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PubMed1991

Assembly and structure of calcium-induced thick vimentin filaments.

Using a viscometric assay and various electron microscopic procedures (negative staining, rotary shadowing, ultrathin sectioning) we have determined the influences of different kinds of ions and of ionic strength on the structures formed by assembly of soluble subunits of vimentin from bovine lens tissue or from Escherichia coli transformed with Xenopus vimentin cDNA. In contrast to the assembly of typical, i.e., 8 to 14-nm, intermediate-sized filaments (IFs) at elevated (e.g., 160 mM) concentrations of monovalent cations and at millimolar Mg2+ concentrations, filaments formed in the presence of Ca2+ ions (e.g., 5 mM) appeared at a lower rate, attained lower viscosity and were considerably thicker and shorter. The largest diameter measured was that for the recombinant amphibian protein: 24.2 +/- 8.5 nm in negative staining, 28.7 +/- 5.6 nm in sections. These thick Ca(2+)-induced filaments, however, revealed the same approximately 2 nm protofilament composition and approximately 20 nm cross-striation pattern as typical IFs, indicative of a similar molecular arrangement. The significance of this unusual structural IF protein assembly is discussed.

Animals↗
PubMed1994

The three-dimensional structure of human erythrocyte aquaporin CHIP.

Water-permeable membranes of several plant and mammalian tissues contain specific water channel proteins, the 'aquaporins'. The best characterized aquaporin is CHIP, a 28 kDa red blood cell channel-forming integral protein. Isolated CHIP and Escherichia coli lipids may be assembled into 2-D crystals for structural analyses. Here we present (i) a structural characterization of the solubilized CHIP oligomers, (ii) projections of CHIP arrays after negative staining or metal-shadowing, and (iii) the 3-D structure at 1.6 nm resolution. Negatively stained CHIP oligomers exhibited a side length of 6.9 nm with four-fold symmetry, and a mass of 202 +/- 3 kDa determined by scanning transmission electron microscopy. Reconstituted into lipid bilayers, CHIP formed 2-D square lattices with unit cell dimensions a = b = 9.6 nm and a p422(1) symmetry. The 3-D map revealed that CHIP tetramers contain central stain-filled depressions about the fourfold axis. These cavities extend from both sides into the transbilayer domain of the molecule leaving only a thin barrier to be penetrated by the water pores. Although CHIP monomers behave as independent pores, we propose that their particular structure requires tetramerization for stable integration into the bilayer.

Aquaporin 1↗
PubMed1986

Electron microscopic visualization of the capsule of Pasteurella multocida.

The results of electron microscopic visualization of capsules of Pasteurella multocida by means of negative staining and section methods are described. Using ruthenium red or a complex of platinum in negative staining, relatively homogeneous capsules were visualized, using alcian-blue, coarse-fibrous structured capsules appeared. The incubation with antiserum partially revealed narrow capsule zones in the sections. Ferritin-linked IgG labelled amorphous material between the bacteria or floccular appendages on the bacterial surface. Gold-linked Con A deposited on thick cord structures, on loose material between the bacteria and sometimes on their cell wall.

Cell Wall↗
PubMed1982

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants.

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.

Cell Membrane↗
PubMed2006

Use of s-100 and chromogranin a antibodies as immunohistochemical markers on detection of malignancy in aortic body tumors in dog.

To define the characteristics of malignancy we performed routine histology and an immunohistochemical study on seventeen aortic body tumors in dogs. We essayed tumors using a panel of immunohistochemical markers: neuron specific enolase (NSE), chromogranin A (CrA) and S-100. Among 17 cases, the neoplastic cells were positive for NSE (17 cases, 100%), S-100 (9 cases, 53%), and CrA (8 cases, 47%), respectively. The sustentacular cells density and chief cell staining intensity were both inversely related to tumor grade. The most relevant data was consistent with a negative staining of S-100 correlated with absence or decreased number of sustentacular cells in tumors grade III. This report indicates that the immunohistochemical panel has utility for the diagnosis of chemodectoma and the negative staining to CrA and S-100 markers in tumors grade III expresses an indication of malignant behaviour of the tumor.

Age Factors↗
PubMed2003

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes.

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the "attachment organelle" of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus.

Animals↗
PubMed1986

A structural basis for R- and T-banding: a scanning electron microscopy study.

The structure of reverse (R)-banded and telomeric (T)-banded chromosomes was studied by examination of the same chromosomes first in the light microscope (LM) followed by the scanning electron microscope (SEM). This procedure demonstrated a structural basis to both the R- and T-banding techniques. A direct correlation was shown between the LM staining patterns and the structural patterns observed in the SEM. In the R-banded chromosomes the positively stained R-bands, viewed by LM, corresponded to highly fibrous three-dimensional regions in the SEM. The negatively stained R-interbands corresponded to flatter regions from which material appeared to have been extracted. These structural observations strongly support the suggestion that chromosomal material is preferentially lost from the R-interbands with aggregation of fibres in the R-bands. T-banded chromosomes showed a similar structure to the R-banded chromosomes. The positively stained T-bands located at the telomeres corresponded to regions of highly aggregated fibres. The remainder of the chromosome, corresponding to the negatively stained area, had a flattened and extracted appearance. These similarities in morphology between the T- and R-banded chromosomes support the view that T-bands result from a progressive breakdown of the R-banded chromosome structure.

Azure Stains↗
PubMed1977

The quatenary structure of Pseudomonas cytochrome oxidase studied by electron microscopy.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) was studied by negative staining in the electron microscope. The best resolution was obtained with uranyl oxalate (pH 6.0) as negative stain. Electron micrographs confirm the idea of the dimeric structure of the enzyme. A rough model of cytochrome oxidase was constructed based on different projections of the molecule seen in the electron micrographs. In this model the subunits are identical and sterically equivalent.

Electron Transport Complex IV↗
PubMed1997

Morphological diversity of cultured canine gastric Helicobacter spp.

The cell morphology, the number of flagella, the occurrence of periplasmic fibrils and ultrastructural structures of five groups of cultured canine gastric Helicobacter spp. were compared. The study included four strains of Helicobacter felis, four strains of Helicobacter bizzozeronii, one strain of 'Flexispira', six strains of an unnamed spiral organism 2 and one strain of an unnamed spiral organism 3 which were isolated from gastric biopsies. Cultures were studied with negative staining, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Bacterial dimensions were measured from the negative staining samples and values were tested with ANOVA and Bonferroni tests. The organisms studied differed from each other morphologically. H. felis was a slightly spiraled organism with periplasmic fibrils. 'Flexispira' was a thin and straight organism with periplasmic fibrils. H. bizzozeronii was a tightly spiraled organism. Spiral organism 2 was loosely spiraled and thicker than the other organisms. Spiral organism 3 was a short curved rod having a single bipolar flagellum. The other species had multiple flagella. As a conclusion the canine gastric Helicobacter spp. can be differentiated from each other morphologically with an electron microscope. The morphological differences were mainly found in the structures involved in motility. The importance of the differences may lie in their impact on the colonization in a gastric mucous environment.

Animals↗
PubMed1968

Morphological phenomena associated with penicillinase induction and secretion in Bacillus licheniformis.

Cells of uninduced Bacillus licheniformis (strain 749) in mid-logarithmic phase have no extensive intracytoplasmic membrane. After induction with cephalosporin C, characteristic organelles that contain tubules and vesicles with single-layered membranes and no visible internal substructure can be seen in thin sections in the periplasm. A magnoconstitutive penicillinase producer (749/C) contains similar structures. It is suggested that they represent a penicillinase secretory apparatus. In the first 15 min after induction, negatively stained preparations of induced 749 show large intracellular vesicles without individual contact with the cell surface. Negatively stained 749/C and fully induced 749 contain invaginations comparable to the structures seen in thin sections. When protoplasts of induced 749 and of 749/C are prepared, vesicles and tubules similar to those seen in thin sections of whole cells are liberated from the cell. Growing protoplasts of induced 749 show massive convolutions of the peripheral membrane, multiple layers of membrane, and characteristic long, slender tubules extending from the protoplast surface. These phenomena are not observed in uninduced 749 except for the production of a relatively small number of tubules. In 749/C, there were fewer convolutions than in induced 749, although tubule production was similar. Multiple layers of membrane were not observed in 749/C. The relation of the penicillinase secretory structures to mesosomes and to secretory structures of other organisms is discussed.

Bacillus↗
PubMed2002

PTEN expression is associated with prognosis for patients with advanced endometrial carcinoma undergoing postoperative chemotherapy.

The prognostic significance of PTEN expression in endometrial carcinoma has not been clear. We conducted the present study to clarify the relationship between PTEN expression and prognosis in advanced endometrial carcinoma. Of 784 patients with endometrial carcinoma who underwent primary treatment between 1985 and 2000 at 5 institutions, 98 pure endometrioid carcinomas with retroperitoneal lymph node metastasis were provided for our study. PTEN expression was determined by immunohistochemic staining. Negative or mixed PTEN staining was observed in 64 (65.3%) patients. The survival rate for PTEN-positive patients was significantly higher than that for PTEN-negative or -mixed patients. PTEN-staining status was not associated with patient age, International Federation of Gynecology and Obstetrics (FIGO) stage, myometrial invasion or histologic grade. Of the 98 patients, 87 received radiation therapy (n = 25) or chemotherapy (n = 62) after surgery. PTEN expression did not relate to survival for patients receiving radiation therapy. In contrast, the survival rate for PTEN-positive cases was significantly higher than that for PTEN-negative or -mixed cases when patients underwent chemotherapy (62.4% vs. 11.8%). Subsequent multivariate analysis revealed that PTEN staining was an independent prognostic factor for patients undergoing chemotherapy. PTEN-positive staining was a significant prognostic indicator of favorable survival for patients with advanced endometrial carcinoma who underwent postoperative chemotherapy.

Adult↗
PubMed1990

Method for forming two-dimensional paracrystals of biological filaments on lipid monolayers.

A method is described for electron microscopic observation of two-dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C-coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain. It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two-dimensional paracrystals under physiological conditions of [Mg2+] and pH.

Crystallization↗
PubMed1994

[The expression of mutant p53 gene in gastric carcinoma].

Loss or inactivation of p53 gene--a suppressor oncogene has been considered to be one of the important mechanisms in the development of human tumors. One of the evidences for mutation of allelic gene of p53 is the identification of p53 protein concentrated in the nuclei of related cells. By using ABC immunohistochemical method, we studied the expression of p53 in cryostatic sections of the tumor tissue and adjacent mucosa resected from 38 patients with gastric cancer. p53 was found to be positive in the nuclei with intensive staining in 24 out of 38 cases with carcinoma (63.2%). p53 positive cells were distributed diffusively in the cancer tissue. All the adjacent mucosa specimens except 10 were negatively stained with p53 monoclonal antibody. These 10 specimens including 3 with dysplasia and 4 with metaplasia were only weakly stained. p53 was also found to be positive in 18 out of 23 cancer patients with metastasis in perigastric lymph nodes (78.3%). We also studied in the same section the nucleolar organizer region-associated proteins (AgNORs) with using silver staining technique to find if there is any relationship between p53 gene mutation and the activity of rRNA transcription of tumor cells. The number of AgNORs dots per nucleus detected in gastric cancer sections with positive staining of p53 (9.9 + 2.14) was greater than those with p53 negative staining (7.2 + 1.68). There was a significant statistical difference between the two groups (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1994

Projection structure of the CHIP28 water channel in lipid bilayer membranes at 12-A resolution.

Osmotic water transport across plasma membranes in erythrocytes and several epithelial cell types is facilitated by CHIP28, a water-selective membrane channel protein. In order to examine the structure of CHIP28 in membranes, large (1.5-2.5-microns diameter), highly ordered, two-dimensional (2-D) crystals of purified and deglycosylated erythrocyte CHIP28 were generated by reconstitution of detergent-solubilized protein into synthetic lipid bilayers via detergent dialysis. Fourier transforms computed from low-dose electron micrographs of such crystals preserved in negative stain display order to 12-A resolution. The crystal lattice is tetragonal (a = b = 99.2 +/- 1.4 A) with plane group symmetry p4g. A projection density map at 12-A resolution defines the molecular boundary and organization of the CHIP28 monomers in the membrane plane. The unit cell contains four CHIP28 dimers, each composed of two oblong-shaped (37 x 25 A ) monomers with opposite orientations. The CHIP28 monomers associate to form tetrameric structures around the 4-fold axes normal to the membrane plane where stain is excluded. The 2-D crystals of CHIP28 display order extending beyond the limit typically achieved by negative staining and therefore may be amenable to high-resolution structure analysis by cryo-electron microscopy.

Aquaporin 1↗
PubMed1984

Ultrastructure of paired helical filaments of Alzheimer's neurofibrillary tangle.

Isolated paired helical filaments (PHF) of Alzheimer's disease and senile dementia of the Alzheimer type (AD/SDAT) were studied by negative stain techniques and electron microscopy (EM). The helical structure of the PHF was confirmed by tilt analysis of negatively stained isolated PHF. A substructure of four protofilaments, each 3-5 nm in diameter, was also observed. A comparison of PHF substructure, neurofilaments and microtubules, revealed no similarity among the three structures.

Aged↗
PubMedDate not supplied

Expression of thymidine phosphorylase as an effect prediction factor for uterine cervical squamous cell carcinoma after radiotherapy: an immunohistochemical study.

Prognoses in cases of uterine cervical squamous cell carcinoma treated with radiotherapy were investigated in association with immunohistochemical expression of an angiogenic factor, thymidine phosphorylase (TP). Forty-six cases of uterine cervical squamous cell carcinoma mainly treated with radiotherapy during 1992-2001 at our clinic were studied. All were diagnosed as stages IIB to IVA. Paraffin-embedded biopsy specimens excised before radiotherapy were stained immunohistochemically using anti-TP monoclonal antibody. The extent of staining in both tumor and interstitial cells was graded as (-), (+/-), (+), and (2+). Specimens with TP expression levels of (2+) and (+) were regarded as positively stained and those with TP expression levels of (+/-) and (-) as negatively stained. The efficacy of radiotherapy in both groups was analyzed by the Kaplan-Meier method. With tumor cells, 5-year survival rates for the positive (n= 38) and negative (n= 8) staining groups were 73.9% and 42.9%, respectively; the rate being significantly higher for the TP-positive group (log rank, P= 0.0096). Contrarily, with staining for interstitial cells, the 5-year survival rates for the positive (n= 20) and negative (n= 26) staining groups were 74.1% and 64.6%, respectively, with no significant difference (log rank, P= 0.406). The efficacy of radiotherapy in the group with positive staining of tumor cells was significantly better than in the negative staining group. Immunohistochemical expression of TP in tumor cells is suggested as a useful prognostic factor for uterine cervical squamous cell carcinomas treated with radiotherapy. Choosing therapy for individual cases by referring to factors including TP expression should contribute to an improved prognosis.

Aged↗
PubMed1988

The surface structure of photoreceptor outer segment disks.

The double-membrane disks in the outer segment of photoreceptors in the rabbit retina were isolated structurally intact after crosslinking with glutaraldehyde. The following criteria were used to determine that intact whole disks were isolated: (1) the disk diameter, 1.4 microns; (2) the presence of one incision extending toward the center of the disk; (3) the edge structure; and (4) the presence of particulate material with the dimensions of the particles corresponding to those present in the particulate fracture face of freeze-fractured outer segment disks. The disks were analyzed after negative staining or after freeze-drying and surface replication. The surface of the disks appeared smooth. In damaged disks negative staining revealed the presence of particulate material located below the disk surface. The observations are interpreted to reveal that the lipid bilayers of the disks are located at the disk surfaces shielding the rhodopsin molecules from contact with the cytoplasm present between the disks.

Animals↗
PubMed1996

In situ hybridization study of messenger RNA for estrogen receptor and immunohistochemical detection of estrogen and progesterone receptors in the human ovary.

The expression of estrogen and progesterone receptors (ER and PR), as well as the presence of messenger RNA for estrogen receptor (ER-mRNA), were analyzed by immunohistochemistry and in situ hybridization, respectively, in the ovary of 25 healthy eumenorrheic women. Ovarian biopsies were taken in different phases of the menstrual cycle during laparotomy or operative laparoscopy performed for extraovarian benign diseases. A total of 126 follicles (105 primordial, 13 preantral and eight antral) and 50 corpora lutea (eight active and 42 atretic) was analyzed. Granulosa cells stained positively for ER, PR and ER-mRNA in 13.3, 9.5 and 17.1% of primordial follicles, respectively. The proportions of preantral and antral follicles with ER-positive granulosa cells were 23.1 and 37.5%, respectively; these follicles were positive for PR in 23.1 and 37.5% of cases, and for ER-mRNA in 30.7 and 37.5% of cases, respectively. For thecal cells, 38.5% of preantral and 37.5% of antral follicles were PR-positive, but no more than 25% stained positive for ER and ER-mRNA. Active corpora lutea stained positive for ER, PR and ER-mRNA in 50, 62.5 and 50% of cases, respectively. Corpora albicantes always stained negative. In all subjects the stroma surrounding both follicles and corpora lutea contained several fibroblast-like cells which stained positive for ER, PR and ER-mRNA. Oocytes and blood vessels stained negative in all cases. This study supports the hypothesis that estrogens and progesterone play a role in the intraovarian regulation of follicle growth from the first steps of follicle development, and participate in the regulation of corpus luteum.

Adult↗