Gene/Protein Disease Symptom Drug Enzyme Compound
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 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lymphoducts and blood vessels exist in the stroma, while none can be detected in the cancer nest itself within cancerous tissue. This explains why metastasis of carcinoma cannot occur without the escape of tumor cells through the basement membrane surrounding the cancer nest into the stroma. Accordingly, observation of the continuity of the basement membrane, what we call the cancer nest membrane, is essential for elucidating the first step of metastasis. Since type IV collagen is the most important structure composing the basement membrane, investigation of the immunohistological localization and continuity of type IV collagen is of value in predicting the metastatic aggressiveness of squamous cell carcinoma. We therefore studied biopsy tissues from the advancing lesion of head and neck squamous cell carcinoma in 95 untreated patients. The tissues were fixed in 85% ethanol and embedded in paraffin, and 5-um thin sections prepared were then immunohistochemically stained for type IV collagen by the ABC method for observation of the continuity status of the cancer nest membrane in relation to metastasis. The basement membranes of normal mucosal epithelium and normal interstitial capillaries were utilized as positive controls, and negative controls were obtained by using PBS in place of the primary antibodies for the immunohistochemical reaction. Membrane discontinuity (breaks or absence) correlated significantly with cervical lymph node metastasis, while intact membrane was associated with a low frequency of cervical lymph node metastasis. There was no obvious relation between the clinical T category and the continuity of the membrane; pN (+) carcinomas with membrane discontinuity included even T1 supraglottic and hypopharyngeal carcinomas, as well as T2 or higher oral mucosal carcinomas and T3 or higher glottic carcinomas, suggesting variation with tumor site. Hypopharyngeal and supraglottic carcinoma was associated with membrane discontinuity and a high incidence of cervical lymph node metastasis. On the other hand, glottic and oral carcinoma more often presented with intact membranes and had a lower incidence of metastasis, although carcinomas in these sites that did present with discontinuity of the membrane were associated with a high incidence of cervical metastasis. Therefore, observation of the continuity of the cancer nest membrane by the expression of type IV collagen may be useful in selecting better specific therapies and determining the necessity of prophylactic neck dissection. A correlation between the degree of tumor differentiation and the continuity of the membrane was also found; well-differentiated tumors with discontinuity of the membrane were frequently associated with cervical lymph node metastasis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Immunohistological investigation of type IV collagen in the basement membrane surrounding the cancer nest (cancer nest membrane) of head and neck squamous cell carcinoma--its relation to frequency of cervical lymph node metastasis]. 146 93

Human alpha 2-macroglobulin (alpha 2M) was eluted as a single nondispersed peak from a TSK-G4000SW size exclusion chromatography column equilibrated in 20 mM-sodium phosphate/100 mM-NaCl, pH 7.2 (PBS). The void volume and total accessible volume of the column were 6.08 ml and 14.42 ml. The elution volume (Ve) of native alpha 2M was 9.20 +/- 0.04 ml. The Ve was altered minimally by changing the ionic strength or adding ethanol to the equilibration buffer. Ternary alpha 2M-trypsin, containing 2 mol of proteinase/mol of inhibitor, and alpha 2M-methylamine failed to be eluted in well-defined peaks when the column was equilibrated in PBS. The majority of either preparation was recovered slowly at Ve values greater than 14.5 ml, reflecting significant nonideal interactions with the support structure. Addition of 10% ethanol or increased ionic strength in the equilibration buffer independently caused either form of reacted alpha 2M to be eluted in a distinct peak at decreased Ve, suggesting that the nonideal interactions included hydrophobic and electrostatic adsorption. When the equilibration buffer was 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2, partial resolution of ternary alpha 2M-trypsin and alpha 2M-methylamine was obtained with a single column run. The Ve values of ternary alpha 2M-trypsin and alpha 2M-methylamine in this buffer were 13.15 +/- 0.08 ml and 11.94 +/- 0.14 ml, respectively. The Ve of native alpha 2M was 8.84 +/- 0.03 ml. The resolving capacity of TSK-G4000SW was exploited to purify native alpha 2M rapidly and efficiently from solutions that contained significant amounts of either ternary alpha 2M-trypsin or binary alpha 2M-trypsin (1 mol of proteinase/mol of inhibitor). This purification was complete within the limits of sensitivity of denaturing and nondenaturing polyacrylamide-gel electrophoresis. alpha 2M-plasmin was well resolved from native alpha 2M. The Ve of alpha 2M-plasmin was 12.88 +/- 0.32 ml in 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2. A number of procedures were used to prepare solutions with up to 90% binary alpha 2M-trypsin. The Ve of binary alpha 2M-trypsin in these various solutions was intermediate between the values of native alpha 2M and ternary alpha 2M-trypsin. The conformations of binary and ternary complex, as reflected by mobility in nondenaturing electrophoresis, were identical, confirming previous results. Finally, in the binary alpha 2M-trypsin complex, the single trypsin cleaved more than two, and as many as all four alpha 2M subunits.
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PMID:Purification and characterization of human alpha 2-macroglobulin conformational variants by non-ideal high performance size-exclusion chromatography. 242 74

We developed a rapid technique for preservation of Hoechst 33342/propidium iodide-stained cells, using ethanol as a fixative. Combined staining with these dyes makes possible analysis of cell-cycle phase-specific cell death. The technique relies on exclusion of propidium iodide from the viable cells, whereas Hoechst stains all of the cells. The bivariate histograms resulting from the flow cytometric analysis contain the equivalent of two single-parameter DNA histograms, one of the living and the other of the dead cell population. Preservation of staining involved addition of 25% ethanol in PBS after propidium iodide staining and before Hoechst staining. The separation between the living and the dead cell populations was maintained for over 3 days at 4 degrees C. This technique will be valuable for quantitative evaluation of the cell-cycle phase-specific effects of cytostatic or cytotoxic agents, particularly in situations where a lag period between staining and analysis is unavoidable.
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PMID:Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation. 245 47

We have established an enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (aCL) using standard sera obtained from the Rayne Institute, St. Thomas' Hospital (London, U.K.). In this study, we compared several fundamental requirements for the assay with the standard assay as a reference, such as conditions for antigen application and test samples using our patients' samples. In addition, the specificity of our assay and cross-reactivities of aCL were also evaluated. In the standard assay, the concentration of antigen was optimal in the range of 30-100 micrograms/ml. The antigen-coating temperature was optimal at 4 degrees C for 16 hours. The method based on rapid evaporation of CL-ethanol solution can be used instead of the standard method. On the other hand, there was no significant difference in the results between physical conditions of the antigen (CL-ethanol solution vs. CL-micelles), between washing solutions (saline vs. PBS containing 0.05% Tween-20) and between test samples (sera vs. plasma). The aCL activity in our patients' samples was almost completely inhibited by pre-incubation of sera with either CL or phospholipid reagent for activated partial thromboplastin time (APTT). Interestingly, the aCL activity of the lupus anticoagulant was negative, but aCL-positive samples were also absorbed by the reagent for APTT. No inhibition of the aCL activity, however, was observed when patients' sera were preincubated with ss-DNA.
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PMID:Anticardiolipin antibodies by an enzyme-linked immunosorbent assay: fundamental studies on the conditions for antigen-application and specificity of the assay. 263 59

In newborn rat skin, disulfide bonds exist in the cell membrane of the stratum corneum and in the cytoplasm of the stratum granulosum. However, in the latter region, they can be detected after ethanol fixed epidermis remains for 2 weeks at 4 degrees C. Thus, the idea arose that the visualization of the disulfide bonds in the stratum granulosum may be caused by proteases. Therefore, the in situ effects by both activation and inhibition of epidermal proteases were examined in tissue sections. Cryostat sections which were fixed in cold ethanol (-20 degrees C, 98%) were incubated either in PBS, pH 7.4, containing 10mM 2-ME as a control or in an epidermal homogenate (15,000 x g supernatant fraction containing 2-ME) at 37 degrees C for 30 minutes and 4 hours, respectively, in order to activate the epidermal proteases and then reacted with NEM, 2-ME, and DACM in steps. Many minute fluorescent particles were seen in the stratum granulosum under both of the above conditions. However, with incubation in PBS without 2-ME, no fluorescent particles were seen. In addition, when the tissue was treated with NEM and zinc chloride before incubation, no fluorescent particles were seen. O-phenanthrolin remarkably inhibited the appearance of these fluorescent particles. In contrast, leupeptin and antipain only slightly inhibited the appearance of these fluorescent particles, and pepstatin and phenylmethylsulfonyl fluoride didn't inhibit at all. In general, as NEM and zinc chloride strongly inhibit thiol proteases, these findings suggest that presence of the minute fluorescent particles in the stratum granulosum could be due to the effects of proteases, especially thiol proteases.
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PMID:The substrate of epidermal proteases--effects of in situ activation of epidermal thiol proteases on stratum granulosum cells. 265 3

Encouraged by recent reports of extraction of diagnostically useful DNA from formalin-fixed tissues, we devised a study to determine which fixatives are suitable for DNA hybridization studies. Seven lymph node specimens (6 lymphomas and one reactive hyperplasia) were studied. Specimens were divided into portions, each of which was processed separately. Processing protocols followed were: 1) snap freezing; 2) immersion in formalin, ethanol, glutaraldehyde, B5 and methanol acetic acid (MAA) Michel's transport medium or phosphate buffered saline for 2, 24, 48 and 120 hrs.; 3) paraffin embedding following fixation. DNA was obtained by proteinase K digestion followed by phenolchloroform extraction. DNA was cleaved with restriction endonucleases (Eco RI and Bam HI), separated by agarose gel electrophoresis, after which Southern blot hybridization with radio-labelled probes for J-heavy and T-beta genes was performed. Fixation with formalin, glutaraldehyde and B5 resulted in poor yields of DNA. MAA produced degradation of DNA and Michel's medium and PBS gave low quantities and purity of extracted DNA. Ethanol fixation consistently permitted extraction of large amounts of high molecular weight DNA suitable for hybridization studies and compared favourably with the fresh-frozen 'gold standard'. We conclude that ethanol may be the fixative of choice when transport or storage conditions limit the availability of fresh frozen tissue for DNA hybridization studies.
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PMID:The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. 313 29

A method is described for the immunofluorescent testing of autoantibodies in the sera of patients with vestibulo-cochlear disorders, using fixed decalcified inner ear tissue preparations from guinea pigs. Fixation in cold ethanol and decalcification in EDTa at 4 degrees C preserved the immunological reactivity of the inner ear tissue in use and allowed excellent delineation of its structural details. Counterstaining of the sections with Evans-blue dye aided in a better identification of the inner ear anatomical structures and in a more precise location of the antibodies present. By mounting the sections in p-phenylenediamine-PBS-glycerin solution, the rapid extinction of the fluorescence seen during photomicroscopy was retarded and the intensity of the immunofluorescence was enhanced. Preservation of the slides for documentation was improved by fixing the intermediate layer of the antigen and the labeled antibody in cold ethanol. The inner ear tissue preparations retained their fluorescent staining up to 6 months. Our method is convenient for screening patients with inner ear disorders for autoantibodies against the different cellular elements and for testing the possible presence of antibodies against the inner ear tissue. So far, we believe that the antibodies detected are not tissue (inner ear) specific.
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PMID:Optimizing immunofluorescence for the testing of autoantibodies in inner ear disorders. 329 40

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.
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PMID:Choice of extraction procedure for estimation of anterior pituitary hormone content. 343 4

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation less than 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.
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PMID:Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. 350 96

A Japanese patient with epidermolysis bullosa acquisita (EBA) was autopsied, and direct immunofluorescence (DIF) testing was performed. Using this patient's serum (EBA serum) and three bullous pemphigoid (BP) sera, the anatomical distribution and immunological characteristics of EBA antigen and BP antigen were investigated by indirect immunofluorescence (IIF). EBA antigen showed the same anatomical distribution as BP antigen in DIF and IIF studies; both antigens were limited to the skin, tongue, oesophagus, trachea, cornea and bladder. EBA antigen was located on the dermal side of both NaCl and PBS-separated skin, whereas BP antigen was limited to the epidermal side. Ethanol fixation abrogated the antigenic stability of BP antigen, but not that of EBA antigen. No difference was found when acetone or formalin fixation was used. The separation methods and prefixation in ethanol could be useful techniques applicable to the classification of the bullous disorders which manifest circulating anti-BMZ antibodies.
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PMID:Anatomical distribution and immunological characteristics of epidermolysis bullosa acquisita antigen and bullous pemphigoid antigen. 352 10


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