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PubMed1991

Morphological and ultrastructural studies of Mycoplasma flocculare and Mycoplasma hyopneumoniae in vitro.

Cells of Mycoplasma flocculare were found to vary in size and shape, especially in the later phases of growth, whereas those of Mycoplasma hyopneumoniae were fairly uniform irrespective of growth phases. Filamentous cells were present in cultures of M flocculare in the stationary and declining phases, but were never found in cultures of M hyopneumoniae. The filamentous and bizarre forms observed when mycoplasmas were suspended in phosphotungstic acid probably result from the action of the hypotonic solution. The surface of all cells was covered by a fuzzy coat consisting of fine hairs or bristles. An electron-lucent region was usually seen in cells negatively stained after centrifugation, but was only occasionally seen in cells negatively stained directly from the medium. Intracytoplasmic membranes were present in sectioned cells. No attachment organelle was found in cells of either species.

Animals↗
PubMed2006

Three-dimensional structure of the gamma-secretase complex.

Gamma-secretase belongs to an atypical class of aspartic proteases that hydrolyzes peptide bonds within the transmembrane domain of substrates, including amyloid-beta precursor protein and Notch. gamma-Secretase is comprised of presenilin, nicastrin, APH-1, and PEN-2 which form a large multimeric membrane protein complex, the three-dimensional structure of which is unknown. To gain insight into the structure of this complex enzyme, we purified functional gamma-secretase complex reconstituted in Sf9 cells and analyzed it using negative stain electron microscopy and 3D reconstruction techniques. Analysis of 2341 negatively stained particle images resulted in the three-dimensional representation of gamma-secretase at a resolution of 48 angstroms. The structure occupies a volume of 560 x 320 x 240 angstroms and resembles a flat heart comprised of two oppositely faced, dimpled domains. A low density space containing multiple pores resides between the domains. Some of the dimples in the putative transmembrane region may house the catalytic site. The large dimensions are consistent with the observation that gamma-secretase activity resides within a high molecular weight complex.

Amyloid Precursor Protein Secretases↗
PubMed1994

The effect of fetomaternal bleeding on the risk of adverse pregnancy outcome in patients with elevated second-trimester maternal serum alpha-fetoprotein levels.

OBJECTIVE: Unexplained maternal serum alpha-fetoprotein elevation has been associated with an increased risk of intrauterine growth retardation, preterm delivery, and intrauterine fetal death. The purpose of this study was to determine whether patients with evidence of recent fetomaternal bleeding as a cause of elevated maternal serum alpha-fetoprotein level are at a lower risk for adverse pregnancy outcome than those without such evidence. STUDY DESIGN: Patients with elevated maternal serum alpha-fetoprotein levels who had a singleton viable fetus without ultrasonographically detectable anomalies were offered inclusion in this study. Study participants had blood drawn for fetal cell analysis before amniocentesis. The pregnancy outcomes of patients with evidence of fetomaternal bleeding were compared with those of patients without. RESULTS: Of 229 patients, 109 (47.6%) had evidence of fetomaternal bleeding as a possible cause of elevated maternal serum alpha-fetoprotein. Of these, 86 (78.9%) had a normal pregnancy outcome compared with 84 of 120 (70.0%) with a negative stain for fetal cells (p not significant). There was no significant difference in the incidence of preterm delivery (14 [12.8%] vs 15 [12.5%]), intrauterine growth retardation (5 [4.6%] vs 9 [7.5%]), or intrauterine fetal death (4 [3.7%] vs 8 [6.6%]) when patients with a positive stain for fetal cells were compared with those with a negative stain. CONCLUSION: Among patients with elevated maternal serum alpha-fetoprotein levels, those with evidence of recent fetomaternal bleeding do not appear to be at decreased risk for adverse pregnancy outcome compared with those without such evidence.

Female↗
PubMed1987

Scanning transmission electron microscopic study of alpha-ketoglutarate dehydrogenase complex from Escherichia coli.

The alpha-ketoglutarate dehydrogenase complex was resolved into its three component enzymes: alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase. Subcomplexes were prepared in vitro by incubating the resolved E2, a 24-subunit cube-shaped molecule, with E1 (dimeric). The morphology and mass of the subcomplexes were determined by scanning transmission electron microscopy of negatively stained and of freeze-dried specimens. Images of both negative stained and freeze-dried subcomplexes were consistent with E1 binding at or near the midpoints of the edges of the E2 molecule. Mass analysis of the freeze-dried specimen showed that at least 95% of E1 remains in the dimeric state (or as two closely juxtaposed monomers) when it binds to E2.

Escherichia coli↗
PubMed1984

Diffraction patterns from stained and unstained helices: consistency or contradiction?

Addressing the issue of how best to integrate negative staining electron microscopy with X-ray fiber diffraction of macromolecular helices, we investigated staining properties of helical models using computational simulations. We compared diffraction patterns of stained and unstained representations of F-actin, myosin S1-decorated actin, and microtubules, to test the reliability of selecting well preserved specimens as those whose optical diffraction patterns most closely match the X-ray patterns of hydrated fibers. We conclude that if the stain layer is considerably thicker than the outer diameter of the specimen, this criterion is likely to be reliable. However, specimens in such thick stain layers may be resolution-limited by dynamic scattering effects such as beam-broadening. With relatively thin staining (the more practically relevant situation) "edge effects" in the stain distribution result in differences between diffraction patterns of stained and unstained specimens. Diffraction pattern changes due to molecular distortions such as shrinkage or flattening were similarly modeled. Since differences between diffraction patterns of stained and unstained helices may be due to either "edge effects" or molecular distortions, it does not appear possible a priori to distinguish between these effects. Comparison of experimental data for the (2.3 nm)-1 layer-line of tobacco mosaic virus (as model system) reveals major differences between the X-ray diffraction pattern of hydrated sols and the Fourier transform of HREM images of negatively stained specimens.

Microscopy, Electron↗
PubMed1997

Three-dimensional structure of NADH-dehydrogenase from Neurospora crassa by electron microscopy and conical tilt reconstruction.

NADH-dehydrogenase (Complex I) is the first complex of the mitochondrial respiratory chain. It is an amphipatic molecule located in the inner mitochondrial membrane and is composed of at least 35 unique subunits encoded by both mitochondrial and nuclear DNA. The whole complex was isolated in detergent from the fungus Neurospora crassa. It is very stable in its isolated form and was analysed as such by electron microscopy. Its mass, determined by dark-field scanning electron microscopy was estimated as 1.12 MDa. The complex was imaged by transmission electron microscopy, by negative staining and by cryo-electron microscopy. A three-dimensional model, with a resolution estimated at 35 A, was calculated from images of negatively stained complexes by the random conical tilt reconstruction technique. This model confirms the general L-shape of the molecule, with arms of equal length and corroborates the hypothesis of a subdivision of the whole complex into three functional domains. Immuno-labelling of the 49 kDA subunit of the peripheral arm allowed its localization within the complex. This is a first step in the subunit mapping of Complex I and the understanding of its activity.

Animals↗
PubMed1993

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1.

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.

Cell Membrane↗
PubMed1996

Performance of a slow-scan CCD camera for macromolecular imaging in a 400 kV electron cryomicroscope.

The feasibility and limitations of a 1024 x 1024 slow-scan charge-coupled device (CCD) camera were evaluated for imaging in a 400kV electron cryomicroscope. Catalase crystals and amorphous carbon film were used as test specimens. Using catalase crystals, it was found that the finite (24 microns) pixel size of the slow-scan CCD camera governs the ultimate resolution in the acquired images. For instance, spot-scan images of ice-embedded catalase crystals showed resolutions of 8 A and 4 A at effective magnifications of 67,000 x and 132,000 x, respectively. Using an amorphous carbon film, the damping effect of the modulation transfer function (MTF) of the slow-scan CCD camera on the specimen's Fourier spectrum relative to that of the photographic film was evaluated. The MTF of the slow-scan CCD camera fell off more rapidly compared to that of the photographic film and reached the value of 0.2 at the Nyquist frequency. Despite this attenuation, the signal-to-noise ratio of the CCD data, as determined from reflections of negatively-stained catalase crystals, was found to decrease to approximately 50% of that of photographic film data. The phases computed from images of the same negatively-stained catalase crystals recorded consecutively on both the slow-scan CCD camera and photographic film were found to be comparable to each other within 12 degrees. Ways of minimizing the effect of the MTF of the slow-scan CCD camera on the acquired images are also presented.

Animals↗
PubMed1993

Immunocytochemical investigation of the rat cementoblast phenotype.

Recent studies have suggested that cementoblasts may be derived from osteoblast progenitor cells, although the cementoblast phenotype has not been extensively characterized. This immunocytochemical study was carried out to investigate the expression by rat cementoblasts of a number of proteins which are characteristic of the osteoblast phenotype. Paraffin sections from developing rat tooth germs and from fully formed adult rat teeth with surrounding tissues, were incubated with antibodies to type I & III collagen, osteocalcin, transforming growth factor beta (TGE beta), and insulin-like growth factor 1 (IGF1). Frozen sections and unfixed resin-embedded sections were stained for alkaline phosphatase activity. Cementum and bone matrix were strongly positive for type I collagen, although there was only weak staining for type III collagen. Cementum was also positive for osteocalcin, which was particularly strong in the matrix of acellular cementum. Most osteoblasts and cementoblasts of the cellular cementum showed intense staining for TGF beta and IGF1, although some cementocytes and osteocytes were negatively stained. The osteoblast- specific anti-E11 mAb reacted strongly with cementoblasts and newly formed cementocytes in the cellular cementum. Cells associated with acellular cementum did not express TGF beta, IGF1 or stain positively with anti-E11 antibody at any time during root development. Cementoblasts were weakly or negatively stained for alkaline phosphatase in contrast to the osteoblasts examined, which may reflect the low level of synthetic activity in cementoblasts. These results demonstrate that osteoblasts and cementoblasts of cellular cementum share many phenotypic characteristics, and also suggest that there may be phenotypic differences between cementoblasts associated with cellular and acellular cementum.

Animals↗
PubMed1979

Studies on the ultrastructural morphology of Bacteroides nodosus.

The morphology of Bacteroides nodosus was examined with the electron microscope. B nodosus stained with solium phosphotungstate and uranyl acetate possessed fimbriae and in addition organisms negatively stained with sodium phosphotungstate often possessed rings on their surface. Phage-like particles were also observed in negatively stained preparations. In thin sections, B nodosus had a multilayered cell envelope and the type of cell division characteristic of Gram-negative bacteria. The cytoplasmic region contained a diffuse nucleoid area, ribosomes and, sometimes, concentrically arranged membranous lamellae. Fimbriae and capsular material were also seen in sections of B nodosus fixed with glutaraldehyde-osmium. Their visualisation appeared to be enhanced when ruthenium red was incorporated n the glutaraldehyde-osmium fixative but only when sections were stained with heavy metal salts, indicating that the fimbriae and capsule were not predominantly polysaccharide in nature.

Bacteroides↗
PubMed1981

Pseudoleukemia. Ineffective regulation of granulopoiesis.

A neonate with Down's syndrome confirmed by chromosomal analysis had leukocytosis, with a WBC count of 48,000/cu mm, at age 13 days. Differential counts showed 70% to 90% blast forms by light microscopy. The blast-cell granules stained negatively with Sudan black B and alpha-napthyl butyrate esterase. Myeloperoxidase activity evaluated light and electron microscopy was negative. The granules stained positively with PAS, beta-glucuronidase, and tartrate-sensitive acid phosphatase. Indirect immunofluorescence for terminal-deoxynucleotidyl-transferase was negative. Our results show that the blast cells of pseudoleukemia may be of lymphoid origin.

Bone Marrow Cells↗
PubMed2002

Prognostic significance of S100A4 expression in gallbladder cancer.

The calcium-binding protein S100A4 has been characterized as a metastasis-inducing molecule, and regulates cell motility and invasiveness of cancer cells. In order to clarify the significance of the expression of S100A4 as a prognostic factor in gallbladder cancer, S100A4 expression in resected gallbladder cancers were examined using an immunohistochemical staining technique. The relationship between S100A4 expression and clinicopathological factors including prognosis were evaluated. Twenty-five of 60 cases (42%) demonstrated positive staining for S100A4. There was no statistically significant association between S100A4 and histological grade, T, N, M factor, presence of stone, or stage. Kaplan-Meier method showed the 5-year survival rate of the group staining positive for S100A4 (31.5%) to be statistically poorer than that of the group staining negative for S100A4 (78.2%). Also in T2 cases, the 5-year survival rate of the group staining positive for S100A4 (57.1%) was statistically poorer than that of the group staining negative for S100A4 (83.3%). On univariate analysis, positive staining for S100A4 was a significant prognostic factor, and the hazard ratio was 4.05. On multivariate analysis, positive staining for S100A4 is also a significant predictor of prognosis second to T factor. These results indicate that positive staining for S100A4 is useful in assessing the prognosis of patients with gallbladder cancer as well as TNM factors.

Aged↗
PubMed1987

Absence of myelin basic protein in granular cell tumors.

Thirty-one granular cell tumors were studied for the presence of myelin basic protein (MBP) using a monoclonal antibody with a characterized specificity and an immunoperoxidase technique. The tumors studied were from all areas of the body, from both sexes, and from nine decades of age. All the tumors stained negatively for MBP. One brief report using a monoclonal antibody had noted positive staining for MBP in granular cell tumors. A more recent study using polyclonal antibodies found negative staining for MBP in Schwann cell neoplasms including granular cell tumors. This study, using an immunohistochemical technique, demonstrated the absence of an MBP marker in granular cell tumors of diverse location despite the fact that granular cell tumors are widely held to be of Schwann cell origin.

Antibodies, Monoclonal↗
PubMed2004

[Effect of cyclooxygenase-2,and matrix metalloproteinase-2 expression on prognosis of lung cancer].

BACKGROUND & OBJECTIVE: Expression of cyclooxygenase-2 (COX-2)and matrix metalloproteinase-2 (MMP-2)increased in most tumor tissues. Inhibitors of both COX-2 and MMP-2 have been experimentally and clinically proved effective against the development of tumor. Our study was designed to investigate the expression of COX-2 and MMP-2, and the effect of those proteins on lymphoid metastasis and prognosis of lung cancer. METHODS: SP immunohistochemistry was used to determine the expression of COX-2 and MMP-2 in 42 patients with lung cancer. Kaplan-Meier and log-rank test were used to compare the survival time of positive staining group and negative staining group. RESULTS: The positive rate of COX-2 was 31%, and that of MMP-2 was 62%. The survival time in positive COX-2 group was significantly shorter than that in negative COX-2 group (P=0.019). The lymphoid metastasis rate of positive MMP-2 group was significantly higher than that of negative MMP-2 group (P=0.009). The survival time in positive MMP-2 group was significantly shorter than that in negative MMP-2 group (P=0.001). No relationship was found between the expression of COX-2 and MMP-2 (P=0.257). CONCLUSION: The expression of COX-2 and MMP-2 are closely correlate with the prognosis of lung cancer, and the over-expression of those proteins suggest poor prognosis in patients with lung cancer.

Adenocarcinoma↗
PubMed1983

On the state of aggregation of newly secreted procollagen.

Procollagen and partially processed procollagen from cultures of primary chicken embryo tendon cells appeared as segment-long-spacing (SLS)-like aggregates when drops of medium were negatively stained and examined by electron microscopy. Similar aggregates were obtained after negative staining of medium partially purified by gel filtration and also after staining thin sections of fixed, dehydrated, and embedded pellets formed by prolonged ultracentrifugation of whole culture medium. In contrast to results from electron microscopy, analysis by velocity density gradient sedimentation or sedimentation equilibrium indicated the exclusive presence of procollagen or partially processed procollagen monomers in solution. These contradictory data can be reconciled if procollagen exists in monomeric form when greatly diluted (as in culture medium), and in specific aggregated form (SLS) at high concentration. We believe that cells in vivo secrete procollagen in high, local concentration packaged in the SLS form. We propose that such zero-D arrayed packages are the precursors of native collagen fibrils.

Animals↗
PubMed1997

AOM-induced mouse colon tumors do not express full-length APC protein.

While evidence in both sporadic and inherited human colorectal cancer and MIN mice implicate the tumor suppressor gene, APC, in the causation of colorectal carcinogenesis, this gene has not been confirmed to be involved in rodent chemically-induced colon cancer models (RCCM). These experimental models are widely used to elucidate mechanisms involved in colon carcinogenesis (initiation, promotion and progression) as well as studies on chemoprevention (dietary and other) and intervention. To validate the RCCM as relevant models for sporadic human colorectal cancer, and to facilitate research on the role of the APC gene in colon carcinogenesis, we investigated the role of APC in azoxymethane (AOM)-induced colorectal tumors in mice. Using an antibody that recognizes the carboxy terminus of APC, we have characterized the pattern of staining observed in normal mouse intestinal tissue, in MIN mouse intestinal adenomas and in AOM-induced mouse colon tumors. The APC protein was localized in the cytoplasm of normal colonic epithelial cells. In the small intestine there was APC immunoreactivity along the villous and staining of the Paneth cells at the base of the glands. In the proximal and distal colonic crypts there appeared to be a gradient of staining which increased towards the luminal surface. This gradient was not as apparent in the small intestinal villi. Nuclei and mucus in the goblet cells showed no immunoreactivity. MIN mouse small bowel and colonic adenomas, known to have lost APC, stained negatively for APC. AOM-induced adenomas and carcinomas also consistently stained negatively using this antibody. This study demonstrates for the first time the loss of wild-type APC protein in AOM-induced mouse colon tumors and suggests that alterations in expression of this tumor suppressor gene, which is so commonly mutated in human colon cancer, is also involved in this animal model of colon cancer.

Adenoma↗
PubMed1997

The prognostic application of cytokeratin typing of nonsmall cell lung carcinoma. A retrospective study.

BACKGROUND: In a previous study, the authors used a variety of anticytokeratin monoclonal antibodies to show that a large proportion of lung tumors cytologically diagnosed as squamous cell carcinoma contain cells expressing simple epithelial cytokeratins, suggesting that these tumors have their origin in adenocarcinoma. These findings raised the possibility that cytokeratin (CK) typing might have a diagnostic capacity not attainable by standard histopathology. The aim of the current study was to assess the value of CK typing for this purpose by determining the correlation between the diagnosis of lung tumors based on CK typing and the survival rate of the patients. METHODS: Paraffin embedded tissue sections of 66 nonsmall cell lung carcinoma (NSCLC) specimens were examined. These included 18 adenocarcinomas, 32 squamous cell carcinomas, and 16 undifferentiated carcinomas, all diagnosed surgically and histopathologically, and further classified as either Stage I or II. CK typing was performed using the streptavidin-biotin-peroxidase method, employing the following anti-CK monoclonal antibodies: Ks.B.17 (which reacts with CK 18), A3-3 (which reacts with CK 13), and E5-9 (which reacts with CK 10). RESULTS: Comparison between the 5-year survival rates (5 ysr) of patients with different NSCLC indicated that all types of Stage II tumors had a much poorer prognosis than Stage I tumors. Differences found in the 5 ysr among patients with different types of Stage I tumors were not statistically significant (adenocarcinomas, 33% 5 ysr; squamous cell carcinomas, 59% 5 ysr; undifferentiated carcinomas, 36% 5 ysr; all diagnosed by conventional histopathology). Similarly, no significant differences were noted in 5 ysr between patients with tumors stained positively or negatively with monoclonal antibodies A3-3 or E5-9 (anti-CK 13 and anti-CK 10, respectively). In contrast, highly significant differences (P = 0.002) were found in the 5 ysr between patients with Stage I tumors positively or negatively stained with monoclonal antibody Ks.B.17 (23% vs. 75% 5 ysr, respectively) regardless of the histologic types of tumors. Especially informative was a combination of immunohistochemical and histologic diagnoses, with best survival rates (87% 5 ysr) in Ks.B.17 negative tumors histologically diagnosed as Stage I squamous cell carcinomas and worst survival rates (14% 5 ysr) in Ks.B.17 positive tumors diagnosed as adenocarcinomas. CONCLUSIONS: The current study showed that CK 18 typing of lung tumors can provide a more accurate diagnosis and therefore facilitate the planning of more suitable therapeutic approaches.

Adenocarcinoma↗
PubMed1994

Localization of thrombospondin and its cysteine-serine-valine-threonine-cysteine-glycine-specific receptor in human breast carcinoma.

BACKGROUND: Thrombospondin (TSP), a cell-matrix adhesion protein, and cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG), a major TSP cell adhesive domain, have recently been shown to play a role in tumor cell metastasis. In this study we immunohistochemically localized TSP and its newly discovered CSVTCG-specific receptor in normal, benign, and neoplastic breast tissues. EXPERIMENTAL DESIGN: Paraffin sections of normal, benign, and neoplastic breast tissue were examined immunohistochemically for the presence of TSP and its CSVTCG-specific receptor using the avidin-biotin complex immunoperoxidase staining procedure. RESULTS: Positive staining using polyclonal antibodies for TSP and its tumor cell adhesion receptor, isolated from a human adenocarcinoma of the lung, was observed in all primary breast ductal carcinomas examined (N = 11). In contrast, all benign lesions and normal breast tissue stained negative for TSP and its receptor with the exception of two fibrocystic breast samples with hyperplasia. One of the samples showed strong TSP staining of ductal apocrine cells, whereas the other showed apical receptor staining of hyperplastic ductal cells. The negatively staining normal and benign tissues consisted of 1 normal breast, 1 gynecomastia, 5 fibroadenomas, and 6 fibrocystic samples. Positive staining for TSP in ductal carcinoma was only localized in the dense stromal collagen adjacent to tumor, whereas the TSP receptor localized to the tumor cells. Consistent with these immunohistochemical staining results was the observation that protein extracts of breast carcinoma cells contained receptor with no detectable TSP as revealed by Western blotting. Capillary endothelium was focally positive for receptor in regions proximal to ductal epithelium in 8 of 11 neoplastic tissues and in 6 of 14 benign samples. CONCLUSIONS: Our results indicate that increasing expression of stromal TSP and the CSVTCG-specific TSP receptor in ductal epithelium correlates with neoplastic transformation. In addition, our results indicate that both malignant and benign breast tissue can stimulate surrounding capillaries to express the TSP receptor, whereas only carcinoma has the capacity to stimulate surrounding nonendothelial stromal cells, such as myofibroblasts, to secrete a TSP-rich matrix that may contribute to the desmoplastic stromal reaction characteristic of ductal carcinoma tumor. The TSP-rich matrix may then promote tumor cell attachment, migration, and angiogenesis, factors important in tumor growth. The receptor-rich capillary endothelium may promote the cell adhesive interactions important in tumor intravasation. Taken together the results of this study provide a rational basis for a role of TSP in tumor angiogenesis and metastasis.

Amino Acid Sequence↗