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PubMed2003

A panel of immunohistochemical stains assists in the distinction between ovarian and renal clear cell carcinoma.

The diagnosis of primary clear cell carcinoma of the ovary or kidney is usually straightforward. However, problems in ascertaining the site of the primary tumor may arise when there is widespread metastatic disease or when clear cell carcinoma is present in both the ovary and kidney. In this study, the value of a panel of antibodies in distinguishing between an ovarian and renal clear cell carcinoma was evaluated. The panel comprised cytokeratin (CK)7 and 20, vimentin, estrogen receptor (ER), CD10, and renal cell carcinoma (RCC) marker. Ovarian clear cell carcinomas (n=14) were positive with CK7 (14/14), vimentin (6/14), ER (2/14), and RCC marker (2/14). All were negative with CD10 and CK20. Renal clear cell carcinomas (n=14) were positive with CD10 (14/14), RCC marker (14/14), vimentin (7/14), CK7 (2/14), and CK20 (1/14). All were negative with ER. This panel allows clear cell carcinomas of the ovary and kidney to be distinguished with a high degree of certainty and is a useful adjunct to histologic examination. Primary ovarian clear cell carcinomas are characterized by CK7 positivity, whereas primary renal neoplasms are characterized by positivity for CD10 and RCC marker and negative staining with CK7.

Antibodies↗
PubMed1996

Functional heterogeneity of human term cytotrophoblasts revealed by differential sensitivity to extracellular Ca2+ and nucleotides.

We have prepared purified cytotrophoblasts from human term placentas and examined the sensitivity of fura-2 loaded cells to the nucleotides ATP and UTP and to changes in extracellular Ca2+ concentration ([Ca2+]o). Purified cytotrophoblasts were obtained by collagenase digestion and separation according to density using self-generated Percoll gradients. The cytotrophoblast fraction was free of red cell and largely free of white cell contamination (as assessed by uniformly negative staining for vimentin and the failure of > 90% of fura-2 loaded cells to respond to the chemotactic peptide fMet-Leu-Phe). Purified cells secreted progesterone in a linear fashion over several hours in the presence of 25-hydroxycholesterol. The cells ranged in size from approximately 7.5 to 50 microns in diameter as described previously for purified cytotrophoblasts, and an analysis of cells for sensitivity to [Ca2+]o or nucleotides suggested functional heterogeneity within the cytotrophoblast population. Small cells (7.5-10 microns) were negative for cytokeratin-8 and, after loading with fura-2, were insensitive to extracellular nucleotides but sensitive to elevations in [Ca2+]o. Medium-sized cells (12-20 microns) were largely cytokeratin-positive (70% of cells) and sensitive to both ATP and UTP but largely insensitive to [Ca2+]o. Large cells (25-50 microns) were uniformly cytokeratin-positive (100% of cells) and, after fura-2 loading, sensitive to both [Ca2+]o and extracellular ATP or UTP. We examined the likely origin of small, medium and large cytotrophoblasts using an immunomagnetic cell sorting procedure that separates villous cytotrophoblasts (which do not express major histocompatibility class I antigens) from extravillous cytotrophoblasts. This procedure resulted in the selective sedimentation of almost all medium and large cells, leading to the conclusion that the small cells were villous cytotrophoblasts whereas medium and large cells were predominantly extravillous in origin. The data suggest that small, medium and large cytotrophoblasts have distinct roles in the function of the term placenta.

Adenosine Triphosphate↗
PubMed1987

Thrombomodulin as a marker for vascular tumors. Comparative study with factor VIII and Ulex europaeus I lectin.

Thrombomodulin (TM) is a newly described endothelial cell-associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Various vascular tumors were characterized with immunoperoxidase staining with the use of a polyclonal anti-TM serum. The staining patterns of TM were compared with those of Factor VIII-related antigen (FVIII-RAG) and Ulex europaeus agglutinin-I (UEA-I), which have been used as markers for endothelial cells. The results showed that TM is a specific and a highly sensitive marker for angiosarcomas in comparison with FVIII-RAG or UEA-I. In contrast, UEA-I is more sensitive for benign vascular tumors than TM or FVIII-RAG. The other mesenchymal tumors of nonvascular origin showed negative staining for three endothelial markers. These results indicate that TM is a new specific and sensitive tool for the diagnosis of angiosarcomas.

Antigens↗
PubMed1978

Morphology and molecular composition of isolated postsynaptic junctional structures.

The solubilization of isolated brain synaptosomal plasma membranes by various detergents was studied and in each case found to depend upon detergent concentration. By using conditions sufficient to extract maximally protein and phospholipid from the membranes, postsynaptic junctional particles were isolated with each of four detergents and their ultrastructural appearances and protein contents compared. Two basic structural forms were identified. One, isolated with Triton X-100, consists of a planar array of dense-staining particles ca. 20 nm in diameter. It resembles the postsynaptic density seen in undigested synaptosomal plasma membranes. The other, isolated with sodium deoxycholate, contains less protein. It has the same overall shape and dimensions as the postsynaptic density, but consists of a branching network of short 5 nm fibres (the postsynaptic junctional lattice) making up an array of contiguous polygons, each ca. 20 nm across. The interior of these polygonal elements seems to be hydrophobic since it cannot be penetrated by metallic salts used for negative staining. It is suggested that the dense-staining 20 nm subunits observed at the postsynaptic junctional site may be composed of hydrophobic proteins inserted into the hollow cores of the lattice polygons. Electrophoretic analysis of the proteins present in the various postsynaptic junctional preparations identified two major common components with molecular masses of 275000 and 47500. The latter is tentatively identified as actin. Components comigrating respectively with alpha- and beta-tubulin are present, and the relation of the lattice structure to subjacent microtubules is discussed.

Animals↗
PubMed1984

Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties.

A method for the rapid purification of caldesmon, an F-actin binding protein of smooth muscle, has been developed. Caldesmon remains native after heating at 90 degrees C, a property that provides the basis for the purification in high yield of both caldesmon and tropomyosin, another heat-stable protein of smooth muscle. Caldesmon purified by this procedure is a highly asymmetric protein with a sedimentation coefficient of approximately 2.7 S and a Stokes radius of about 91 A. The protein exists as two polypeptide chains of Mr = 135,000 and 140,000, with each Mr polypeptide being resolvable into several isoelectric species. Estimates based on densitometry of stained gels suggest that caldesmon is more abundant in smooth muscle than filamin or alpha-actinin. Purified caldesmon bound to F-actin in the pH range 6-8. Binding was unaffected by Ca2+ or Mg2+ at up to millimolar levels. Binding was saturable, with a polypeptide molar ratio of about one caldesmon to six actins at saturation. F-actin binding was not inhibited by saturating levels of tropomyosin. Caldesmon dramatically increased the viscosity of F-actin. Light microscopy and electron microscopy of negatively stained material revealed that caldesmon induced the formation of massive F-actin bundles which contained up to hundreds of filaments. Electron microscopy of sectioned caldesmon-saturated F-actin mixtures revealed large bundles which appeared to include linear arrays of regularly spaced actin filaments cut transversely, exhibiting a center to center spacing of 15 nm. Possible structural implications based on the existence of these structures is presented.

Actins↗
PubMed2000

Membrane interaction of influenza virus M1 protein.

The M1 protein of influenza virus is thought to make contact with the cytoplasmic tails of the glycoprotein spikes, lipid molecules in the viral membrane, and the internal ribonucleoprotein particles. Here we show electron micrographs of negatively stained virus particles in which M1 is visualized as a 60-A-long rod that touches the membrane but apparently is not membrane inserted. Photolabeling with a membrane restricted reagent resulted in labeling of the transmembrane region of haemagglutinin but not of M1, also suggesting that most of M1 is not embedded into the hydrophobic core of the viral membrane. Finally, in vitro reconstitution experiments using soluble M1 protein and synthetic liposomes or Madin-Darby canine kidney cell membranes suggest that M1 can bind to negatively charged liposomes and to the cellular membranes and that this binding can be prevented under high-salt conditions. Although none of these experiments prove that there does not exist a minor fraction of M1 that is membrane inserted, it appears that most of M1 in the virus is membrane associated through electrostatic interactions.

Animals↗
PubMed1995

Localisation and immunological properties of a 24-kDa surface protein of Haemophilus ducreyi.

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Amino Acid Sequence↗
PubMed1979

C-band polymorphism in human chromosome no. 6.

Chromosome analysis of G-banded cells from 92 individuals consecutively referred to the Cytogenetics Laboratory show that the paracentromeric band in the short arm (6p11), which stains negatively with G-banding and darkly with C-banding, shows a marked increase in size in about 9% of chromosome no. 6. The results of this study and those from a family in which two variants of chromosome no. 6 are segregating are compatible with a Mendelian pattern of inheritance.

Azure Stains↗
PubMed1982

Comparison of an enzyme immunoassay with electron microscopic procedures for detecting rotavirus.

The sensitivity and specificity of an enzyme immunoassay (Rotazyme), an ongrid immunoelectron microscopy procedure, and conventional negative stain electron microscopic techniques were compared. By using partially purified human rotavirus and simian rotavirus (SA-11) of known particle concentration, the enzyme immunoassay was essentially equivalent to the immunoelectron microscopic procedure and significantly more sensitive than conventional electron microscopic techniques. The level of sensitivity was approximately 10(6) particles per ml for simian rotavirus SA-11 and 10(7) particles per ml for human rotavirus. In an evaluation of 455 clinical samples by these techniques, a sensitivity of 98% and specificity of 92% were demonstrated. Samples negative by the immunoelectron microscopic procedure and positive by enzyme immunoassay could be confirmed by a blocking assay.

Animals↗
PubMed2003

Full-length rat amylin forms fibrils following substitution of single residues from human amylin.

Pancreatic amyloid deposits, composed of the 37 amino acid residue peptide amylin, represent an integral part of type 2 diabetes mellitus pathology. Human amylin (hA) forms fibrils in vitro and is toxic to cultured pancreatic islet beta-cells. In contrast, rat amylin (rA) which differs from hA by only six amino acid residues in the central region of the peptide, residues 18-29, does not form fibrils and is not cytotoxic. To elucidate the role of individual residues in fibril formation, we have generated a series of full-length rA variants and examined their ability to form fibrils in vitro. Single-residue substitutions with amino acids from corresponding positions of the hA sequence, i.e. R18H, L23F, or V26I, were sufficient to render rA competent for fibril formation albeit at a small yield. Combining two or three of these substitutions generally increased the ability to produce fibrils. Variant rA fibril morphologies were examined by negative stain electron microscopy and found to be similar to those generated by hA itself. Bulk assays, i.e. involving thioflavin-T fluorescence and sedimentation, showed that the amount of fibril formation was relatively small for these rA variants when compared to hA under the same conditions. Fibril growth was demonstrated by time-lapse atomic force microscopy, and MALDI-TOF mass spectrometry was used to verify that fibrils consisted of full-length peptide. Our observations confirm previous reports that the three proline residues play a dominant negative role in fibril formation. However, their presence is not sufficient to completely abolish the ability of rA to form fibrils, as each of the other three implicated residues (i.e. R18, L23 and V26) also has a dominant modulating effect.

Amino Acid Sequence↗
PubMed1965

Electron microscopy of chromatophores of Rhodopseudomonas spheroides.

Gibson, K. D. (St. Mary's Hospital Medical School, London, England). Electron microscopy of Rhodopseudomonas spheroides. J. Bacteriol. 90:1059-1072. 1965.-Fixed and stained chromatophores and whole cells of anaerobically grown Rhodopseudomonas spheroides were examined in thin sections in the electron microscope. Both purified chromatophores and intracellular membrane-bound vesicles had exactly the same appearance, namely that of spheres or ellipsoids with a thin electron-dense shell surrounding an electron-lucent interior. The distribution of diameters in the two types of structure was also found to be the same, and was compatible with a normal distribution, with a mean of 570 A and a standard deviation 40 A. Negatively stained chromatophores appeared like discs or collapsed spheres. The presence of invaginations of the cytoplasmic membrane in this species was confirmed, and a new structure resembling a twin chromatophore was observed. The bearing of these results on theories of the origin of chromatophores is discussed, and it is concluded that they offer some support for each one of the three main theories about the origin of particulate organelles.

Bacterial Chromatophores↗
PubMed1992

[A case of sarcoidosis with primary acute pulmonary cavitation].

A 23-year-old man was admitted to our hospital on June 24, 1991, because of worsening chest X-ray findings of sarcoidosis. In August 1990, he was referred to our outpatient office, because of BHL and nodular lesions on chest X-ray film performed at his company 4 months earlier. At that time, serum ACE was elevated to 34.0 IU/l, and Ga scintigraphy showed abnormal uptake in bilateral lacrimal and salivary glands, mediastinal and hilar lymph nodes, and in the lung fields. TBLB specimen showed noncaseating epithelioid granuloma with giant cells and negative stains for acid-fast bacilli. Although it was planned to follow this patient without medication, he did not return to our outpatient department. In June 1991, because of worsening of lesions in the lung at annual checkup at his company, he was referred and admitted for steroid therapy. Chest X-ray film on admission showed BHL, multiple nodular lesions in both lung fields, and bullous change in the left upper lobe. Chest CT on admission showed three cavitating lesions within preexisting nodules. PPD skin test was negative, and sputum smears and cultures were repeatedly negative for pyogenic bacteria and acid-fast bacilli. Therapy was initiated with prednisolone 30 mg daily. Four months later, there was marked resolution of BHL and nodular lesions, and the cavitating lesions were no longer visible on chest X-ray film. From the clinical and radiological observations, it is concluded that the cavitating lesions in the present case were primary acute pulmonary cavitation in sarcoidosis, distinct from infection, bullae, or cystic bronchiectasis which are seen in the chronic and fibrotic stages of sarcoidosis.

Adult↗
PubMed2006

The utility of GLUT-1 immunolocalization in cell blocks: An adjunct to the fine needle aspiration diagnosis of cystic squamous lesions of the head and neck.

BACKGROUND: Cytologic distinction of metastatic cystic squamous cell carcinoma (SCC) from benign squamous cell lesions, especially the ones with superimposed inflammatory atypia, can be very challenging. The authors evaluated the usefulness of glucose transporter-1 (GLUT-1) immunostaining as an adjunct to fine-needle aspirations of squamous lesions of the head and neck. METHODS: Immunohistochemical staining for GLUT-1 was performed on paraffin-embedded cell blocks of 28 cases with the following cytologic diagnoses: 1) metastatic SCC (11 cases); 2) atypical squamous cells, SCC [corrected] cannot be excluded (6 cases); and 3) cytologic findings consistent with branchial cleft cyst (BCC) (11 cases). RESULTS: All 11 cases with an unequivocal cytologic diagnosis of metastatic SCC were positive for GLUT-1. Tissue follow-up confirmed metastatic SCC in all 11 cases. The squamous cells in all 11 cases with cytologic findings consistent with BCC were negative for GLUT-1; tissue follow-up confirmed diagnoses of BCC in 8 cases. In the remaining 3 cases, excision was not performed, and, clinically, no recurrences were identified at 28, 20, and 16 months of follow-up. Of the 6 cases with cytologic diagnosis of atypical squamous cells, 3 were negative and 3 were positive for GLUT-1. Subsequent excisional biopsies in these cases revealed 4 cases of metastatic SCC (3 positive and 1 negative for GLUT-1), 1 case of BCC (negative for GLUT-1), and 1 case of thyroglossal duct cyst (negative for GLUT-1). CONCLUSION: GLUT-1 immunostaining is a useful adjunct in differentiating benign and malignant squamous lesions in cell-block material. Although negative staining for GLUT-1 does not exclude malignancy, positive immunoreactivity for GLUT-1 may aid in accurate diagnosis of malignancy in cytomorphologically equivocal squamous lesions of the head and neck.

Biopsy, Fine-Needle↗
PubMed1995

Proliferation Markers and EGF in ACTH-Secreting Adenomas and Carcinomas of the Pituitary.

Pituitary carcinomas are only defined by their metastatic growth, which may be intracranial or systemic. To establish further morphological and immunohistochemical differences between pituitary carcinomas and adenomas, 19 ACTH-secreting adenomas (10 non invasive and 9 invasive) and 2 ACTH-secreting carcinomas with their metastases were studied for expression of the intermediate filaments keratin and vimentin and the tumor-associated antigens Ki67, proliferating cell nuclear antigen (PCNA), epidermal growth factor (EGF), cathepsin D, p53, and carcinoembryonic antigen (CEA). Immunohistochemistry was performed using avidin-biotin techniques on formalin-fixed, paraffin-embedded tissue. With the exception of one noninvasive pituitary adenoma, one carcinoma, and the metastases, all tumors contained keratin; none contained vimentin. All tumors stained negative for CEA and p53. Eleven (58.5%) adenomas and both pituitary carcinomas contained Ki67-positive nuclei; 14 (74%) adenomas and one carcinoma revealed PCNA. No correlation was found between the two markers. Seven (38%) adenomas showed a labeling index <1 % for cathepsin D, whereas none of the carcinomas or metastases did so. EGF was found in 7 (38%) adenomas and in both carcinomas. A tendency to a higher rate of EGF positivity in the invasive adenomas was observed. The metastases showed a higher labeling index, and far more intense staining results for Ki67, PCNA, and EGF than the primary tumor. The metastases also had a higher proliferation rate and growth factor content than the carcinoma itself.

Journal Article↗
PubMed1992

Ultrastructure of Warthin-Starry stain-positive bacteria in abscess-forming reticular lymphadenitis.

This study describes the ultrastructure of Warthin-Starry (WS) stain-positive bacteria in abscess-forming reticular lymphadenitis (ARL) compatible with cat scratch disease (CSD). Sections containing WS-positive bacteria were re-embedded in Epon, and semithin sections were examined by electron microscopy. Silver particles were aggregated on the outer surfaces of the bacteria. Stereoscopic observations clearly showed that the bacteria were pleomorphic, rod-shaped, arranged in a row or at an angle, and frequently showed septal formation. Electron microscopy of ultrathin sections revealed that the cell wall possessed an outer membrane characteristic of gram-negative bacteria. The results indicate that rod-shaped bacteria with the WS-positive, gram-negative staining cause ARL histopathologically consistent with CSD.

Abscess↗
PubMed1978

Paracrystalline arrays of protein-synthesis elongation factor Tu. Comparison with polymerized actin.

Homogeneous protein synthesis elongation factor Tu from Escherichia coli forms aggregates at high concentrations of ammonium sulfate which have a filamentous appearance in the light microscope. Electron microscopy of negatively stained preparations shows that these aggregates are paracrystalline, including three different forms. On the basis of analyses by optical diffraction, this polymorphism can be explained in terms of three different tubular foldings of the same basic two-dimensional surface lattice. This can be compared with that underlying the structure of actin filaments, thus providing a crucial test of the putative relationship between the elongation factor and actin [Rosenbusch, J. P. et al. (1976) J. Supramol. Struct. 5, 391-396]. The differences between the surface lattices, in conjunction with the negative results of sensitive immunochemical tests for possible cross-reactivities between the two proteins, suggest that any such relationship is very remote.

Actins↗
PubMed1994

[Immunocryoultramicrotomy (gelatin embedding and polyvinyl alcohol embedding methods): its procedures, usefulness, and limitations--application to human blood cells].

Cryoultramicrotomy applying immunogold staining (immunocryoultramicrotomy) is a useful method to demonstrate the localization of antigens in biological specimens. Recently, we have developed a modified method for immunocryoultramicrotomy using gelatin as an embedding medium (gelatin embedding method). In this study, we compared the results obtained by the gelatin embedding method with those obtained by a conventional method applying polyvinyl alcohol embedding (PVA embedding method). Both methods were easy to perform, and yielded fairly satisfactory results in terms of detecting antigens and preserving ultrastructures. According to the gelatin embedding method, membranes enclosing organelles revealed a negatively stained image, and organelles were clearly delineated by an electron-lucent layer. In contrast, a positively stained image was obtained by the PVA embedding method; however, the delineation of organelles was somewhat inferior to that of the gelatin embedding method. Although these methods were useful to detect various biological antigens, they had some limitations in sensitivity. Prior glutaraldehyde fixation was requisite for cryosectioning and preserving ultrastructures, but it caused the loss of antigenicity to some extent. This should be taken into account when evaluating the results obtained by immunocryoultramicrotomy. In this paper, we present the detailed procedures of the gelatin embedding method, as well as the PVA embedding method, and demonstrate the localization of myeloperoxidase, lysozyme and CD3 molecules in human blood cells using both methods.

Antibodies, Monoclonal↗
PubMed1988

Synthesis and secretion of lipoproteins by human hepatocytes in culture.

Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver fragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precursor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [3H]glycerol, more than 92% of the [3H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.

Apolipoprotein A-I↗