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At least 649 records · Page 36Linked to original sources
PubMed2000

Gelsolin inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils.

Amyloid beta-protein (Abeta) is present in soluble form in the plasma and cerebrospinal fluid (CSF) of normal people and patients with Alzheimer's disease (AD). However, in AD patients, Abeta gets fibrillized as the main constituent of amyloid plaques in the brain. Soluble synthetic Abeta also forms amyloid-like fibrils when it is allowed to age. The mechanism that prevents soluble Abeta from fibrillization in biological fluids is not clear. We recently reported that gelsolin, a secretory protein, binds to Abeta, and that gelsolin/Abeta complex is present in the plasma [V.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Biochem. Biophys. Res. Commun. 258 (1999) 241-246.]. We now studied the effect of gelsolin on Abeta fibrillization. Congo red staining and electron microscopic examination in negative staining of aged samples of Abeta alone and Abeta incubated with gelsolin showed that gelsolin inhibits the fibrillization of synthetic Abeta 1-40 and Abeta 1-42 at gelsolin to Abeta molar ratio of 1:40. In addition, gelsolin also defibrillized the preformed fibrils of Abeta 1-40 and Abeta 1-42 in a time-dependent manner. These results suggest that gelsolin functions as an anti-amyloidogenic protein in the plasma and CSF, where it prevents Abeta from fibrillization, and helps to maintain it in the soluble form.

Amyloid beta-Peptides↗
PubMed2005

Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes.

The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.

Amino Acid Sequence↗
PubMed1981

Characterization and fractionation of rat liver nuclear matrix.

The rat liver nuclear matrix retains the shape of the nucleus and reveals a sponge-like structure in negative staining and scanning electron microscopy. A fibrous layer (dense lamina) with associated pore complexes is preserved on the surface of the nuclear matrix. In negative staining as well as in high resolution scanning electron microscopy this layer is perceived as a network consisting of alveoli of 10 to 30 nm in diameter with pore complexes differing in arrangement of the annular granules. In sections a fibrous layer 15 to 30 nm in width granules of 7 to 10 nm in diameter can be observed. Structures similar to the pore complexes are revealed not only at the fibrous layer but also inside the nucleus and in close proximity to the nucleoli. Four fractions of the nuclear matrix have been isolated by successive extraction with 0.05 M EDTA and 0.025 N NaOH. Almost 80% of the total matrix protein dissolves in dilute alkali. 4 to 5% of the total matrix protein is soluble in EDTA. From insoluble residue two fractions can be isolated: one rich in pore complexes and another one retaining the shape of the nucleus with spongy or alveolar structure. The latter fraction is regarded to constitute a proper framework or skeleton of the nucleus.

Amino Acids↗
PubMed1977

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation.

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.

Animals↗
PubMed1999

Expression of pepsinogen C in gynecomastias and male breast carcinomas.

Pepsinogen C is a proteolytic enzyme involved in the digestion of proteins in the stomach; it is also synthesized by a significant percentage of female breast carcinomas. In addition, it has been demonstrated that pepsinogen C is one of the few proteins induced by androgens in breast carcinoma cells. Here we evaluate the expression of pepsinogen C by immunoperoxidase staining in normal breast tissue from 3 male patients, 15 gynecomastia tissues, 2 male in situ breast carcinomas, and 68 male invasive breast carcinomas. Pepsinogen C immunostaining values were quantified in male breast tumors using the HSCORE system, which considers both the intensity and the percentage of cells staining at each intensity. The results indicated positive immunohistochemical staining for pepsinogen C in all gynecomastia tissues, the two in situ ductal carcinomas, and 52 of 68 invasive breast carcinomas (76.4%). The three normal breast tissues analyzed showed negative staining for pepsinogen C, whereas invasive tumors showed clear differences among them with regard to the intensity and percentage of staining cells. In addition, pepsinogen C scores were significantly higher in well-differentiated (grade I, 188.7) and moderately differentiated (grade II, 145.8) tumors than in poorly differentiated (grade III, 98.5) tumors (p = 0. 032). Similarly, significant differences in pepsinogen C content were found between estrogen receptor (ER)-positive tumors and ER-negative tumors (158.5 vs. 44.3, respectively; p = 0.009). Patients with pepsinogen C-positive tumors reached longer relapse-free and overall survival periods than did those with tumors with negative staining, but no statistical differences were observed between survival curves calculated for these two groups of patients. This results demonstrate expression of pepsinogen C by gynecomastias and by a high percentage of male breast carcinomas and may indicate an important role of pepsinogen C in the pathophysiology of male breast diseases.

Adolescent↗
PubMedDate not supplied

The limitations of immunoenzyme approaches to distinguish between 'specific' and 'non-specific' antibody-forming cells, with particular respect to immunocytochemical studies on the in situ immune response.

After antigenic stimulation, specific antibody-forming cells can be detected in situ in tissue sections of lymphoid organs using an antigen specific immunoenzyme approach. Immunoglobulin-forming cells staining positively with a particular antigen-enzyme conjugate are considered to be specific antibody-forming cells (provided that the right control conjugates show a negative staining). Immunoglobulin-forming cells, staining negatively with the same antigen-enzyme conjugate, may do so for two reasons: (i) they are in fact specific antibody-forming cells, but are not detected as such because of limitations of the technique; (ii) they are real-nonspecific immunoglobulin producing cells. In this paper, the limitations of the technique are discussed.

Animals↗
PubMed1975

Cytochemical studies on sarcoplasmic reticulum of heart and skeletal muscle.

Ultrastructural aspects and cytochemical localization of Ca uptake and of -SH groups involved in the calcium transport of vesicular fragments of sarcoplasmic reticulum (SR) from rabbit skeletal muscle and from dog myocardium were studied by positive and various negatively-staining techniques. Size, shape, and membrane structure as well as proportion of calcium-containing vesicles vary a great deal depending on the method applied. On negatively stained material sarcoplasmic reticulum derivatives are identifiable in both skeletal muscle as well as in heart preparations by the presence on the surface of the vesicular membrane of 40-A particles and the capability of accumulating calcium. The amount of these vesicles is lower in cardiac SR than in the skeletal SR; however, no qualitative differences can be observed. Vesicles of cardiac SR labeled with the electron dense -SH group reagent, Hg-phenyl azoferritin, also bind asymmetrically the marker only at the outer surface of the membrane-like microsomes of skeletal muscle.

Animals↗
PubMed2004

[Effect of fixation methods on immunocytochemical localization of NMDAR1 on cultured cortical neuron membrane].

OBJECTIVE: To investigate the changes in subcellular localization of NMDAR1 on cultured cortical neurons in response to different methods for fixation of neuron cells. METHODS: Subcellular localization of NMDAR1 in cultured cortical neurons fixed with different fixatives and procedures was studied by immunocytochemical avidin-biotin peroxidase (ABC) method. RESULTS: Neurons fixed with -20 degrees Celsius pure acetone and -20 degrees Celsius pure methanol for 5 min presented typical positive staining of NR1 subunit located on the polar membrane of the neurons where the dendrites originated and on the stem of the dendrites. The neurons fixed with 4% formaldehyde alone for 20 min or in the presence of 0.5% glutaraldehyde (1:1) for 10 min resulted in false-negative staining. Fixation of the neurons with 95% ethanol for 10 min yielded false-negative staining on the membrane and false-positive staining in the nuclei. CONCLUSIONS: Each antigen may have its specific demand for fixation method in immunocytochemistry, and for NMDAR1 antigen, mild fixation is recommended as by -20 degrees Celsius pure acetone and -20 degrees Celsius pure methanol for 5 min. NMDAR1 distributes on the polar membrane of the neurons where the dendrites originate and on the dendritic stem.

Animals↗
PubMed1998

Immunohistochemical staining for desmogleins 1 and 2 in keratinocytic neoplasms with squamous phenotype: actinic keratosis, keratoacanthoma and squamous cell carcinoma of the skin.

Desmosomes are intercellular junctions that have been shown to be down-regulated in certain types of carcinoma and that may play a role in suppression of invasion and metastasis. This paper describes an immunohistochemical study of three types of epidermal neoplasms with monoclonal antibody to desmoglein in order to determine how desmosomal staining correlates with the clinical, biological and histopathological features of these neoplasms. Actinic keratosis (AK) is the most common keratinocytic premalignant neoplasm that was reported to have a 10-20% rate of malignant transformation into squamous cell carcinoma (SCC). Keratoacanthoma (KA) is a benign neoplasm that involutes spontaneously after a few months of rapid growth. SCC is a malignant tumour capable of metastasis. Electron microscope studies of KA and SCC showed significantly reduced staining for desmosomes in SCC but not in KA. We have examined staining for desmoglein using the monoclonal antibody 33-3D, a mouse IgM monoclonal antibody, that recognizes the cytoplasmic domains of desmoglein (Dsg)1 and Dsg2 on frozen sections. Immunohistochemical staining of normal skin with this antibody revealed strong pericellular localization of the antigen, outlining the cell membranes of the keratinocytes. A series of 30 AKs, 12 KAs and 24 SCCs was stained immunohistochemically with 33-3D monoclonal antibody. All examined KAs showed extensive pericellular staining for Dsg. By contrast, juxtanuclear staining for Dsg was noted in 12 SCCs, and completely negative staining in seven SCCs. The five remaining SCCs showed focal pericellular staining for the Dsg marker. The most common finding in AK was focal pericellular staining for Dsg, with complete absence of staining in dysplastic areas (25 cases). In five cases negative pericellular staining in dysplastic areas was associated with juxtanuclear accumulation of the Dsg marker. A strong negative correlation between Dsg staining and degree of dysplasia was obtained. The Dsg pattern in KA is similar to normal epidermis and shows a clear difference between KA and SCC. AK has a limited loss of Dsg expression in a SCC-like pattern that is congruent with its premalignant nature. As the stain works on frozen tissue, it may be helpful for rapid differentiation in selected cases in cutaneous oncology and Mohs micrographic surgery. This antibody may also have great potential for the detection of the effects of chemopreventive agents in skin cancer.

Animals↗
PubMed1993

The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity.

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.

Animals↗
PubMed1964

FINE STRUCTURE OF THE GRAM-NEGATIVE BACTERIUM ACETOBACTER SUBOXYDANS.

The morphological features of the cell wall, plasma membrane, protoplasmic constituents, and flagella of Acetobacter suboxydans (ATCC 621) were studied by thin sectioning and negative staining. Thin sections of the cell wall demonstrate an outer membrane and an inner, more homogeneous layer. These observations are consistent with those of isolated, gram-negative cell-wall ghosts and the chemical analyses of gram-negative cell walls. Certain functional attributes of the cell-wall inner layer and the structural comparisons of gram-negative and gram-positive cell walls are considered. The plasma membrane is similar in appearance to the membrane of the cell wall and is occasionally found to be folded into the cytoplasm. Certain features of the protoplasm are described and discussed, including the diffuse states of the chromatinic material that appear to be correlated with the length of the cell and a polar differentiation in the area of expected flagellar attachment. Although the flagella appear hollow in thin sections, negative staining of isolated flagella does not substantiate this finding. Severe physical treatment occasionally produces a localized penetration into the central region of the flagellum, the diameter of which is much smaller then that expected from sections. A possible explanation of this apparent discrepancy is discussed.

Acetobacter↗
PubMed1983

A survey of 50 S ribosomal subunits by dark field electron microscopy.

A survey of dark field electron micrographs of the 50 S ribosomal subunit of E. coli has been performed and supplemented, for comparative purposes, by examination of negatively stained or metal shadowed specimens in the bright field mode. Attention was directed to the so-called "crown" and "kidney" views. The elongated appendage seen in negatively stained crown profiles was not observed in unstained or positively stained samples examined in dark field; these showed only symmetrical crown profiles regardless of changes in buffer type and drying method and of the presence or absence of uranyl acetate treatment and glutaraldehyde fixation. The crown view occasionally displayed a bifurcation in one of the lateral lobes, while the kidney profile showed a groove near the base of the convex edge. Uranyl acetate treatment produced delicate stripes which may give an indication of the surface RNA distribution.

Escherichia coli↗
PubMed1990

Testicular sex cord stromal tumour with granulosa cell differentiation: detection of steroid hormone receptors as a possible basis for tumour development and therapeutic management.

A testicular sex cord stromal tumour with granulosa cell differentiation, typical of granulosa cell tumours of the adult type, was investigated immunohistologically on snap frozen and paraffin wax embedded material. The predominance of vimentin and the additional expression of cytokeratin subtypes 8 and 18, as well as the negative staining for epithelial membrane antigen, accorded with results previously reported, for ovarian granulosa cell tumours; the lack of expression of desmoplakin, however, was a distinctive feature. Together with negative staining for leucocyte common antigen, the antigen pattern facilitates the differential diagnosis between granulosa cell tumour and undifferentiated carcinoma or gonadal lymphoma, although its suitability for differentiating within the group of gonadal stromal tumours seems to be limited. The small growth fraction, shown by the monoclonal antibody Ki-67, is typical of the clinical behaviour of granulosa cell tumours. The expression of oestrogen and progesterone receptors, also recently found in testicular Leydig cell tumours, may provoke new approaches to the management of testicular granulosa cell tumours, as well as a new hypothesis on the development of these tumours.

Aged↗
PubMed1985

Role of macrophages in atherosclerosis. Sequential observations of cholesterol-induced rabbit aortic lesion by the immunoperoxidase technique using monoclonal antimacrophage antibody.

To elucidate the role of macrophages in atherosclerosis, sequential observations were carried out on cholesterol-induced aortic lesions of the rabbit with the immunoperoxidase technique by use of monoclonal antimacrophage antibody. Animals on cholesterol diets for 8 weeks or longer showed increased accumulations of lipid-filled macrophages in the intima. At a very early stage, when no grossly visible alterations were observed, macrophages were seen clinging to the endothelial surface with apparent penetration of the endothelium. A single line of three or four vacuolated macrophages were found in otherwise normal subendothelial spaces. In flat lesions consisting of a few layers of foam cells, lipid-laden macrophages were the cells that predominated. In advanced plaque lesions, negatively staining, most presumably, smooth muscle foam cells became noticeable and increased in number. At this stage, macrophage foam cells predominated in the superficial layer of the lesion, whereas negatively staining foam cells were prevalent in deeper areas. Macrophages were also spotted within areas of necrosis, and they were occasionally observed near the necrotic core of the atheroma. During 24 to 74 weeks after termination of the cholesterol diet, the number of cells with specific macrophage staining markedly diminished. The results support the concept that circulating monocytes are the prime source of foam cells in the initial phase of atherogenesis. It seems likely that the major role of macrophages in atherogenesis is to remove lipids from areas of lesion formation. The failure of macrophages to perform their scavenger role successfully may be a major factor in the accumulation of lipid-rich debris in advanced lesions. Relative paucity of macrophages may also be an important factor explaining why regression of atherosclerosis occurs rarely in the rabbits.

Animals↗
PubMed1984

Polarity of the 14-strand fibers of sickle cell hemoglobin determined by cross-correlation methods.

Images of negatively stained fibers of sickle cell hemoglobin have been analyzed by cross-correlation methods. These methods are used to compensate for the curvature and variable repeat distance characteristic of negatively stained fibers. Averaged images obtained by the correlation procedure display considerably more detail than the filtered images obtained earlier by Fourier methods. The averaged images are sufficiently detailed that the back-projection method for obtaining cross-sections can now be applied directly to the correlation-averaged images. This method was used previously to deduce the 14-strand structure on Fourier-filtered images that incorporated only the near-equatorial maxima. In this way the 14-strand structure has been reconfirmed without utilizing the partial Fourier reconstructions employed earlier that might conceivably have introduced spurious features. In addition, application of the correlation procedure with and without inversion of the reference reveals a consistent polarity in all of the fibers examined. The confirmation of the 14-strand structure by a new procedure and the determination of fiber polarity would appear to eliminate the alternative model of the fibers with a 16-strand structure with equal numbers of strands (eight) of each polarity.

Crystallography↗
PubMed1985

Three-dimensional molecular shape determination from a limited number of projections.

A real space method allowing the reconstruction of negatively stained crystalline objects from a limited number of projections is presented. The method is based on the assumption that only two density levels are required to describe an ideally negatively stained object (that of the volume occupied by the object and that of the volume occupied by the stain). The method is illustrated by the reconstruction of the asymmetric unit of catalase microcrystals using only the three principal projections. It is shown that other projections can then be predicted to a good approximation.

Animals↗
PubMed1971

Structure of the Chromatium sulfur particle and its protein membrane.

Sulfur particles extracted from Chromatium vinosum strain D were found to be bounded by a unique proteinaceous membrane. Ultrastructural examination of the membrane in Epon sections and bovine serum albumin sections and examination of negatively stained, sulfur-free membrane ghosts revealed a monomolecular sheet composed of 2.5-nm globular components. The internal sulfur was found to bind large amounts of a variety of negative stains and to form myelin-like structures upon rupture of the surrounding membrane.

Bacterial Chromatophores↗
PubMed1984

Further evidence for the B-cell nature of hairy cells. A study using immunostaining of splenic tissue with a wide panel of monoclonal antibodies.

Phenotypic characterization of neoplastic cells from 5 patients with hairy cell leukemia (HCL) was performed with 29 monoclonal and 6 polyclonal (anti-Ig) antibodies using immunoperoxidase staining of fresh frozen splenic tissue. Monotypic Ig was expressed in 4 cases, one case was non-expressive. Strong staining was obtained in all cases by monoclonal antibodies (MAs) specific for 3 pan-B-lymphocyte antigens (by anti-B 1, To 15, anti-Leu 12). Five other B-cell related antigens detectable with appropriate MAs (BA-1, anti-B2, DAKO-C3 b R, Tü 1, 38.13) were absent in all cases. The stainings with 13 T-cell associated MAs (OKT 3, OKT 4, anti-Leu 3 a, OKT 6, OKT 8, Tü 68, OKT 10, anti-Lyt 2, Tü 71, OKT 11, anti-Lyt 3, Tü 14, Tü 33) were all negative. Stainings with 4 MAs recognizing myelocytic and/or monocytic antigens (OKM 1, anti-Mo 1, anti-Mo 2, 3 C4) were also negative. We included 14 frozen biopsies with B-type chronic lymphatic leukemia (B-CLL) into our immunohistological study in order to establish phenotypic differences between HCL and B-CLL. Five MAs (Tü 1, anti-Lyt 2, Tü 71, BA-1 and anti-B2) gave consistently negative staining in HCL cases but positive staining in most or all B-CLL cases. The study provides significant evidence for the B-cell nature of HCL and also establishes important phenotypic differences between HCL and B-CLL.

Adult↗