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PubMed1995

Fluorescence image analysis of the MCF-7 cycle related changes in chromatin texture. Differences between AT- and GC-rich chromatin.

This paper reports on quantitative in situ changes in chromatin structure that occur throughout the cell cycle of the human breast cancer epithelial cell line, MCF-7. Texture parameters were measured by image cytometry on nuclei stained by DNA specific fluorochromes. These parameters calculated from the co-occurrence and run length matrices of grey level images were previously shown to be related to condensation, organization and distribution of DNA. In some experiments, cells were triple stained for DNA/Ki-67/PCNA, and compartmentalization in the cycle was ascertained from the Ki-67/PCNA pattern expression. In these experiments, Hoechst dye was used to stain DNA. Chromatin of cells traversing G1 phase progressively decondensed and became homogeneously distributed. In addition, these G1 cells had more condensed chromatin than cells in G0 phase (as determined by Ki-67 negative staining). During the S and G2 phases, chromatin condensation took place and an increasing reticulated organization was quantified. Similar profile of changes in chromatin texture was found in experiments done with cells double stained by AT-specific Hoechst dye and the GC-specific mithramycin dye. GC-rich chromatin texture-associated parameters greatly varied comparing to those of AT-rich chromatin during the G0/G1 phase as well as in the first mid-S phase. Conversely, variation of the AT-associated parameters was much greater in the second half of S phase as compared to the GC-associated parameters that barely varied during this period. This study well establishes the correlation between in situ chromatin texture and proliferation state because the latter is assessed by proliferation-associated antigens. Moreover, changes in chromatin texture are independently ascribed to the AT- and GC-rich regions suggesting that these 2 types of chromatin are involved to different extents in transcriptional and replicational tasks.

Base Composition↗

PubMed1989

An immunohistochemical analysis of T cells in primary B cell malignant lymphoma of the brain.

An immunohistochemical study was performed on small lymphoid cells present in frozen tissue sections of seven cases of primary B cell malignant lymphomas of the brain by using monoclonal antibodies to T cell (Leu-1, OKT-11, Leu-3a, and Leu-2a) and B cell (BA-1 and Leu-12) surface markers. In all the seven cases, positive reaction for Leu-1 and OKT-11 was seen in the majority of the small lymphoid cells which were dispersed among the lymphoma cells or clustered around blood vessels. The large neoplastic cells were unstained by these antibodies. Staining for T cell subsets with antibodies to Leu-3a and Leu-2a showed heterogeneous staining in each case. The ratio of Leu-3a+ to Leu-2a+ cells was less than one in six cases, demonstrating a suppressor/cytotoxic phenotype predominance. Most of these small lymphoid cells were negatively stained by antibodies to BA-1 and Leu-12. From these findings, it was shown that the small lymphoid cells observed in primary B cell lymphomas of the brain were of T cell lineage, distinct from the neoplastic cells, and probably reactive in nature. The implications of these findings are discussed.

Brain Neoplasms↗

PubMed2002

Three-dimensional electron cryo-microscopy study of the extrinsic domains of the oxygen-evolving complex of spinach: assignment of the PsbO protein.

Three independent three-dimensional reconstructions of the spinach photosystem II-light-harvesting complex supercomplex were derived from single particle analyses of non-stained, vitrified samples imaged by electron microscopy. Each reconstruction was found to differ significantly in the composition of the lumenal oxygen-evolving complex extrinsic proteins. From difference mapping, aided by electron microscopy of negatively stained selectively washed samples, regions of density were assigned to the PsbO and PsbP/PsbQ proteins. Interpretation of the density assigned to the PsbO protein was explored using computer-aided structural predictions. PsbO is calculated to be mainly a beta-protein (38% beta) composed of two domains within an overall elongated shape (Pazos, F., Heredia, P., Valencia, A., and De Las Rivas, J. (2001) Proteins Struct. Funct. Genet. 45, 372-381). The positioning and fitting of the proposed structural model for the PsbO protein within the three-dimensional map indicated that there is a single copy per reaction center. Moreover, the structural model derived for PsbO, together with difference mapping, indicates that this protein stretches across the surface of the reaction center with its N- and C-terminal domains located toward the CP47 and CP43 side, respectively. This structural assignment is discussed in terms of the recent x-ray-derived cyanobacterial model of PSII (Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739-743).

Cryoelectron Microscopy↗

PubMed1989

Association of Reoviridae particles in an enteric syndrome of poults observed in turkey flocks during 1988.

An enteric syndrome of turkey poults, characterized by enteritis, crop mycosis, intestinal changes (pale, thin-walled ballooning with watery contents), and rickets, occurred during 1988 in 74 turkey flocks from different farms belonging to 9 California turkey growers. The flocks ranged in size from 9,000 to 120,000 birds. Pools of intestine sections from 618 birds, representing 78 field cases, were examined. Histopathological examination of the intestines showed a mild to severe atrophy with a reduced depth of crypts, which was more prominent in the distal part of the small intestine. Viral isolation attempts with primary cell cultures of chicken embryo kidney cells were negative. Examination by electron microscopy of negatively stained intestinal specimens revealed the presence of Reoviridae particles of 58.8 to 80 nm in diameter. Enzyme-linked immunosorbent assay results on the intestinal pools for mammalian and group A avian rotaviruses were negative. A statistically significant relationship was found for the presence of Reoviridae particles in the intestines of 10-21-day-old birds. Of the 7 most common pathological conditions analyzed, 2, rickets and intestinal changes (thin-walled ballooning intestine with watery contents), showed a statistically significant association with the presence of Reoviridae particles.

Age Factors↗

PubMed1994

Methods in pathology. Optimization of proliferating cell nuclear antigen immunohistochemical staining by microwave heating in zinc sulfate solution.

Immunohistochemical staining of routinely processed formalin-fixed, paraffin-embedded archival tissue sections with proliferating cell nuclear antigen (PCNA) monoclonal antibody has facilitated the understanding of the regulation of cell proliferation. However, false-negative staining due to masking or poor preservation of antigen epitopes by fixatives or other unknown conditions has been a major problem in PCNA immunohistochemical studies. Microwave heating (MWH) has been used to retrieve antigen in archival tissue. However, optimal staining conditions have not yet been defined. We therefore investigated the effect of MWH with or without a metal solution (zinc sulfate) on routinely processed paraffin-embedded tissue to determine the optimal conditions for retrieval of PCNA. The results were compared with routine immunohistochemical staining. We found that MWH in 1% zinc sulfate solution successfully retrieved PCNA; the optimal MWH time was 7.5 mins. In contrast, when MWH was performed in water, the specific intensity of PCNA expression was not increased because MWH not only enhanced the intensity of the antigen staining but also increased the background staining. In summary, MWH technique in zinc-sulfate solution allowed successful PCNA retrieval from formalin-fixed archival tissue and thus facilitated accurate immunohistochemical evaluation of cell proliferative activity in cancers.

Antibodies, Monoclonal↗

PubMed1999

Immunohistochemical detection of p53 protein accumulation in head and neck cancer: correlation with p53 gene alterations.

The genetic and functional status of the p53 gene may be an important factor in guiding therapeutic strategies for patients with cancer. The purpose of this study was to determine whether p53 immunohistochemistry (IHC) accurately reflects the mutational status of the p53 gene and to determine whether p53 IHC independently predicts tumor responsiveness to radiation therapy for patients with HNSCC. p53 IHC was performed using the monoclonal antibody DO7 on tumors from 85 patients with HNSCC treated with primary or adjuvant radiation. The p53 status in all of these tumors was previously assessed by direct sequence analysis of exons 5 through 9: 49 tumors were p53 wild-type, and 36 harbored p53 gene mutations. All patients were well characterized with respect to locoregional recurrence, distant spread, and survival. Positive p53 staining was observed in 53 of the 85 cases (62%). Only 27 (51%) of these 53 IHC-positive cases harbored gene mutations in exons 5 through 9; 23 (72%) of the 32 IHC-negative cases did not harbor mutations. The overall correlation rate between IHC and sequencing was 59% (P < .04, chi2). Discordant results were observed for 35 (41%) cases, including 26 IHC-positive cases and 9 IHC-negative cases. In 7 of 9 cases, false-negative staining was due to a nonsense or splice-site mutation. p53 IHC was not predictive of overall survival (P = .37) or disease-free survival (P = .95). In a sizable number of cases, p53 IHC does not reflect the mutational status of the p53 gene. Specific types of alterations (eg, truncating mutations) and other factors may contribute to this poor correlation. Moreover, p53 IHC does not appear to be an independent predictor of tumor responsiveness to radiation in patients with HNSCC.

Carcinoma, Squamous Cell↗

PubMed1997

Molecular nature of a sperm acrosomal antigen recognized by HS-13 monoclonal antibody.

Among the numerous monoclonal antibodies generated against human sperm antigens, HS-13 monoclonal antibody was shown to react with an intra-acrosomal antigen from human, mouse and rat. In this study, HS-13 was used as the affinity ligand for the purification of the cognate antigen from human sperm by immunoaffinity chromatography. The purified cognate antigen from human sperm, designated as HSAg-13, was found to be a protein with a molecular weight of approximately 80 kDa on SDS-PAGE in the presence of reducing reagents. This monoclonal antibody was used as the probe to study the tissue distributions and developmental expression of the cognate antigen from human, mouse and rat by immunohistochemical assays. It was concluded that the antigen recognized by HS-13 antibody is highly sperm specific and found only in sperm and mature testis, but not in any other somatic tissues examined in human and mouse. The antigen was shown to be expressed at the postmeiotic stages of spermatogenesis in mouse and rat. By using indirect immunofluorescent staining assay, HS-13 was shown to react only with the methanol-fixed acrosome-intact sperm but not with the live sperm. Following calcium ionophore A23187 treatment, acrosome-reacted sperm showed either negative staining or residual staining in the equatorial region, suggesting the intra-acrosomal location of HSAg-13. The spontaneous acrosome reaction following overnight incubation in BWW medium resulted in a statistically significant decrease of antibody-stained human sperm. In view of excellent correlations for the scoring of acrosome-intact sperm with that of fluorescence-labeled Pisum sativum agglutinin (PSA) probe, HS-13 monoclonal antibody can be routinely used for monitoring sperm capacitation and acrosome reaction.

Acrosome↗

PubMed2006

Increased expression of pAKT is associated with radiation resistance in cervical cancer.

Phosphorylated AKT (pAKT) is a major contributor to radioresistance in human cancers. The aim of this study was to investigate the association of pAKT expression and radiation resistance in cervical cancer. A retrospective review was made of the records of 27 women who received primary radiation therapy due to locally advanced cervical cancer (LACC) with FIGO stage IIB-IVA. Nine patients regarded as radiation resistant developed local recurrences with a median progression free interval of 9 months. Eighteen patients did not show local recurrences, and were regarded as a radiation-sensitive group. Using pretreatment paraffin-embedded tissues, we evaluated pAKT expression by immunohistochemistry. A significant association was found between the level of pAKT expression and local recurrence. Immunohistochemical staining for pAKT was significantly more frequent in the radiation-resistant than in the radiation-sensitive group (P=0.004). The mean progression-free survival was 86 months for patients with pAKT-negative staining (19 cases) and 44 months for patients with pAKT-positive expression (eight cases) (P=0.008). These results suggest that signalling from phosphatidylinositide 3-kinase/pAKT can lead to radiation resistance, and that evaluation of pAKT may be a prognostic marker for response to radiotherapy in LACC.

Adult↗

PubMed1976

Early myocardial ischaemia: evaluation of the histochemical haematoxylin-basic fuchsin-picric acid (HBFP) staining technique.

A series of experiments, carried out to evaluate the histochemical method for the morphological diagnosis of early stages of myocardial ischaemis (HBFP) is reported. The experiments were performed on dog hearts in which ischaemia was induced by coronary artery ligation for different periods of time. The original procedure or modifications of the HBFP-technique, including different staining, rinsing, and differentiation times, the use of different commercial brands of chemicals and preparatory changes of routine histological procedures such as fixation, embedding, cutting, and mounting, did not give satisfactory results. False positive and negative staining was frequent. Very equivocal results were obtained on serial sections of ischaemic tissue samples. Therefore this method was regarded as unreliable and non-reproducible.

Animals↗

PubMed2001

Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro.

PURPOSE: To isolate, culture, and characterize goblet cells from the conjunctiva of rats. METHODS: Conjunctival tissue was surgically removed from Sprague-Dawley rats. Goblet cells were then isolated from the nictitating membrane and fornix using explant cultures. Cells derived from the explants were grown and propagated in RPMI medium supplemented with 10% fetal bovine serum. They were characterized using an enzyme-linked lectin assay (ELLA) with the lectin Ulex europaeus agglutinin-1 (UEA-1), Western blot analysis, PCR, light and electron microscopy, specialized histochemistry and indirect immunofluorescence microscopy. RESULTS: Goblet cells were successfully isolated from conjunctival explants by scraping nongoblet cells from the culture vessel. To date, cultures have been passaged a minimum of three times without the loss of their specific cellular markers. Cells identified as goblet cells fulfilled the following criteria: positive staining for alcian blue/periodic acid Schiff reagent, cytokeratin (CK)-7, the lectins UEA-I and Helix pomatia agglutinin (HPA), MUC5AC, and M(3) muscarinic receptor; detection of MUC5AC mRNA using RT-PCR; and negative staining for CK-4, M(1) muscarinic receptor, and Banderia simplicifolia lectin. The authors also measured, using the ELLA, substantial amounts of UEA-I-detectable high-molecular-weight glycoproteins and MUC5AC released into the medium. CONCLUSIONS: Cultured goblet cells retain many characteristics of goblet cells in vivo and thus may serve as a useful tool in delineating the pathobiology of the ocular surface.

Animals↗

PubMed2003

BRCA2 mutations and androgen receptor expression as independent predictors of outcome of male breast cancer patients.

PURPOSE: Germline mutations of the BRCA2 gene are involved in the development of a considerable number of male breast cancer cases. Although phenotypic differences have been observed between sporadic and BRCA-related breast carcinomas, conflicting data exist on the differences in prognosis of women with hereditary and sporadic breast cancer. The purpose of the study was to investigate the prognostic value of BRCA2 status in male breast carcinoma (MBC). EXPERIMENTAL DESIGN: We studied 43 male breast cancer patients, including 12 with BRCA2 mutations. Tumor samples were characterized immunohistochemically using antibodies to estrogen receptor, progesterone receptor, and androgen receptor (AR). RESULTS: BRCA2-related tumors presented at the earlier age compared with sporadic tumors (P = 0.005). Patients positive and negative for BRCA2 mutations did not differ with respect to tumor size, lymph node involvement, histological grade, and sex hormone receptor status. Five-year disease-free survival (DFS) and overall survival (OS) were significantly decreased in BRCA2-positive patients (67% versus 28% for BRCA2-negative versus positive patients, respectively, P = 0.017 for DFS; 86% versus 25%, P = 0.006 for OS). Shorter survival was also correlated with expression of AR in tumor tissue (74% versus 33% for patients with tumors staining negatively and positively for AR, P = 0.029 for DFS; 71% versus 57%, P = 0.05 for OS). CONCLUSIONS: The BRCA2 mutations and AR expression in tumor tissue are independent adverse factors for MBC prognosis. BRCA2-related MBC presents at the earlier age compared with non-BRCA2-related cancer, but do not differ with respect to other clinicopathological features.

Adult↗

PubMed1991

[Monoclonal antibody MIA15-5, which inhibits cell motility and metastatic potential, has a great effect on the prognosis of the post-surgical patients with lung cancer].

The functional monoclonal antibody, selected based on the inhibition of cell motility, was found to be directed to specific carbohydrate structure Fuc alpha 1----2 Gal beta 1----R. This monoclonal antibody, MIA15-5 was established after immunization of mice with adenocarcinoma line of the lung PC7, and selected based on inhibition of U937, HEL, and MAC10 cell migration due to the transwell assay. MIA15-5 reacted with 30-40% of high metastatic variant BL6 of mouse melanoma B16 line and metastatic deposition to lung after injection of BL6 cells was strongly inhibited if MIA15-5 was injected within 3 hours, but was not inhibited by injection of other anti-H antibodies under the same conditions. Injection of MIA15-5 had much less inhibitory effect if administered 1 day after BL-6 injection, and no effect at 3 days. In immunohistochemical staining of 149 formalin fixed specimens of patients with lung cancer, especially adenocarcinoma has the highly frequency of MIA15-5 (65.4%, 49 of 75 cases) and the five year survival rate of the patients with negative staining of MIA15-5 was 58.6% and much better than one with positive staining, 20.9%.

Adenocarcinoma↗

PubMed1982

Rapid and reliable method for diagnostic electron microscopy of faeces.

Four methods are described for examining viruses in faeces by electron microscopy using negative staining. Faeces samples from a total of 180 patients with diarrhoea illnesses were processed for electron microscopy by the direct staining technique, pseudoreplica technique, microsolute concentration technique and by ultracentrifugation. Virus particles (including rotaviruses and adenoviruses) were found in fifty-five (31%) samples when the results of all methods were combined. Bacteriophages were found in thirty of the samples which were read virus negative. Herpesviruses were also found in the faecal samples of two patients with diarrhoea illness. Parvovirus-like particles were identified in one sample which was rotavirus and adenovirus positive. Calicivirus-like particles were found in a sample which was adenovirus positive. With the direct staining technique thirty-six (20%) samples contain virus particles; with microsolute concentration technique forty-eight (27%) samples contain virus particles and with ultracentrifugation thirty-eight (21%) contain virus particles. It is believed the microsolute concentration technique is rapid and more reliable than the pseudoreplica technique and ultracentrifugation method, and is a more preferable method for the diagnosis of faecal sample by electron microscopy.

Adenoviridae↗

PubMed1991

Expression of annexin VI (p68, 67 kDa-calelectrin) in normal human tissues: evidence for developmental regulation in B- and T-lymphocytes.

This paper describes the tissue distribution of annexin VI, a Ca(2+)-dependent phospholipid binding protein, and a member of the annexin super-gene family. In order to determine whether annexin VI expression correlated with a particular functional phenotype, an extensive series of non-pathological human tissues were examined, in which annexin VI was detected either immunohistochemically or by immunofluorescence, using a rabbit polyclonal anti(human annexin VI)-IgG of known specificity. Although most tissues investigated were found to express annexin VI, the protein was usually confined to highly specific cell types within each tissue, the staining generally appearing cytoplasmic and diffuse. There was particularly good correlation between annexin VI expression and hormone secreting cells, with positive staining in the islet cells of the pancreas, the Leydig cells of the testis and the cells of the adrenal cortex. A notable exception was the parathyroid gland, which lacked detectable annexin VI. Although the protein was absent in most epithelia, it was expressed strongly in certain secretory epithelia; e.g. the ductal epithelial cells of the salivary glands and non-lactating breast, and the sweat glands and their ducts. The observation that the epithelial cells of lactating breast failed to stain for annexin VI suggests functional regulation of protein expression in this tissue. However, the most interesting finding was that annexin VI expression appeared to be developmentally regulated in B- and T-lymphocyte differentiation, with negative staining in the proliferating B cells of the germinal centre of the lymph nodes, but strong staining in the mature small lymphocytes of the cortex, mantle zone and paracortex.

Adrenal Cortex↗

PubMed1991

Basaloid-squamous carcinoma of the esophagus with marked deposition of basement membrane substance.

A 71-year-old Japanese man with basaloid-squamous carcinoma of the esophagus is reported. The carcinoma contained basaloid cells, a few small cornified foci, and a large amount of eosinophilic hyaline substance, which reacted positively upon periodic acid-Schiff, type IV collagen, and laminin staining. Ultrastructural examination revealed markedly replicated basement membranes (BM). The morphological findings suggested that this tumor secreted abundant BM substance. Small nests of cancer cells were attached to the dysplastic esophageal epithelium. The tumor cells exhibited negative staining for mucin, secretory component, lactoferrin, and carcinoembryonic antigen. These findings, as well as the observed keratinization and attachment between the carcinoma nests and mucosal epithelium, indicate that the tumor originated in the mucosal epithelium of the esophagus.

Aged↗

PubMed1990

The relation of prostatic acid phosphatase and prostate specific antigen with tumour grade in prostatic adenocarcinoma: an immunohistochemical study.

This study examines the relationship between the presence of prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) in tissue, the serum PAP and PSA levels and tumour grade in prostatic adenocarcinoma. Thirty-three tumours were studied by the indirect immunoperoxidase method for PAP and PSA production. Thirty-one of them were stained positive for both markers. The remaining two which previous histopathological studies suggested were transitional cell carcinoma, stained negative. In primary tumours there was a weak trend for poor immunoreactivity in high grade neoplasms but the correlation was not significant. The elevated serum PAP and PSA levels did not correlate with the intensity of staining in the tissue.

Acid Phosphatase↗

PubMed1975

Tannic acid-stained microtubules with 12, 13, and 15 protofilaments.

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.

Animals↗

PubMed1995

p53 immunoreactivity and mutation of the p53 gene in smooth muscle tumours of the uterine corpus.

Mutation and overexpression of p53 have been described in uterine malignant mixed Müllerian tumours and in endometrial adenocarcinoma, where it has been associated with a poor prognosis. This study examines p53 expression and mutation of the p53 gene in benign and malignant smooth muscle tumours of the uterine corpus. p53 expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 23 leiomyosarcomas, 10 tumours of uncertain malignant potential (TUMPs), and 18 leiomyomas. Single-stranded conformational polymorphism, (SSCP) analysis of exons 5-8 of the p53 gene was performed on 13 leiomyosarcomas, nine TUMPs, and eight leiomyomas. With microwave antigen retrieval, p53 immunoreactivity was seen in 13/23 microwave treatment, staining was abolished in three leiomyosarcomas, all immunoreactive TUMPs, and the single positive leiomyoma. SSCP analysis revealed mutation in three leiomyosarcomas. There was one mutation in exon 5 in a case with positive immunohistochemistry. Two cases with negative staining showed mutation, one in exon 7 and one in exon 8. Mutation was present in exon 7 in 4/9 and in exon 6 in 1/9 TUMPs. All of these cases showed positive immunohistochemistry. There was no significant difference in outcome between cases with and without positive immunohistochemistry. p53 expression is seen in a significant proportion of uterine leiomyosarcomas. Microwave antigen retrieval increases the proportion of positive cases and also results in positive staining in TUMPs. Mutation of the p53 gene occurs in only a minority of leiomyosarcomas and in a significant proportion of TUMPs. Positive immunohistochemistry does not, however, correlate with the presence of mutation and other factors may be responsible for p53 detection in many cases.

Base Sequence↗