stromal cell tumors from carcinosarcomas. To assess Sertoli cell differentiation in a rare malignant biphasic testicular tumor, we compared the immunophenotypic profile of the tumor with that of Sertoli cell nodules and adenomas and mullerian carcinosarcomas. DESIGN: Immunohistochemical staining was performed on 6 testes (4 with hyperplastic Sertoli cell nodules, 2 with Sertoli cell adenomas) and 7 carcinosarcomas (6 involving the uterus, 1 involving the uterus and ovary) using primary monoclonal antibodies AE1/AE3, CAM 5.2, CA 19.9, and antibodies directed against epithelial membrane antigen, carcinoembryonic antigen (monoclonal and polyclonal), S100, placental alkaline phosphatase, and inhibin. These staining results were compared with those of the index case. RESULTS: All testes showed positive staining for inhibin and vimentin in the Sertoli cells of the nodules and adenomas. One Sertoli cell nodule showed focal staining with AE1/AE3 and CAM 5.2. Both adenomas showed focal positive staining for S100. All nodules and adenomas were negative for epithelial membrane antigen, monoclonal and polyclonal carcinoembryonic antigen, CA 19.9, and placental alkaline phosphatase. In contrast, the carcinomatous areas of the carcinosarcomas were all negative for inhibin but exhibited positive staining for AE1/AE3, CAM 5.2, and epithelial membrane antigen. The carcinosarcomas showed variable expression of vimentin, S100, carcinoembryonic antigen, CA 19.9, and placental alkaline phosphatase. The epithelial component of the tumor from the index case showed strong diffuse staining for inhibin and vimentin and only very faint focal staining with AE1/AE3 and CAM 5.2. The epithelial component was negative for epithelial membrane antigen, monoclonal and polyclonal carcinoembryonic antigen, S100, CA 19.9, and placental alkaline phosphatase. CONCLUSIONS: The immunohistochemical findings in the index case support the diagnosis of Sertoli cell tumor with a heterologous sarcomatous component over carcinosarcoma. Inhibin seems to be the best single marker for Sertoli cell differentiation. To our knowledge, only 1 other case of this rare testicular tumor has been reported in the literature.     

Serologic assay for secretory component distinguishes mechanical from hepatocellular cholestasis in humans

In rats, serum secretory component (SC) is elevated in mechanical but not hepatocellular cholestasis. To determine if serum SC might distinguish cholestatic syndromes in humans, serum samples were obtained from control subjects and patients with mechanical and hepatocellular cholestasis. Equal volumes of serum were assayed for SC by immunoblotting with an antibody specific for human SC. Quantitative densitometry of these immunoblots showed that in mechanically obstructed patients serum SC was reversibly elevated to a level approximately 10-fold higher than that of patients with hepatocellular cholestasis (P < 0.001). When comparing the two cholestatic groups, levels of serum alkaline phosphatase, but not bilirubin and alanine aminotransferase, were significantly higher in the group with mechanical cholestasis (P < 0.01). When comparing individual patients, serum SC was more reliable than alkaline phosphatase in distinguishing the two cholestatic syndromes (P < 0.05). Thus, serum SC may distinguish mechanical from hepatocellular cholestasis in humans.     

Effect of proton pump inhibitor, omeprazole, on expression of rat parietal cell specific carbohydrate antigen, type 2 chain N-acetyllactosamine

The effect of a proton pump inhibitor, omeprazole, on the rat gastric mucosal expression of carbohydrate antigens was studied. Type 2 chain N-acetyllactosamine was detected specifically on the apicocanalicular cell membranes of parietal cells. Pretreatment of rats with omeprazole profoundly suppressed the antigen expression, which followed the inhibition of gastric acid secretion. When omeprazole was discontinued, the antigen was reexpressed, which preceded the restoration of acid secretion. The antigen-negative tissues became antigen-positive when they were desialylated. Gastric membrane vesicles from the normal and omeprazole-treated rats were antigen-positive and -negative, respectively. SDS-PAGE revealed that a glycoprotein with an apparent molecular weight of 64-78 kDa carried type 2 chain N-acetyllactosamine. In the omeprazole-treated rats, the same molecular weight glycoprotein was positively immunostained only after desialylation. We concluded that: (1) the expression of type 2 chain N-acetyllactosamine was closely correlated with gastric acid secretion, and (2) the inhibition of acid secretion was accompanied by the sialylation of the parietal cell membrane glycoprotein.     

Trichodysplasia and amelogenesis imperfecta

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes is described. The visualization of hybrids is done using the indoxyl-nitro blue tetrazolium alkaline phosphatase reaction. The addition of polyvinyl alcohols of high molecular weight (40-100 kDa) to the reaction medium enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a 20-fold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.     

Reproducibility of polypeptide spot positions in two-dimensional gels run using carrier ampholytes in the isoelectric focusing dimension

The reproducibility of complex protein patterns in two-dimensional (2-D) gels run with carrier ampholytes in the first dimension has been investigated. Two different laboratories collaborated in the study and 18 or 19 gels were run in each laboratory for comparison. The electrophoresis chemicals, running devices, and samples were standardized in both labs. The resulting 37 gels were scanned with a charge-coupled device (CCD) camera and spots were located, counted, quantified, and matched using a commercially available image analysis system. Subsequently, the reproducibility of spot position was determined. To perform the statistical analysis, the test gels were initially each matched to a master reference gel. Next, three sets of 12 gels (the image analysis software database could analyze only 12 gels at a time) were analyzed and the isoelectric point (pI) and molecular weight (M(r)) positional variation of all the spots that matched across the gels in each set was determined. The resulting statistical analysis indicates very high reproducibility of the carrier ampholyte technique.